Posts Tagged ‘virus’

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Once herpes simplex infects a person, the virus goes into hiding inside nerve cells, hibernating there for life, periodically waking up from its sleep to reignite infection, causing cold sores or genital lesions to recur. Research from Harvard Medical School showed that the virus uses a host protein called CTCF, or cellular CCCTC-binding factor, to display this type of behavior. Researchers revealed with experiments on mice that CTCF helps herpes simplex regulate its own sleep-wake cycle, enabling the virus to establish latent infections in the body’s sensory neurons where it remains dormant until reactivated. Preventing that latency-regulating protein from binding to the virus’s DNA, weakened the virus’s ability to come out of hiding.


Herpes simplex virus’s ability to go in and out of hiding is a key survival strategy that ensures its propagation from one host to the next. Such symptom-free latency allows the virus to remain out of the reach of the immune system most of the time, while its periodic reactivation ensures that it can continue to spread from one person to the next. On one hand, so-called latency-associated transcript genes, or LAT genes, turn off the transcription of viral RNA, inducing the virus to go into hibernation, or latency. On the other hand, a protein made by a gene called ICP0 promotes the activity of genes that stimulate viral replication and causes active infection.


Based on these earlier findings, the new study revealed that this balancing act is enabled by the CTCF protein when it binds to the viral DNA. Present during latent or dormant infections, CTCF is lost during active, symptomatic infections. The researchers created an altered version of the virus that lacked two of the CTCF binding sites. The absence of the binding sites made no difference in early-stage or acute infections. Similar results were found in infected cultured human nerve cells (trigeminal ganglia) and infected mice model. The researchers concluded that the mutant virus was found to have significantly weakened reactivation capacity.


Taken together, the experiments showed that deleting the CTCF binding sites weakened the virus’s ability to wake up from its dormant state thereby establishing the evidence that the CTCF protein is a key regulator of sleep-wake cycle in herpes simplex infections.





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Phosphorylation-dependent interaction between antigenic peptides and MHC class I

Curator: Larry H. Bernstein, MD, FCAP



Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self.

Nat Immunol. 2008 Nov;9(11):1236-43.  Epub 2008 Oct 5.
Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of ‘transformed self’. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide-HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.
Figure 1
Bioinformatic characterization of the HLA-A2–restricted phosphopeptide repertoire. (a) Distribution of phosphorylated residues among naturally processed (A2 phosphopeptide) and predicted HLA-A2 binding phosphopeptides (Phosphosite, EMBL). The frequency of phosphorylated residues at each position is displayed for naturally processed HLA-A2 associated phosphopeptides, and for peptides in EMBL and Phosphosite datasets that contain phosphorylation sites and are predicted, according to criteria described in Methods, to bind HLA-A2. (b) Representation of positively charged residues (Arg or Lys) at P1 among naturally processed HLA-A2 associated phosphopeptides, phosphopeptides from the EMBL or Phosphosite datasets that are predicted to bind HLA-A2 and contain a p-Ser residue at the P4 position, and datasets of naturally processed non-phosphorylated peptides (B-LCL) and known HLA-A2 binding peptides (Immune Epitope). Selection criteria for the latter two datasets are described in Methods. * = P<0.001, NS= not significant. (c, d) Representation of subdominant residues at the P2 anchor position (c) and the PC (P9) position (d) in naturally processed HLA-A2 associated phosphopeptides and in datasets of naturally processed non-phosphorylated peptides and known HLA-A2 binding peptides.
Changes in protein expression or metabolism due to intracellular infection or cellular transformation modify the repertoire of peptides generated and therefore displayed by class I MHC molecules, resulting in presentation of “altered self” to the immune system. T cell receptor (TCR)-mediated recognition of specific MHC-bound peptides by CD8 T lymphocytes results in cytolytic activity and release of pro-inflammatory cytokines, which are key components of anti-viral and anti-tumor immunity. Evidence suggests that peptides containing post-translational modifications (PTM), including deamidation, cysteinylation, glycosylation, and phosphorylation, contribute to the pool of MHC-bound peptides presented at the cell surface and represent potential targets for T cell recognition2. Indeed, the majority of naturally occurring PTM-bearing peptides defined to date can be discriminated from their unmodified homologs specifically by T cells2-4.  …..
Recent studies have highlighted protein phosphorylation as a process with the capacity to generate unique peptides bound to class I MHC molecules. Significant numbers of different phosphorylated peptides are presented by several HLA-A and HLA-B alleles that are prevalent in humans3,4, demonstrating their widespread potential as antigens. Moreover, CD8+ T lymphocytes recognize these phosphopeptides in a manner that is both peptide sequence-specific and phosphate-dependent3, 4. Thus, phosphopeptides can be immunologically distinguished from their non-phosphorylated counterparts. Consistent with their presentation by class I MHC molecules, most phosphorylated peptides are derived from proteins that function intracellularly, and processing of both model and naturally occurring phosphopeptides is dependent on transport into the endoplasmic reticulum (ER) by transporter associated with antigen processing (TAP)3, 5. Furthermore, rapid degradation by the proteasome, a process that regulates the activity of many transcription factors, cell growth modulators, signal transducers and cell cycle proteins6-8, is frequently dependent on target protein phosphorylation9-11. ….
Phosphopeptide antigens are of significant therapeutic interest because deregulation of protein kinase activity, normally tightly controlled, is one of the hallmarks of malignant transformation and is thought to contribute directly to oncogenic signaling pathways involved in cell growth, differentiation and survival13-15. In addition, mutation-induced deregulation of a limited number of critical kinases can often lead to activation of several signaling cascades and increases in the extent of protein phosphorylation within the cell16-18. These considerations strongly suggest that alterations in protein phosphorylation during malignancy represent a distinctive immunological signature of “transformed self”. Consistent with this notion, the phosphopeptides presented by HLA-A*0201….

Nα-Terminal Acetylation for T Cell Recognition: Molecular Basis of MHC Class I–Restricted Nα-Acetylpeptide Presentation

As one of the most common posttranslational modifications (PTMs) of eukaryotic proteins, Nα-terminal acetylation (Nt-acetylation) generates a class of Nα-acetylpeptides that are known to be presented by MHC class I at the cell surface. Although such PTM plays a pivotal role in adjusting proteolysis, the molecular basis for the presentation and T cell recognition of Nα-acetylpeptides remains largely unknown. In this study, we determined a high-resolution crystallographic structure of HLA (HLA)-B*3901 complexed with an Nα-acetylpeptide derived from natural cellular processing, also in comparison with the unmodified-peptide complex. Unlike the α-amino–free P1 residues of unmodified peptide, of which the α-amino group inserts into pocket A of the Ag-binding groove, the Nα-linked acetyl of the acetylated P1-Ser protrudes out of the groove for T cell recognition. Moreover, the Nt-acetylation not only alters the conformation of the peptide but also switches the residues in the α1-helix of HLA-B*3901, which may impact the T cell engagement. The thermostability measurements of complexes between Nα-acetylpeptides and a series of MHC class I molecules derived from different species reveal reduced stability. Our findings provide the insight into the mode of Nα-acetylpeptide–specific presentation by classical MHC class I molecules and shed light on the potential of acetylepitope-based immune intervene and vaccine development.

Produced by Ag processing and proteasomal degradation of intracellular proteins, polypeptides serve as CTL epitopes presented by MHC class I molecules, which play a critical role in cellular immunity (1). Eukaryotic proteins bearing various posttranslational modifications (PTMs) can generate a group of modified Ags, which contribute to a special repertoire of MHC-associated peptides presented at the cell surface as potential targets for TCR-mediated recognition. A modified peptide may become a new Ag because of the distinguished antigenicity compared with its unmodified homolog. A variety of natural peptide Ags containing modification have been observed that can be immunologically discriminated by T cells from their unmodified homologs as “altered self” (2). Thus, the significance of PTMs on epitopes and the application of modified peptides in vaccine development for immunotherapy against cancer and autoimmune diseases have been increasingly appreciated (3, 4).

The molecular bases of the presentation of peptides with several PTMs by MHC class I molecules have been successfully explicated. For instance, the formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of H2-M3 (5); both the glycan and the phosphate moieties of the central region of the glycopeptides (6, 7) and the phosphopeptides (8, 9), respectively, are exposed to enable TCR binding, and the deimination (citrullination) of arginine on a peptide presented by two HLA-B27 subtypes induces distinct peptide conformations (10).

Nα-terminal acetylation (Nt-acetylation) is one of the most common PTMs, occurring on the vast majority of eukaryotic proteins. In humans, >80% of the different varieties of intracellular proteins are irreversibly Nt-acetylated by Nα-acetyltransferases, often after the removal of the initiator methionine. Only a subset of the penultimate residues (Ala, Ser, Thr, Cys, and Val) or the retained initiator methionine can be acetylated at the α-amino (NH2) groups (11). A recent study found that acetylated N-terminal residues of eukaryotic proteins act as specific degradation signals (Ac-N-degrons) that are recognized by specific ubiquitin ligases (12). A subsequent systematic analysis demonstrated that Nt-acetylation can also represent an early determining factor in the cellular sorting for prevention of protein targeting to the secretory pathway (13). These findings suggested that Nt-acetylation–mediated inhibition of secretion could contribute to the retention of proteins in the cytosol where they may subsequently be ubiquitinylated through the specific recognition of their Ac-N-degrons and thereby generating Nt-acetylated proteasomal digestion products (14). Hence, these Nt-acetylated polypeptides in the form of MHC-associated neoantigens stand a good chance to be recognized by T cells. This has indeed been illuminated in an Nt-acetylated MHC class II–restricted peptide derived from myelin basic protein, which stimulates murine T cells to elicit experimental autoimmune encephalomyelitis, whereas the nonacetylated form does not (15). A structural study subsequently suggested that the Nt-acetylation of this peptide is essential for MHC class II binding (16).

For MHC class I, the first Nt-acetylated natural ligand was identified more than a decade ago (17). However, the mode of interaction of this acetylated peptide with class I molecules remained largely enigmatic. To understand this, we determined the crystal structures of a naturally occurring Nt-acetylated self-peptide (NAc-SL9) and two nonmodified variants (SL9 and HL8), respectively, in complex with HLA-B*3901. Taken together with the thermostability analyses of Nα-acetylpeptides complexed with a series of class I molecules of human and murine origin, we elucidated that Nt-acetylation exerts a destabilizing effect on peptide–MHC (pMHC) complex, thereby influencing TCR recognition.


Our results here provide the structural and thermodynamic insights into the presentation of Nt-acetylated peptides by MHC class I molecules. The structure of the Nα-acetylpeptide in complex with HLA-B*3901 outlines a molecular interpretation of the reduced stability of MHC class I–bound Nt-acetylated peptides and also highlights a potential influence of Nt-acetylation on antigenic identity and T cell recognition. In addition, the structure elucidation of HLA-B*3901, the predominant B39 subtype, also is valuable in studying immune diseases associated with this MHC allele.

In a previous report, the Nt-formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of the murine MHC class I H2-M3 playing an anchoring role for MHC class I binding (Supplemental Fig. 2A) (5). In our study, the methyl and carbonyl groups of the acetyl are rotated upwards like two arms that push the peptide-binding groove open (Fig. 2G, Supplemental Fig. 2B), thereby altering its immunogenicity at the expense of the pMHC stability. The thermostability we tested from seven human and one murine complexes indicates a general feature of Nα-acetylpeptide in weakening the binding affinity to MHC class I, which could be revealed by the gel-filtration chromatography of pMHC refolding assays as well (Supplemental Fig. 3). Their instability would partially explain why, as yet, such epitopes are rarely found. Within N-terminal residues of eukaryotic proteins, Ser is the most frequently acetylated in vivo (11). The Ala, Thr, Cys, and Val residues can also be Nt-acetylated and have small side chains like Ser. Thus, the rotation of P1 residues observed in the pHLA-B*3901 complex with an acetylated P1-Ser could very well be a general mode in Nα-acetylpeptide binding. In contrast, the long side chain of Met precludes it from being rotated into pocket A, but a certain reorientation is presumed to take place in the acetylated P1-Met based on the thermal instability (Fig. 6H). Besides the accommodation of the acetyl moiety, Nt-acetylation is presumed to decrease the stability of the pHLA-B*3901 complex as a result of the conformational switch of the Arg62. Arg62 in the α1-helix is largely conserved in almost all HLA-B and -C allotypes (Table V). For other HLA class I (Table V, Fig. 8), the long charged side chains of the residues in position 62 (Glu62 of A24 and Gln62 of A11 and so on) also may interact with the acetyl. Hence, the residue in position 62 plays a key role in the interaction between acetyl group and the H chain, which may influence not only the Nα-acetylpeptide binding to HLA molecules but also the TCR docking.

The discoveries that intracellular proteins with Ac-N-degrons are inhibited from being secreted (13) and then are degraded via ubiquitylation (12) raise many questions on the biological significance of acetylation-mediated proteolysis (14). The Nt-acetylated peptides with the size of MHC class I ligands (8–11 aa) as neoepitopes for CD8+ T cells, represent one of the possible roles of the Nt-acetylated digestion products. The vast armory of intracellular proteins that are frequently Nt-acetylated can create a large pool of Nα-acetylpeptides for Ag presentation and T cell surveying. The Nt-acetylation potentially impacts the TCR-MHC interaction in three different aspects: 1) the direct interaction of the solvent-exposed acetyl moiety; 2) the altered conformation of the central region of the peptide main chain; and 3) the conformational switches of the MHC residues. The Nt-acetylation creation of a distinctive pMHC landscape and participation in a potential binding element for TCR engagement described in our results highlights needs for further investigation into the Nα-acetylpeptide–specific TCR repertoires.  ……

see…J Immunol 2014; 192:5509-5519



The Cellular Redox Environment Alters Antigen Presentation*

Jonathan A. Trujillo,§12Nathan P. Croft,1Nadine L. Dudek,1Rudragouda ChannappanavarAlex TheodossisAndrew I. Webb,…., Jamie Rossjohn,‡‡,§§5Stanley Perlman,§6 and Anthony W. Purcell,7
The Journal of Biological Chemistry 289; 27979-27991.


Background: Modification of cysteine residues, including glutathionylation, commonly occurs in peptides bound to and presented by MHC molecules.

Results: Glutathionylation of a coronavirus-specific T cell epitope results in diminished CD8 T cell recognition.

Conclusion: Cysteine modification of a T cell epitope negatively impacts the host immune response.

Significance: Cross-talk between virus-induced oxidative stress and the T cell response probably occurs, diminishing host cell recognition of infected cells.

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5–10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.

Antigen Presentation     Antigen Processing     Glutathionylation     Mass Spectrometry (MS)     Oxidation-Reduction (Redox)     Redox Regulation     T-cell     Viral Immunology

Small fragments of proteins (peptides) derived from both intracellular and extracellular sources are displayed on the surface of cells by molecules encoded within the major histocompatibility complex (MHC). These peptides are recognized by T lymphocytes and provide the immune system with a surveillance mechanism for the detection of pathogens and cancer cells. The fidelity with which antigen presentation communicates changes in the intracellular proteome is critical for immune surveillance. Not only do antigens expressed at vastly different abundances need to be represented within the array of peptides selected and presented at the cell surface (collectively termed the immunopeptidome (1, 2)), but changes in their post-translational state also need to be conveyed within this complex mixture of peptides. For example, changes in antigen phosphorylation have been linked to cancer, and the presentation of phosphorylated peptides has been shown to communicate the cancerous state of cells to the immune system (36). Other types of post-translational modification play a central role in the pathogenesis of autoimmune diseases (7), such as arginine citrullination in arthritis (810), deamidation of glutamine residues in wheat proteins in celiac disease (1115), and cysteine oxidation in type 1 diabetes (16, 17). Cysteine is predicted to be present in up to 14% of potential T cell epitopes based on its prevalence in various pathogen and host proteomes (18). However, reports of cysteine-containing epitopes are much less frequent due to technical difficulties associated with synthesis and handling of cysteine-containing peptides and their subsequent avoidance in many epitope mapping studies (19). Cysteine can be modified in numerous ways, including cysteinylation (the disulfide linkage of free cysteine to peptide or protein cysteine residues), oxidation to cysteine sulfenic (oxidation), sulfinic (dioxidation) and sulfonic acids (trioxidation), S-nitrosylation, and S-glutathionylation. Such modifications may occur prior to or during antigen processing; however, the role of cysteine modification in T-cell-mediated immunity has not been systematically addressed.

In addition to constitutive presentation of a subset of oxidatively modified peptides, it is anticipated that changes in the proportion of these ligands will occur upon infection because oxidative stress, triggering of the unfolded protein response, and modulation of host cell synthesis by the virus are hallmarks of this process (2027). For example, host cell stress responses modulate expression, localization, and function of Toll-like receptors, a key event in the initiation of the immune response (28). Oxidative stress would also be predicted to affect protein function through post-translational modification of amino acids, such as cysteine. Indeed, because of the reactive nature of cysteine and the requirements for cells to regulate the redox state of proteins to maintain function, a number of scavenging systems for redox-reactive intermediates exist. The tripeptide glutathione (GSH) is one of the key intracellular antioxidants, acting as a scavenger for reactive oxygen species. Reduced GSH is equilibrated with its oxidized form, GSSG, with normal cytosolic conditions being that of the reduced state in a ratio of ∼50:1 (GSH/GSSG) (29). Modification of proteins and peptides with GSH (termed S-glutathionylation) occurs following reaction of GSSG with the thiol group of cysteine in a reaction catalyzed by the detoxifying enzyme, glutathione S-transferase (GST). A variety of cellular processes and signaling pathways, such as the induction of innate immunity, apoptosis, redox homeostasis, and cytokine production, are modulated by this GST-catalyzed post-translational modification (3032). S-Glutathionylation can eventuate via oxidative stress, whereby the intracellular levels of GSSG increase.

Given that viruses are known to induce oxidative stress (3335), the intracellular environment of viral infection may lead to an increase inS-glutathionylated cellular proteins and viral antigens. For instance, HSV infection induces an early burst of reactive oxygen species, resulting in S-glutathionylation of TRAF family members, which in turn is linked to downstream signaling and interferon production (36). The potential for modification of viral antigens subsequent to reactive oxygen species production is highlighted by S-glutathionylation of several retroviral proteases, leading to host modulation of protease function (37). Indeed large scale changes in protein S-glutathionylation are observed in HIV-infected T cell blasts (38), suggesting that functional modulation of both host and viral proteins occurs via this mechanism. Whether these S-glutathionylated proteins inhibit or enhance immune responses to the unmodified epitope or generate novel T-cell epitopes that are subsequently recognized by the adaptive immune system is unclear.

Here, we investigate the frequency of modification of cysteine-containing MHC-bound peptides by interrogating a large database of naturally processed self-peptides derived from B-lymphoblastoid cells, murine tissues, and cytokine-treated cells. In addition, the functional consequences of Cys modification of T cell epitopes was investigated using an established model of infection that involves an immunodominant cysteine-containing epitope derived from a neurotropic strain of mouse hepatitis virus, strain JHM (JHMV)8(3941). We describe S-glutathionylation of this viral T cell epitope and the functional and structural implications of redox-modulated antigen presentation. Collectively our studies suggest that S-glutathionylation plays a key, previously unappreciated role in adaptive immune recognition.



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Plant to Animal Viral Transmission

Larry H. Bernstein, MD, FCAP, Curator



Algae Virus May Be Linked to Human Cognitive Decline


A research team led by scientists at the University of Nebraska-Lincoln (UNL) has provided the first direct evidence that an algae-infecting virus can invade and potentially replicate within some mammalian cells. Known as Acanthocystis turfacea chlorella virus 1, or ATCV-1, the pathogen is among a class of chloroviruses long believed to take up residence only in green algae. That thinking changed with a 2014 study from Johns Hopkins University and UNL that found gene sequences resembling those of ATCV-1 in throat swabs of human participants.

The new study (“Response of mammalian macrophages to challenge with the Chlorovirus ATCV-1), published in the Journal of Virology, introduced ATCV-1 to macrophage cells that serve critical functions in the immune responses of mice, humans, and other mammals. By tagging the virus with fluorescent dye and assembling three-dimensional images of mouse cells, the authors determined that ATCV-1 successfully infiltrated them.

The authors also measured a threefold increase in ATCV-1 within 24 hours of introducing the virus. The relatively modest spike nevertheless suggests that ATCV-1 can replicate within the macrophage cells, according to co-author David Dunigan, Ph.D.

Though a few studies have documented viruses jumping from one biological kingdom to another, chloroviruses were previously thought to have a limited “host range” that stopped well short of the animal kingdom, said Dr. Dunigan.

“A few years ago, no one I know would have made a prediction like this,” noted Dr. Dunigan, research professor of plant pathology and member of the Nebraska Center for Virology. “You probably would’ve been laughed out of the room. But we are now in the middle of something that is so very interesting.”

The macrophage cells underwent multiple changes characteristic of those breached by a virus, he continued. These changes eventually included a form of programmed death that virologists consider an innate “scorched earth” defense against the spread of viruses, which require living cells to survive and replicate.

Before dying, the cells exhibited multiple signs of stress that tentatively support links to mild cognitive impairments first reported in the 2014 paper. The new study measured a post-viral rise in interleukin 6, which previous research has linked with diminished spatial learning and certain neurological diseases. The authors also reported an increase in nitric oxide, an important signaling molecule that has been associated with memory impairments when produced in excess.

The 2014 investigation, which was initially designed to test the cognitive functioning of human participants, found that those with the ATCV-1 DNA performed slightly worse on measures of visual processing and visual motor speed. Mice inoculated with the virus showed similar deficits in memory and attention while navigating mazes. The 2014 paper further suggested that ATCV-1 altered the expression of more than 1,000 genes in the rodent hippocampus, an area of the brain tied to memory and spatial navigation.

The new study’s authors are continuing their collaboration with Johns Hopkins in the hope of ultimately confirming whether and how the virus contributes to any cognitive deficits suggested by the initial studies.

“It is still unclear whether the factors induced by the cell-based virus challenge could also be induced in the whole animal, and whether the induced factors cause cognitive impairments in the animal or the human,” said co-author Tom Petro, Ph.D., professor of microbiology and immunology at the University of Nebraska Medical Center.

Dr. Dunigan said he and his colleagues are also searching for other cellular responses to ATCV-1 while investigating how these responses might drive systemic changes in mice.


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Viruses, Vaccines and Immunotherapy

Writer and Curator: Larry H. Bernstein, MD, FCAP

This discussion is exclusively concerned with viruses, the infectious particles of coated DNA or RNA, that have been a major cause of epidemics.  There will be a presentation of mechanisms of disease and disease resistance, and of diagnosis and treatment.  The preventive treatment of viral disease has a long history that began with the discovery by Edward Jenner (1772) that the milkmaids did not get “smallpox”, but were protected by a history of “cowpox”, which led to the vaccine (1796). Jenner’s work is widely regarded as the foundation of immunology.  Rabies may be the oldest infectious disease known to man.  Louis Pasteur created the first vaccine in 1885.
Today we appreciate the advances in vaccination as a matter of public health with – measles, mumps, rubella, and the astounding work on polio. The making of a vaccine for influenza has been an evolutionary struggle.

Edward Jenner

Edward Jenner

For many centuries, smallpox devastated mankind.  It was common knowledge that survivors of smallpox became immune to the disease. As early as 430 BC, survivors of smallpox were called upon to nurse the afflicted. Interestingly, reminiscent of Florence Nightingale, Dr. Sydenham (1624–1689) treated his patients by allowing no fire in the room, leaving the windows permanently open.  Prior to vaccination, innoculation or variolation was used by subcutaneous insertion by a lancet wet with fresh matter taken from a ripe pustule of some person who suffered from smallpox. This method was introduced by Circassian traders (to immunize the Sultan’s Harem as children)  in the Ottoman Turkish Empire in 1670 .

The earliest evidence of skin lesions resembling those of smallpox is found on faces of mummies from the time of the 18th and 20th Egyptian Dynasties (1570–1085 bc). The first stages of the decline of the Roman Empire (ad 108) coincided with a large-scale epidemic: the plague of Antonine, which accounted for the deaths of almost 7 million people. The Arab expansion, the Crusades, and the discovery of the West Indies all contributed to the spread of the disease. With the rapid pace of vaccine development in recent decades, the historic origins of immunization are often forgotten.

Unknown in the New World, smallpox was introduced by the Spanish and Portuguese conquistadors. The disease decimated the local population and was instrumental in the fall of the empires of the Aztecs and the Incas. Similarly, on the eastern coast of North America, the disease was introduced by the early settlers and led to a decline in the native population. The devastating effects of smallpox also gave rise to one of the first examples of biological warfare. During the French-Indian War (1754–1767), Sir Jeffrey Amherst, the commander of the British forces in North America, suggested the deliberate use of smallpox to diminish the American Indian population hostile to the British. Another factor contributing to smallpox in the Americas was the slave trade because many slaves came from regions in Africa where smallpox was endemic. Since the attack on the World Trade Center on September 11, 2001, the threat of biological warfare and bioterrorism has reemerged. Smallpox has been identified as a possible agent of bioterrorism.
(Google sources)

Hepatitis C/B

Virus-host interactomics: new insights and opportunities for antiviral
drug discovery
B de Chassey, L Meyniel-Schicklin, J Vonderscher1, P André and V Lotteau
de Chassey et al. Genome Medicine 2014, 6:115

The current therapeutic arsenal against viral infections remains limited,
with often poor efficacy and incomplete coverage, and appears inadequate
to face the emergence of drug resistance. Our understanding of viral biology
and pathophysiology and our ability to develop a more effective antiviral
arsenal would greatly benefit from a more comprehensive picture of the
events that lead to viral replication and associated symptoms. Towards this
goal, the construction of virus-host interactomes is instrumental, mainly
relying on the assumption that a viral infection at the cellular level can be
viewed as a number of perturbations introduced into the host protein network
when viral proteins make new connections and disrupt existing ones. Here,
we review advances in interactomic approaches for viral infections, focusing
on high-throughput screening (HTS) technologies and on the generation of
high-quality datasets. We show how these are already beginning to offer
intriguing perspectives in terms of virus-host cell biology and the control of
cellular functions, and we conclude by offering a summary of the current
situation regarding the potential development of host-oriented antiviral

Innate and adaptive immune responses in HCV infections
Markus H. Heim, Robert Thimme
Journal of Hepatology Update: Hepatitis C
J Hepatol 2014; 61: j S14–S25.

Hepatitis C virus has been identified a quarter of a decade ago as a
leading cause of chronic viral hepatitis that can lead to cirrhosis
and hepatocellular carcinoma. Only a minority of patients can
clear the virus spontaneously during acute infection. Elimination
of HCV during acute infection correlates with a rapid induction of
innate, especially interferon (IFN) induced genes, and a delayed
induction of adaptive immune responses. However, the majority
of patients is unable to clear the virus and develops viral persistence
in face of an ongoing innate and adaptive immune
response. The virus has developed several strategies to escape
these immune responses. For example, to escape innate
immunity, the HCV NS3/4A protease can efficiently cleave and
inactivate two important signalling molecules in the sensory
pathways that react to HCV pathogen-associated molecular patterns
(PAMPs) to induce IFNs, i.e., the mitochondrial anti-viral
signalling protein (MAVS) and the Toll-IL-1 receptor-domaincontaining
adaptor-inducing IFN-b (TRIF). Despite these escape
mechanisms, IFN-stimulated genes (ISGs) are induced in a large
proportion of patients with chronic infection. Of note, chronically
HCV infected patients with constitutive IFN-stimulated gene
(ISG) expression have a poor response to treatment with pegylated
IFN-a (PegIFN-a) and ribavirin. The mechanisms that protect
HCV from IFN-mediated innate immune reactions are not entirely
understood, but might involve blockade of ISG protein translation
at the ribosome, localization of viral replication to cell compartments
that are not accessible to anti-viral IFN-stimulated effector
systems, or direct antagonism of effector systems by viral
proteins. Escape from adaptive immune responses can be
achieved by emergence of viral escape mutations that avoid recognition
by antibodies and T cells. In addition, chronic infection is
characterized by the presence of functionally and phenotypically
altered NK and T cell responses that are unable to clear the virus
but most likely contribute to the ongoing liver disease. In this
review, we will summarize current knowledge about the role of
innate and adaptive immune responses in determining the outcome
of HCV infection.

Host-virus interactions in acute hepatitis C
Key Points
• Hepatitis C virus (HCV) has a very high replicative
capacity. Within days after infection, viral titres of
>106 IU/ml can be measured in the serum
• The innate immune system reacts to HCV infections
with the induction of interferon (IFN)-stimulated genes
in the liver. This initial type I and/or type III IFN driven
response controls viral replication to some extent, but
can not eliminate HCV completely
• 4-8 weeks after infection, HCV specific T cells are
recruited to the liver. HCV replication is inhibited by
noncytolytic (IFN-γ mediated) and cytolytic mechanisms.
In about 30% of patients, the immune reaction during
acute hepatitis C is strong enough to eliminate HCV
• In the acute phase of the infection, HCV is highly
vulnerable to therapy with recombinant IFN-α. Over
90% of patients can be cured with IFN-α monotherapy

Severity of Hepatitis C Virus (Genotype-3) Infection Positively
Correlates with Circulating MicroRNA-122 in Patients Sera
S Kumar, YK Chawla, S Ghosh, and A Chakraborti
Disease Markers 2014; Article ID 435476, 6 pages

Introduction. Hepatitis C virus (genotype-3) causes acute and
chronic hepatitis infection predomination in India. The infectious
phase of the virus requires various host factors for its survival
and subsequent viral particle production. Small RNA molecules like
microRNA-122 (miR-122) are one such factormostly present in the
liver and play a supportive role in viral replication. Objective. In
this study, diagnostic potential of miR-122 is evaluated in the sera
of chronic hepatitis C patients. Methods. miRNAs were isolated
fromthe sera samples of patients as well as controls and miR-122
expression was quantified by real-time PCR. Results. A significant
augmentation was observed in the level of circulating miR-122
(median level, 0.66 versus 0.29, p = 0.001) in patients compared to
controls with ROC value of 0.929 ± 0.034 (p < 0.001). Interestingly,
miR-122 level also depicted a significant positive correlation
with serum ALT (p = 0.53), AST (p = 0.44), and viral load
(p = 0.52). Conclusion. The study thus unveiled the role of miR-122 as a
plausible diagnostic biomarker during HCV genotype-3 infection in India.

MicroRNA Expression Profiling in PBMCs: A Potential Diagnostic
Biomarker of Chronic Hepatitis C
Chiu-Chun Chang, Chun-Che Lin, Wan-Ling Hsieh, Hsin-Wu Lai, et al.
Disease Markers 2014; Article ID 367157, 9 pages

The expression levels of miR-16, miR-193b, miR-199a, miR-222, and
miR-324 in PBMCs were significantly higher in CHC patients
compared with healthy controls and significantly different between
CHC patients withHCV genotype 1 (GT-1) and non-genotype-1
(non-GT-1).Multivariate logistic regression analysis also showed
that patients with high expression levels of the six target miRNAs
had an approximately 7.202-fold risk of CHC compared with those
with low expression levels of the target miRNAs.We concluded
that the expression levels of miR-16, miR-193b, miR-199a, miR-222,
and miR-324 target miRNAs in PBMCs of CHC may act as
significant risk biomarkers for the development of CHC.

Neutrophil-to-Lymphocyte Ratio as a Predictor of Response to
Peginterferon plus Ribavirin Therapy for Chronic Hepatitis C
Ming-Te Kuo, Tsung-Hui Hu, Sheng-Nan Lu, CH Hung, Jing-Houng Wang, et al.
Disease Markers 2014; Article ID 462958, 6 pages

We aimed to determine whether neutrophil-to-lymphocyte ratio (NLR)
could be a predictor of antiviral response in chronic hepatitis C patients.
A total of 602 consecutive patients (genotype 1, n = 263; genotype 2,
n = 297; others/unknown, n = 42) receiving response-guided
therapy with peginterferon plus ribavirin were recruited. NLR was
related to clinical and virological features and to treatment outcome.
Rapid virological response (RVR) and sustained virological response
(SVR) were achieved in 436 (73%) and 458 (76%) of the patients,
respectively. Higher NLR ( ≥1.42) was found to be associated with
higher prevalence of DM (p = 0.039) and higher hepatitis C viral
load (p = 0.002) and white cell count (p < 0.001). NLR was
significantly lower in patients with RVR and SVR compared
to those without (𝑃 = 0.032 and 0.034, resp.). However, NLR
was not an independent factor by multivariate analysis. In the
subgroup analysis, higher NLR (≥1.42) (odds ratio, 0.494,
p = 0.038) was an independent poor predictor of SVR in
genotype 2 patients but was not in genotype 1 patients. In
conclusion, NLR is a simple and easily accessible marker to
predict response to peginterferon plus ribavirin therapy
for chronic hepatitis C genotype 2.

Therapy of Hepatitis C — Back to the Future
T. Jake Liang and Marc G. Ghany
N Engl J Med

The results of several phase 3 studies of interferon-free combination
regimens of DAAs reported in the Journal now2-4 and recently5-9
unequivocally show the superiority of two regimens over the standard-
of-care treatment (a combination of peginterferon, ribavirin, and
a protease inhibitor) for HCV genotype 1 infection. A previous editorial
in the Journal highlighted the significantly improved response rates
(rates of sustained virologic response of 93% to 99%) with a
coformulated regimen of sofosbuvir (a nucleotide NS5B inhibitor)
and ledipasvir (an NS5A inhibitor) among patients with HCV genotype
1 infection, as compared with the rates with the previously approved
interferon-based single-DAA combination therapy.

Sofosbuvir and Ribavirin in HCV Genotypes 2 and 3
S Zeuzem, GM Dusheiko, R Salupere, et al.
N Eng J Med May 2014

In clinical trials, treatment with a combination of the nucleotide
polymerase inhibitor sofosbuvir and the antiviral drug ribavirin was
associated with high response rates among patients with hepatitis C
virus (HCV) genotype 2 infection, with lower response rates among
patients with HCV genotype 3 infection.
We conducted a study involving patients with HCV genotype 2 or 3
infection, some of whom had undergone previous treatment with
an interferon-based regimen. We randomly assigned 91 patients with
HCV genotype 2 infection and 328 with HCV genotype 3 infection, in
a 4:1 ratio, to receive sofosbuvir–ribavirin or placebo for 12 weeks.
On the basis of emerging data from phase 3 trials indicating that
patients with HCV genotype 3 infection had higher response rates
when they were treated for 16 weeks, as compared with 12 weeks,
the study was unblinded, treatment for all patients with genotype 3
infection was extended to 24 weeks, the placebo group was terminated,
and the goals of the study were redefined to be descriptive and not
include hypothesis testing. The primary end point was a sustained
virologic response at 12 weeks after the end of therapy.
Of the 419 patients who were enrolled and treated, 21% had cirrhosis
and 58% had received previous interferon-based treatment. The criterion
for a sustained virologic response was met in 68 of 73 patients (93%; 95%
confidence interval [CI], 85 to 98) with HCV genotype 2 infection who
were treated for 12 weeks and in 213 of 250 patients (85%; 95% CI, 80 to 89)
with HCV genotype 3 infection who were treated for 24 weeks. Among
patients with HCV genotype 3 infection, response rates were 91%
and 68% among those without and those with cirrhosis, respectively.
The most common adverse events were headache, fatigue, and pruritus.
Therapy with sofosbuvir–ribavirin for 12 weeks in patients with HCV
genotype 2 infection and for 24 weeks in patients with HCV genotype 3
infection resulted in high rates of sustained virologic response. (Funded
by Gilead Sciences; VALENCE number, NCT01682720.)

New developments in the management of hepatitis C virus infection:
focus on boceprevir
Marina Berenguer, F Xavier López-Labrador
Biologics: Targets and Therapy 2012:6 249–256

Chronic hepatitis C virus infection is an important public health problem,
and the standard treatment (combination of pegylated interferon-α and
ribavirin) has an effectiveness rate of only 40%–50%. Novel virus-specific
drugs have recently been designed, and multiple compounds are under
development. The approval for the clinical use of direct-acting antivirals
in 2011 (boceprevir [BOC] and telaprevir, viral NS3 protease inhibitors)
has increased recovery rates by up to 70%. Therefore, a highly effective
treatment has been envisioned for the first time. This paper focuses on
BOC and the implementation of new BOC-based treatment regimes.

ABT-450/r–Ombitasvir and Dasabuvir with or without Ribavirin for HCV
P Ferenci, D Bernstein, J Lalezari, D Cohen, Y Luo, C Cooper, et al.
N Engl J Med May 4, 2014.

The interferon-free regimen of ABT-450 with ritonavir (ABT-450/r),
ombitasvir, and dasabuvir with or without ribavirin has shown efficacy
in inducing a sustained virologic response in a phase 2 study involving
patients with hepatitis C virus (HCV) genotype 1 infection. We conducted
two phase 3 trials to examine the efficacy and safety of this regimen in
previously untreated patients with HCV genotype 1 infection and no cirrhosis.
We randomly assigned 419 patients with HCV genotype 1b infection
(PEARL-III study) and 305 patients with genotype 1a infection (PEARL-IV
study) to 12 weeks of ABT-450/r–ombitasvir (at a once-daily dose of
150 mg of ABT-450, 100 mg of ritonavir, and 25 mg of ombitasvir), dasabuvir
(250 mg twice daily), and ribavirin administered according to body weight
or to matching placebo for ribavirin. The primary efficacy end point was a
sustained virologic response (an HCV RNA level of <25 IU per milliliter)
12 weeks after the end of treatment.
The study regimen resulted in high rates of sustained virologic response
among patients with HCV genotype 1b infection (99.5% with ribavirin
and 99.0% without ribavirin) and among those with genotype 1a infection
(97.0% and 90.2%, respectively). Of patients with genotype 1b infection,
1 had virologic failure, and 2 did not have data available at post-treatment
week 12. Among patients with genotype 1a infection, the rate of virologic
failure was higher in the ribavirin-free group than in the ribavirin
group (7.8% vs. 2.0%). In both studies, decreases in the hemoglobin
level were significantly more common in patients receiving ribavirin.
Two patients (0.3%) discontinued the study drugs owing to adverse events.
The most common adverse events were fatigue, headache, and nausea.
Twelve weeks of treatment with ABT-450/r–ombitasvir and dasabuvir
without ribavirin was associated with high rates of sustained virologic
response among previously untreated patients with HCV genotype 1
infection. Rates of virologic failure were higher without ribavirin than
with ribavirin among patients with genotype 1a infection but not among
those with genotype 1b infection. (Funded by AbbVie; PEARL-III and
PEARL-IV numbers, NCT01767116 and NCT01833533.)

Individualized treatment of chronic hepatitis C with pegylated interferon
and ribavirin
Roberto J Carvalho-Filho and Olav Dalgard
Pharmacogenomics and Personalized Medicine 2010:3 1–13

Chronic infection with hepatitis C virus (HCV) is a major public health
problem, with perhaps 180 million people infected worldwide. A significant
proportion of these will eventually develop clinical complications, such as
cirrhosis, liver decompensation and hepatocellular carcinoma. Sustained
virological response (SVR) to antiviral therapy is associated with
improvement in liver histology and survival free of liver-related complications.
Great effort has been made to improve SVR rate by adapting the duration
of therapy according to HCV genotype and to on-treatment response. Rapid
virological response (RVR, undetectable HCV RNA at week 4) usually has
a high positive predictive value for achieving SVR and early virological
response (EVR, > 2 log reduction or undetectable HCV RNA at week 12)
exhibits a high negative predictive value for non-response. Individualized
approach can improve cost-effectiveness of HCV antiviral therapy by
reducing side effects and the costs of therapy associated with unnecessary
exposure to treatment and through extending therapy for those with
unfavorable features. This article summarizes recent data on strategies
of individualized treatment in naïve patients with mono-infection by
the different HCV genotypes. The management of common side effects,
the impact of HCV infection on health-related quality of life and the
potential applications of host genomics in HCV therapy are briefly discussed.

Simeprevir for the treatment of hepatitis C virus infection
L Izquierdo, F Helle, C François, S Castelain, G Duverlie, E Brochot
Pharmacogenomics and Personalized Medicine 2014:7 241–249

Simeprevir (TMC435, Olysio™), a second-generation hepatitis C virus
(HCV) protease inhibitor, has been recently approved for the treatment
of genotype 1 chronic hepatitis C in combination with pegylated interferon
and ribavirin. This molecule has very different characteristics from first-
generation protease inhibitors. Results from trials show that simeprevir
is highly effective and safe, with few adverse events. We discuss the
specific features of this new treatment option for HCV infection, in terms
of in vitro data, pharmacological data, and clinical trials. We also discuss
the impact of Q80K polymorphism at baseline. Studies evaluating interferon-
free regimens with simeprevir are ongoing. Future combinations of two or
more direct-acting antiviral agents, targeting different viral enzymes and
with synergistic antiviral effects, will be approved, allowing treatment of
pan-genotypic HCV with optimized sustained virologic responses. Simeprevir
will undoubtedly be part of future treatment strategies.

Boceprevir and personalized medicine in hepatitis C virus infection
F Habersetzer, C Leboeuf, M Doffoël, TF Baumert
Pharmacogenomics and Personalized Medicine 2012:5 125–137

Boceprevir was the first agent, along with telaprevir, of a novel class of
direct-acting antivirals that entered clinical practice for the treatment
of chronic hepatitis C. Boceprevir is an antiprotease that directly blocks
hepatitis C virus (HCV) replication. Two studies in patients with HCV
genotype 1 infection have shown that addition of boceprevir to the
standard of care, ie, pegylated interferon-alfa (PEG-IFN-α) and ribavirin,
markedly increased the rate of sustained virological response. A sustained
virological response was obtained in about 70% of patients who had never
been treated, as well as in 69%–75% and 40% of previous relapsers and
nonresponders to PEG-IFN-α-ribavirin, respectively. Side effects were
observed in almost all treated patients. Anemia, the most frequent adverse
event related to administration of boceprevir, occurred in about 50% of
patients. The decision to add boceprevir to the standard of care is made
on an individual basis, and takes into account the prognosis of the liver
disease, the efficacy of therapy, as it could be at best predicted, and the
side effects that may arise, taking into account the comorbidities of the
patient. Ultimately, the treatment must be accepted by the patient, who
should fully understand the benefits and risks. Boceprevir trials were
designed with the concept of individualized and response-guided therapy
which establishes treatment decisions on how rapidly patients respond
to treatment. Individualized therapy for chronic hepatitis C is based on
patient and viral characteristics to make the best choice about whether a
person will benefit from therapy and to evaluate on-treatment predictors
of response to shorten therapy in patients with a rapid response as well as
in patients who did not respond sufficiently to expect HCV eradication.
This review focuses on the main results obtained so far, their impact on
the treatment of patients with chronic hepatitis C, and potential
therapeutic perspectives.

Ribavirin at the Era of Novel Direct Antiviral Agents for the Treatment
of Hepatitis C Virus Infection: Relevance of Pharmacological Monitoring
P Pradat, V Virlogeux, Marie-Claude Gagnieu, F Zoulim, and F Bailly
Adv Hepatol 2014, Article ID 493087, 13 pages

Ribavirin is often used for the treatment of hepatitis C virus (HCV)
infection. Although its mechanisms of action remain to be clearly
elucidated, ribavirin plays a beneficial role for achieving virological
response and decreasing the rate of virological relapse after
treatment cessation. However, ribavirin may induce side effects
leading to early treatment discontinuation. Among them,
hemolytic anemia is the most frequent and results from
intraerythrocyte accumulation. Pharmacological studies have
shown that early ribavirin exposure assessed by the area under
the curve (AUC) at day 0 and ribavirin trough concentration
during the first three months of therapy were correlated with
sustained virological response (SVR). These studies highlighted
the relevance of ribavirin pharmacologic monitoring and early
dose adaptation during therapy. Although the role of ribavirin
within new direct acting antiviral (DAA) combinations will
probably decrease in the future, its potential benefit in difficult-
to-treat patients such as patients with severe hepatopathy or
patients who failed triple therapy including patients with
multiresistance will need to be further investigated.

Clinical utility of pharmacogenomics in the management
of hepatitis C
J Trinks, ML Hulaniuk, MA Redal, D Flichman
Pharmacogenomics and Personalized Medicine 2014:7 339–347

Hepatitis C virus (HCV) was identified for the first time more than
20 years ago. Since then, several studies have highlighted the
complicated aspects of this viral infection in relation to its worldwide
prevalence, its clinical presentation, and its therapeutic response.
Recently, two landmark scientific breakthroughs have moved us
closer to the successful eradication of chronic HCV infection. First,
response rates in treatment-naïve patients and in prior non-responders
to pegylated-interferon-α and ribavirin therapy are increasing as a direct
consequence of the development of direct-acting antiviral drugs. Secondly,
the discovery of single-nucleotide polymorphisms near the interleukin
28B gene significantly related to spontaneous and treatment-induced
HCV clearance represents a milestone in the HCV therapeutic landscape.
The implementation of this pharmacogenomics finding as a routine test
for HCV-infected patients has enhanced our understanding of viral
pathogenesis, has encouraged the design of ground-breaking antiviral
treatment regimens, and has become useful for pretreatment decision
making. Nowadays, interleukin 28B genotyping is considered to be a
key diagnostic tool for the management of HCV-infected patients and
will maintain its significance for new combination treatment schemes
using direct-acting antiviral agents and even in interferon-free regimens.
Such pharmacogenomics insights represent a challenge to clinicians,
researchers, and health administrators to transform this information
into knowledge with the aim of elaborating safer and more effective
therapeutic strategies specifically designed for each patient. In conclusion,
the individualization of treatment regimens for patients with hepatitis C,
that may lead to a universal cure in future years, is becoming a reality
due to recent developments in biomarker and genomic medicine.
In light of these advances, we review the scientific evidence and clinical
implications of recent findings related to host genetic factors in the
management of HCV infection.

Hepatitis B virus and Homo sapiens proteome-wide analysis: A
profusion of viral peptide overlaps in neuron-specific human proteins
Rosalia Ricco and Darja Kanduc
Biologics: Targets & Therapy 2010:4 75–81

The primary amino acid sequence of the hepatitis B virus (HBV)
proteome was searched for identity spots in the human proteome
by using the Protein Information Resource database. We find that
the HBV polyprotein shares sixty-five heptapeptides, one octapeptide,
and one nonapeptide with the human proteins. The viral matches
are disseminated among fundamental human proteins such as
adhesion molecules, leukocyte differentiation antigens, enzymes,
proteins associated with spermatogenesis, and transcription factors.
As a datum of special interest, a number of peptide motifs are shared
between the virus- and brain-specific antigens involved in neuronal
protection. This study may help to evaluate the potential cross
reactions and side effects of HBV antigen-based vaccines.

Dual effects of interleukin-18: inhibiting hepatitis B virus replication
in HepG2.2.15 cells and promoting hepatoma cells metastasis
Y Zhang, Y Li, Y Ma, S Liu, Y She, P Zhao, M Jing, et al.
Am J Physiol Gastrointest Liver Physiol 301: G565–G573, 2011

Interleukin-18 (IL-18) has been reported to inhibit hepatitis B
virus (HBV) replication in the liver of HBV transgenic mice;
however, the molecular mechanism of its antiviral effect has not
been fully understood. In the present study, it was shown that
IL-18 and its receptors (IL-18R) were constitutively expressed
in hepatoma cell lines HepG2 and HepG2.2.15 as well as
normal liver cell line HL-7702. We demonstrated that IL-18
directly inhibited HBV replication in HepG2.2.15 cells via
downregulating the activities of HBV core and X gene promoters.
The suppressed HBV replication by IL-18 could be rescued by
the administration of BAY11-7082, an inhibitor of transcription
factor NF-B. On the other hand, it was of interest that IL-18
promoted HepG2 cell metastasis and migration dose dependently
in both wound-healing assays and Transwell assays. The underlying
mechanism could be partially attributable to the increased activities
of extracellular matrix metalloproteinase (MMP)-9, MMP-3, and
MMP-2 by IL-18, which upregulated the mRNA levels of MMP-3
and MMP-9 in a NF-B-dependent manner. Furthermore, it was
confirmed that expression of IL-18/IL-18R and most MMPs were
remarkably upregulated in hepatocellular carcinoma (HCC) liver
cancer tissue specimens, suggesting that IL-18/IL-18R-triggered
signaling pathway was closely related to HCC metastasis
in vivo. Therefore, we revealed the dual effects of IL-18 in
human hepatocytes: it not only inhibited HBV replication but
also promoted hepatoma cells metastasis and migration. NF-B
played a critical role in both effects. Our work contributed to a
deeper understanding of the biological function of IL-18 in
human hepatocytes.


The export receptor Crm1 forms a dimer to promote nuclear
export of HIV RNA
David S Booth, Yifan Cheng, Alan D Frankel
eLife 2014;3:e04121

The HIV Rev protein routes viral RNAs containing the Rev Response
Element (RRE) through the Crm1 nuclear export pathway to the
cytoplasm where viral proteins are expressed and genomic RNA is
delivered to assembling virions. The RRE assembles a Rev oligomer
that displays nuclear export sequences (NESs) for recognition by
the Crm1-RanGTP nuclear receptor complex. Here we provide the
first view of an assembled HIV-host nuclear export complex using
single particle electron microscopy. Unexpectedly, Crm1 forms a
dimer with an extensive interface that enhances association with
Rev-RRE and poises NES binding sites to interact with a Rev oligomer.
The interface between Crm1 monomers explains differences between
Crm1 orthologs that alter nuclear export and determine cellular tropism
for viral replication. The arrangement of the export complex identifies
a novel binding surface to possibly target an HIV inhibitor and may point
to a broader role for Crm1 dimerization in regulating host gene expression.

RNA-directed remodeling of the HIV-1 Rev protein orchestrates assembly
of the Rev-Rev response element complex
B Jayaraman, DC Crosby, C Homer, I Ribeiro, D Mavor, AD Frankel
eLife 2014;

The HIV-1 protein Rev controls a critical step in viral replication by
mediating the nuclear export of unspliced and singly-spliced viral
mRNAs. Multiple Rev subunits assemble on the Rev Response Element
(RRE), a structured region present in these RNAs, and direct their
export through the Crm1 pathway. Rev-RRE assembly occurs via
several Rev oligomerization and RNA-binding steps, but how these
steps are coordinated to form an export-competent complex is unclear.
Here, we report the first crystal structure of a Rev dimer-RRE complex,
revealing a dramatic rearrangement of the Rev-dimer upon RRE binding
through re-packing of its hydrophobic protein-protein interface. Rev-RNA
recognition relies on sequence-specific contacts at the well-characterized
IIB site and local RNA architecture at the second site. The structure
supports a model in which the RRE utilizes the inherent plasticity of
Rev subunit interfaces to guide the formation of a functional complex.

HIV-1 envelope glycoprotein structure
Alan Merk and Sriram Subramaniam
Curr Opin Struct Biol. 2013 April ; 23(2): 268–276.

The trimeric envelope glycoprotein of HIV-1, composed of gp120 and
gp41 subunits, remains a major target for vaccine development. The
structures of the core regions of monomeric gp120 and gp41 have
been determined previously by X-ray crystallography. New insights
into the structure of trimeric HIV-1 envelope glycoproteins are now
coming from cryo-electron tomographic studies of the gp120/gp41
trimer as displayed on intact viruses and from cryo-electron microscopic
studies of purified, soluble versions of the ectodomain of the trimer.
Here, we review recent developments in these fields as they relate to
our understanding of the structure and function of HIV-1 envelope

The QSAR and docking calculations of fullerene derivatives as HIV-1
protease inhibitors
Noha A. Saleh
Spectrochim Acta Part A: Molec and Biomol Spectrosc 2015; 136: 1523–1529

The inhibition of HIV-1 protease is considered as one of the most
important targets for drug design and the deactivation of HIV-1.
In the present work, the fullerene surface (C60) is modified by
adding oxygen atoms as well as hydroxymethylcarbonyl (HMC)
groups to form 6 investigated fullerene derivative compounds.
These compounds have one, two, three, four or five O atoms +
HMC groups at different positions on phenyl ring. The effect of
the repeating of these groups on the ability of suggested compounds
to inhibit the HIV protease is studied by calculating both Quantitative
Structure Activity Relationship (QSAR) properties and docking simulation.
Based on the QSAR descriptors, the solubility and the hydrophilicity
of studied fullerene derivatives increased with increasing the number
of oxygen atoms + HMC groups in the compound. While docking
calculations indicate that, the compound with two oxygen atoms +
HMC groups could interact and binds with HIV-1 protease active site.
This is could be attributed to the active site residues of HIV-1 protease
are hydrophobic except the two aspartic acids. So that, the increase in the
hydrophilicity and polarity of the compound is preventing and/or
decreasing the hydrophobic interaction between the compound and
HIV-1 protease active site.

In silico identification of Novel HIVProtease inhibitors (PIs) using
ZINC drug Database
KK Srivastava, S Srivastava, T Alam, Rituraj
Int J Pharma Sci and Res (IJPSR) Dec 2014; 5(12):947-952.

The Human immunodeficiency virus type-1 protease is one of the
most important target of highly active anti-retrovirus therapy
(HAART) for the treatment of all acquired immune deficiency
syndrome (AIDS). Protease inhibitor Darunavir is most recent
included as a PI in the list of HARRT, more effective against mutant
type and wild type of Protease with increased no. of H-bonding then
precursors approved by FDA, So herein we taken Darunavir as a base
structure for virtually identification of more/similar efficient drug
like leads then Darunavir using PDB structure (3BGR) of Protease
from PDB database ‘RCSB’ versus chemical compounds database
‘ZINC’ using Schrodinger and Discovery Studio software. Using
molecular constraint search with similarity coefficient ‘Tanimoto’,
1,65,000 ligands were extracted and docking analysis resulted in
some efficient in docking and in other computational medicinal
parameters, we are reporting such leads, and, they may further
undergo through high end extensive virtual investigation and beyond.



In this work, we have tried to recognized some more/similar
potent drug like leads instead ‘Darunavir’ may be more effective,
we used five different RT crystallographic structures for better
identification/verification for our results, ZINC78487241,
ZINC09060710, ZINC72320180, ZINC78487244 & ZINC78487242
are showing very fine computed properties therefore, this study
verifies the importance of small drug like molecules libraries as
like ‘’and their use certainly help scientific groups
to enhance their capabilities in drug discovery with reducing time,
including drug discovery process prior synthesis. Meanwhile all
herein identified molecules may further investigate instead “in silico”.

Development and Customization of a Color-Coded Microbeads-Based
Assay for Drug Resistance in HIV-1 Reverse Transcriptase
L Gu, Ai Kawana-Tachikawa, T Shiino, H Nakamura, M Koga, et al.
PLoS ONE 9(10): e109823.

Other Virus Diseases

A global phylogenetic analysis in order to determine the host species
and geography dependent features present in the evolution of avian
H9N2 influenza hemagglutinin
Andrew R. Dalby and Munir Iqbal
PeerJ 2014.

A complete phylogenetic analysis of all of the H9N2 hemagglutinin
sequences that were collected between 1966 and 2012 was carried out
in order to build a picture of the geographical and host specific evolution
of the hemagglutinin protein. To improve the quality and applicability
of the output data the sequences were divided into subsets based upon
location and host species. The phylogenetic analysis of hemagglutinin
reveals that the protein has distinct lineages between China and the
Middle East, and that wild birds in both regions retain a distinct form
of the H9 molecule, from the same lineage as the ancestral hemagglutinin.
The results add further evidence to the hypothesis that the current
predominant H9N2 hemagglutinin lineage might have originated in
Southern China. The study also shows that there are sampling problems
that affect the reliability of this and any similar analysis. This raises
questions about the surveillance of H9N2 and the need for wider
sampling of the virus in the environment. The results of this analysis
are also consistent with a model where hemagglutinin has predominantly
evolved by neutral drift punctuated by occasional selection events. These
selective events have produced the current pattern of distinct lineages in
the Middle East, Korea and China.

Describing the hexapeptide identity platform between the influenza A
H5N1 and Homo sapiens proteomes
Darja Kanduc
Biologics: Targets & Therapy 2010:4 245–261

We searched the primary sequence of influenza A H5N1 polyprotein
for hexamer amino acid sequences shared with human proteins using
the Protein International Resource database and the exact peptide
matching analysis program. We find that the viral polyprotein shares
numerous hexapeptides with the human proteome. The human
proteins involved in the viral overlap are represented by antigens
associated with basic cell functions such as proliferation, development,
and differentiation. Of special importance, many human proteins
that share peptide sequences with influenza A polyprotein are
antigens such as reelin, neurexin I-α, myosin-IXa, Bardet–Biedl
syndrome 10 protein, Williams syndrome transcription factor,
disrupted in schizophrenia 1 protein, amyotrophic lateral sclerosis 2
chromosomal region candidate gene 17 protein, fragile X mental
retardation 2 protein, and jouberin. That is, the viral-vs-human
overlap involves human proteins that, when altered, have been
reported to be potentially associated with multiple neurological
disorders that can include autism, epilepsy, obesity, dystonia,
ataxia–telangiectasia, amyotrophic lateral sclerosis, sensorineural
deafness, sudden infant death syndrome, Charcot-Marie-Tooth
disease, and myelination. The present data are discussed as a
possible molecular basis for understanding influenza A viral
escape from immunosurveillance and for defining anti-influenza
immune-therapeutic approaches devoid of collateral adverse events.

Middle East respiratory syndrome coronavirus neutralising serum
antibodies in dromedary camels: a comparative serological study

CBEM Reusken, BL Haagmans, MA Müller, C Gutierrez,
Gert-Jan Godeke, et al.
Lancet Infectious Diseases Oct 2013; 13(10): 859 – 866

A new betacoronavirus—Middle East respiratory syndrome coronavirus
(MERS-CoV)—has been identified in patients with severe acute
respiratory infection. Although related viruses infect bats, molecular
clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in
contact with dromedary camels or goats. We investigated possible
animal reservoirs of MERS-CoV by assessing specific serum antibodies
in livestock.
We took sera from animals in the Middle East (Oman) and from
elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40),
goats (n=40), dromedary camels (n=155), and various other camelid
species (n=34) were tested for specific serum IgG by protein microarray
using the receptor-binding S1 subunits of spike proteins of MERS-CoV,
severe acute respiratory syndrome coronavirus, and human coronavirus
OC43. Results were confirmed by virus neutralization tests for MERS-CoV
and bovine coronavirus.  50 of 50 (100%) sera from Omani camels and 15
of 105 (14%) from Spanish camels had protein-specific antibodies against
MERS-CoV spike. Sera from European sheep, goats, cattle, and other
camelids had no such antibodies. MERS-CoV neutralising antibody titers
varied between 1/320 and 1/2560 for the Omani camel sera and between
1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-
neutralization by bovine coronavirus antibodies. MERS-CoV or a related
virus has infected camel populations. Both titres and seroprevalences in
sera from different locations in Oman suggest widespread infection.

Cosic’s Resonance Recognition Model for Protein Sequences and
Photon Emission Differentiates Lethal and Non-Lethal Ebola
Strains: Implications for Treatment
Nirosha J. Murugan, Lukasz M. Karbowski, Michael A. Persinger
Open Journal of Biophysics, 2015, 5, 35-43

The Cosic Resonance Recognition Model (RRM) for amino acid
sequences was applied to the classes of proteins displayed by four
strains (Sudan, Zaire, Reston, Ivory Coast) of Ebola virus that
produced either high or minimal numbers of human fatalities.
The results clearly differentiated highly lethal and non-lethal strains.
Solutions for the two lethal strains exhibited near ultraviolet (~230 nm)
photon values while the two asymptomatic forms displayed near i
nfrared (~1000 nm) values. Cross-correlations of spectral densities of
the RRM values of the different classes of proteins associated with the
genome of the viruses supported this dichotomy. The strongest coefficient
occurred only between Sudan-Zaire strains but not for any of the other
pairs of strains for sGP, the small glycoprotein that intercalated with
the plasma cell membrane to promote insertion of viral contents into
cellular space. A surprising, statistically significant cross-spectral
correlation occurred between the “spike” glycoprotein component (GP1)
of the virus that associated the anchoring of the virus to the mammalian
cell plasma membrane and the Schumann resonance of the earth whose
intensities were determined by the incidence of equatorial thunderstorms.
Previous applications of the RRM to shifting photon wavelengths emitted
by melanoma cells adapting to reduced ambient temperature have validated
Cosic’s model and have demonstrated very narrow wave-length (about 10 nm)
specificity. One possible ancillary and non-invasive treatment of people
within which the fatal Ebola strains are residing would be whole body
application of narrow band near-infrared light pulsed as specific
physiologically-patterned sequences with sufficient radiant flux density
to perfuse the entire body volume.

Nov 23rd, 2014
Replication of Ebola Virus

Ebola Virus do not replicate through any kind of cell division; rather,
they use a combination of host and virally encoded enzymes, alongside
host cell structures, to produce multiple copies of viruses. These then self-assemble into viral macromolecular structures in the host cell. The
virus completes a set of steps when infecting each individual cell.

Replication of Ebola Virus

Following are the steps during the replication of Ebola Virus:


First of all, there is attachment of virus to host receptors through
GP glycoprotein which is endocytosed into vesicles in the host cell.
Host DC-SIGN and DC-SIGNR play a role in virion attachment.

Viral Entry (Penetration)

The virion enters early endosomes by Macropinocytosis or
clathrin-mediated endocytosis.


In this process, ruffled segments of the host’s plasma membrane
protrude outward from the cell and form invaginations where
the virus utilizes glycoproteins in order to attach to the surface
of the plasma membrane. Macropinocytosis is a process in which
the Eukaryotic host cells form macropinosomes, segments of plasma
membranes that extend out from the cell approximately 0.2-10 µm, in
order to incorporate the virus into the cell. The formation of macro-
pinosomes occurs spontaneously, as a result of the activation of various
growth factors, or simultaneously with the intake of cellular molecules
or extracellular fluid.

Clathrin-mediated endocytosis

Clathrin-mediated endocytosis is the other means by which Ebolavirus
enters the host cell. This process is very similar to macropinocytosis
in that the plasma membrane forms invaginations that engulf the cell.
However, clathrin-mediated endocytosis is different in that proteins on
the surface of the host’s surface, and in particular clathrin, facilitate
the attachment of the virus to the host’s cell surface. Glycoproteins
are still used to attach the virus to the cell surface, and the NP-C1
cholesterol transporter still facilitates the fusion of the virus with
endosomes and lysosomes and still allows the virus to escape into
the cytoplasm. Without the NPC1 cholesterol transporter, Ebolavirus
cannot leave the vesicle in order to replicate and cause infection in other cells.

To penetrate the cell, the viral membrane fuses with vesicle membrane,
and the nucleocapsid is released into the cytoplasm.

In some culture cells, GP glycoprotein can be processed by host
Cathepsin L andCathepsin B into 19kDa GP1. But this processing is
not happening in all cells or for all ebolavirus. 19kDA GP1 interacts
with host NPC1, which is highly expressed in dendritic cells.

Fusion of virus membrane with the vesicle membrane is triggered
by either low pH orNPC1 binding.

Sequential Transcription

During transcription, the RNA genome is transcribed into seven
monocistronic mRNAs whose length is determined by highly
conserved start and stop signals.

The transcription process begins with the binding of the polymerase
complex to a single binding site located within the leader region of
the genome. The complex then slides along the RNA template and
sequentially transcribes the individual genes in their 3’ to 5’ order.
Encapsidated, negative-sense genomic ssRNA is used as a template
for the synthesis (3′-5′) of polyadenylated, monocistronic mRNAs
and, using the host cell’s ribosomes, tRNA molecules, etc.,
the mRNA is translated into individual viral proteins.


As viral protein levels rise, a switch occurs from translation to replication.
Using the negative-sense genomic RNA as a template, a complementary
+ssRNA is synthesized; this is then used as a template for the synthesis
of new genomic (-)ssRNA, which is rapidly encapsidated. Replication
presumably starts when enough nucleoprotein is present to encapsidate
neo-synthetized antigenomes and genomes.


The newly formed nucleocapsids and envelope proteins associate at the
host cell’s plasma membrane; budding occurs, destroying the cell.

These viruses recruit components of the cellular ESCRT (endosomal
sorting complex required for transport) system to mediate host-assisted
viral budding. SCRT complexes are normally used by the cell for
biological functions involving membrane remodeling, such as intra-luminal vesicle formation, autophagy or terminal stages of cytokinesis.
The ESCRT family consists of ESCRT-0, ESCRT-I, ESCRT-II which are
primarily involved in cargo sorting and membrane deformation, and
ESCRT-III which cleaves the bud neck from its cytosolic face.  In the
last step, vps4 disassembles the complex. The budding reaction
catalyzed by the ESCRT machinery has reversed topology when
compared with most other budding processes in the cell, such as
endocytosis and formation of transport vesicles.

Release –   Finally, the virion is released.

Favipiravir elicits antiviral mutagenesis during virus replication in vivo

Armando Arias, Lucy Thorne, Ian Goodfellow
eLife 2014;3:e03679.

Lethal mutagenesis has emerged as a novel potential therapeutic
approach to treat viral infections. Several studies have demonstrated
that increases in the high mutation rates inherent to RNA viruses lead
to viral extinction in cell culture, but evidence during infections in vivo
is limited. In this study, we show that the broad-range antiviral nucleoside
favipiravir reduces viral load in vivo by exerting antiviral mutagenesis
in a mouse model for norovirus infection. Increased mutation frequencies
were observed in samples from treated mice and were accompanied with
lower or in some cases undetectable levels of infectious virus in faeces
and tissues. Viral RNA isolated from treated animals showed reduced
infectivity, a feature of populations approaching extinction during antiviral
mutagenesis. These results suggest that favipiravir can induce norovirus
mutagenesis in vivo, which in some cases leads to virus extinction,
providing a proof-of-principle for the use of favipiravir derivatives or
mutagenic nucleosides in the clinical treatment of noroviruses.

Individualization of antiretroviral therapy
Rebecca Pavlos, Elizabeth J Phillips
Pharmacogenomics and Personalized Medicine 2012:5 1–17

Antiretroviral therapy (ART) has evolved considerably over the last
three decades. From the early days of monotherapy with high toxicities
and pill burdens, through to larger pill burdens and more potent
combination therapies, and finally, from 2005 and beyond where we
now have the choice of low pill burdens and once-daily therapies.
More convenient and less toxic regimens are also becoming available,
even in resource-poor settings. An understanding of the individual
variation in response to ART, both efficacy and toxicity, has evolved
over this time. The strong association of the major histocompatibility
class I allele HLA-B*5701 and abacavir hypersensitivity, and its
translation and use in routine HIV clinical practice as a predictive
marker with 100% negative predictive value, has been a success story
and a notable example of the challenges and triumphs in bringing
pharmacogenetics to the clinic. In real clinical practice, however, it is
going to be the exception rather than the rule that individual
biomarkers will definitively guide patient therapy. The need for
individualized approaches to ART has been further increased by the
importance of non-AIDS comorbidities in HIV clinical practice. In the
future, the ideal utilization of the individualized approach to ART will
likely consist of a combined approach using a combination of knowledge
of drug, virus, and host (pharmacogenetic and pharmacoecologic [factors
in the individual’s environment that may be dynamic over time])
information to guide the truly personalized prescription. This review
will focus on our knowledge of the pharmacogenetics of the efficacy
and toxicity of currently available antiretroviral agents and the current
and potential utility of such information and approaches in present
and future HIV clinical care.

Additional Considerations

PLOS Biology Paper Wins Omenn Prize for Viral Evasion Story
By Roli Roberts  Posted: June 19, 2014

We talk to the authors of a PLOS Biology research article published in
May 2013 that won the Omenn Prize for the best article published in 2013.
The Omenn Prize is awarded annually by the Evolution, Medicine, & Public
Health Foundation to authors of articles related to “evolution in the context
of medicine and public health,” and the winner was picked from a tough
long-list of 47 papers. Four other papers, including two from our sister
journal PLOS Pathogens, were cited for “honorable mention”
(Graves et al. Huijben et al.).

The PLOS Biology paper looks at how an essential mammalian protein –
the transferrin receptor, TfR1 – evolves in the face of contrasting selective
pressures. TfR1 is a protein that sits on the membrane of our cells and
mediates the regulated uptake of iron. TfR1 is stuck in the horns of a
dilemma. On the one hand, it has to be able to bind its functional partners –
the iron-loaded plasma protein transferrin, and a negative regulator protein
called HFE; this requirement constrains the sequence and structure of TfR1
through evolutionary time. On the other hand, it has to evade viruses that
exploit its handy cell-surface location, such as arenaviruses and the rodent
retrovirus MMTV.

TfR1 can carry on binding transferrin and HFE while dodging viruses

TfR1 can carry on binding transferrin and HFE while dodging viruses

TfR1 can carry on binding transferrin and HFE while dodging viruses.

The authors compared sequences of TfR1 from various mammalian hosts
and then expressed them on the surface of cells to check a) their ability
to confer vulnerability or resistance to MMTV and arenaviruses such as
Machupo, Junin and Guanarito virus and b) their ability to bind to
transferrin. This image from the paper summarises the central finding –
how TfR1 (green) manages to square this circle by evolving rapidly (red)
to change the outer surfaces that are hijacked by viruses while keeping
constant the central surfaces that it uses to bind transferrin and HFE
(purple, blue).

First author Ann Demogines and lead author Sara Sawyer – both from
the University of Texas at Austin – told us how the study first arose and
then evolved into the paper that you can now read on our website.

Sawyer recalls the exact point at which the project started: “In the first year
of my faculty position, Welkin Johnson invited me to give a talk at the New
England Primate Research Center.  While I was there, I had a 45 minute
meeting with his colleague, Mike Farzan [also a co-author]. Mike had just
discovered TfR1 as the cellular receptor for arenaviruses, and suggested to
me that this might be a molecule that is engaged in an evolutionary arms race.
While ideas like this often arise out of conversations between scientists, I
remember having a gut reaction that this was something worth pursuing.”

Positively selected amino acids (red) hit the virus binding sites on TfR1 (blue, grey).

Positively selected amino acids (red) hit the virus binding sites on TfR1 (blue, grey).

Positively selected amino acids (red) hit the virus binding sites on
TfR1 (blue, grey)

Demogines, who received $5000 from the Foundation, describes
how the spectacular arrangement of the evolutionarily selected sites
emerged: “I am still amazed to this day by the results of the evolutionary
analysis.  We were able to take DNA sequence from just 7 species and
computationally predict six sites under selection.  These sites were
scattered on the linear diagram of the protein, and didn’t make much
sense to us. But, when we placed them onto the 3D crystal structure
they formed a beautiful ridge going straight down the outer surface of
the receptor.  That was a great day in the lab.  We knew this had to
mean something!”

Demogines goes on to think about the implications of her paper and
related studies: “This work really gets me excited about the future of
evolutionary analysis applied in biomedical research.  As we collect the
genome sequences from more and more species, especially rodents and
bats which are major reservoirs for zoonotic and potentially zoonotic
viruses, we should be able to do this type of analysis more and more.
This type of analysis has many applications: allowing us to identify
critical cofactors involved in the viral lifecycle, viral binding sites, and
potentially novel drug targets. It can also be used to study interactions
with bacterial pathogens, although this has not yet been extensively explored.”

If you’d like to find out more about this elegant study, why not read the
article itself, or the accompanying Primer written by John Coffin:

“Dual Host-Virus Arms Races Shape an Essential Housekeeping Protein”
by Ann Demogines, Jonathan Abraham, Hyeryun Choe, Michael Farzan and
Sara L. Sawyer.

“Virions at the Gates: Receptors and the Host–Virus Arms Race” by
John M. Coffin.
Angiomotin Functions in HIV-1 Assembly and Budding
G Mercenne, SL Alam, J Arii, MS Lalonde and WI Sundquist
eLife 2015;

Many retroviral Gag proteins contain PPXY late assembly domain motifs that recruit proteins of the NEDD4 ubiquitin E3 ligase family to facilitate virus release. Overexpression of NEDD4L can also stimulate HIV-1 release but in this case the Gag protein lacks a PPXY motif, suggesting that NEDD4L may function through an adaptor protein. Here, we demonstrate that the cellular protein Angiomotin (AMOT) can bind both NEDD4L and HIV-1 Gag. HIV-1 release and infectivity are stimulated by AMOT overexpression and inhibited by AMOT,depletion, whereas AMOT mutants that cannot bind NEDD4L cannot function in virus release. Electron microscopic
analyses revealed that in the absence of AMOT assembling Gag molecules fail to form a fully spherical enveloped particle. Our experiments indicate that AMOT and other motin family members function together with NEDD4L to help complete immature virion assembly prior to ESCRT-mediated virus budding.

The DEAH-Box RNA Helicase DHX15 Activates NF-B and MAPK Signaling Downstream of MAVS During Antiviral Responses

K Mosallanejad, Y Sekine, S Ishikura-Kinoshita, K Kumagai, T Nagano, et al.
Sci. Signal., 29 Apr 2014;  7(323), p. ra40]

During infection with an RNA virus, the DExD/H-box RNA helicases RIG-I (retinoic acid–inducible gene I) and MDA5 (melanoma differentiation–associated gene 5) activate the interferon regulatory factor 3 (IRF3), nuclear factor B (NF-B), c-Jun amino-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways through an unknown mechanism involving the adaptor protein MAVS (mitochondrial antiviral signaling). We used a Drosophila misexpression screen to identify DEAH-box polypeptide 15 (DHX15) as an activator of the p38 MAPK pathway. Human DHX15 contributed to the activation of the NF-B, JNK, and p38 MAPK pathways, but not the IRF3 pathway, in response to the synthetic double-stranded RNA analog poly(I:C) (polyinosinic-polycytidylic acid), and DHX15 was required for optimal cytokine production in response to poly(I:C) and infection with RNA virus. DHX15 physically interacted with MAVS and mediated the MAVS-dependent activation of the NF-B and MAPK pathways. Furthermore, DHX15 was required for poly(I:C)- and RNA virus–dependent, MAVS-mediated apoptosis. Thus, our findings indicate that, in RIG-I–like receptor signaling, DHX15 specifically stimulates the NF-B and MAPK pathways downstream of MAVS and contributes to MAVS-mediated cytokine production and apoptosis.

Mitochondrial Membrane Potential Is Required for MAVS-Mediated Antiviral Signaling
T Koshiba, Kai Yasukawa, Y Yanagi, and Shun-ichiro Kawabata
Sci. Signal., 1 Feb 2011; 4(158), p. ra7]

Mitochondria, dynamic organelles that undergo cycles of fusion and fission, are the powerhouses of eukaryotic cells and are also involved in cellular innate antiviral immunity in mammals. Mitochondrial antiviral immunity depends on activation of the cytoplasmic retinoic acid–inducible gene I (RIG-I)–like receptor (RLR) signaling pathway and the participation of a mitochondrial outer membrane adaptor protein called MAVS (mitochondrial antiviral signaling). We found that cells that lack the ability to undergo mitochondrial fusion as a result of targeted deletion of both mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) exhibited impaired induction of interferons and proinflammatory cytokines in response to viral infection, resulting in increased viral replication. In contrast, cells with null mutations in either Mfn1 or Mfn2 retained their RLR-induced antiviral responses. We also found that a reduced mitochondrial membrane potential (m) correlated with the reduced antiviral response. The dissipation in m did not affect the activation of the transcription factor interferon regulatory factor 3 downstream of MAVS, which suggests that m and MAVS are coupled at the same stage in the RLR signaling pathway. Our results provide evidence that the physiological function of mitochondria plays a key role in innate antiviral immunity.

The Ubiquitin-Specific Protease USP15 Promotes RIG-I–Mediated Antiviral Signaling by Deubiquitylating TRIM25
Eva-Katharina Pauli, YK Chan, ME Davis, S Gableske, MK Wang, KF F, et al.
Sci. Signal., 7 Jan 2014; 7(307), p. ra3

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys63)–linked ubiquitin chains to the RNA sensor retinoic acid–inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-β. Conversely, Lys48-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys48-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I–dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I–mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling.

Antiviral Mitochondrial Action
Editor’s Summary
Sci. Signal., 1 Feb 2011;  4(158), p. ra7

Mitochondria are the energy generators of the cell, but they also act as platforms upon which complexes of proteins respond to RNA-containing viruses within the cytosol. Through genetic and pharmacological means, Koshiba et al. present evidence that suggests that the contribution of mitochondria to these antiviral responses is not as passive as originally thought. Indeed, their data suggest that successful resistance to viral infections depends on maintenance of the internal physiological functions of mitochondria coupled with the functions of the external protein complexes.

Prolonging Antiviral Signaling
Editor’s Summary
Sci. Signal., 7 Jan 2014; 7(307), p. ra3

As part of the innate immune response to infection by RNA viruses, signaling by the cytosolic RNA sensor retinoic acid–inducible gene-I (RIG-I) stimulates the production of type I interferons (IFNs). RIG-I activity depends on its Lys63-linked polyubiquitylation by the ubiquitin E3 ligase TRIM25. As part of a negative feedback mechanism, the linear ubiquitin assembly complex (LUBAC) mediates the Lys48-linked polyubiquitylation and degradation of TRIM25, leading to inhibition of RIG-I signaling. Pauli et al. found that the deubiquitylase USP15 (ubiquitin-specific protease 15) counteracted the function of LUBAC by removing polyubiquitin from TRIM25, thus promoting its stabilization and enhancing the RIG-I–dependent antiviral response. Loss of USP15 resulted in decreased type I IFN production and enhanced viral replication in infected cells. Together, these data suggest that USP15 fine-tunes the antiviral response by sustaining the production of IFNs.

NIH-funded study uncovers range of molecular alterations in head and neck cancers, new potential drug targets

TCGA tumor genome sequencing analyses offer new insights into the effects of HPV and smoking, and find genomic similarities with other cancers

human papillomavirus (HPV) in head and neck cancer

human papillomavirus (HPV) in head and neck cancer

TCGA researchers have uncovered new details about the potential role of the human papillomavirus (HPV) in head and neck cancer. HPV-related head and neck cancers have been growing in number

Bethesda, Md., Wed., Jan. 28, 2015 – Investigators with The Cancer Genome Atlas (TCGA) Research Network have discovered genomic differences – with potentially important clinical implications – in head and neck cancers caused by infection with the human papillomavirus (HPV). HPV is the most common sexually transmitted virus in the United States, and the number of HPV-related head and neck cancers has been growing. Almost every sexually active person will acquire HPV at some point in their lives, according to the Centers for Disease Control and Prevention.

The researchers also uncovered new smoking-related cancer subtypes and potential new drug targets, and found numerous genomic similarities with other cancer types. Taken together, this study’s findings may provide more detailed explanations of how HPV infection and smoking play roles in head and neck cancer risk and disease development, and offer potential novel diagnostic and treatment directions.

The study is the most comprehensive examination to date of genomic alterations in head and neck cancers. The results were published online Jan. 28, 2015 in the journal Nature. TCGA is jointly supported and managed by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI), both parts of the National Institutes of Health.

The U.S. Food and Drug Administration-approved HPV vaccines should be able to prevent the cancers caused by HPV infection in head and neck cancers and elsewhere, including anal cancer, whose incidence has also been increasing.
However, these vaccines work by preventing new infections, and the long interval between infection and cancer development make it important to understand the molecular changes that bring about these HPV-positive head and neck cancers – as well as those that lead to the HPV-negative cancers – and to develop new approaches for treating them.

“The rapid increase in HPV-related head and neck cancers, noticeably in oropharyngeal tumors, has created an even greater sense of urgency in the field,” said D. Neil Hayes, M.D., M.P.H, senior author of the study report and associate professor of medicine at the University of North Carolina (UNC) and the UNC Lineberger Comprehensive Cancer Center at Chapel Hill. Oropharyngeal cancer starts in the oropharynx, which is the part of the throat just behind the mouth. “We’re uncovering differences between tumors with and without HPV infection, and these new data are allowing us to rethink how we approach head and neck cancers.”

In the study, researchers performed genomic analyses on 279 tumors – head and neck squamous cell carcinomas (HNSCC) – from untreated patients. Approximately 80 percent of tumor samples were from individuals who smoked. The majority of samples were oral cavity cancers (61 percent) and larynx cancers (26 percent).

While only about 25 percent of head and neck cancers are linked to HPV infection, TCGA researchers confirmed that many patients with HPV-associated tumors have specific alterations of the gene FGFR3 and mutations in the PIK3CA gene, which are also found in a much broader set of mutations in smoking-related tumors. In contrast, while the EGFR (epidermal growth factor receptor) gene is frequently altered in HPV-negative tumors in smokers, it is rarely abnormal in HPV-positive tumors. Such insights may help in developing potential therapies and biomarkers, noted Dr. Hayes.

Head and neck cancers comprise a constellation of tumors of the mouth, throat, larynx, nasal cavity, salivary gland and elsewhere that have frequently been attributed to tobacco and alcohol use in most patients. Some 90 percent are squamous cell carcinomas, which occur in the surface layers of cells in the body. An estimated 55,000 people developed head and neck cancer in the United States in 2014. Approximately 12,000 Americans die from the diseases each year. Head and neck cancers are common worldwide, with more than 600,000 cases diagnosed each year.

“The rising worldwide incidence of head and neck cancers makes these types of large integrated genomic analyses by TCGA vital to establish a more detailed understanding of disease causes and behavior, and for the development of new treatment approaches,” said NIH Director Francis S. Collins, M.D., Ph.D.

Scientists found that more than 70 percent of head and neck cancers had alterations in genes for growth factor receptors (EGFR, FGFR, IGFR, MET, ERBB2, DDR2), signaling molecules (PIK3CA, HRAS) and cell division regulation (CCND1). These genes may play roles in pathways that control cell growth and proliferation, and for which therapies are either available or in development.

The investigators also discovered new clues about drug resistance in head and neck cancers. They found that genes affecting about 40 percent of such cancers form key parts of a pathway that helps determine cell survival and drug resistance. They showed that extra copies of the genes FADD and BIRC2, or mutations in or the absence of the CASP8 gene in smoking-related cancers – all which affect the process of programmed cell death – may underlie the resistance of cancer cells to current treatments. Similarly, the absence of the TRAF3 gene, or extra copies of a gene for the growth-promoting E2F1 protein in HPV-related cancers, may also increase resistance.

The findings showed similarities between head and neck cancer genomes and other cancers, including squamous cell lung and cervical, indicating possible common paths of cancer development, and potential treatment opportunities. “It is surprising to see that head and neck tumor genomes are remarkably similar to cervical and squamous lung cancer genomes. They are from very different organs, but they show similar losses and gains of genetic material across tumors,” Dr. Hayes noted. These common genetic abnormalities belong to a pathway that protects cells from damage and stress.

“These novel findings help establish a genomic map of various head and neck cancers, provide new insights into other similar cancers and  may further our understanding of how viruses can impact disease,” said NHGRI Director Eric D. Green, M.D., Ph.D.

“While many head and neck cancers are preventable, they are increasingly common throughout the world, and often challenging to effectively treat over the long term,” said NCI Director Harold Varmus, M.D. “This type of broad analysis provides important new clues for future research and treatment directions.”

The TCGA Research Network has generated data and published analyses on a number of cancers, all of which can be found on the TCGA website,

The TCGA Research Network consists of more than 150 researchers at dozens of institutions across the nation.  A list of participants is available at More details about The Cancer Genome Atlas, including Quick Facts, Q&A, graphics, glossary, a brief guide to genomics and a media library of available images can be found at

NHGRI is one of the 27 institutes and centers at the National Institutes of Health. The NHGRI Extramural Research Program supports grants for research and training and career development at sites nationwide. Additional information about NHGRI can be found at

NCI leads the National Cancer Program and the NIH effort to dramatically reduce the burden of cancer and improve the lives of cancer patients and their families, through research into prevention and cancer biology, the development of new interventions, and the training and mentoring of new researchers. For more information about cancer, please visit the NCI website at or call NCI’s Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).

The NIDCD supports and conducts research and research training on the normal and disordered processes of hearing, balance, taste, smell, voice, speech, and language and provides health information, based upon scientific discovery, to the public. For more information about NIDCD programs, see the NIDCD website.

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 institutes and centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases.

Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation
Siqi Liu1, Xin Cai1, Jiaxi Wu1, Qian Cong2, Xiang Chen1,3, Tuo Li1, Fenghe Du1,3, Junyao Ren1, Youtong Wu1, Nick Grishin2,3, Zhijian J. Chen1,3,*

1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
3Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.

During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cyclic guanosine monophosphate–adenosine monophosphate synthase, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here, we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases inhibitor of nuclear factor κB subunit IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.

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Genomics of Bacterial and Archaeal Viruses


Reporter: Larry H Bernstein, MD, FCAP of Bacterial and Archaeal Viruses/

Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere
M Krupovic, D Prangishvili, RW Hendrix, and DH Bamford
Over the past few years, the viruses of prokaryotes have been transformed in the view of microbiologists from simply being convenient experimental model systems into being a major component of the biosphere. They are
  • the global champions of diversity,
  • they constitute a majority of organisms on the planet,
  • they have large roles in the planet’s ecosystems,
  • they exert a significant—some would say dominant—force on
  • the evolution of their bacterial and archaeal hosts, and
  • they have been doing this for billions of years,
  • possibly for as long as there have been cells.
This transformation in status  or, rather, our expanded appreciation of the importance of these viruses in the biosphere is due to a few significant developments in both understanding and technology.
(i) It has become clear that the population sizes of these viruses are astoundingly large. This realization grew out of electron microscopic enumerations of tailed phage virions in costal seawater, and numerous measurements in other environments have been made since then. A current estimate based on these measurements is that
  • there are  1031 individual tailed phage virions in the global biosphere—
  • enough to reach for 200 million light years if laid end to end—and measurements of population turnover suggest that
  • it takes roughly 1024 productive infections per second to maintain the global population.
(ii) Advances in DNA sequencing technology have led to dramatic qualitative improvements in how we understand the
  • genetic structure of viral populations,
  • the mechanisms of viral evolution, and
  • the diversity of viral sequences.
The majority of newly determined gene and protein sequences of these viruses has no relatives detectable in the public sequence databases, and
  • analysis of metagenomic data provides strong evidence that
  • there is more genetic diversity in the genes of the viruses of prokaryotes
    • than in any other compartment of the biosphere.
(iii) Facilitated by these conceptual and technical advances, studies of bacterial and archaeal viruses as important components of global biology have flourished. These viruses are revealed as important players in
  • carbon and energy cycling in the oceans and other natural environments and
  • as major agents in the ecology and evolution of their cellular hosts.
(iv) The isolation and characterization of new viruses have accelerated. This has been especially important for the archaeal viruses, where the discovery of new viruses and of new virus types had lagged behind bacteriophage discovery. For the bacteriophages, the isolation of newly discovered viruses has helped improve the still extremely sparse coverage of sequence diversity and the narrow phylogenetic range of hosts represented by current data.
(v) High-resolution structures determined
  • for capsid proteins and other virion proteins,
  • together with information about virion assembly mechanisms,
  • have allowed surprising inferences about ancestral connections among genes whose DNA sequences and encoded protein sequences
    • have diverged to the point that they are no longer detectably related.
English: Schematic diagram of the hexon of a v...

English: Schematic diagram of the hexon of a virus capsid (Photo credit: Wikipedia)

English: Adsorption of virions to cells. Portu...

English: Adsorption of virions to cells. Português do Brasil: Adsorção de vírus a células. (Photo credit: Wikipedia)

Polio virus (picornavirus)

Polio virus (picornavirus) (Photo credit: Sanofi Pasteur)

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Curator/Reporter Aviral Vatsa PhD, MBBS

Based on: A review by (Wink et al., 2011)

This post is in continuation to Part 1 by the same title.

In part one I covered the basics of role of redox chemistry in immune reactions, the phagosome cauldron, and how bacteria bacteria, virus and parasites trigger the complex pathway of NO production and its downstream effects. While we move further in this post, the previous post can be accessed here.


Regulation of the redox immunomodulators—NO/RNS and ROS

In addition to eradicating pathogens, NO/RNS and ROS and their chemical interactions act as effective immunomodulators that regulate many cellular metabolic pathways and tissue repair and proinflammatory pathways. Figure 3 shows these pathways.

Figure 3. Schematic overview of interactive connections between NO and ROS-mediated metabolic pathways. Credit: (Wink et al., 2011)

Regulation of iNOS enzyme activity is critical to NO production. Factors such as the availability of arginine, BH4, NADPH, and superoxide affect iNOS activity and thus NO production. In the absence of arginine and BH4 iNOS becomes a O2_/H2O2 generator (Vásquez-Vivar et al., 1999). Hence metabolic pathways that control arginine and BH4 play a role in determining the NO/superoxide balance. Arginine levels in cells depend on various factors such as type of uptake mechanisms that determine its spatial presence in various compartments and enzymatic systems. As shown in Fig3 Arginine is the sole substrate for iNOS and arginase. Arginase is another key enzyme in immunemodulation. AG is also regulated by NOS and NOX activities. NOHA, a product of NOS, inhibits AG, and O2–increases AG activity. Importantly, high AG activity is associated with elevated ROS and low NO fluxes. NO antagonises NOX2 assembly that in turn leads to reduction in O2_ production. NO also inhibits COX2 activity thus reducing ROS production. Thus, as NO levels decline, oxidative mechanisms increase. Oxidative and nitrosative stress can also decrease intracellular GSH (reduced form) levels, resulting in a reduced antioxidant capability of the cell.

Immune-associated redox pathways regulate other important metabolic cell functions that have the potential for widespread impact on cells, organs, and organisms. These pathways, such as mediated via methionine and polyamines, are critical for DNA stabilization, cell proliferation, and membrane channel activity, all of which are also involved in immune-mediated repair processes.

NO levels dictate the immune signaling pathway

NO/RNS and ROS actively control innate and adaptive immune signaling by participating in induction, maintenance, and/or termination of proinflammatory and anti-inflammatory signaling. As in pathogen eradication, the temporal and spatial concentration profiles of NO are key factors in determining immune-mediated processes.

Brune and coworkers (Messmer et al., 1994) first demonstrated that p53 expression was associated with the concentrations of NO that led to apoptosis in macrophages. Subsequent studies linked NO concentration profiles with expression of other key signaling proteins such as HIF-1α and Akt-P (Ridnour et al., 2008; Thomas et al., 2008). Various levels of NO concentrations trigger different pathways and expectedly this concentration-dependent profile varies with distance from the NO source.NO is highly diffucible and this characteristic can result in 1000 fold reduction in concentration within one cell length distance travelled from the source of production. Time course studies have also shown alteration in effects of same levels of NO over time e.g. NO-mediated ERK-P levels initially increased rapidly on exposure to NO donors and then decreased with continued NO exposure (Thomas et al., 2004), however HIF-1α levels remained high as long as NO levels were elevated. Thus some of the important factors that play critical role in NO effects are: distance from source, NO concentrations, duration of exposure, bioavailability of NO, and production/absence of other redox molecules.

Figure and legend credits: (Wink et al., 2011)

Fig 4: The effect of steady-state flux of NO on signal transduction mechanisms.

This diagram represents the level of sustained NO that is required to activate specific pathways in tumor cells. Similar effects have been seen on endothelial cells. These data were generated by treating tumor or endothelial cells with the NO donor DETANO (NOC-18) for 24 h and then measuring the appropriate outcome measures (for example, p53 activation). Various concentrations of DETANO that correspond to cellular levels of NO are: 40–60 μM DETANO = 50 nM NO; 80–120 μM DETANO = 100 nM NO; 500 μM DETANO = 400 nM NO; and 1 mM DETANO = 1 μM NO. The diagram represents the effect of diffusion of NO with distance from the point source (an activated murine macrophage producing iNOS) in vitro (Petri dish) generating 1 μM NO or more. Thus, reactants or cells located at a specific distance from the point source (i.e., iNOS, represented by star) would be exposed to a level of NO that governs a specific subset of physiological or pathophysiological reactions. The x-axis represents the different zone of NO-mediated events that is experienced at a specific distance from a source iNOS producing >1 μM. Note: Akt activation is regulated by NO at two different sites and by two different concentration levels of NO.

Species-specific NO production

The relationship of NO and immunoregulation has been established on the basis of studies on tumor cell lines or rodent macrophages, which are readily available sources of NO. However in humans the levels of protein expression for NOS enzymes and the immune induction required for such levels of expression are quite different than in rodents (Weinberg, 1998). This difference is most likely due to the human iNOS promotor rather than the activity of iNOS itself. There is a significant mismatch between the promoters of humans and rodents and that is likely to account for the notable differences in the regulation of gene induction between them. The combined data on rodent versus human NO and O2– production strongly suggest that in general, ROS production is a predominant feature of activated human macrophages, neutrophils, and monocytes, and the equivalent murine immune cells generate a combination of O2– and NO and in some cases, favor NO production. These differences may be crucial to understanding how immune responses are regulated in a species-specific manner. This is particularly useful, as pathogen challenges change constantly.

The next post in this series will cover the following topics:

The impact of NO signaling on an innate immune response—classical activation

NO and proinflammatory genes

NO and regulation of anti-inflammatory pathways

NO impact on adaptive immunity—immunosuppression and tissue-restoration response

NO and revascularization

Acute versus chronic inflammatory disease


1. Wink, D. A. et al. Nitric oxide and redox mechanisms in the immune response. J Leukoc Biol 89, 873–891 (2011).

2. Vásquez-Vivar, J. et al. Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase. J. Biol. Chem. 274, 26736–26742 (1999).

3. Messmer, U. K., Ankarcrona, M., Nicotera, P. & Brüne, B. p53 expression in nitric oxide-induced apoptosis. FEBS Lett. 355, 23–26 (1994).

4. Ridnour, L. A. et al. Molecular mechanisms for discrete nitric oxide levels in cancer. Nitric Oxide 19, 73–76 (2008).

5. Thomas, D. D. et al. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic. Biol. Med. 45, 18–31 (2008).

6. Thomas, D. D. et al. Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 101, 8894–8899 (2004).

7. Weinberg, J. B. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol. Med. 4, 557–591 (1998).

Further reading on NO:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

Author: Aviral Vatsa, PhD, September 23, 2012

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.

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Curator and Reporter: Aviral Vatsa PhD, MBBS

Based on: A review by Wink et al., 2011

This is the first part of a two part post

Nitric oxide (NO), reactive nitrogen species (RNS) and reactive oxygen species (ROS) perform dual roles as immunotoxins and immunomodulators. An incoming immune signal initiates NO and ROS production both for tackling the pathogens and modulating the downstream immune response via complex signaling pathways. The complexity of these interactions is a reflection of involvement of redox chemistry in biological setting (fig. 1)

Fig 1. Image credit: (Wink et al., 2011)

Previous studies have highlighted the role of NO in immunity. It was shown that macrophages released a substance that had antitumor and antipathogen activity and required arginine for its production (Hibbs et al., 1987, 1988). Hibbs and coworkers further strengthened the connection between immunity and NO by demonstrating that IL2 mediated immune activation increased NO levels in patients and promoted tumor eradication in mice (Hibbs et al., 1992; Yim et al., 1995).

In 1980s a number of authors showed the direct evidence that macrophages made nitrite, nitrates and nitrosamines. It was also shown that NO generated by macrophages could kill leukemia cells (Stuehr and Nathan, 1989). Collectively these studies along with others demonstrated the important role NO plays in immunity and lay the path for further research in understanding the role of redox molecules in immunity.

NO is produced by different forms of nitric oxide synthase (NOS) enzymes such as eNOS (endothelial), iNOS (inducible) and nNOS (neuronal). The constitutive forms of eNOS generally produce NO in short bursts and in calcium dependent manner. The inducible form produces NO for longer durations and is calcium independent. In immunity, iNOS plays a vital role. NO production by iNOS can occur over a wide range of concentrations from as little as nM to as much as µM. This wide range of NO concentrations provide iNOS with a unique flexibility to be functionally effective in various conditions and micro-environements and thus provide different temporal and concentration profiles of NO, that can be highly efficient in dealing with immune challenges.

Redox reactions in immune responses

NO/RNS and ROS are two categories of molecules that bring about immune regulation and ‘killing’ of pathogens. These molecules can perform independently or in combination with each other. NO reacts directly with transition metals in heme or cobalamine, with non-heme iron, or with reactive radicals (Wink and Mitchell, 1998). The last reactivity also imparts it a powerful antioxidant capability. NO can thus act directly as a powerful antioxidant and prevent injury initiated by ROS (Wink et al., 1999). On the other hand, NO does not react directly with thiols or other nucleophiles but requires activation with superoxide to generate RNS. The RNS species then cause nitrosative and oxidative stress (Wink and Mitchell, 1998).

The variety of functions achieved by NO can be understood if one looks at certain chemical concepts. NO and NO2 are lipophilic and thus can migrate through cells, thus widening potential target profiles. ONOO-, a RNS, reacts rapidly with CO2 that shortens its half life to <10 ms. The anionic form and short half life limits its mobility across membranes. When NO levels are higher than superoxide levels, the CO2-OONOintermediate is converted to NO2 and N2O3 and changes the redox profile from an oxidative to a nitrosative microenvironment. The interaction of NO and ROS determines the bioavailability of NO and proximity of RNS generation to superoxide source, thus defining a reaction profile. The ROS also consumes NO to generate NO2 and N2O3 as well as nitrite in certain locations. The combination of these reactions in different micro-environments provides a vast repertoire of reaction profiles for NO/RNS and ROS entities.

The Phagosome ‘cauldron’

The phagosome provides an ‘isolated’ environment for the cell to carry out foreign body ‘destruction’. ROS, NO and RNS interact to bring about redox reactions. The concentration of NO in a phagosome can depend on the kind of NOS in the vicinity and its activity and other localised cellular factors. NO and is metabolites such as nitrites and nitrates along with ROS combine forces to kill pathogens in the acidic environment of the phagosome as depicted in the figure 2 below.

Fig 2. The NO chemistry of the phagosome. (image credit: (Wink et al., 2011)

This diagram depicts the different nitrogen oxide and ROS chemistry that can occur within the phagosome to fight pathogens. The presence of NOX2 in the phagosomes serves two purposes: one is to focus the nitrite accumulation through scavenging mechanisms, and the second provides peroxide as a source of ROS or FA generation. The nitrite (NO2−) formed in the acidic environment provides nitrosative stress with NO/NO2/N2O3. The combined acidic nature and the ability to form multiple RNS and ROS within the acidic environment of the phagosome provide the immune response with multiple chemical options with which it can combat bacteria.


There are various ways in which NO combines forces with other molecules to bring about bacterial killing. Here are few examples

E.coli: It appears to be resistant to individual action of NO/RNS and H2O2 /ROS. However, when combined together, H2O2 plus NO mediate a dramatic, three-log increase in cytotoxicity, as opposed to 50% killing by NO alone or H2O2 alone. This indicates that these bacteria are highly susceptible to their synergistic action.

Staphylococcus: The combined presence of NO and peroxide in staphylococcal infections imparts protective effect. However, when these bacteria are first exposed to peroxide and then to NO there is increased toxicity. Hence a sequential exposure to superoxide/ROS and then NO is a potent tool in eradicating staphylococcal bacteria.

Mycobacterium tuberculosis: These bacterium are sensitive to NO and RNS, but in this case, NO2 is the toxic species. A phagosome microenvironment consisting of ROS combined with acidic nitrite generates NO2/N2O3/NO, which is essential for pathogen eradication by the alveolar macrophage. Overall, NO has a dual function; it participates directly in killing an organism, and/or it disarms a pathway used by that organism to elude other immune responses.


Many human parasites have demonstrated the initiation of the immune response via the induction of iNOS, that then leads to expulsion of the parasite. The parasites include Plasmodia(malaria), Leishmania(leishmaniasis), and Toxoplasma(toxoplasmosis). Severe cases of malaria have been related with increased production of NO. High levels of NO production are however protective in these cases as NO was shown to kill the parasites (Rockett et al., 1991; Gyan et al., 1994). Leishmania is an intracellualr parasite that resides in the mamalian macrophages. NO upregulation via iNOS induction is the primary pathway involved in containing its infestation. A critical aspect of NO metabolism is that NOHA inhibits AG activity, thereby limiting the growth of parasites and bacteria including Leishmania, Trypanosoma, Schistosoma, HelicobacterMycobacterium, and Salmonella, and is distinct from the effects of RNS. Toxoplasma gondii is also an intracellular parasite that elicits NO mediated response. INOS knockout mice have shown more severe inflammatory lesions in the CNS that their wild type counterparts, in response to toxoplasma exposure. This indicates the CNS preventative role of iNOS in toxoplasmosis (Silva et al., 2009).


Viral replication can be checked by increased production of NO by induction of iNOS (HIV-1, coxsackievirus, influenza A and B, rhino virus, CMV, vaccinia virus, ectromelia virus, human herpesvirus-1, and human parainfluenza virus type 3) (Xu et al., 2006). NO can reduce viral load, reduce latency and reduce viral replication. One of the main mechanisms as to how NO participates in viral eradication involves the nitrosation of critical cysteines within key proteins required for viral infection, transcription, and maturation stages. For example, viral proteases or even the host caspases that contain cysteines in their active site are involved in the maturation of the virus. The nitrosative stress environment produced by iNOS may serve to protect against some viruses by inhibiting viral infectivity, replication, and maturation.

To be continued in part 2 …


Gyan, B., Troye-Blomberg, M., Perlmann, P., Björkman, A., 1994. Human monocytes cultured with and without interferon-gamma inhibit Plasmodium falciparum parasite growth in vitro via secretion of reactive nitrogen intermediates. Parasite Immunol. 16, 371–3

Hibbs, J.B., Jr, Taintor, R.R., Vavrin, Z., 1987. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science 235, 473–476.

Hibbs, J.B., Jr, Taintor, R.R., Vavrin, Z., Rachlin, E.M., 1988. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157, 87–94.

Hibbs, J.B., Jr, Westenfelder, C., Taintor, R., Vavrin, Z., Kablitz, C., Baranowski, R.L., Ward, J.H., Menlove, R.L., McMurry, M.P., Kushner, J.P., 1992. Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleu

Rockett, K.A., Awburn, M.M., Cowden, W.B., Clark, I.A., 1991. Killing of Plasmodium falciparum in vitro by nitric oxide derivatives. Infect Immun 59, 3280–3283.

Stuehr, D.J., Nathan, C.F., 1989. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169, 1543–1555.

Wink, D.A., Hines, H.B., Cheng, R.Y.S., Switzer, C.H., Flores-Santana, W., Vitek, M.P., Ridnour, L.A., Colton, C.A., 2011. Nitric oxide and redox mechanisms in the immune response. J Leukoc Biol 89, 873–891.

Wink, D.A., Mitchell, J.B., 1998. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 25, 434–456.

Wink, D.A., Vodovotz, Y., Grisham, M.B., DeGraff, W., Cook, J.C., Pacelli, R., Krishna, M., Mitchell, J.B., 1999. Antioxidant effects of nitric oxide. Meth. Enzymol. 301, 413–424.

Xu, W., Zheng, S., Dweik, R.A., Erzurum, S.C., 2006. Role of epithelial nitric oxide in airway viral infection. Free Radic. Biol. Med. 41, 19–28.

Yim, C.Y., McGregor, J.R., Kwon, O.D., Bastian, N.R., Rees, M., Mori, M., Hibbs, J.B., Jr, Samlowski, W.E., 1995. Nitric oxide synthesis contributes to IL-2-induced antitumor responses against intraperitoneal Meth A tumor. J. Immunol. 155, 4382–4390.

Further reading on NO:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

Author: Aviral Vatsa, PhD, September 23, 2012

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.


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