Attenuation and Restoration of Severe Acute Respiratory Syndrome Coronavirus Mutant Lacking 2'-O-Methyltransferase Activity

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and, more recently, Middle Eastern respiratory syndrome CoV (MERS-CoV) underscores the importance of understanding critical aspects of CoV infection and pathogenesis. Despite significant insights into CoV cross-species transmission, replication, and virus-host interactions, successful therapeutic options for CoVs do not yet exist. Recent identification of SARS-CoV NSP16 as a viral 2′-O-methyltransferase (2′-O-MTase) led to the possibility of utilizing this pathway to both attenuate SARS-CoV infection and develop novel therapeutic treatment options. Mutations were introduced into SARS-CoV NSP16 within the conserved KDKE motif and effectively attenuated the resulting SARS-CoV mutant viruses both in vitro and in vivo. While viruses lacking 2′-O-MTase activity had enhanced sensitivity to type I interferon (IFN), they were not completely restored in their absence in vivo. However, the absence of either MDA5 or IFIT1, IFN-responsive genes that recognize unmethylated 2′-O RNA, resulted in restored replication and virulence of the dNSP16 mutant virus. Finally, using the mutant as a live-attenuated vaccine showed significant promise for possible therapeutic development against SARS-CoV. Together, the data underscore the necessity of 2′-O-MTase activity for SARS-CoV pathogenesis and identify host immune pathways that mediate this attenuation. In addition, we describe novel treatment avenues that exploit this pathway and could potentially be used against a diverse range of viral pathogens that utilize 2′-O-MTase activity to subvert the immune system

Envelope 2 protein phosphorylation sites S75 & 277 of hepatitis C virus genotype 1a and interferon resistance: A sequence alignment approach

<p>Abstract</p> <p>Background</p> <p>Hepatitis C is a major health problem affecting more than 200 million individuals in world including Pakistan. Current treatment regimen consisting of interferon alpha and ribavirin does not always succeed to eliminate virus completely from the patient's body.</p> <p>Results</p> <p>Interferon induced antiviral protein kinase R (PKR) has a role in the hepatitis C virus (HCV) treatment as dsRNA activated PKR has the capacity to phosphorylate the serine and threonine of E2 protein and dimerization viral RNA. E2 gene of hepatitis C virus (HCV) genotype 1 has an active role in IFN resistance. E2 protein inhibits and terminates the kinase activity of PKR by blocking it in protein synthesis and cell growth. This brings forward a possible relation of E2 and PKR through a mechanism via which HCV evades the antiviral effect of IFN.</p> <p>Conclusion</p> <p>A hybrid in-silico and wet laboratory approach of motif prediction, evolutionary and structural anlysis has pointed out serine 75 and 277 of the HCV E2 gene as a promising candidate for the serine phosphorylation. It is proposed that serine phosphorylation of HCV E2 gene has a significant role in interferon resistance.</p

Lethal Influenza Virus Infection in Macaques Is Associated with Early Dysregulation of Inflammatory Related Genes

The enormous toll on human life during the 1918–1919 Spanish influenza pandemic is a constant reminder of the potential lethality of influenza viruses. With the declaration by the World Health Organization of a new H1N1 influenza virus pandemic, and with continued human cases of highly pathogenic H5N1 avian influenza virus infection, a better understanding of the host response to highly pathogenic influenza viruses is essential. To this end, we compared pathology and global gene expression profiles in bronchial tissue from macaques infected with either the reconstructed 1918 pandemic virus or the highly pathogenic avian H5N1 virus A/Vietnam/1203/04. Severe pathology was observed in respiratory tissues from 1918 virus-infected animals as early as 12 hours after infection, and pathology steadily increased at later time points. Although tissues from animals infected with A/Vietnam/1203/04 also showed clear signs of pathology early on, less pathology was observed at later time points, and there was evidence of tissue repair. Global transcriptional profiles revealed that specific groups of genes associated with inflammation and cell death were up-regulated in bronchial tissues from animals infected with the 1918 virus but down-regulated in animals infected with A/Vietnam/1203/04. Importantly, the 1918 virus up-regulated key components of the inflammasome, NLRP3 and IL-1β, whereas these genes were down-regulated by A/Vietnam/1203/04 early after infection. TUNEL assays revealed that both viruses elicited an apoptotic response in lungs and bronchi, although the response occurred earlier during 1918 virus infection. Our findings suggest that the severity of disease in 1918 virus-infected macaques is a consequence of the early up-regulation of cell death and inflammatory related genes, in which additive or synergistic effects likely dictate the severity of tissue damage

Gene Expression Patterns of Dengue Virus-Infected Children from Nicaragua Reveal a Distinct Signature of Increased Metabolism

Dengue is a widespread viral disease for which over 3 billion people are at risk. There are no drug treatments or vaccines available for this disease. It is also difficult for physicians to predict which patients are at highest risk for the severe manifestations known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We used genome-wide transcriptional profiling analysis to study peripheral blood responses to dengue among patients from Nicaragua. We found that patients with severe manifestations involving shock had very different transcriptional profiles from dengue patients with mild and moderate illness. We then compared our results with other microarray experiments on dengue patients available from public databases and confirmed that dengue is often associated with large changes to the metabolic processes within cells. This approach could identify prognostic markers for severe dengue as well as provide a better understanding of the pathophysiology associated with different grades of disease severity

Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

In this work, we demonstrate that that both human whole skin and freshly isolated skin fibroblasts are productively infected with Dengue virus (DENV). In addition, primary skin fibroblast cultures were established and subsequently infected with DENV-2; we showed in these cells the presence of the viral antigen NS3, and we found productive viral infection by a conventional plaque assay. Of note, the infectivity rate was almost the same in all the primary cultures analyzed from different donors. The skin fibroblasts infected with DENV-2 underwent signaling through both TLR3 and RIG-1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 IRF7, when compared with mock-infected fibroblasts. Our data suggest that fibroblasts might even participate producing mediators involved in innate immunity that activate and contribute to the orchestration of the local innate responses. This work is the first evaluating primary skin fibroblast cultures obtained from different humans, assessing both their susceptibility to DENV infection as well as their ability to produce molecules crucial for innate immunity

Copy Number Variation of CCL3-like Genes Affects Rate of Progression to Simian-AIDS in Rhesus Macaques (Macaca mulatta)

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10−6) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se

Genomic Analysis Reveals a Potential Role for Cell Cycle Perturbation in HCV-Mediated Apoptosis of Cultured Hepatocytes

The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death–related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-β1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation

STAT2 Mediates Innate Immunity to Dengue Virus in the Absence of STAT1 via the Type I Interferon Receptor

Dengue virus (DENV) is a mosquito-borne flavivirus, and symptoms of infection range from asymptomatic to the severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication. We have previously reported that signal transducer and activator of transcription (STAT) 1-deficient mice are resistant to DENV-induced disease, but little is known about this STAT1-independent mechanism of protection. To determine the molecular basis of the STAT1-independent pathway, mice lacking STAT1, STAT2, or both STAT1 and STAT2 were infected with a virulent mouse-adapted strain of DENV2. In the first 72 hours of infection, the single-deficient mice lacking STAT1 or STAT2 possessed 50–100 fold higher levels of viral RNA than wild type mice in the serum, spleen, and other visceral tissues, but remained resistant to DENV-induced death. In contrast, the double-deficient mice exhibited the early death phenotype previously observed in type I and II IFN receptor knockout mice (AG129), indicating that STAT2 is the mediator of the STAT1-independent host defense mechanism. Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression. Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1. Collectively, these results help elucidate the nature of the poorly understood STAT1-independent host defense mechanism against viruses by identifying a functional type I IFN/STAT2 signaling pathway following DENV infection in vivo

Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

BACKGROUND: Double-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58(IPK)), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58(IPK) is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58(IPK) activation was hitherto unknown. PRINCIPAL FINDINGS: Human heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58(IPK) from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2α phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN β production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58(IPK) activation and subsequent inhibition of PKR-mediated host response during IAV infection. SIGNIFICANCE: Our findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58(IPK) mediated inhibition of PKR activity during IAV infection

Global mRNA Degradation during Lytic Gammaherpesvirus Infection Contributes to Establishment of Viral Latency

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3′ end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment