HLA class I molecules consistently present internal influenza epitopes

Cytotoxic T lymphocytes (CTL) limit influenza virus replication and prevent morbidity and mortality upon recognition of HLA class I presented epitopes on the surface of virus infected cells, yet the number and origin of the viral epitopes that decorate the infected cell are unknown. To understand the presentation of influenza virus ligands by human MHC class I molecules, HLA-B*0702-presented viral peptides were directly identified following influenza infection. After transfection with soluble class I molecules, peptide ligands unique to infected cells were eluted from isolated MHC molecules and identified by comparative mass spectrometry (MS). Then CTL were gathered following infection with influenza and viral peptides were tested for immune recognition. We found that the class I molecule B*0702 presents 3–6 viral ligands following infection with different strains of influenza. Peptide ligands derived from the internal viral nucleoprotein (NP_418–426 and NP_473–481) and from the internal viral polymerase subunit PB1 (PB1_329–337) were presented by B*0702 following infection with each of 3 different influenza strains; ligands NP_418–426, NP_473–481, and PB1_329–337 derived from internal viral proteins were consistently revealed by class I HLA. In contrast, ligands derived from hemagglutinin (HA) and matrix protein (M1) were presented intermittently on a strain-by-strain basis. When tested for immune recognition, HLA-B*0702 transgenic mice responded to NP_418–426 and PB1_329–337 consistently and NP_473–481 intermittently while ligands from HA and M1 were not recognized. These data demonstrate an emerging pattern whereby class I HLA reveal a handful of internal viral ligands and whereby CTL recognize consistently presented influenza ligands.     

Antiviral Activity of Umifenovir In Vitro against a Broad Spectrum of Coronaviruses,Including the Novel SARS-CoV-2 Virus

An escalating pandemic of the novel SARS-CoV-2 virus is impacting global health, and effective antivirals are needed. Umifenovir (Arbidol) is an indole-derivative molecule, licensed in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It has been shown that umifenovir has broad spectrum activity against different viruses. We evaluated the sensitivity of different coronaviruses, including the novel SARS-CoV-2 virus, to umifenovir using in vitro assays. Using a plaque assay, we revealed an antiviral effect of umifenovir against seasonal HCoV-229E and HCoV-OC43 coronaviruses in Vero E6 cells, with estimated 50% effective concentrations (EC50) of 10.0 ± 0.5 µM and 9.0 ± 0.4 µM, respectively. Umifenovir at 90 µM significantly suppressed plaque formation in CMK-AH-1 cells infected with SARS-CoV. Umifenovir also inhibited the replication of SARS-CoV-2 virus, with EC50 values ranging from 15.37 ± 3.6 to 28.0 ± 1.0 µM. In addition, 21–36 µM of umifenovir significantly suppressed SARS-CoV-2 virus titers (≥2 log TCID50/mL) in the first 24 h after infection. Repurposing of antiviral drugs is very helpful in fighting COVID-19. A safe, pan-antiviral drug such as umifenovir could be extremely beneficial in combating the early stages of a viral pandemic.     

High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein

The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010–001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a T_m increase of more than 10 °C. NVR-010–001-E2 binds at the dimer–dimer interface of the core proteins, forms a new interaction surface promoting protein–protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010–001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein–protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.Hepatitis B virus (HBV) infection is a major global health concern. It is estimated that 240 million people live with chronic hepatitis B (CHB) with a high risk of developing severe liver disease or liver cancer. Globally, 780,000 deaths per year are associated with HBV infection (1). Current approved treatment options (IFN alpha products or nucleoside analogs) are indicated for treatment of only a subset of CHB patients and are curative in only a very small proportion of patients, resulting in an urgent need for new types of therapies that can increase HBV cure rates.The HBV core protein is a viral protein with no known related protein present in human cells. The core protein performs multiple essential roles at different stages of the virus life cycle; it interacts with other host and viral proteins and has to be able to form a capsid stable enough to protect viral RNA and DNA and be able to release viral DNA at the right time in the virus life cycle. HBV core protein is therefore an excellent target for the development of new, virus-selective, safe and effective antiviral agents to improve treatment options for this disease (2, 3). The HBV core protein consists of 183–185 amino acids that form an N-terminal (amino acids 1–149) capsid assembly domain and a C-terminal nucleic acid binding domain (amino acids 150–185). The viral capsid in infectious HBV particles is formed from 120 copies of assembled core protein dimers enclosing the viral DNA (4, 5). Small molecules that target the HBV core protein assembly domain can disrupt functional HBV capsid assembly and can be potent inhibitors of HBV replication (6–11). Most of these compounds have the ability to induce aggregation of the HBV core N-terminal assembly domain in vitro. Representative HBV core inhibitors from the heteroaryldihydropyrimidine (HAP) series of compounds, BAY 41–4109 and GLS4, have also shown antiviral activity in vivo, in mouse models of HBV replication (12–14). However, drug-like properties of current leads have not been optimized, and new classes of HBV core protein targeting compounds need to be developed to maximize antiviral efficacy as well as pharmacokinetic and safety profiles. Lead optimization has been hampered by the lack of high-resolution crystal structures. Crystal structures of assembled wild-type HBV capsid have only been obtained at resolutions above 3 Å, and structures including bound small-molecule inhibitors have been determined at 4.2- and 5.05-Å resolution. Considering these resolution limits together with the technical and time challenges in achieving these structures, the previously published methods are insufficient to support structure-guided lead optimization (5, 15, 16).Here we present the structure of NVR-010–001-E2, a potent inhibitor of HBV replication from the HAP series of antiviral agents, bound to its target site on the HBV core protein at 1.95-Å resolution. The impact of mutations in the binding site on antiviral activity of NVR-010–001-E2 is consistent with the compound–protein interactions observed in the crystal structure and provides a starting point for the evaluation of possible pathways to drug resistance development. The data presented here provide a structural explanation for the ability of a small molecule to induce protein assembly and exemplify an efficient method to facilitate structure-guided optimization of HBV core inhibitors.     

Detailed Analyses of Molecular Interactions between Favipiravir and RNA Viruses In Silico

There are currently no antiviral agents for human metapneumovirus (HMPV), respiratory syncytial virus (RSV), mumps virus (MuV), or measles virus (MeV). Favipiravir has been developed as an anti-influenza agent, and this agent may be effective against these viruses in vitro. However, the molecular mechanisms through which the agent affects virus replication remain to be fully elucidated. Thus, to clarify the detailed molecular interactions between favipiravir and the RNA-dependent RNA polymerase (RdRp) of HMPV, RSV, MuV, MeV, and influenza virus, we performed in silico studies using authentic bioinformatics technologies. As a result, we found that the active form of favipiravir (favipiravir ribofuranosyl-5′-triphosphate [F-RTP]) can bind to the RdRp active sites of HMPV, RSV, MuV, and MeV. The aspartic acid residue of RdRp active sites was involved in the interaction. Moreover, F-RTP was incorporated into the growing viral RNA chain in the presence of nucleotide triphosphate and magnesium ions. The results suggested that favipiravir shows two distinct mechanisms in various viruses: RdRp active site inhibition and/or genome replication inhibition.     

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3′-to-5′ exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3′ end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN–nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.

The 29.9-kb single-stranded RNA genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the global COVID-19 pandemic, is replicated and transcribed by the viral RNA-dependent RNA polymerase (RdRp, nsp12) (1–3). Unlike the high-fidelity cellular replicative DNA polymerases, viral RdRp enzymes, including the CoV RdRp, do not contain a proofreading exonuclease domain to ensure high fidelity. The resulting higher mutation rate (10⁻⁴ to 10⁻⁶ substitutions per nucleotide per round of replication) is generally thought to promote rapid viral adaptation in response to selective pressure (4–6). However, the lack of proofreading activity in RdRp poses a particular challenge for the replication of CoVs, which feature the largest known RNA virus genomes (27 to 32 kb, up to twice the length as the next-largest nonsegmented RNA viral genomes) (7, 8). It has been reported that SARS-CoV nsp12 is the fastest viral RdRp known but with an error rate more than one order of magnitude higher than the generally admitted error rate of viral RdRps (9), clearly necessitating a unique proofreading mechanism.To mitigate the low fidelity of RdRp, all coronaviruses encode a 3′-to-5′ exoribonuclease (ExoN) in multifunctional nsp14 (10–12), which forms a complex with nsp10 critical for the ExoN activity, and additionally contains a C-terminal guanine N7 methyl transferase (N7-MTase) domain. Mutations of SARS-CoV-2 nsp14 exhibit strong association with increased genome-wide mutation load (13, 14), and genetic inactivation of ExoN in engineered SARS-CoV and murine hepatitis virus (MHV) leads to 15- to 20-fold increases in mutation rates (7, 15, 16). Furthermore, in a mouse model, SARS-CoV with inactivated ExoN shows a mutator phenotype with decreased fitness and lower virulence over serial passage, suggesting a potential strategy for generating a live, impaired-fidelity coronavirus vaccine (17). Alternatively, recent studies show that ExoN inactivation abrogates replication of SARS-CoV-2 and Middle East Respiratory Syndrome CoV (18), hinting at additional functions for ExoN in viral replication. Indeed, ExoN activity has been reported to mediate the extensive viral RNA recombination required for subgenomic messenger RNA (mRNA) synthesis during normal replication of CoVs, including SARS-CoV-2 (19), and it was shown to be required for resistance to the antiviral innate immune response for MHV (20). ExoN inactivation also significantly increases the sensitivity of CoVs to nucleoside analogs that target RdRp, which is consistent with the biochemical activity of ExoN to excise mutagenic or chain-terminating nucleotides misincorporated by RdRp (21–23). These observations combine to suggest that chemical inhibition of ExoN could be an effective antiviral strategy against CoVs. In this study, we determined high-resolution crystal structures of the SARS-CoV-2 ExoN–nsp10 complex and studied its biochemical activities. Furthermore, we used molecular dynamics (MD) simulations to better understand the dynamics of nsp14, nsp10, and their interaction with RNA.     

Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5⁺ ILC2 responses, and induced a dominant IL-13⁺ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ–deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR⁺ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

Innate lymphoid cells (ILCs) and T cells represent critical populations of cells that have diverse roles in inflammation and protection (1, 2). Both cell populations consist of subsets that differ in cytokine expression and function. While T cells are important for viral clearance, they can also exacerbate lung immunopathology (1, 3–5) Among ILC subsets, ILC2s play a critical role in pulmonary immunity, particularly in maintaining the lung barrier surface (6–9). During infection, ILC2s respond to the epithelial cell–derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin, and produce the type 2 cytokine IL-5 (10–12). This, in turn, can lead to increased eosinophil recruitment and airway hyperreactivity (AHR) (8, 13–15). Like T cells, ILC2s can play both beneficial and detrimental roles during viral lung infection (6–8).It is known that host cytokines can regulate the activity of ILC and T cell subsets. For example, we previously found that interferon (IFN)-γ deficiency results in enhanced ILC2 activity and increased survival from challenge with the 2009 pandemic strain A/California/04/2009 (CA04) influenza A virus (8). However, our current studies have shown no effect of IFN-γ following challenge with the Puerto Rico/8/1934 (PR8) influenza A virus, a strain that is a commonly used model for the highly virulent 1918 pandemic influenza virus. Although both strains are H1N1 influenza A viruses, they have striking differences in expression of functional PB1-F2, a viral proapoptotic protein that is associated with immunopathology and mortality (16). While the PR8 viral strain expresses full-length PB1-F2, the PB1-F2 gene in the CA04 strain is truncated and nonfunctional (16–20). As a result, the PR8 virus exhibits significantly increased virulence compared to the CA04 viral strain. However, the impact of PB1-F2 on the lymphocyte function that is critical for protection during influenza is not known. A better understanding of the role of pathogen virulence factors in regulating immune cell activity during influenza may aid in designing future therapies for human use.We hypothesized that the PB1-F2 virulence protein can differentially regulate ILC2 and T cell activity in conjunction with host IFN-γ signaling. To test this hypothesis, we have investigated pulmonary immunity in wild-type (WT) and IFN-γ–deficient BALB/c mice infected with PB1-F2 gene reassortant PR8 and CA04 viruses. Our findings demonstrate that viral virulence genes, together with host factors, play critical roles in regulating both ILC2 and T cell responses during influenza, and this, in turn, determines host survival.     

Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTR_P162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5B_P162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.     

Viral Infections and Systemic Lupus Erythematosus: New Players in an Old Story

A causal link between viral infections and autoimmunity has been studied for a long time and the role of some viruses in the induction or exacerbation of systemic lupus erythematosus (SLE) in genetically predisposed patients has been proved. The strength of the association between different viral agents and SLE is variable. Epstein–Barr virus (EBV), parvovirus B19 (B19V), and human endogenous retroviruses (HERVs) are involved in SLE pathogenesis, whereas other viruses such as Cytomegalovirus (CMV) probably play a less prominent role. However, the mechanisms of viral–host interactions and the impact of viruses on disease course have yet to be elucidated. In addition to classical mechanisms of viral-triggered autoimmunity, such as molecular mimicry and epitope spreading, there has been a growing appreciation of the role of direct activation of innate response by viral nucleic acids and epigenetic modulation of interferon-related immune response. The latter is especially important for HERVs, which may represent the molecular link between environmental triggers and critical immune genes. Virus-specific proteins modulating interaction with the host immune system have been characterized especially for Epstein–Barr virus and explain immune evasion, persistent infection and self-reactive B-cell “immortalization”. Knowledge has also been expanding on key viral proteins of B19-V and CMV and their possible association with specific phenotypes such as antiphospholipid syndrome. This progress may pave the way to new therapeutic perspectives, including the use of known or new antiviral drugs, postviral immune response modulation and innate immunity inhibition. We herein describe the state-of-the-art knowledge on the role of viral infections in SLE, with a focus on their mechanisms of action and potential therapeutic targets.     

Comprehensive Profiling of Mutations to Influenza Virus PB2 That Confer Resistance to the Cap-Binding Inhibitor Pimodivir

Antivirals are used not only in the current treatment of influenza but are also stockpiled as a first line of defense against novel influenza strains for which vaccines have yet to be developed. Identifying drug resistance mutations can guide the clinical deployment of the antiviral and can additionally define the mechanisms of drug action and drug resistance. Pimodivir is a first-in-class inhibitor of the polymerase basic protein 2 (PB2) subunit of the influenza A virus polymerase complex. A number of resistance mutations have previously been identified in treated patients or cell culture. Here, we generate a complete map of the effect of all single-amino-acid mutations to an avian PB2 on resistance to pimodivir. We identified both known and novel resistance mutations not only in the previously implicated cap-binding and mid-link domains, but also in the N-terminal domain. Our complete map of pimodivir resistance thus enables the evaluation of whether new viral strains contain mutations that will confer pimodivir resistance.     

Interference of hepatitis C virus replication in cell culture by antisense peptide nucleic acids targeting the X-RNA

The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is the essential catalytic enzyme for viral genome replication. It initiates minus-strand RNA synthesis from a highly conserved 98-nt sequence, called the X-RNA, at the 3'-end of the plus-strand viral genome. In this study, we evaluated the antiviral effects of peptide nucleic acids (PNAs) targeting the X-RNA. Our in vitro RdRp assay results showed that PNAs targeting the three major stem-loop (SL) domains of X-RNA can inhibit RNA synthesis initiation. Delivery of X-RNA-targeted PNAs by fusing the PNAs to cell-penetrating peptides (CPPs) into HCV-replicating cells effectively suppressed HCV replication. Electrophoretic mobility shift assays revealed that the PNA targeting the SL3 region at the 5'-end of X-RNA dissociated the viral RdRp from the X-RNA. Furthermore, delivery of the SL3-targeted PNA into HCV-infected cells resulted in the suppression of HCV RNA replication without activation of interferon β expression. Collectively, our results indicate that the HCV X-RNA can be effectively targeted by CPP-fused PNAs to block RNA-protein and/or RNA-RNA interactions essential for viral RNA replication and identify X-RNA SL3 as an RdRp binding site crucial for HCV replication. In addition, the ability to inhibit RNA synthesis initiation by targeting HCV X-RNA using antisense PNAs suggests their promising therapeutic potential against HCV infection.     

ISG15 conjugation system targets the viral NS1 protein in influenza A virus–infected cells

ISG15 is an IFN-α/β–induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a “loss of function” mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.     

Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virus–host interactions underlying HCV infection.Compositional studies of virions provide powerful clues for understanding the functions of viral proteins; assembly and entry pathways; and, more broadly, mechanisms of virus–host interactions. Increasingly sensitive MS techniques have enabled the detection of viral and cellular proteins that are incorporated in virions even at very low levels (1).Hepatitis C virus (HCV) is a positive-sense ssRNA virus of the Flaviviridae family. This bloodborne pathogen causes chronic liver infection that develops into cirrhosis and hepatocellular carcinoma and is the leading indication for liver transplantation. Over 185 million people are chronically infected with HCV (2). Despite great advances in the ability to study this virus in vitro, significant gaps remain in our understanding of the infectious particle and the virus–host interactions required for HCV propagation.The protein composition of HCV is not known. Although the capsid protein, Core, and the E1 and E2 envelope glycoproteins are thought to be the major constituents of the virion, it remains to be determined if nonstructural viral proteins are packaged as well. A growing body of literature suggests that cellular proteins are important components of HCV. Indeed, this virus closely associates with LDL and very-LDL components, forming a chimeric lipoviral particle (LVP). Biochemical and ultrastructural studies demonstrated that infectious HCV particles are coated with endogenous apolipoproteins that play key roles in viral attachment and entry, explaining the higher infectivity of lipoprotein-associated HCV (2).The heterogeneous size and appearance of extracellular HCV, ranging from 40 to >100 nm in diameter, suggests that the set of associated proteins (both viral and cellular), as well as their stoichiometry, might vary across the virion population. Additionally, the specific infectivity of HCV changes according to the cell system/host that the virus is produced in, highlighting a strong contribution of the host to the makeup of the virus particles (2). Therefore, a proteomic analysis of HCV represents an attractive means of discovering novel virus–host interactions with possible implications for understanding exploitation/subversion strategies that this chronic virus uses to persist within the host.We recently described two methods for producing and affinity-purifying high titers of cell culture-derived HCV (HCVcc) that enabled ultrastructural analysis of HCV virions (3). The first was based on the construction of an infectious clone with tags fused at the N terminus of E2, whereas the second relied on the use of potent HCV-neutralizing Abs.In this study, we used both affinity purification approaches to perform proteomic analysis of extracellular HCV virions. We established that Core¹⁷⁷ (amino acids 1–177) is the form incorporated in mature HCV particles and detected a number of virion-associated viral and cellular proteins. Functional characterization of HCV-associated cellular proteins identified new host factors, including a nuclear pore complex (NPC) protein, that participate in HCV infection.     

Suppression of hepatitis C virus replication by protein kinase C-related kinase 2 inhibitors that block phosphorylation of viral RNA polymerase

Summary.  Hepatitis C virus (HCV) infection is a serious threat to human health worldwide. In spite of the continued search for specific and effective anti-HCV therapies, the rapid emergence of drug-resistance variants has been hampering the development of anti-HCV drugs designed to target viral enzymes. Targeting host factors has therefore emerged as an alternative strategy offering the potential to circumvent the ever-present complication of drug resistance. We previously identified protein kinase C-related kinase 2 (PRK2) as a cellular kinase that phosphorylates the HCV RNA-dependent RNA polymerase (RdRp). Here, we report the anti-HCV activity of HA1077, also known as fasudil, and Y27632, which blocks HCV RdRp phosphorylation by suppressing PRK2 activation. Treatment of a Huh7 cell line, stably expressing a genotype 1b HCV subgenomic replicon RNA, with 20 μ m each of HA1077 and Y27632 reduced the HCV RNA level by 55% and 30%, respectively. A combination of the inhibitors with 100 IU/mL interferon α (IFN-α) significantly potentiated the anti-HCV drug activities resulting in approximately a 2-log₁₀ viral RNA reduction. We also found that IFN-α does not activate PRK2 as well as its upstream kinase PDK1 in HCV-replicating cells. Furthermore, treatment of HCV-infected cells with 20 μ m each of HA1077 and Y27632 reduced the levels of intracellular viral RNA by 70% and 92%, respectively. Taken together, the results identify PRK2 inhibitors as potential antiviral drugs that act by suppressing HCV replication via inhibition of viral RNA polymerase phosphorylation.     

Regulation of Avian Leukosis Virus Subgroup J Replication by Wnt/β-Catenin Signaling Pathway

Wnt/β-catenin signaling is a highly conserved pathway related to a variety of biological processes in different cells. The regulation of replication of various viruses by Wnt/β-catenin signaling pathway has been reported. However, the interaction between the Wnt/β-catenin pathway and avian leukosis virus is unknown. In the present study, we investigated the effect of modulating the Wnt/β-catenin pathway during avian leukosis virus subgroup J (ALV-J) infection. The activation of the Wnt/β-catenin pathway by GSK-3 inhibitor increased ALV-J mRNA, viral protein expression, and virus production in CEF cells. This increase was suppressed by iCRT14, one of the specific inhibitors of the Wnt/β-catenin signaling pathway. Moreover, treatment with iCRT14 reduced virus titer and viral gene expression significantly in CEF and LMH cells in a dose-dependent manner. Inhibition Wnt/β-catenin signaling pathway by knockdown of β-catenin reduced virus proliferation in CEF cells also. Collectively, these results suggested that the status of Wnt/β-catenin signaling pathway modulated ALV-J replication. These studies extend our understanding of the role of Wnt/β-catenin signaling pathway in ALV-J replication and make a new contribution to understanding the virus–host interactions of avian leukosis virus.