Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein.Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 g/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.Hepatitis C virus (HCV), a positive-strand RNA virus belonging to the Flaviviridae family, is the major cause of non-A, non-B viral hepatitis. HCV has infected approximately 200 million people worldwide and current estimates suggest that as many as 3 million individuals are newly infected each year (4). Approximately 80% of those infected fail to clear the virus; a chronic infection ensues, frequently leading to severe chronic liver disease, cirrhosis, and hepatocellular carcinoma (2, 41). Current treatments for chronic infection are ineffective for approximately 50% of patients, and there is a pressing need to develop preventative and therapeutic vaccines.Due to the error-prone nature of the RNA-dependent RNA polymerase and the high replicative rate in vivo (30, 46), HCV exhibits a high degree of genetic variability. Crucially, this propensity for genetic change allows the virus to respond to and overcome a variety of selective pressures, including host immunity and antiviral therapy (18,26,37,44,53). HCV can be classified into six genetically distinct genotypes and further subdivided into at least 70 subtypes, which differ by approximately 30% and 15% at the nucleotide level, respectively (59, 61). A significant challenge for the development of vaccines will lie in identifying protective epitopes that are conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope proteins, the natural targets for the neutralizing response, are two of the most variable proteins (10).The envelope proteins E1 and E2 are responsible for cell binding and entry (5,8,16,51,57). They are N-linked glycosylated (23,31,43,62) transmembrane proteins with a Nterminal ectodomain and a C-terminal hydrophobic membrane anchor (12,21,22). In vitro ex...

Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.Hepatitis C virus (HCV) is the sole member of the Hepacivirus genus within the family Flaviviridae. It is a major cause of community-acquired and posttransfusion hepatitis. More than 170 million people worldwide are seropositive for HCV, and only 20% of those infected are able to clear the virus. In the remaining 80% of individuals, the virus persists and can

Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1ΔE1E2-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1W529A was unaffected by the presence of neutralizing antibodies that inhibit E2–CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission.

BackgroundThe outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions.Methodology/Principal findingsWe obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action.Conclusions/SignificanceThe full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.

The hepatitis C virus (HCV) p7 protein plays a critical role during particle formation in cell culture and is required for virus replication in chimpanzees. The discovery that it displayed cation channel activity in vitro led to its classification within the "viroporin" family of virus-coded ion channel proteins, which includes the influenza A virus (IAV) M2 protein. Like M2, p7 was proposed as a potential target for much needed new HCV therapies, and this was supported by our finding that the M2 inhibitor, amantadine, blocked its activity in vitro. Since then, further compounds have been shown to inhibit p7 function but the relationship between inhibitory effects in vitro and efficacy against infectious virus is controversial. Here, we have sought to validate multiple p7 inhibitor compounds using a parallel approach combining the HCV infectious culture system and a rapid throughput in vitro assay for p7 function. We identify a genotype-dependent and subtype-dependent sensitivity of HCV to p7 inhibitors, in which results in cell culture largely mirror the sensitivity of recombinant protein in vitro; thus building separate sensitivity profiles for different p7 sequences. Inhibition of virus entry also occurred, suggesting that p7 may be a virion component. Second site effects on both cellular and viral processes were identified for several compounds in addition to their efficacy against p7 in vitro. Nevertheless, for some compounds antiviral effects were specific to a block of ion channel function. Conclusion: These data validate p7 inhibitors as prototype therapies for chronic HCV disease. H epatitis C virus (HCV) chronically infects over3% of the population causing severe liver disease. Despite intensive efforts, no vaccine exists and asymptomatic acute infection results in most carriers being unaware of their positive status. Combination antiviral therapy is available based on interferon ␣ (IFN␣) and ribavirin (Rib). This treatment is expensive, poorly tolerated, and its outcome is largely determined by virus genotype 1 ; resistance of many genotype 1 isolates results in a sustained response for only 50% of patients overall, despite good response rates for other virus strains. New, virus-specific, therapies are in production, yet face resistance caused by HCV sequence variation. 2 Future therapies will likely require combinatorial approaches, targeting multiple virus-specific processes.HCV is the prototype member of the Hepacivirus genus within the Flaviviridae. It is enveloped and possesses a positive strand RNA genome of ϳ9.6 kb. An internal ribosome entry site (IRES) present in the 5Ј untranslated region (UTR) drives translation of a single polyprotein that is cleaved to yield 10 mature proteins. The core and envelope (E) glycoproteins, with the RNA genome, comprise the virion, while nonstructural (NS) proteins modulate host metabolism and replication of the viral RNA. 3 Viral insensitivity to IFN/Rib maps to regions within the NS proteins that confer resistance to the innate immune response, 4,5 whereas t...