Summary Viral nucleic acids often trigger an innate immune response in infected cells. Many viruses, including hepatitis C virus (HCV), have evolved mechanisms to evade intracellular recognition. Nevertheless, HCV-permissive cells can trigger a viral RNA-, TLR7- and cell contact-dependent compensatory interferon response in nonpermissive plasmacytoid dendritic cells (pDCs). Here we report that these events are mediated by transfer of HCV RNA-containing exosomes from infected cells to pDCs. The exosomal viral RNA transfer is dependent on the endosomal sorting complex (ESCRT) machinery and on Annexin A2, an RNA-binding protein involved in membrane vesicle trafficking, and it is suppressed by exosome release inhibitors. Further, purified concentrated HCV RNA-containing exosomes are sufficient to activate pDCs. Thus, vesicular sequestration and exosomal export of viral RNA may serve both as a viral strategy to evade pathogen-sensing within infected cells and as a host strategy to induce an unopposed innate response in replication-nonpermissive by-stander cells.

An amphipathic ␣-helical peptide (C5A) derived from the membrane anchor domain of the hepatitis C virus (HCV) NS5A protein is virocidal for HCV at submicromolar concentrations in vitro. C5A prevents de novo HCV infection and suppresses ongoing infection by inactivating both extra-and intracellular infectious particles, and it is nontoxic in vitro and in vivo at doses at least 100-fold higher than required for antiviral activity. Mutational analysis indicates that C5A's amphipathic ␣-helical structure is necessary but not sufficient for its virocidal activity, which depends on its amino acid composition but not its primary sequence or chirality. In addition to HCV, C5A inhibits infection by selected flaviviruses, paramyxoviruses, and HIV. These results suggest a model in which C5A destabilizes viral membranes based on their lipid composition, offering a unique therapeutic approach to HCV and other viral infections.HCV ͉ amphipathic peptide ͉ antiviral peptide ͉ NS5A ͉ HIV H epatitis C virus (HCV), a member of the Flaviviridae family (1), is a single-stranded positive-sense RNA virus that causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (2, 3). HCV infects Ͼ170 million people worldwide and is the most common cause of liver transplantation in the United States (3). There is no vaccine available for HCV, and the only currently approved treatment (combination therapy with IFN and ribavirin) has limited efficacy and serious side effects (4, 5). Thus, development of new classes of antiviral compounds with improved efficacy and toxicity profiles is urgently needed.The development of HCV replicon technology several years ago (6) greatly accelerated the pace of antiviral drug discovery, leading to the development of HCV protease and polymerase inhibitors that are currently under clinical evaluation (7,8). The landscape for drug discovery improved further with the establishment of a cell culture model of HCV infection in 2005 (9-11), making it possible to search for inhibitors of every step in the HCV life cycle and agents that target the virus itself. We now report the discovery of several HCV-derived synthetic peptides that inhibit HCV infection in the cell culture infection system. One of those inhibitory peptides, an amphipathic ␣-helical 18-mer derived from the membrane anchor domain of the HCV nonstructural protein NS5A that was particularly potent against HCV and selected other virus infections, serves as the basis of this report. Results Identification of Antiviral Peptides.A peptide library of 441 overlapping peptides (18-mers offset by 11 amino acids) covering the entire HCV polyprotein (H77 strain, genotype 1a) was screened (20 M) for the ability to inhibit HCV infection (JFH-1) in a focus reduction assay using Huh-7.5.1 cells (Fig. 1). Thirteen peptides were shown to inhibit HCV focus formation by Ͼ90%. Validation of the antiviral activity of the 13 inhibitory peptides was performed by comparing the ability of each peptide (20 M) to inhibit the expansion of HCV RNA in Huh-7.5.1 cells ...

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.

Over 170 million people are chronically infected by the hepatitis C virus (HCV) and at risk for dying from liver cirrhosis and hepatocellular carcinoma. Current therapy is expensive, associated with significant side effects, and often ineffective. Discovery of antiviral compounds against HCV traditionally involves a priori target identification followed by biochemical screening and confirmation in cell-based replicon assays. Typically, this results in the discovery of compounds that address a few predetermined targets and are prone to select for escape variants. To attempt to identify antiviral compounds with broad target specificity, we developed an unbiased cell-based screening system involving multiple rounds of infection in a 96-well format. Analysis of a publicly available library of 446 clinically approved drugs identified 33 compounds that targeted both known and previously unexplored aspects of HCV infection, including entry, replication, and assembly. Discovery of novel viral and cellular targets in this manner will broaden the therapeutic armamentarium against this virus, allowing for the development of drug mixtures that should reduce the likelihood of mutational escape.cell-based assay | antivirals | entry | replication | assembly H epatitis C virus (HCV) (Flaviviridae) is an enveloped, positivestranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma (1). HCV establishes persistent infection, and more than 170 million people are chronically infected worldwide (2). Chronic infection is associated with chronic hepatitis, cirrhosis, and hepatocellular carcinoma (3). Although the mechanisms by which HCV causes liver disease are not entirely understood, immunologically mediated events play an important role in HCV clearance and pathogenesis (4).The HCV plus-stranded RNA genome (9.6 kb) encodes a single polyprotein that is cleaved into structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (5). Infection is initiated by virus-particle binding to cellular receptors, internalization through receptor-mediated endocytosis, and delivery of the viral genome to the cytosol after endosomal acidification (6). Delivery and/or translation of incoming viral genomes depends on cellular autophagy-related factors (7), enabling viral gene expression, replication (8), and production of progeny virus, which depends on VLDL biosynthesis (9-11).Currently, there is no vaccine against HCV, and the standard therapy (pegylated IFN-alfa plus ribavirin) is associated with significant side effects and is only effective in a fraction of the patients (12). Establishment of HCV replicons (13, 14) greatly contributed to the discovery of antiviral compounds that target the viral NS3-4A serine protease and NS5B RNA polymerase (15). Although they are extremely potent, these agents select for resistant variants because of the error-prone RNA polymerase activity of HCV (16).The development of HCV infection models (17-19) that reproduce the entire life cycle of HCV in...

We have previously shown that IFN-␤ inhibits hepatitis B virus (HBV) replication by noncytolytic mechanisms that either destabilize pregenomic (pg)RNA-containing capsids or prevent their assembly. Using immortalized murine hepatocyte cell lines stably transfected with a doxycycline (dox)-inducible HBV replication system, we now show that replication-competent pgRNA-containing capsids are not produced when the cells are pretreated with IFN-␤ before HBV expression is induced with dox. Furthermore, the turnover rate of preformed HBV RNA-containing capsids is not changed in the presence of IFN-␤ or IFN-␥ under conditions in which further pgRNA synthesis is inhibited by dox removal. In summary, these results demonstrate that types 1 and 2 IFN activate hepatocellular mechanism(s) that prevent the formation of replicationcompetent HBV capsids and, thereby, inhibit HBV replication.he hepatitis B virus (HBV) is a noncytopathic hepatotropic DNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma (1). Viral clearance and disease pathogenesis during HBV infection are tightly associated with the appearance of a vigorous T cell response to all of the viral proteins (2-6). CD8ϩT cells are the main immune effector cells during HBV infection, because viral clearance and liver disease are blocked by depletion of CD8ϩT cells in acutely infected chimpanzees (7).Noncytolytic T cell functions play a role in HBV clearance, because HBV DNA largely disappears from the liver and blood long before the peak of liver disease in chimpanzees acutely infected with 10 8 genome equivalents of HBV (7,8), and this occurs as soon as IFN-␥ is produced in the liver of the infected animal (7, 9, 10), suggesting that IFN-␥ may inhibit HBV replication. In fact, we have shown (11) that adoptively transferred HBV-specific CD8ϩ T cells inhibit viral replication by a noncytopathic IFN-␥-mediated mechanism in an HBV transgenic mouse model. Similarly, intrahepatic induction of IFN-␣͞␤ inhibits HBV replication noncytopathically in transgenic mice (12, 13), and we have previously demonstrated that this occurs by reducing the intracellular content of HBV RNAcontaining capsids without altering either HBV gene expression, translation, capsid maturation, or virus secretion (13). Importantly, IFN-␤ and -␥ inhibit HBV replication in immortalized hepatocyte cell lines derived from HBV transgenic mice (14), confirming that these cytokines mediate noncytolytic inhibition of HBV replication. Furthermore, the antiviral activity is induced within the first 3 h of IFN-␤ signaling (15), consistent with the rapid clearance kinetics of HBV RNA-containing capsids in HBV transgenic mice (13) and immortalized HBV transgenic hepatocytes (14). However, our previous studies did not reveal whether the antiviral effect of IFN-␤ prevented the formation of replication-competent HBV RNAcontaining capsids or degraded existing capsids.To address these questions, in the current study, we examined the antiviral mechanism whereby IFN-␤ and -␥ inhibit HBV replication in ...