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.

Lambda interferon (IFN-)

We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (ϳ40-to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ϳ60 and ϳ45 nm in diameter. The ϳ60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ϳ45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-lowdensity, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ϳ60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Although a preventive vaccine is available, the therapeutic options for chronically infected patients are limited. It has been shown that RNA interference can prevent HBV gene expression and replication in vivo when HBV expression vectors are delivered simultaneously with small interfering RNA (siRNA) or siRNA expression constructs. However, the therapeutic potential of siRNAs to interrupt ongoing HBV replication in vivo has not been established. Here, we show that expression of HBVspecific siRNAs in the liver of HBV transgenic mice by recombinant adenoviruses can suppress preexisting HBV gene expression and replication to almost undetectable levels for at least 26 days. These results demonstrate that efficiently delivered siRNAs should be able to silence HBV in chronically infected patients.adenovirus vector ͉ RNA interference C hronic hepatitis B virus (HBV) infection causes Ͼ1 million deaths each year due to cirrhosis of the liver and hepatocellular carcinoma (www.cdc.gov͞hepatitis and www.who.int͞ mediacentre͞factsheets͞fs204͞en). Hence, there is a need for alternative ways to treat this persistent infection. RNA interference (RNAi) has rapidly emerged as a technology for regulating mammalian gene expression because it provides a means of sequence-directed degradation of specific RNAs (1-8). In the case of HBV, published in vivo hydrodynamic transfection studies have shown that simultaneous delivery of HBV expression plasmids and HBV-specific small interfering RNAs (siRNAs) (or siRNA-expressing constructs) to the mouse liver can prevent the induction of HBV gene expression and replication (9-11). To expand on those studies, we have examined the therapeutic potential of RNAi for the treatment of chronic HBV infection where ongoing viral gene expression and replication are established in the liver before siRNA delivery.Several variables that have not been previously addressed could affect the utility of RNAi for the treatment of an ongoing HBV infection. First, in the context of an established infection, viral RNAs may be protected within complexes or nucleocapsid structures as has been reported for hepatitis D virus (1), Rous sarcoma virus (12), and respiratory syncytial virus (RSV) (13). This is particularly relevant to HBV, which replicates in capsids after encapsidation of the viral pregenomic RNA. A second variable is how efficiently preexisting viral RNA can be reduced and to what extent RNA suppression can block viral DNA replication. Finally, established viral gene expression may allow for the induction of viral RNAi-defense mechanisms as reported for plant viruses (14) and flockhouse nodavirus (15).It has been shown that RNAi is capable of reducing mammalian gene expression in vivo. Genes such as fas (16), caspase-8 (17), agouti-related peptide (18), tyrosine hydroxylase (19), and ras (20, 21) have been targeted for degradation in mice, and corresponding changes in phenotype were observed. Yet, the actual degree o...

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 ...