Hepatitis A virus particles released from cells can hijack and become wrapped in host-derived membranes by using proteins that facilitate the budding of many enveloped viruses, calling into question the traditional distinction between enveloped and non-enveloped viruses. This study of hepatitis A virus (HAV) challenges a basic tenet in virology, the classic distinction between 'enveloped' and 'non-enveloped' viruses. HAV, a picornavirus consisting of a single-stranded RNA packaged in a protein shell, is classified as a non-enveloped virus, lacking a lipid-bilayer membrane. Stanley Lemon and colleagues now show that HAV particles released from cells can co-opt the ESCRT (endosomal sorting-complexes required for transport) machinery, becoming cloaked in host-derived membranes. HAV's hijacked envelope may facilitate the virus's spread within the liver, but could also explain how passive transfer of antibody works as post-exposure prophylaxis of hepatitis A, a phenomenon that has been known clinically for decades but has not been explained. Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane1, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis2,3,4. HAV infects in a stealth-like manner and replicates efficiently in the liver5. Virus-specific antibodies appear only after 3–4 weeks of infection, and typically herald its resolution3,4. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure6, when virus replication is well established in the liver5. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (‘eHAV’) resemble exosomes7, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT)8, namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between ‘enveloped’ and ‘non-enveloped’ viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.

The hepatitis E virus (HEV) sheds into feces as nonenveloped virions but circulates in the blood in a membrane-associated, quasi-enveloped form (eHEV). Since the eHEV virions lack viral proteins on the surface, we investigated the entry mechanism for eHEV. We found that compared to nonenveloped HEV virions, eHEV attachment to the cell was much less efficient, requiring a longer inoculation time to reach its maximal infectivity. A survey of cellular internalization pathways identified clathrin-mediated endocytosis as the main route for eHEV entry. Unlike nonenveloped HEV virions, eHEV entry requires Rab5 and Rab7, small GTPases involved in endosomal trafficking, and blocking endosomal acidification abrogated eHEV infectivity. However, low pH alone was not sufficient for eHEV uncoating, suggesting that additional steps are required for entry. Supporting this concept, eHEV infectivity was substantially reduced in cells depleted of Niemann-Pick disease type C1, a lysosomal protein required for cholesterol extraction from lipid, or in cells treated with an inhibitor of lysosomal acid lipase. These data support a model in which the quasi-envelope is degraded within the lysosome prior to virus uncoating, a potentially novel mechanism for virus entry.IMPORTANCEThe recent discovery of quasi-enveloped viruses has shifted the paradigm of virus-host interactions. The impact of quasi-envelopment in the virus life cycle and pathogenesis is largely unknown. HEV is a highly relevant model to study these questions. HEV circulates as quasi-enveloped virions in the blood that are hidden from neutralizing antibodies. eHEV particles most likely are responsible for the cell-to-cell spread of the virus. Given the increasing concerns about persistent HEV infection and its potential for transmission via the blood supply, understanding how eHEV infects cells is important for understanding its pathogenesis and developing therapies. Our data provide evidence that eHEV uses a potentially novel mechanism for cellular entry. Several steps critical to eHEV entry were identified and may provide a basis for developing treatments for hepatitis E. Because quasi-enveloped viruses resemble exosomes, these data also may provide insights into the exosome-mediated intercellular communications.

Significance: Hepatitis E virus (HEV) is a main cause of acute hepatitis worldwide. Recent evidence suggests that HEV-infected cells release a secreted form of ORF2 protein (ORF2 ^S ) but its origin and function are unknown. Here we demonstrate that ORF2 ^S and ORF2 ^C (the actual capsid protein) are different translation products and that ORF2 ^S is not essential for the HEV life cycle but inhibits antibody-mediated neutralization of HEV. Our results have important implications for understanding the HEV replication cycle and immune evasion mechanisms. The identified internal start codon in this study is highly conserved in most HEV strains, suggesting that the production of ORF2 ^S is an evolutionary conserved function for HEV.

The VP35 protein of Ebola virus is a viral antagonist of interferon. It acts to block virus or double-stranded RNA-mediated activation of interferon regulatory factor 3, a transcription factor that facilitates the expression of interferon and interferon-stimulated genes. In this report, we show that the VP35 protein is also able to inhibit the antiviral response induced by alpha interferon. This depends on the VP35 function that interferes with the pathway regulated by double-stranded RNA-dependent protein kinase PKR. When expressed in a heterologous system, the VP35 protein enhanced viral polypeptide synthesis and growth in Vero cells pretreated with alpha/beta interferon, displaying an interferon-resistant phenotype. In correlation, phosphorylation of PKR and eIF-2α was suppressed in cells expressing the VP35 protein. This activity of the VP35 protein was required for efficient viral replication in PKR ^+/+ but not PKR ^−/− mouse embryo fibroblasts. Furthermore, VP35 appears to be a RNA binding protein. Notably, a deletion of amino acids 1 to 200, but not R312A substitution in the RNA binding motif, abolished the ability of the VP35 protein to confer viral resistance to interferon. However, the R312A substitution rendered the VP35 protein unable to inhibit the induction of the beta interferon promoter mediated by virus infection. Together, these results show that the VP35 protein targets multiple pathways of the interferon system.

Hepatitis A virus (HAV) is an hepatotropic human picornavirus that is associated only with acute infection. Its pathogenesis is not well understood because there are few studies in animal models using modern methodologies. We characterized HAV infections in three chimpanzees, quantifying viral RNA by quantitative RT-PCR and examining critical aspects of the innate immune response including intrahepatic IFN-stimulated gene expression. We compared these infection profiles with similar studies of chimpanzees infected with hepatitis C virus (HCV), an hepatotropic flavivirus that frequently causes persistent infection. Surprisingly, HAV-infected animals exhibited very limited induction of type I IFN-stimulated genes in the liver compared with chimpanzees with acute resolving HCV infection, despite similar levels of viremia and 100-fold greater quantities of viral RNA in the liver. Minimal IFN-stimulated gene 15 and IFIT1 responses peaked 1–2 wk after HAV challenge and then subsided despite continuing high hepatic viral RNA. An acute inflammatory response at 3–4 wk correlated with the appearance of virus-specific antibodies and apoptosis and proliferation of hepatocytes. Despite this, HAV RNA persisted in the liver for months, remaining present long after clearance from serum and feces and revealing dramatic differences in the kinetics of clearance in the three compartments. Viral RNA was detected in the liver for significantly longer (35 to >48 wk) than HCV RNA in animals with acute resolving HCV infection (10–20 wk). Collectively, these findings indicate that HAV is far stealthier than HCV early in the course of acute resolving infection. HAV infections represent a distinctly different paradigm in virus–host interactions within the liver.