Protein composition of the hepatitis A virus quasi-envelope

Abstract

The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasienveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAVassociated proteins (n = 105) were significantly enriched for components of the endolysosomal system (>60%, P < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.exosome | multivesicular body | ESCRT | extracellular vesicle | picornavirus T he Picornaviridae are a large and diverse family of positivestrand RNA viruses that include numerous pathogens (1). Classically considered "nonenveloped," these viruses package their single-stranded genomes within stable icosahedral protein capsids that are released from cells following cell lysis. However, recent work has revealed that several picornaviruses gain egress from cells in a nonlytic fashion within the lumen of extracellular vesicles shed from the cell. Most notably, hepatitis A virus (HAV; genus Hepatovirus), an important cause of enterically transmitted hepatitis in humans, replicates without cytopathic effect and is released from cells as membrane-wrapped, "quasienveloped" (eHAV) virions similar in size and density to exosomes (2). eHAV virions have been identified in sera collected from persons with acute hepatitis A, as well as in supernatant fluids of infected cell cultures. These virions are completely cloaked in host-derived membranes that protect the capsid from neutralizing antibody until the quasi-envelope is degraded wi...

A decade of RNA virus metagenomics is (not) enough

“…These results have led to a hypothesis in which part of the RNA virus origins involve selfish vesicular transport that likely expanded with the rise of intracellular eukaryotic transport (Greninger, 2015;Koonin et al, 2006;Kuehn and Kesty, 2005). The list of RNA viruses that have acquired a cellular envelope continues to grow Feng et al, 2013;McKnight et al, 2017). New concepts in bacterial vesicular transport might be consistent the close evolutionary link between the eukaryotic RNA virus polymerases and bacterial retroelements (Jan, 2017;Kuehn and Kesty, 2005).…”

Unconventional secretion of hepatitis A virus

“…Then, secretion of these luminal contents proceeds via the Golgi apparatus and exocytic vesicles to the outside of the cell. Of course, such classifications beg to be violated, and in PNAS, McKnight et al (1) consolidate a mechanism by which hepatitis A virus (HAV) does just that.…”

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus

“…However, the mechanism for exit (and entry) of these viruses without disruption of the cell's plasma membrane is not well understood. Only recently has unconventional secretion of virioncontaining extracellular vesicles been described (Bird et al, 2014;Chen et al, 2015;Feng et al, 2013;McKnight et al, 2017;Robinson et al, 2014). Delivery of such a 'payload' containing multiple virions has the potential to propagate infection that might otherwise be hindered by frequent detrimental mutations in the viral genome that are present due to the inherently errorprone replication of RNA (Biebricher and Eigen, 2005;Schulte et al, 2015).…”

“…It has been proposed that multiple capsid-enclosed viral genomes that are transferred by vesicles en bloc into a single cell are the sole contributor to replication kinetics in the new host (Chen et al, 2015). Indeed, this is the case for exosomes secreted from HAV-infected cells (McKnight et al, 2017). However, the delivery of (+) and (-) vRNA and viral replication proteins by extracellular vesicles into cells suggests that viral replication of PV may be facilitated by the delivery of (+) RNA, (-) RNA, and the viral proteins necessary for replication (e.g.…”

“…Changes in immune responses and susceptibility to infection have also been observed in PVinfected cells (Yoshikawa et al, 2006). The chaperone protein HSP90, which is a facilitating factor in viral replication of hepatitis A virus (McKnight et al, 2017), hepatitis C virus (HCV) (Okamoto et al, 2006) and influenza A (Chase et al, 2008), was identified only in infectious vesicles, in both microvesicles and exosomes. Recent studies on Enterovirus 71, a member of the picornaviridae family, showed that virions transported in exosomes were able to replicate in new target cells, while the inflammatory type I interferon response was suppressed due to miRNAs co-delivered by these exosomes (Fu et al, 2017).…”