Distinct Entry Mechanisms for Nonenveloped and Quasi-Enveloped Hepatitis E Viruses

Abstract

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 quasienveloped viruses resemble exosomes, these data also may provide insights into the exosome-mediated intercellular communications.

Extracellular vesicles are the Trojan horses of viral infection

“…Only after engagement with the host cell did HAV and HEV particles within vesicles become sensitive to neutralizing antibodies, supporting the model of disruption of the vesicle membrane during infection [7••,19]. The mechanism of membrane disruption is currently unknown but recent findings with HEV suggest uptake of vesicles into acidified endosomes where endosomal lipases and lipid-extracting proteins (e.g.…”

“…Both the small exosomes containing HAV, HEV and the larger autophagosome-derived extracellular vesicles containing poliovirus, Coxsackievirus and rhinovirus, when isolated intact from the extracellular environment and added to new susceptible host cells, demonstrated a high degree of infectivity [7••,10,11••,19]. Notably for poliovirus, Coxsackievirus and rhinovirus, infections with vesicles were still receptor-mediated, that is viruses had to bind their respective receptors, suggesting that the vesicle membrane around the viral particles was disrupted after the vesicles engaged with the new host cell [7••,11••].…”

“…The mechanism of membrane disruption is currently unknown but recent findings with HEV suggest uptake of vesicles into acidified endosomes where endosomal lipases and lipid-extracting proteins (e.g. Niemann–Pick transporter) permeabilize the membrane [19]. Notably, many non-enveloped virus capsid proteins including the adenovirus protein VI, the rotavirus VP5 and the poliovirus VP4 proteins have intrinsic membrane disrupting activities that can be triggered upon acidification of their environment [21–23].…”

Lipid Tales of Viral Replication and Transmission

“…This suggests that the vesicle membranes are ruptured, during or after uptake into the new host cell, as prior to that the viruses are also resistant to neutralizing antibodies [63,80], the latter a potentially significant immune evasion mechanism for this mode of transmission by non-enveloped viruses. Nevertheless, this raises the important question of how this membrane is eventually ruptured.…”

“…Nevertheless, this raises the important question of how this membrane is eventually ruptured. Recent evidence points to endosomal lipases and cholesterol transfer proteins such as the Niemann-Pick transporter playing a role in the process [80]. In addition, the viral cargo itself could facilitate this rupture as many non-enveloped viral capsid proteins including the adenovirus protein VI, rotavirus VP5 and the VP4 proteins from rhinovirus and HAV, under acidified conditions (such as those that would be encountered within endosomes), undergo conformational changes that are sufficient to penetrate and disrupt membranes [81-84].…”