Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

Abstract

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wildtype ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wildtype Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles. INTRODUCTIONHepatitis E virus (HEV), a member of the genus Hepevirus in the family Hepeviridae, is the causative agent of acute or fulminant hepatitis E, which occurs in many parts of the world, principally as a water-borne infection in developing countries and zoonotically in industrialized countries (Chandra et al., 2008;Colson et al., 2010;Dalton et al., 2008;Purcell & Emerson, 2008;Tei et al., 2003;Yazaki et al., 2003). HEV is a non-enveloped small virus with a diameter of 27-32 nm, present in the bile and faeces of infected hosts, while HEV particles in the circulating blood and culture supernatant are found to be associated with lipids (Takahashi et al., 2008b(Takahashi et al., , 2010Yamada et al., 2009a). The HEV genome is a positive-sense, ssRNA composed of approximately 7200 nt, which is capped and polyadenylated (Kabrane-Lazizi et al., 1999;Tam et al., 1991). The genome consists of a 59 UTR, three ORFs, a 39 UTR and a poly(A) tail at the 39 terminus (Emerson & Purcell, 2007). ORF1 encodes non-structural proteins including a methyltransferase, papain-like cysteine protease, helicase and RNA-dependent RNA polymerase (Agrawal et al., 2001;Koonin et al., 1992). ORF2 and ORF3 overlap, and the ORF2 and ORF3 proteins are translated from a bicistronic subgenomic RNA 2.2 kb in length (Graff et al., 2006;Ichiyama et al., 2009). The ORF2 protein is the viral capsid protein of 660 a...

The PSAP Motif within the ORF3 Protein of an Avian Strain of the Hepatitis E Virus Is Not Critical for Viral InfectivityIn Vivobut Plays a Role in Virus Release

“…Our inability to detect a synergistic effect on protein release when the ORF2 and ORF3 proteins are coexpressed in LMH cells differs from published results in which particle release is abolished when mutations of the PSAP motif within the ORF3 protein are analyzed (10,45,46,62). It is important to mention that these published studies were in the context of the full-length viral genome, whereas our study looked at ORF2 and ORF3 proteins independently of other viral proteins and RNA.…”

“…Recent work suggests that a PXXP motif within the HEV ORF3 protein may play a similar role during HEV particle formation (10,45,46,62). In the case of enveloped viruses, expression of the structural proteins is sufficient to produce virus-like particles (VLPs) which can be released from the cell.…”

“…Alpha-1-microglobulin secretion is upregulated via interaction with tumor suppressor gene 101 (TSG101) (56). Most recently, the ORF3 protein interaction with TSG101 is thought to direct virion release through the host proteins forming multivesicular bodies (10,45,46,56,62). The avian HEV ORF3 protein contains a singular proline-rich amino acid motif PREPSAPP.…”

Molecular Epidemiology of Hepatitis E Virus (Hev) in South America: Current Status

“…The third ORF encodes for a small phosphoprotein of 123 aa associated with cytoskeleton, and is involved in virus replication and virion morphogenesis. Recently, its role in virus release from infected cells has been demonstrated (Yamada et al 2009;Nagashima et al 2011). In addition, ORF3 is necessary for viral infection in Rhesus macaques in vivo, but not in vitro (Graff et al 2005).…”

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

“…The biogenesis of the membrane-associated, quasi-enveloped HEV particles (eHEV) involves a specific interaction between pORF3 and Tsg101, a cellular component of the endosomal sorting complex required for transport (ESCRT) that is commonly involved in the budding of many classic enveloped viruses (17)(18)(19). As a result, pORF3 is incorporated into the eHEV virion but is not found in virions in the feces (20). Neither polyclonal anti-pORF2 nor anti-pORF3 antibodies capture eHEV, except following detergent treatment, indicating that viral proteins are completely hidden within the eHEV membrane (16,21).…”