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Replacement of the hepatitis E virus ORF3 protein PxxP motif with heterologous late domain motifs affects virus release via interaction with TSG101.

ABSTRACT: The ORF3 protein of hepatitis E virus (HEV) contains a "PSAP" amino acid late domain motif, which allows for interaction with the endosomal sorting complexes required for transport (ESCRT) pathway aiding virion release. Late domain motifs are interchangeable with other viral late domain motifs in several enveloped viruses, however, it remains unknown whether HEV shares this functional interchangeability and what implications this might have on viral replication. In this study, by substituting heterologous late domain motifs (PPPY, YPDL, and PSAA) for the HEV ORF3 late domain (PSAP), we demonstrated that deviation from the PSAP motif reduces virus release as measured by viral RNA in culture media. Virus release could not be restored by insertion of a heterologous late domain motif or by supplying wild-type ORF3 in trans, suggesting that the HEV PSAP motif is required for viral exit which cannot be bypassed by the use of alternative heterologous late domains.

SUBMITTER: Kenney SP

PROVIDER: S-EPMC4679584 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Replacement of the hepatitis E virus ORF3 protein PxxP motif with heterologous late domain motifs affects virus release via interaction with TSG101.

Kenney Scott P SP Wentworth Jacquelyn L JL Heffron Connie L CL Meng Xiang-Jin XJ

Virology 20151027

The ORF3 protein of hepatitis E virus (HEV) contains a "PSAP" amino acid late domain motif, which allows for interaction with the endosomal sorting complexes required for transport (ESCRT) pathway aiding virion release. Late domain motifs are interchangeable with other viral late domain motifs in several enveloped viruses, however, it remains unknown whether HEV shares this functional interchangeability and what implications this might have on viral replication. In this study, by substituting he ...[more]

PMID: 26457367

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Project description:UnlabelledHepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-β mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-β promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-β expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion.ImportanceHepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA. This enhancement may play a role in HEV invasion, as vp13 is essential for HEV infection in vivo. The results of this study provide insights into virus-cell interactions during HEV infection. In addition to revealing its possible roles in HEV interference with cellular signaling, these results suggest that the second half of the vp13 sequence can be ligated into the genomes of attenuated live viruses to induce an innate immune response for better protective immunity, as well as a marker for differentiation of vaccinated animals from those infected with the corresponding wild-type viruses.

Dataset Information

Replacement of the hepatitis E virus ORF3 protein PxxP motif with heterologous late domain motifs affects virus release via interaction with TSG101.

Publications

Replacement of the hepatitis E virus ORF3 protein PxxP motif with heterologous late domain motifs affects virus release via interaction with TSG101.

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