Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Abstract

Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.Hepatitis E virus (HEV) is transmitted via the fecal-oral route, predominantly through contaminated water. HEV causes acute self-limiting hepatitis and in much of the developing world is responsible for sporadic infections, as well as large epidemics, of acute hepatitis E, especially in Asia and Africa.Four genotypes comprising a single serotype of mammalian HEV have been identified (26,28). HEV was recently classified as the sole member of the genus Hepevirus, family Hepeviridae (3); sequence analysis suggests it is most closely related to rubella virus, an enveloped virus in the family Togaviridae. However, HEV does not contain a lipid envelope (26). The virion is 27 to 30 nm in diameter (1, 26) and has a sedimentation coefficient of 183S (2). The HEV genome is a positivesense RNA, approximately 7.2 kb in length, with a short, capped 5Ј noncoding region and a 3Ј noncoding region preceding a poly(A) tail. Viral genomic RNA is infectious for some cultured cells and nonhuman primates: transfection with capped recombinant genomes results in production of infectious virions in vitro and acute hepatitis and/or seroconversion in vivo (6-8). The coding region of HEV contains three partially overlapping open reading frames (ORFs): ORF1 encodes the nonstructural proteins, ORF2 encodes the capsid protein, and ORF3, which overlaps the N terminus of ORF2, encodes a small protein of 114 amino acids (aa) (13, 15) that might be a regulatory protein (7,19,33,44). The ORF2 and ORF3 proteins are encoded by a bicistronic subgenomic RNA (13,35,45).HEV does not grow sufficiently well either in cell culture or in nonhuman primates to permit direct biochemical analysis. Therefore, various in vitro systems have been utili...

Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody

“…In the present study, we show that mutations at residues on p239 within this potential receptor-binding region (E549A, K554A and G591A) lead to a significant loss of binding to host cells (Supplementary information, Figure S10). In addition, mutation of residue N562 in this region to Gln has been shown to result in loss of HEV infectivity in both cultured cells and rhesus macaques [30]. Meanwhile, this residue was also reported to be one of the several essential residues for host cell interaction [9].…”

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

“…Probably, this protein of about 660 amino acids (aa), is the only structural protein of the virus which assemble into a highly structured multimer (60 copies) Mushahwar et al 1996;Pavio et al 2010). ORF2 is putatively translated from a subgenomic RNA (Purcell and Emerson, 2001) and contains 3 putative N-glycosylation sites and an endoplasmic reticulum signal peptide (Graff et al 2008).…”

Development of a novel immunoperoxidase monolayer assay for detection of swine Hepatitis E virus antibodies based on stable cell lines expressing the ORF3 protein

“…ORF2 is located at the 3' end of the genome and encodes a capsid protein that encapsulates the viral RNA genome and contains an endoplasmic reticulum (ER) signal peptide and 3 putative N-glycosylation sites (Graff et al, 2008), which have been the focus of studies of antigenic sites (Ma et al, 2009;Pan et al, 2010). However, recent studies compared the polypeptide ends of the ORF1, ORF2 and ORF3 proteins.…”