Mutations within potential glycosylation sites in the capsid protein of hepatitis E virus prevent the formation of infectious virus particles.

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.

A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus.

Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.

The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques.

An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis-reactive element.

In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons.

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells.

In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein.

Hepatitis E virus (HEV) RNA replication occurred in seven of nine primate cell cultures transfected with in vitro transcripts of an infectious cDNA clone. Cell-to-cell spread did not occur in cell cultures, but rhesus monkeys inoculated with lysates of HEV-transfected PLC/PRF/5 and Huh-7 cells became infected with HEV. A replicon with the ORF2 and ORF3 genes deleted and replaced with the green fluorescent protein gene also replicated in the same primate cells that supported the replication of the full-length genome.

Importance of amino acid 216 in nonstructural protein 2B for replication of hepatitis A virus in cell culture and in vivo.

Clinical isolates of hepatitis A virus (HAV) replicate inefficiently in cell culture unless mutations are acquired throughout the genome. An Ala-to-Val substitution in the nonstructural protein 2B (2B-216) was known to have a major impact on replication in cell culture. Analysis of chimeric viruses confirmed that the 2B-A[216]V change was critical for efficient replication and that Leu or Ile could substitute for Val.