Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo

Abstract

To determine the initiation strategy of the hepatitis E virus (HEV) open reading frame 3 (ORF3), we constructed five HEV mutants with desired mutations in the ORF1 and ORF2 junction region and tested their levels of in vivo infectivity in pigs. A mutant with a C-terminally truncated ORF3 is noninfectious in pigs, indicating that an intact ORF3 is required for in vivo infectivity. Mutations with substitutions in the first in-frame AUG in the junction region or with the same T insertion at the corresponding position of HEV genotype 4 did not affect the virus infectivity or rescue, although mutations with combinations of the two affected virus recovery efficiency, and a single mutation at the third in-frame AUG completely abolished virus infectivity in vivo, indicating that the third in-frame AUG in the junction region is required for virus infection and is likely the authentic initiation site for ORF3. A conserved double stem-loop RNA structure, which may be important for HEV replication, was identified in the junction region. This represents the first report of using a unique homologous pig model system to study the molecular mechanism of HEV replication and to systematically and definitively identify the authentic ORF3 initiation site.

FIG. 1.

Schematic diagram of the predicted genomic organization and nucleotide sequence alignment of the noncoding junction region between ORF1 and ORF2 of HEV. (A) Swine HEV (genotype 3), Sar-55 strain (genotype 1), Mexico strain (genotype 2), and T1 strain (genotype 4). Nucleotide position numbers correspond to the sequence of clone pSHEV-3 of genotype 3 swine HEV. Dots denote sequence identity, and dashes denote deletions. The stop codon of ORF1 is indicated by carets, and the four AUGs are indicated by asterisks. AUG1, -2, and -3 are in-frame and serve as the potential ORF3 start codons, and AUG4 serves as the ORF2 start codon among HEV genotypes 1 to 3. In genotype 4, a single-nucleotide T insertion 2 nt downstream of AUG2 (marked with circle) results in the predicted ORF3 initiation at AUG3 and the predicted ORF2 initiation at AUG1, -2, and/or -4. The presumed coding regions between AUG1 and AUG3 for ORF3 in HEV genotypes 1 to 3 and between AUG1 and AUG4 for ORF2 in genotype 4 HEV are denoted by the dashed-line box based on our hypothesis that AUG3 is the authentic ORF3 initiation site and that AUG4 is the authentic ORF2 initiation site for all HEV genotypes. (B) Avian HEV (putative genotype 5). There exist three AUGs that could potentially serve as the initiation sites for different ORFs. In addition to AUG2 and AUG3 as the potential ORF3 and ORF2 start codons, respectively, a novel small ORF X is predicted to start at AUG1.

FIG. 2.

Construction of HEV mutants derived from the wild-type genotype 3 swine HEV consensus cDNA clone pSHEV-3. (A) Alignment of nucleotide sequences in the junction regions among the mutants and their group assignments for the in vivo infectivity study in pigs. T7p represents a T7 RNA polymerase core promoter, and A₁₅ represents a 15-nt poly(A) tail. The unique AflII and AvrII restriction sites on pSHEV-3 used for the cloning purpose are also shown. For an explanation of the symbols, see the legend for Fig. 1, panel A. (B) In vitro transcription of full-length capped RNAs from the wild-type infectious cDNA clone pSHEV-3 and five HEV mutant clones. Five microliters of the transcription mixture was separated in a 1% agarose gel to check the quality of the transcription before being intrahepatically inoculated into the liver of pigs. An arrow indicates the in vitro RNA transcripts of expected size.

FIG. 3.

Determination of the in vivo infectivity of wild-type and mutant cDNA clones of genotype 3 HEV in pigs. Fecal viral shedding, viremia, and seroconversion to IgG anti-HEV antibodies in pigs intrahepatically inoculated with capped RNA transcripts from wild-type swine HEV clone pSHEV-3 (A), PBS buffer (B), mutant-AGA (C), mutant-1 (D), mutant-3 (E), mutant-5 (F), and mutant-19 (G). Results of RT-PCR for the presence (closed circles) or absence (open circles) of HEV RNA in fecal and serum samples are indicated. IgG anti-HEV antibody is plotted as the ELISA optical density (A₄₀₅). The ELISA cutoff value, indicated by a dashed line, is 0.30.

FIG. 4.

A conserved double stem-loop RNA secondary structure is predicted by the mfold program in the junction region between ORF1 and ORF2 of HEV. (A) Genotype 3 HEV and its derived mutants. (B) The other four genotypes of HEV. The four AUGs (AUG1, -2, -3, and 4) in genotypes 1 through 4 HEV strains and the three AUGs in avian HEV putative genotype 5 are indicated. The contiguous AAC/U triplets are marked with asterisks. The mutations introduced in mutant-1, -3, and -5 are indicated by italic letters.