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

Abstract

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. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.

FIG. 1.

Schematic diagram of part of ORF3 overlap regions indicating introduced mutations and location of a conserved nucleotide sequence. The nucleotide and amino acid sequences for pSK-HEV-2 are shown (GenBank no. AF444002). Box: region conserved among human HEV strains (GenBank nos. AY115488, AP003430, AB074918, AB073912, AF082843, AF060668, AF060669, AB074920, AB097812, D10330, M94177, AF076239, E17109, X99441, AF459438, M73218, D11092, L25547, X98292, AJ272108, AF444003, AY230202, M74506, AB091395, AB091394, AB080575, AB074915, AB074917, and AB097811). The shaded box indicates seven contiguous nucleotides identical in human and avian HEV(GenBank no. AY043166).

FIG. 2.

Confocal immune fluorescence microscopy demonstrating that ORF2 protein (green) is not detected in HuH-7 cells transfected with ΔORF2 genomes and ORF3 (red) is not produced in cells transfected with an ORF3-null mutant. Note that the generation of ORF2 granules is a slow and asynchronous process so that cells within one transfected sample exhibited the total range of staining patterns and the cells shown portray a single example in each case. Cells transfected with wild-type genomes and stained on day 7 posttransfection with MAb α-ORF2 (A) or MAb α-ORF3 (B) (merge image [C]). Cells transfected with ΔORF2 genomes and stained on day 7 posttransfection with MAb α-ORF2 (D) or MAb α-ORF3 (E). Cells transfected with an ORF3-null mutant and stained on day 7 posttransfection with α-ORF2 MAb (F), α-ORF3 MAb (G), α-ORF2 polyclonal antibody (H), or α-ORF3 MAb (I). All photographs were taken with the 63× objective lens, but panels A to C were reduced compared to panels D to I for graphic layout.

FIG. 3.

Synonomous mutations that prevent synthesis of ORF2 and ORF3 proteins change the predicted secondary structure of the RNA. The first methionine codon in ORF3 is boxed. Six introduced synonymous mutations are indicated in boldface, and the conserved region is shaded. The predicted secondary structures with the lowest energy are presented.

FIG. 4.

An expressed ORF3 peptide-luciferase fusion protein exhibits less luciferase activity than does authentic luciferase expressed from reading frame 2. The luciferase activity detected in cells transfected with mutants expressing luciferase reading frame 2 protein (▴) or reading frame 3 fusion protein (•) is compared to that in mock-transfected cells (−), in cells transfected with the luciferase reading frame 2 mutant encoding an inactive RdRp (▪), and in a luciferase reading frame 3 mutant in which the conserved nucleotide sequence was deleted (♦).

FIG. 5.

Rhesus macaques transfected with the S80A mutant either did not get infected or experienced a highly attenuated infection compared to macaques transfected with the S80L mutant. Dotted line, ALT cutoff for hepatitis; ♦, genomes; shaded, ALT. Macaques were transfected intrahepatically with genomes containing serine 80 mutated to alanine (A and B) or to leucine (C and D).