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

Abstract

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. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.

FIG. 1.

Schematic representation of HEV intergenotypic chimeras and of HEV replicons expressing luciferase. The braces indicate the 3′-terminal region [nt 7086 to the poly(A) tract] that was exchanged to generate chimeras. The EcoRI site was used for cloning. The replicons contained the firefly luciferase gene (1,658 nt long) inserted in frame with the initiation codon of ORF2 and replacing the 5′-terminal 672 nt of ORF2. ORF3 was truncated to encode only the N-terminal 14 amino acids. A mutation introduced into the RdRp gene changed the GDD motif to GAD to generate a replication-deficient replicon.

FIG. 2.

(A) Sequence alignment of the 3′-terminal regions of HEV constructs. Numbering corresponds to the sequence of pSar55. A dash denotes a deletion. ORF2 ends at the indicated stop codon. (B) Predicted secondary structures (MFOLD program) of the region from nt 7086 to the poly(A) tract of Sar55 (parent), the 3′-terminal intergenotypic chimeras Sar/3′swine and Sar/3′Mex, and the 3′-terminal position 7106 mutants E/luc-7106, E/luc-7106/7144, and E/luc-7106/7097. The secondary structure with the lowest energy is presented. Nucleotide 7106 is indicated, highlighting the only difference between Sar55 and the 7106 mutant. Nucleotides 7097 and 7144 are highlighted in the structures containing mutations at these sites. The stop codon of ORF2 is indicated, and the 3′ NCR sequences are presented as white letters on a black background.

FIG. 3.

Confocal microscopy of Huh-7 cells 7 days after transfection with transcripts synthesized in vitro from (A1 and A2) pSar/3′Mex, (B1 and B2) pSar/3′swine, and (C1 and C2) pSar55. Two examples of transfected cells are shown for each virus, representing the different staining patterns most commonly found. (D) Mock-transfected Huh-7 cells. (d) Mock-transfected cells in phase contrast. The merged images of cells doubly labeled for ORF2 protein (green) and ORF3 protein (red) are shown. ORF2 was visualized with Alexa Fluor 488-conjugated anti-human secondary antibody, and ORF3 was visualized with Alexa Fluor 568-conjugated anti-rabbit secondary antibody. Colocalization of the two proteins would appear as yellow in the merged images.

FIG. 4.

Representative experiment showing luciferase activities in lysates of Huh-7 cells. Values are presented as means and standard deviations from two parallel transfections. (A) Huh-7 cells were transfected with capped (E/luc and E/luc-GAD) and uncapped (E/lucu and E/luc-GADu) replicon RNAs and with a negative control transcription mixture lacking T7 RNA polymerase (mock). (B) Huh-7 cells were transfected with capped replicon RNA containing different 3′-terminal sequences (the intergenotypic chimeras E/luc-3′Mex and E/luc-3′swine, mutant E/luc-7106, the wild-type parental replicon E/luc, and the replication-deficient parental replicon E/luc-GAD) or were mock transfected. (C) Huh-7 cells were transfected with capped replicon RNA containing different mutations in the 3′-terminal region (E/luc-7106, E/luc-7106/7097, and E/luc-7106/7144), E/luc, and E/luc-GAD or were mock transfected as described above.

FIG. 5.

Serological (anti-HEV) and biochemical (ALT) responses and numbers of viral genomes in the serum of rhesus macaques transfected with transcripts synthesized in vitro from pSar55 (wild type), pSar/3′Mex, or pSar/3′swine. Horizontal bars indicate the presence of anti-HEV, as detected in an ELISA. The broken line represents twice the mean preinoculation level of ALT for individual animals.