Deletions of the hypervariable region (HVR) in open reading frame 1 of hepatitis E virus do not abolish virus infectivity: evidence for attenuation of HVR deletion mutants in vivo

Abstract

Hepatitis E virus (HEV) is an important human pathogen, although little is known about its biology and replication. Comparative sequence analysis revealed a hypervariable region (HVR) with extensive sequence variations in open reading frame 1 of HEV. To elucidate the role of the HVR in HEV replication, we first constructed two HVR deletion mutants, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a genotype 1 human HEV replicon. Evidence of HEV replication was detected in Huh7 cells transfected with RNA transcripts from mutant hHVRd2, as evidenced by expression of enhanced green fluorescent protein. To confirm the in vitro results, we constructed three avian HEV mutants with various HVR deletions: mutants aHVRd1, with deletion of aa 557 to 585 (Delta557-585); aHVRd2 (Delta612-641); and aHVRd3 (Delta557-641). Chickens intrahepatically inoculated with capped RNA transcripts from mutants aHVRd1 and aHVRd2 developed active viral infection, as evidenced by seroconversion, viremia, and fecal virus shedding, although mutant aHVRd3, with complete HVR deletion, was apparently attenuated in chickens. To further verify the results, we constructed four additional HVR deletion mutants using the genotype 3 swine HEV as the backbone. Mutants sHVRd2 (Delta722-781), sHVRd3 (Delta735-765), and sHVRd4 (Delta712-765) were shown to tolerate deletions and were infectious in pigs intrahepatically inoculated with capped RNA transcripts from the mutants, whereas mutant sHVRd1 (Delta712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype in infected pigs. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.

FIG. 1.

Schematic diagram showing the relative positions of the HVR of ORF1 from representative isolates in four major genotypes of mammalian HEV (genotypes 1 to 4, in parentheses), along with putative functional domains: MET, methyltransferase; P, papain-like cysteine protease; Y, Y domain; H, HVR; X, X domain; HEL, helicase; RDRP, RNA-dependent RNA polymerase. The amino acid sequences of different HEV strains were aligned using the Clustal W method of the MegAlign program (DNAStar, Inc.). The amino acid sequence in the HVR of the genotype 1 Sar55 human HEV strain is shown at the top, and only differences are indicated for the other strains. Amino acid sequences identical to the genotype 1 HEV Sar55 sequence are indicated by dots, and deletions are indicated by dashes. Representative strains from each genotype used in this study are underlined.

FIG. 2.

(A) Schematic diagram showing the HVR (aa 707 to 777) in ORF1 of the genotype 1 human HEV (strain Sar55) replicon expressing EGFP. MET, methyltransferase; P, papain-like cysteine protease; Y, Y domain; H, HVR; X, X domain; HEL, helicase; RDRP, RNA-dependent RNA polymerase. The amino acid sequence of each HVR deletion mutant is aligned with that of the wild-type Sar55 HEV replicon to show the relative positions of the in-frame amino acid deletions: mutants hHVRd1 (aa 711 to 777) and hHVRd2 (aa 747 to 761). (B) Fluorescence microscopy of Huh7 liver cells at 6 days posttransfection with similar amounts of capped RNA transcripts from the wild-type Sar55 replicon with the EGFP gene (A), HVR deletion mutants hHVRd1 (B) and hHVRd2 (C), and mock-transfected cells (D).

FIG. 3.

(A) Schematic diagram showing the HVR (aa 557 to 641) in ORF1 of avian HEV, along with putative functional domains: MET, methyltransferase; P, papain-like cysteine protease; H, HVR; HEL, helicase; and RDRP, RNA-dependent RNA polymerase. The amino acid sequence of each HVR deletion mutant is aligned with that of the wild-type strain of avian HEV to show the relative positions of the in-frame amino acid deletions: mutants aHVRd1 (aa 557 to 585), aHVRd2 (aa 612 to 641), and aHVRd3 (aa 557 to 641). (B) Seroconversion to IgG anti-HEV in chickens inoculated with capped RNA transcripts from the wild-type avian HEV infectious clone and its derived HVR deletion mutants. IgG anti-HEV was plotted as the ELISA optical density (OD) (A₄₀₅), and the ELISA cutoff value was 0.30. Chickens 5357, 5361, and 5368 were each inoculated with RNA transcripts from HVR deletion mutant aHVRd1 (group A); chickens 5353, 5372, and 5388 with RNA transcripts from mutant aHVRd2 (group B); and chickens 5354, 5355, and 5385 with RNA transcripts from mutant sHVRd3 (group C). Chickens 5362, 5367, and 5374 (group D) were each inoculated with RNA transcripts from the wild-type avian HEV infectious cDNA clone as positive controls, and chickens 5352, 5358, and 5369 (group E) were intrahepatically inoculated with PBS buffer as negative controls.

FIG. 4.

(A) Schematic diagram showing the HVR (aa 707 to 790) in ORF1 of the genotype 3 swine HEV. MET, methyltransferase; P, papain-like cysteine protease; Y, Y domain; H, HVR; X, X domain; HEL, helicase; and RDRP, RNA-dependent RNA polymerase. The amino acid sequence of each HVR deletion mutant is aligned with that of the wild-type strain of the genotype 3 swine HEV to show the relative positions of the in-frame amino acid deletions: mutants sHVRd1 (aa 712 to 790), sHVRd2 (aa 722 to 781), sHVRd3 (aa 735 to 765), and sHVRd4 (aa 712 to 765). (B) Seroconversion to IgG anti-HEV in pigs inoculated with capped RNA transcripts from the wild-type swine HEV infectious clone and its derived HVR deletion mutants. IgG anti-HEV was plotted as the ELISA optical density (OD) (A₄₀₅), and the ELISA cutoff value was 0.30. Pigs 286, 296, and 608 were each inoculated with capped RNA transcripts from HVR deletion mutant sHVRd1 (group A); pigs 291, 295, and 613 with RNA transcripts from mutant sHVRd2 (group B); pigs 292, 604, and 606 with RNA transcripts from mutant sHVRd3 (group C); pigs 288, 293, and 300 with RNA transcripts from mutant sHVRd4 (group D); and pigs 289, 294, and 612 with RNA transcripts from wild-type genotype 3 swine HEV infectious clone pSHEV-3 (group E). Pigs 603, 609, and 611 were intrahepatically inoculated with PBS buffer as negative controls (group F).