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. 2023 Mar 18;15(3):784.
doi: 10.3390/v15030784.

Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus

Huimin Bai  1 , Yasushi Ami  2 , Yuriko Suzaki  2 , Yen Hai Doan  3 , Masamichi Muramatsu  4 , Tian-Cheng Li  4
Affiliations

Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus

Huimin Bai et al. Viruses. .

Abstract

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837CRNA, T2885CRNA, and T2837C/T2885CRNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836TRNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.

Keywords: HEV-1 mutant; Mongolian gerbil; ORF4 mutant; PLC/PRF/5 cells; genotype 1; hepatitis E virus; reverse genetic system.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
HEV-1 (LC061267) genome organization and the position of the nucleotide mutations. ORF1 to ORF4 and the putative functional domains observed in ORF1 are depicted. Hel: helicase, HVR: hypervariable region, MT: methyltransferase, PCP: papain-like cysteine protease, RdRp: RNA-dependent RNA polymerase, X: X-domain, and Y: Y-domain. The numbers indicate the nucleotide position from the 5′-end. Mutated nucleotides are shown by bold and italics.
Figure 2
Figure 2
Generation and replication of the original and ORF4-defective HEV-1 in PLC/PRF/5 cells. PLC/PRF/5 cells were transfected with capped HEV-1RNA, A2836TRNA, T2837CRNA, T2885CRNA, and T2837RNA/T2885RNA, respectively. The culture supernatant was collected every 4 days, and the viral RNA was measured by RT-qPCR. The viral RNA copy numbers are shown in the HEV-1RNA-transfected cells (≤), A2836TRNA-transfected cells (○), T2837CRNA-transfected cells (◊), T2885CRNA-transfected cells (△), and T2837C/T2885CRNA-transfected cells (×).
Figure 3
Figure 3
Infectivity of the original and ORF4-defective HEV-1s in PLC/PRF/5 cells. PLC/PRF/5 cells were inoculated with HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, and T2837C/T2885Cp0 containing the same RNA copy number, respectively. The culture supernatant was collected every 4 days, and the viral RNA was measured by RT-qPCR. The RNA titers are shown in the HEV-1p0-infected cells (≤), A2836Tp0-infected cells (○), T2837Cp0-infected cells (◊), T2885Cp0-infected cells (△), and T2837C/T2885Cp0-infected cells (×).
Figure 4
Figure 4
Infection of original and ORF4-defective viruses in Mongolian gerbils. Fifteen Mongolia gerbils were randomly separated into five groups (n = 3 per group). Individual gerbils are indicated by ○, △, and ☐. Each group received HEV-1p0, A2836Tp0, T2837Cp0, T2885Cp0, or T2837C/T2885Cp0 via intraperitoneal injection. The kinetics of the viral RNA in the fecal specimens were measured by RT-qPCR (a). The serum samples were collected at the end of the experiment (day 28 post-inoculation [p.i.]), and the anti-HEV-IgG antibody titers were determined by an ELISA with the virus-like particles (VLPs) of HEV-1 as the antigens (b). Dotted lines: the cut-off values. The minimum endpoints of the antibody titers are blackened.
Figure 5
Figure 5
Detection of viral proteins in HEV-1-infected PLC/PRF/5 cells. A cynomolgus monkey’s serum bearing the anti-HEV-1 IgG antibody titer 1:3,276,800 by ELISA was used for Western blot analyses. The minimum endpoints of the antibody titers are blackened (a). HEV-1p0-, T2837Cp0-, and T2837C/T2885Cp0-infected PLC/PRF/5 cells were harvested on day 48 p.i., and the virus proteins were detected by Western blot analysis (b). ORF4 was expressed by an E. coli expression system, and the related protein was analyzed by SDS-PAGE (c) and a Western blot analysis with monkey anti-HEV-1 serum (d) and anti-HEV-7 serum (e). M: molecular weight, NC: no-infected PLC/PRF/5 cells (b) or no-transformed BL21 (DE3) cells (c–e). pET32a (+): vector pET32a (+)-transformed BL21 (DE3). pET32aORF4: pET32aORF4-transformed BL21 (DE3).
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This research was supported by grants from the Research Program on Hepatitis (nos. JP22fk0210109, JP22fk0210075) from the Japan Agency for Medical Research and Development (AMED).

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