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An analysis of two open reading frames (ORF3 and ORF4) of rat hepatitis E virus genome using its infectious cDNA clones with mutations in ORF3 or ORF4
- Tanggis,, Kobayashi, Tominari, Takahashi, Masaharu, Jirintai, Suljid, Nishizawa, Tsutomu, Nagashima, Shigeo, Nishiyama, Takashi, Kunita, Satoshi, Hayama, Emiko, Tanaka, Takeshi, Mulyanto,, Okamoto, Hiroaki
- Virus research 2018 v.249 pp. 16-30
- Orthohepevirus A, messenger RNA, leucine, liver, protein synthesis, complementary DNA, open reading frames, genome, proline, mutation, clones, mutants, virus replication, rats, tissues
- Rat hepatitis E virus (ratHEV) genome has four open reading frames (ORFs: ORF1, ORF2, ORF3 and ORF4). The functions of ORF3 and ORF4 are unknown. An infectious cDNA clone (pUC-ratELOMB-131L_wt, wt) and its derivatives including ORF3-defective (ΔORF3) and ORF4-defective (ΔORF4) mutants, were constructed and their full-length RNA transcripts transfected into PLC/PRF/5 cells. ΔORF3 replicated as efficiently as wt in cells. However, ≤1/1000 of the number of progenies were detectable in the culture supernatant of ΔORF3-infected cells compared with wt-infected cells. ORF4 protein was not detectable in ratHEV-infected cells or in the liver tissues of ratHEV-infected rats. No marked differences were noted between wt and ΔORF4 regarding the viral replication and protein expression. ORF3 mutants with proline-to-leucine mutations at amino acids (aa) 93, 96 and/or 98 in ORF3 were constructed and transfected into PLC/PRF/5 cells. Wt and an ORF3 mutant with leucine at aa 98 (ORF3-L98) replicated efficiently (density 1.15–1.16 g/cm³), while ORF3-L93 + L96 exhibited a decreased viral release and banded at 1.26–1.27 g/cm³, similar to ΔORF3. In conclusion, the ORF3 protein, especially its proline residues at aa 93 and 96, is essential for the release of membrane-associated ratHEV particles, and ORF4 is unnecessary for the replication of ratHEV.