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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.