Recombinant hepatitis E virus genomes infectious for primates: Importance of capping and discovery of a cis-reactive element

Abstract

Hepatitis E virus recombinant genomes transcribed in vitro from two cDNA clones differing by two nucleotides were infectious for chimpanzees. However, one cDNA clone encoded a virus that was attenuated for chimpanzees and unable to infect rhesus monkeys. The second cDNA clone encoded a virus that infected both chimpanzees and rhesus monkeys and caused acute hepatitis in both. One mutation differentiating the two clones identified a cisreactive element that appeared to overlap the 3 end of the capsid gene and part of the 3 noncoding region. Capping of the RNA transcripts was essential for infectivity.

Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

“…The infectious cDNA clone of HEV strain Sar-55, pSK-E2 (GenBank accession no. AF444002) and plasmid CMV-Sar were described previously (16,27). Plasmid CMV-MT29 was generated by replacing the first 29 nucleotides of Sar-55 subgenomic RNA with those of Kernow-C1 HEV in the plasmid CMV-Sar.…”

“…Full-length viral cDNA was transcribed with T7 polymerase, and capped transcripts were transfected into S10-3 or deer cells with DMRIE-C (Invitrogen) as described previously (27) and detailed in SI Materials and Methods. LLC-PK1 cells were killed by all transfection methods tried.…”

Hepatitis E: Molecular Virology and Pathogenesis

“…7,8 Further understanding of the HEV replication came from the development of infectious cDNA clone of HEV which helped in elucidating the importance of 5 0 end cap, RdRp activity and nonstructural polyprotein processing. 9,10 Mutational and strand-specific PCR analysis using the HEV replicon helped in delineating the bi-cistronic sub-genomic RNA based expression strategy for ORF2 and ORF3 proteins and the presence of an additional untranslated region (UTR) in the intergenic region with probable cis reactive element thought to function as sub-genomic promoter. [11][12][13][14] Till date only one cis-acting element has been characterized, that in the 3 0 UTR, which was shown to form stable secondary structures and help in the localization of RdRp.…”

“…The 3 0 UTR of HEV has been shown to form cis-active stem-loop structures which localized the viral RNA dependent RNA polymerase in in vitro binding studies and henceforth implicated in the initiation of virus replication. 10,15 The poly A tail at the 3 0 end is approximately 150-200 nucleotides long and is crucial for RdRp binding to the 3 0 UTR. An additional UTR was recently established with the elucidation of sub-genomic RNA start site downstream of the ORF1 stop site.…”

“…26 Capping activity of HEV 5 0 UTR has been shown to be essential for infectivity in vivo and replication in vitro by using a HEV infectious cDNA clone and a HEV replicon expressing green fluorescent protein, respectively. 10,27 Conservation of the X domain, which has been exclusively found in association with viral papain-like proteases, suggests that HEV encoded protease bears similarity to proteases observed in other positive-strand RNA viruses like alphaviruses and RubV. The catalytic residues predicted by computer alignment for HEV protease were Cys483 and His590.…”