The In Vitro-Synthesized RNA from a cDNA Clone of Hepatitis E Virus Is Infectious

Abstract

Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with Hepatitis E virus (HEV) is established as an etiological agent of the epidemic and sporadic forms of waterborne hepatitis (6, 25). The first well-characterized HEV epidemic was reported in Delhi, India, in 1955 (35). Several other epidemics have since been described in developing countries on nearly every continent (19,21). HEV has a positive-strand polyadenylated RNA genome ϳ7.2 kb in length (27) containing three open reading frames (ORFs). Nonstructural ORF1 (5Ј end) codes for a polyprotein of 1,693 amino acids (pORF1) and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicase, and an RNAdependent RNA polymerase (RdRp). The second ORF (3Ј end) codes for the major viral capsid protein of 660 amino acid (pORF2), while the third and smallest ORF (ORF3) codes for a 123-amino-acid-long polypeptide (pORF3) whose function is unknown (32). Apart from its coding region, the viral genome has 27-and 68-nucleotide (nt)-long noncoding regions at its 5Ј and 3Ј ends, respectively (32). The genome sequences of HEV have been reported from different geographical isolates and show a high degree of homology at both the nucleotide and amino acid levels (3,4,12,32,33). Expression of structural proteins pORF2 and pORF3 in prokaryotic and eukaryotic systems has been reported by different investigators (7,11,14,23,29). Earlier, we have expressed and characterized pORF2 and pORF3 in animal cells. pORF2 is an 88-kDa glycoprotein which is expressed intracellularly, as well as on the cell surface (14, 37). The ORF3 protein (pORF3) is a 13.5-kDa phosphoprotein which is phosphorylated by the cellular mitogen-activated protein kinase and associates with the cytoskeleton (36). We have also recently expressed the ORF1 polyprotein in both prokaryotic and eukaryotic systems (2).In the absence of a reliable in vitro culture system...

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

“…These data, taken in conjunction with the demonstration that capped Sar-55 transcripts infected chimpanzees, whereas uncapped transcripts did not, suggested that a cap is required for viral viability. The in vivo transfection results provide an explanation for the failure of Panda et al (19) to infect rhesus monkeys with HEV transcripts from an Indian strain, because they used only uncapped transcripts. However, it is not clear how they were able to infect cultured Hep G2 cells with uncapped transcripts.…”

“…Recently it was reported that transfection of cultured Hep G2 cells with noncapped RNA transcripts from a full-length cDNA clone of an Indian strain of HEV resulted in productive replication (19). However, RNA transcripts from the cDNA clone were not infectious for rhesus monkeys when inoculated intrahepatically, as has been done successfully for other positivesense RNA viruses.…”

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.…”

“…28 ORF1 polyprotein processing has been observed in transfection studies of infectious HEV cDNA clone. 9 The exact cleavage sites of the ORF1 polyprotein are still not known. Another study demonstrating partial cleavage of the full-length 185 kDa ORF1 polyprotein had been shown.…”

Construction of an infectious cDNA clone of genotype 1 avian hepatitis E virus: characterization of its pathogenicity in broiler breeders and demonstration of its utility in studying the role of the hypervariable region in virus replication

“…The Korean avian HEV belongs to genotype 1. The construction of infectious cDNA clones of HEV provided useful tools to elucidate the structural and functional relationship of HEV genes and facilitate the study of HEV pathogenesis in animal models (Huang et al, 2005a;Okamoto, 2011;Panda et al, 2000;Yamada et al, 2009b). Thus far, only the genotype 2 avian HEV infectious clone is available (Huang et al, 2005b;Kwon et al, 2011).…”