Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1)

Hepatitis E virus (HEV) causes enterically transmitted epidemic and sporadi c viral hepatitis affecting millions of people in the developing world. Dif ferent geographical isolates of HEV show a high degree of homology at the n ucleotide and amino acid levels. The similar to 7.2 kb RNA genome has three open reading frames of which ORF1 is predicted to code for the viral nonst ructural polyprotein. The expression, processing and properties of the nons tructural ORF1 polyprotein have not been reported so far. In this study, th e complete HEV ORF1 was reconstructed from overlapping fragments amplified by polymerase chain reaction (PCR) of total RNA isolated from the bile flui d of a rhesus monkey experimentally infected with HEV isolate from an epide mic. The complete assembled ORF1 was sequenced using HEV specific primers. The ORF1 polyprotein was expressed in E. coli, in a cell free translation s ystem and in HepG2 cells, and was characterized by western blotting and imm unoprecipitation using acute phase patient serum as well as polyclonal anti bodies raised against defined parts of the ORF1 polyprotein. The nonstructu ral polyprotein of HEV was expressed as a 186 kDa protein. No processing wa s observed into discrete units, either in-vitro based on a kinetic analysis , or in HepG2 cells based on immunoprecipitation. J. Med. Virol. 60:275-283 , 2000, (C) 2000 Wiley-Liss, Inc.