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

Ansari, Israrul Haque ; Nanda, Santosh Kumar ; Durgapal, Hemlata ; Agrawal, Shipra ; Mohanty, Sujit Kumar ; Gupta, Dinesh ; Jameel, Shahid ; Panda, Subrat Kumar (2000) Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1) Journal of Medical Virology, 60 (3). pp. 275-283. ISSN 0146-6615

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Hepatitis E virus (HEV) causes enterically transmitted epidemic and sporadic viral hepatitis affecting millions of people in the developing world. Different geographical isolates of HEV show a high degree of homology at the nucleotide and amino acid levels. The ~7.2 kb RNA genome has three open reading frames of which ORF1 is predicted to code for the viral nonstructural polyprotein. The expression, processing and properties of the nonstructural ORF1 polyprotein have not been reported so far. In this study, the complete HEV ORF1 was reconstructed from overlapping fragments amplified by polymerase chain reaction (PCR) of total RNA isolated from the bile fluid of a rhesus monkey experimentally infected with HEV isolate from an epidemic. The complete assembled ORF1 was sequenced using HEV specific primers. The ORF1 polyprotein was expressed in E. coli, in a cell free translation system and in HepG2 cells, and was characterized by western blotting and immunoprecipitation using acute phase patient serum as well as polyclonal antibodies raised against defined parts of the ORF1 polyprotein. The nonstructural polyprotein of HEV was expressed as a 186 kDa protein. No processing was observed into discrete units, either in-vitro based on a kinetic analysis, or in HepG2 cells based on immunoprecipitation.

Item Type:Article
Source:Copyright of this article belongs to John Wiley and Sons, Inc.
Keywords:Hepatitis E Virus (HEV); Polymerase Chain Reaction (PCR); Western Blotting and Immunoprecipitation
ID Code:13078
Deposited On:11 Nov 2010 06:55
Last Modified:23 Feb 2011 05:32

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