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Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle
Published on: February 1, 2017
Expression of the hepatitis E virus ORF1
Archives of Virology
|August 30, 2000
Summary
Hepatitis E virus (HEV) nonstructural polyprotein (NSP) processing was investigated. Cleavage was observed in vivo but not mediated by the predicted protease, suggesting novel or cellular protease involvement.
Area of Science:
- Virology
- Molecular Biology
- Protease Function
Background:
- Hepatitis E virus (HEV) is a plus-strand RNA virus.
- ORF1 encodes nonstructural proteins (NSP) homologous to alphavirus-like superfamily viruses.
- In related viruses, NSP is cleaved by a papain-like cysteine protease (PCP).
Purpose of the Study:
- To determine if HEV NSP is processed by a PCP.
- To investigate the mechanism of HEV NSP processing.
Main Methods:
- Expression of HEV ORF1 in vitro and in vivo using coupled transcription/translation and vaccinia virus systems.
- Site-specific mutagenesis of predicted protease active site residues.
- N- and C-terminus-specific immunoprecipitation and deletion mutagenesis.
Main Results:
- Full-length 185 kDa HEV NSP was expressed, with no processing in vitro.
- Two cleavage products (107 kDa and 78 kDa) were observed in vivo after extended incubation.
- Mutagenesis of a predicted catalytic cysteine did not abolish cleavage, and a key histidine residue was not conserved, questioning PCP activity.
- HEV NSP processing could not be authenticated in infected primary monkey hepatocytes.
Conclusions:
- The predicted PCP in HEV NSP is likely not functional.
- HEV NSP processing may involve a novel viral protease or a cellular protease.
- Further research is needed to elucidate HEV NSP processing mechanisms.
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