Expression and processing of the Hepatitis E virus ORF1 nonstructural polyprotein.
BACKGROUND
The ORF1 of hepatitis E virus (HEV) encodes a nonstructural polyprotein of approximately 186 kDa that has putative domains for four enzymes: a methyltransferase, a papain-like cysteine protease, a RNA helicase and a RNA dependent RNA polymerase. In the absence of a culture system for HEV, the ORF1 expressed using bacterial and mammalian expression systems has shown an approximately 186 kDa protein, but no processing of the polyprotein has been observed. Based on these observations, it was proposed that the ORF1 polyprotein does not undergo processing into functional units. We have studied ORF1 polyprotein expression and processing through a baculovirus expression vector system because of the high level expression and post-translational modification abilities of this system.


RESULTS
The baculovirus expressed ORF1 polyprotein was processed into smaller fragments that could be detected using antibodies directed against tags engineered at both ends. Processing of this approximately 192 kDa tagged ORF1 polyprotein and accumulation of lower molecular weight species took place in a time-dependent manner. This processing was inhibited by E-64d, a cell-permeable cysteine protease inhibitor. MALDI-TOF analysis of a 35 kDa processed fragment revealed 9 peptide sequences that matched the HEV methyltransferase (MeT), the first putative domain of the ORF1 polyprotein. Antibodies to the MeT region also revealed an ORF1 processing pattern identical to that observed for the N-terminal tag.


CONCLUSIONS
When expressed through baculovirus, the ORF1 polyprotein of HEV was processed into smaller proteins that correlated with their proposed functional domains. Though the involvement of non-cysteine protease(s) could not be be ruled out, this processing mainly depended upon a cysteine protease.
Sehgal
Deepak
D
Virology Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India. deepak.sehgal@lycos.com
Thomas
Saijo
S
Chakraborty
Mahua
M
Jameel
Shahid
S
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D016752:Hepatitis E virus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D018411:Spodoptera; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D010455:Peptides; D020815:Polyproteins; D017361:Viral Nonstructural Proteins; D003546:Cysteine Endopeptidases