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The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton
M Zafrullah1, M H Ozdener, S K Panda
1Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Abstract:
Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.
Insights
Hepatitis E virus (HEV) pORF3 protein is a cytoskeleton-associated phosphoprotein. This finding provides insights into the virus
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Hepatitis E virus (HEV) is a significant pathogen, particularly in developing nations.
- Limited in vitro culture systems hinder understanding of HEV's basic biology.
- The function of the pORF3 protein, encoded by HEV's third open reading frame, remains largely unknown.
Purpose of the Study:
- To investigate the biological characteristics and cellular localization of the HEV pORF3 protein.
- To identify the phosphorylation site(s) and associated kinases involved in pORF3 modification.
- To explore the role of hydrophobic domains in pORF3's interaction with cellular structures.
Main Methods:
- Expression of pORF3 in transiently transfected COS-1 and Huh-7 cells.
- Site-directed mutagenesis to identify the phosphorylation site (Ser-80).
- In vitro phosphorylation assays using MAPK and analysis of pORF3 mutants.
- Subcellular fractionation to determine pORF3 localization and the role of hydrophobic domains.
Main Results:
- HEV pORF3 was identified as a phosphoprotein phosphorylated at Ser-80, a site conserved and recognized by MAPK.
- In vitro phosphorylation by MAPK mirrored in vivo modification patterns.
- pORF3 contains two hydrophobic N-terminal domains and associates with the cytoskeleton.
- Deletion of the first hydrophobic domain disrupted cytoskeletal association.
Conclusions:
- HEV pORF3 is a cytoskeleton-associated phosphoprotein.
- Phosphorylation, particularly by MAPK, is a key modification of pORF3.
- Hydrophobic domains are crucial for pORF3's cytoskeletal localization.
- These findings suggest a potential role for pORF3 in the HEV replication cycle.