The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2.
Hepatitis E virus (HEV) is a human RNA virus containing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser(80) of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.
Tyagi
Shweta
S
Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 1100067, India.
Korkaya
Hasan
H
Zafrullah
Mohammad
M
Jameel
Shahid
S
Lal
Sunil K
SK
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002213:Capsid / Q000378:metabolism*; D017353:Gene Deletion; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D012694:Serine / Q000737:chemistry; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
C400971:ORF2 protein, Hepatitis E virus; C400973:ORF3 protein, Hepatitis E virus; D014764:Viral Proteins; D012694:Serine; D001616:beta-Galactosidase