The ORF3 protein of hepatitis E virus interacts with hemopexin by means of its 26 amino acid N-terminal hydrophobic domain II.
Hepatitis E virus (HEV) is a nonenveloped plus-stranded RNA virus that is a major cause of acute hepatitis in many developing countries. Recent work has shown HEV may be endemic in developed countries also. The 5' two-thirds of the 7.2 kb single-stranded RNA genome of HEV encodes ORF1, and the 3' end encodes the structural proteins ORF2 and ORF3. ORF1 is the nonstructural protein involved in viral RNA synthesis, and ORF2 is the major capsid protein, whereas ORF3 is a very small protein of only 123 amino acids. The precise cellular functions of ORF3 protein remain obscure, although it has been postulated to be a viral regulatory protein. To elucidate the role of ORF3 in viral pathogenesis, the yeast two-hybrid system was used to screen a human liver cDNA library for proteins interacting with ORF3. One of the ORF3-interacting partners thus isolated and identified was hemopexin, a 60 kDa acute-phase plasma glycoprotein with a high binding affinity to heme. The two-hybrid result was validated by in vitro pull-down and co-immunoprecipitation assays and finally by intracellular fluorescence resonance energy transfer. Using a deletion mapping approach, the hydrophobic domain II of ORF3 (spanning amino acids 37 to 62) was found to be responsible for binding to Hpx, with amino acids 63 to 77 possibly contributing to the strength of the interaction. The biological significance of this interaction in the virus life cycle has been discussed.
Ratra
Ruchi
R
Virology Group, International Centre for Genetic Engineering and Biotechnology, P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi-110067, India.
Kar-Roy
Anindita
A
Lal
Sunil K
SK
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006466:Hemopexin / Q000378:metabolism*; D047468:Immunoprecipitation; D011485:Protein Binding; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D017931:DNA Primers; C400973:ORF3 protein, Hepatitis E virus; D014764:Viral Proteins; D006466:Hemopexin