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J. Biol. Chem., Vol. 279, Issue 28, 29308-29319, July 9, 2004
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The ORF3 Protein of Hepatitis E Virus Interacts with Liver-specific 
1-Microglobulin and Its Precursor 1-Microglobulin/Bikunin Precursor (AMBP) and Expedites Their Export from the Hepatocyte*
From the Virology Group, International Centre for Genetic Engineering & Biotechnology, P. O. Box 10504, Aruna Asaf Ali Rd., New Delhi 110067, India
Hepatitis E virus (HEV), a plus-stranded RNA virus contains three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein and ORF3 codes for a phosphoprotein of undefined function. Using the yeast two-hybrid system to screen a human cDNA liver library we have isolated, an N-terminal deleted protein, 
1 -microglobulin/bikunin precursor (AMBP) that specifically interacts with the ORF3 protein of HEV. Independently cloned, full-length AMBP was obtained and tested positive for interaction with ORF3 using a variety of in vivo and in vitro techniques. AMBP, a liver-specific precursor protein codes for two different unrelated proteins 1-microglobulin (1m) and bikunin. 1 m individually interacted with ORF3. The above findings were validated by COS-1 cell immunoprecipitation, His6 pull-down experiments, and co-localization experiments followed by fluorescence resonance energy transfer analysis. Human liver cells showing co-localization of ORF3 with endogenously expressing 1 m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of 1m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that m was not degraded in the presence of ORF3. Finally, 1pulse labeling of 1m showed that its secretion was expedited out of the liver cell at faster rates in the presence of the ORF3 protein. Hence, ORF3 has a direct biological role in enhancing 1m export from the hepatocyte.
Received for publication, February 24, 2004 , and in revised form, March 22, 2004.
* This work was supported in part by internal funds from the International Centre for Genetic Engineering & Biotechnology (ICGEB) and the Wellcome Trust. The Confocal Microscopy Facility at ICGEB is funded through an International Senior Research Fellowship of the Wellcome Trust (to S. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Senior Research Fellow of the University Grants Commission.
Senior Research Fellow of the Council of Scientific and Industrial Research, India.
¶ To whom correspondence should be addressed. Tel.: 91-11-2619-5007; Fax: 91-11-2616-2316; E-mail: sunillal{at}icgeb.res.in.
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