Hepatitis E virus (HEV), a plus-stranded RNA viruscontains 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
Hepatitis E virus (HEV)1 is a waterborne pathogen and is responsible for sporadic infections as well as large epidemics of acute viral hepatitis in developing countries (1-4). HEV is a plus-stranded RNA virus with a genome of ϳ7.2 kb, containing three open reading frames called ORF1, ORF2, and ORF3 (5-7). ORF1 (5079 bp) is at the 5Ј-end of the genome and is predicted to code for putative non-structural proteins with sequences homologous to those encoding a viral methyltransferase, a cysteine protease, a RNA helicase, and a RNA-dependent RNA polymerase (5, 7-9). In the absence of a reliable in vitro culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken. ORF2 and ORF3 have been expressed in Escherichia coli, animal cells, baculovirus, yeast, and in vitro in a coupled transcription-translation system (10 -14). ORF2, a 88-kDa glycoprotein, is expressed intracellularly as well as on the cell surface and is the major capsid protein for HEV. It is synthesized as a precursor that is processed through signal sequence cleavage into the mature protein, which is capable of self-association (15, 16). ORF3 encodes a small 13.5-kDa phosphoprotein that is expressed intracellularly, associates with the cytoskeleton and shows no major processing (17, 18). The ORF3 protein dimerizes using a 43-amino acid region, interacts with SH3 domains and activates cellular MAP kinase (19,20). Recently the phosphorylated form of the ORF3 protein has also been shown to interact with the non-glycosylated form of the ORF2 (capsid) protein of HEV (21). These properties of ORF3 clearly indicate that this protein may have multiple roles in HEV pathogenesis. To delineate the functions of this viral protein, studies were conducted to screen and characterize ORF3-interacting host proteins from a human liver cDNA library.Since a few years of its introduction, the yeast two-hybrid system has proven invaluable for studying physical interactions between genetically defined partners, for identifying contacts among the subunits of multiprotein complexes (22-25), and for mapping specific domains involved in protein-protein interactions (20,21,26). In this system, two plasmid-borne gene fusions are co-transformed into yeast cells, and the interaction between these two fusion proteins is measured by the reconstitution...
