The ORF3 protein of hepatitis E virus interacts with liver-specific α₁-microglobulin and its precursor α₁- microglobulin/bikunin precursor (AMBP) and expedites their export from the hepatocyte

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, α₁-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 α₁-microglobulin (α₁m) and bikunin. α₁m 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 α₁m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of α₁m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that α₁m was not degraded in the presence of ORF3. Finally, pulse labeling of α₁m 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 α₁m export from the hepatocyte.