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The ORF3 protein of hepatitis E virus interacts with liver-specific alpha1-microglobulin and its precursor alpha1-microglobulin/bikunin precursor (AMBP) and expedites their export from the hepatocyte.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2004 Jul 09; Vol. 279 (28), pp. 29308-19. Date of Electronic Publication: 2004 Mar 22. - Publication Year :
- 2004
-
Abstract
- 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, alpha(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 alpha(1)-microglobulin (alpha(1)m) and bikunin. alpha(1) m individually interacted with ORF3. The above findings were validated by COS-1 cell immunoprecipitation, His(6) 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 alpha(1) m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of alpha(1)m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that alpha m was not degraded in the presence of ORF3. Finally, (1)pulse labeling of alpha(1)m 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 alpha(1)m export from the hepatocyte.
- Subjects :
- Alpha-Globulins genetics
Animals
Brefeldin A pharmacology
COS Cells
Cell Line, Tumor
Fluorescence Resonance Energy Transfer
Gene Library
Golgi Apparatus metabolism
Hepatitis E virus genetics
Hepatocytes chemistry
Hepatocytes drug effects
Humans
Liver cytology
Membrane Glycoproteins genetics
Monensin pharmacology
Open Reading Frames
Protein Synthesis Inhibitors pharmacology
Protein Transport physiology
Trypsin Inhibitor, Kunitz Soybean genetics
Two-Hybrid System Techniques
Viral Proteins genetics
Alpha-Globulins metabolism
Hepatitis E virus metabolism
Hepatocytes physiology
Liver metabolism
Membrane Glycoproteins metabolism
Trypsin Inhibitor, Kunitz Soybean metabolism
Viral Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 279
- Issue :
- 28
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15037615
- Full Text :
- https://doi.org/10.1074/jbc.M402017200