The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

Abstract

Hepatitis E virus (HEV), a human plus-stranded RNA virus, contains three open reading frames (ORF).Of these, ORF1 encodes the viral nonstructural polyprotein, ORF2 encodes the major capsid protein, and ORF3 codes for a phosphoprotein of undefined function. Recently, using the yeast two-hybrid system to screen a human cDNA liver library, we have isolated and characterized AMBP (␣ 1 -microglobulin/bikunin precursor), which specifically interacts with the ORF3 protein of HEV. The ORF3 protein expedites the processing and secretion of ␣ 1 -microglobulin. When checked individually for interaction, the second processed protein from AMBP, bikunin, strongly interacted with the full-length ORF3 protein. This protein-protein interaction has been validated by immunoprecipitation in both COS-1 and Huh7 cells and by His 6 pull-down assays. In dual-labeling immunofluorescent staining, followed by fluorescence microscopy of transfected human liver cells, ORF3 colocalized with endogenously expressed bikunin. Finally, a 41-amino-acid C-terminal region of ORF3 has been found to be responsible for interacting with bikunin. The importance of this virus-host protein-protein interaction, with reference to the viral life cycle, has been discussed.Hepatitis E is an acute disease endemic in many countries throughout developing parts of the world, in particular on the continents of Africa and Asia, where it causes epidemics and sporadic infections. The causative agent, hepatitis E virus (HEV), is transmitted via the fecal-oral route, predominantly through contaminated water (7,10,25,26). HEV is a plusstranded RNA virus with a 7.2-kb genome containing three open reading frames (ORF), ORF1, ORF2, and ORF3, encoding three different proteins (20,32,35). ORF1 (5,079 bp) is at the 5Ј end of the genome and is predicted to code for the putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (1,20,27,35). 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, and yeast and in vitro in a coupled transcription-translation system (12,15,17,22,23). ORF2, the major capsid protein for HEV, is synthesized as a precursor and is processed through signal sequence cleavage into the mature protein, which is capable of selfassociation (18, 36). ORF3 encodes a small 13.5-kDa phosphoprotein that is expressed intracellularly, associates with the cytoskeleton, and shows no major processing (3, 42). The ORF3 protein dimerizes using a 43-amino-acid region, interacts with SH3 domains, and activates mitogen-activated protein kinase (21, 37). The phosphorylated form of the ORF3 protein has also been shown to interact with the nonglycosylated form of the major capsid protein, ORF2 (38). These properties of ORF3 clearly indicate that this protein may have multiple roles in HEV pathogenesis. Rece...

The Hepatitis E Virus Open Reading Frame 3 Product Interacts with Microtubules and Interferes with Their Dynamics

“…The exact functions of vp13 in HEV infection remain unknown although the findings of a number of studies have shown that it plays a role in cellular signaling pathways (13,17,24,(34)(35)(36)40). During subcellular fractionation of COS-7 cells transfected with a vp13-expressing plasmid, vp13 was found to partition with the cytoskeletal fraction (40).…”

Genotype-Specific Evolution of Hepatitis E Virus

“…Interestingly, ORF3 is not necessary for in vitro infection (20) but is necessary for in vivo infection of macaques (21). It is most likely multifunctional (19) and involved in pathogenesis (22)(23)(24)(25). Most of the coding region in the HEV genome is under purifying selection (26,27), i.e., selection against change in the amino acid sequence.…”

Initiation at the Third In-Frame AUG Codon of Open Reading Frame 3 of the Hepatitis E Virus Is Essential for Viral Infectivity In Vivo

“…It has been reported that recombinant ORF3 protein forms dimers through amino acid residues 81 to 123 (28); interacts with ORF2 protein mediated by amino acid residues 57 to 81 (23); binds various SH3 domain-containing proteins via amino acid residues 75 to 113 and activates mitogen-activated protein kinase (12); and interacts with a liver-specific protein, ␣ 1 -microglobulin/bikunin precursor (AMBP), and its two processed proteins, ␣ 1 m (26) and bikunin (27), through the C-terminal region (amino acid residues 81 to 123). These data indicated that the C-terminal region of the HEV ORF3 protein is a multifunctional domain.…”