The Phosphorylated Form of the ORF3 Protein of Hepatitis E Virus Interacts with Its Non-glycosylated Form of the Major Capsid Protein, ORF2

Abstract

Hepatitis E virus (HEV) is a human RNA virus contain-ing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser 80 of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.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), 1 is transmitted via the fecal-oral route, predominantly through contaminated water (1). HEV is an RNA virus with a positive-sense genome ϳ7.2 kb in length with three open reading frames (ORF1, ORF2, and ORF3) encoding three different proteins (2-4). ORF1 (5079 bp) is at the 5Ј-end of the genome and is predicted to code for putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (3-6). 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 transcriptiontranslation system (7-11). ORF2 encodes the major HEV structural protein, which has been shown to be an 88-kDa glycoprotein that is expressed intracellularly as well as on the cell surface. It is synthesized as a precursor and is processed through signal sequence cleavage into the mature protein, which is capable of self-association (12, 13). When expressed through the baculoviral expression system, ORF2 was shown to assemble into virus-like particles (VLPs), which were cell-associated as w...

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

“…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.…”

“…In an effort to understand the cellular localization of bikunin, we used Huh7 human hepatoma cells, which express bikunin endogenously. These cells were subjected to immunofluorescent staining followed by fluorescence microscopy, as described previously (38). To study colocalization of ORF3 with bikunin, dual-labeling experiments were conducted (Fig.…”

“…Mammalian COS-1 cells were transiently transfected with pMT-ORF3 alone or along with pMT-bikunin (Table 1) as described previously (38). [ 35 S]methionine-labeled cell lysates were immunoprecipitated using ORF3 polyclonal antibodies.…”

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.…”

DISTRIBUTION AND GENETIC VARIABILITY OF AVIAN HEPATITIS E VIRUS ON POULTRY FARMS IN ex-USSR

“…Its causative agent, the aHEV (Hepevirus: Hepeviridae) is a non-enveloped virus with a singlestranded, positive-sense RNA genome (6) that is approximately 6600 bp in size and contains three open reading frames (ORFs) (7). 5´-end ORF1 encodes the nonstructural polypeptide with several domains, including a methyltransferase domain, a papain-like cystein protease domain, a helicase domain and a RNAdependent RNA polymerase domain (8), and ORF2 and ORF3 encode the capsid protein and a posphoprotein, respectively (9)(10)(11).…”