THE ORF3 PROTEIN OF HEPATITIS-E VIRUS IS A PHOSPHOPROTEIN THAT ASSOCIATES WITH THE CYTOSKELETON

Hepatitis E virus (HEV) is a major human pathogen in the developing wo rld. In the absence of an in vitro culture system, very little informa tion exists on the basic biology of the virus. A small protein (simila r to 13.5 kDa) of unknown function, pORF3, is encoded by the third ope n reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants we re created to establish Ser-80 as the phosphorylation site. This resid ue is present within a conserved primary sequence that showed consensu s sites for phosphorylation by p34(cdc2) kinase (cdc2K) and mitogen-ac tivated protein kinase (MAPK). In vitro experiments with hexahistidine -tagged pORF3 expressed either in Escherichia coli or in COS-1 cells s howed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcell ular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on dele tion of hydrophobic domain I (amino acid residues 1 to 32). These resu lts suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within th e HEV replicative cycle.