The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton

Abstract

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (ϳ13.5 kDa) of unknown function, pORF3, is encoded by the third open 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 were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34 cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed 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 subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.

Molecular virology of hepatitis E virus

“…The ORF3 protein has been expressed using various systems that include E. coli (Panda et al, 1995), the yeast Pichia pastoris (Lal et al, 1997) and mammalian cell lines (Jameel et al, 1996). In mammalian cells orf3 expressed a protein of ∼13 kDa, which was phosphorylated at a single Serine residue by cellular mitogen-activated protein kinase (MAPK) (Zafrullah et al, 1997). Cells transfected with HEV genomic RNA prepared in vitro were also found to express a protein of the same size, which could be immunoprecipitated with anti-ORF3 antibodies Graff et al, 2005;.…”

“…Cells transfected with HEV genomic RNA prepared in vitro were also found to express a protein of the same size, which could be immunoprecipitated with anti-ORF3 antibodies Graff et al, 2005;. Subcellular fractionation showed the ORF3 protein to associate with the cytoskeleton (Zafrullah et al, 1997). Confocal microscopy has revealed its punctate and filamentous intracellular distribution; the latter is attributed to its interaction with microtubules (Kannan et al, 2009).…”

“…Of these, D1 is extremely rich in cysteine residues and is required for the ORF3 protein to associate with the cytoskeleton (Zafrullah et al, 1997), to bind microtubules (Kannan et al, 2009) and a MAPK phosphatase (Kar-Roy et al, 2004); however, none of these activities have been linked conclusively to one or more cysteine residues present in this domain. Domain P1 contains two overlapping motifs that have homology to kinase substrates -PMSP that is phosphorylated by MAPK and SPLR that has a loose homology to the SPKK motif that is phosphorylated by cdc2 kinase.…”

“…Domain P1 contains two overlapping motifs that have homology to kinase substrates -PMSP that is phosphorylated by MAPK and SPLR that has a loose homology to the SPKK motif that is phosphorylated by cdc2 kinase. Of these, Ser-80 (now Ser-71 in the 114 aa ORF3 protein) is phosphorylated by MAPK, both in transfected cells as well as in vitro (Zafrullah et al, 1997); whether the ORF3 protein expressed during viral infection is also phosphorylated has not been tested. The P2 domain has two overlapping PXXP motifs, which have been described in many viral and cellular proteins involved in signal transduction, and bind the Src homology 3 (SH3) domains found in other signal-transducing molecules (Pawson, 1995).…”

Antiviral strategies for hepatitis E virus

“…ORF2 encodes the 660-aa capsid protein that consists of a shell domain (S), a middle domain (M, also called P1) and a protruding domain (P, also called P2) (Guu et al, 2009;Yamashita et al, 2009). The protein translated from ORF3 is 114 aa in length, is phosphorylated (Zafrullah et al, 1997) and unique to HEV and closely related viruses.…”