Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain

Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by inte rferon and activated upon autophosphorylation. We previously identified fou r autophosphorylated amino acids and elucidated their participation in PKR activation. Three of these sites are in the central region of the protein, and one is in the kinase domain, Here we describe the identification of fou r additional autophosphorylated amino acids in the spacer region that separ ates the two dsRNA-binding motifs in the RNA-binding domain. Eight amino ac ids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2. This region of E2 is required for its i nhibition of PKR although the mechanism of inhibition is not known. Replace ment of all four of these residues in PKR with alanines did not dramaticall y affect kinase activity in vitro or in yeast Saccharomyces cerevisiae, How ever, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity. A synthetic pe ptide corresponding to this region of PKR was phosphorylated in vitro by PK R, but phosphorylation was strongly inhibited after PKR was preincubated wi th HCV E2. Another synthetic peptide, corresponding to the central region o f PKR and containing serine 242, was also phosphorylated by active PKR, but E2 did not inhibit this peptide as efficiently. Neither of the PKR peptide s was able to disrupt the HCV E2-PKR interaction. Taken together, these res ults show that PKR is autophosphorylated on serine 83 and threonines 88, 89 , and 90, that this autophosphorylation may enhance kinase activation, and that the inhibition of PKR by HCV E2 is not solely due to duplication of an d competition with these autophosphorylation sites.