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Abstract
Vaccinia virus replication is resistant to the anti-viral effect of interferon in various cell lines. Vaccinia has been shown specifically to inhibit the activation of the interferon (IFN)-induced, double-stranded RNA (dsRNA)-dependent, 2$\sp\prime,$5$\sp\prime$-oligoadenylate synthetase pathway and the interferon-induced, double-stranded RNA dependent protein kinase (PKR) pathway. The vaccinia virus PKR inhibitor has been reported to be a protein that interacts in a stoichiometric manner with dsRNA. A M$\sb{\rm r}$ = 25,000, vaccinia-encoded dsRNA-binding protein, p25, was found to copurify with PKR inhibitory activity. The goal of this work was first to identify the vaccinia gene that encodes p25 and determine if p25 is the actual PKR inhibitor, and second, to investigate the role p25 plays in vaccinia replication.
Amino acid sequence analysis of a chymotryptic fragment of p25 revealed similarity to the vaccinia E3L gene. The E3L gene was cloned and expressed in COS cells and in vitro. A M$\sb{\rm r}$ = 25,000 polypeptide that could bind to dsRNA and that reacted with p25 specific anti-serum was produced in each case. Extracts from COS cells expressing p25 exhibited PKR inhibitory activity. These results demonstrate that the E3L gene encodes p25, and that the products of the E3L gene have PKR inhibitory activity.
Further characterization of the E3L proteins revealed the following: (1) A motif, which is conserved in several dsRNA binding proteins, was found to be necessary for the dsRNA binding and PKR inhibitory activity of the E3L proteins; and (2) the E3L proteins were substrates of PKR.
Studies on the replication of a mutant vaccinia virus with the E3L gene deleted indicated first that, in L cells, the E3L gene is necessary for the IFN resistance of vaccinia virus and for the inhibition of PKR and 2$\sp\prime,$5$\sp\prime$-oligoadenylate synthetase pathways in vaccinia-infected L cells. Second, that the dsRNA binding activity of the E3L proteins is required for vaccinia replication in HeLa cells. These results suggest that the E3L gene products play an important role in the vaccinia life cycle.
