A vaccinia virus-encoded double-stranded RNA-binding protein, p25, has been previously implicated in inhibition of the interferon-induced, double-stranded RNAactivated protein kinase. In this study, we have identified the vaccinia viral gene (WR strain) that encodes p25. Amino acid sequence analysis of a chymotryptic fragment of p25 revealed a close match to the vaccinia virus (Copenhagen strain) E3L gene. The WR strain E3L gene was cloned and expressed either in COS-1 cells or in rabbit reticulocyte lysates in vitro. A M, 25,000 polypeptide that could bind to poly(rI) poly(rC)-agarose and that reacted with p25-specific antiserum was produced in each case. In addition, COS cells expressing E3L gene products inhibited activation of the double-stranded RNA-activated protein kinase in extracts from interferon-treated cells. Removal of E3L-encoded products by adsorption with anti-p25 antiserum resulted in loss of kinase inhibitory activity. These results demonstrate that the vaccinia virus E3L gene encodes p25 and that the products of the E3L gene have kinase inhibitory activity. Comparison of the deduced amino acid sequence of the E3L gene products with the protein sequence data base revealed a region closely related to the human interferon-induced, double-stranded RNA-activated protein kinase.

A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA. The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA ofhuman immunodeficiency virus type 1. TRBP inhibited phosphorylation ofthe interferon-induced ribosome-asted protein kinase PKR and of the eukaryotic transtion initiation factor eIF-2a in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR. TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant laking the expression of the dsRNA-binding protein E3L. These results implicate RBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the hnhibition ofPKR activation and stimulate translation in a llized manner.

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Vaccinia virus has evolved multiple mechanisms to counteract the interferon-induced antiviral host cell response. Recently, two vaccinia virus gene products were shown to interfere with the activity of the double-stranded RNA-dependent protein kinase (PKR): the K3L gene product and the E3L gene product. We have evaluated the efficiency by which these gene products inhibit PKR and whether they act in a synergistic manner. The effects of the two vaccinia virus gene products were compared in an in vivo system in which translation of a reporter gene (dihydrofolate reductase or eukaryotic translation initiation factor 2at [eIF-2a]) was inhibited because of the localized activation of PKR. In this system, the E3L gene product, and to a lesser extent the K3L gene product, potentiated translation of the reporter gene and inhibited eIF-2at phosphorylation. Analysis in vitro demonstrated that the E3L gene product inhibited PKR approximately 50to 100-fold more efficiently than the K3L gene product. However, further studies demonstrated that the mechanism of action of these two inhibitors was different. Whereas the E3L inhibitor interfered with the binding of the kinase to double-stranded RNA, the K3L inhibitor did not. We propose that the K3L inhibitor acts through its homology to eIF-2a to interfere with the interaction of eIF-2a with PKR. The two inhibitors did not display a synergistic effect on translation or eIF-2a phosphorylation. In addition, neither K3L nor E3L expression detectably altered cellular protein synthesis.

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Vaccinia virus with the E3L gene deleted was able to replicate in RK-13 but not HeLa cells. This host range phenotype could be complemented by an E3L gene expressed transiently from a plasmid. Analysis of mutants of E3L indicates that the ability to complement deletion of E3L correlates with the ability of mutated proteins to bind double-stranded RNA but not with their ability to migrate to the nucleus.

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