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Journal of Virology, January 2008, p. 840-848, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.01891-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Loss of Protein Kinase PKR Expression in Human HeLa Cells Complements the Vaccinia Virus E3L Deletion Mutant Phenotype by Restoration of Viral Protein Synthesis

Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California 93106,¹ School of Life Sciences, Center for Infectious Disease and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287,² Biomolecular Sciences and Engineering Program, University of California, Santa Barbara, California 93106³

Received 29 August 2007/ Accepted 18 October 2007

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (E3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of E3L virus was increased by nearly 2 log₁₀ in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the E3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in E3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the subunit of protein synthesis initiation factor 2 (eIF-2) was elevated severalfold in E3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2 phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with E3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.

Published ahead of print on 24 October 2007.