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Journal of Virology, December 2006, p. 11817-11826, Vol. 80, No. 23
0022-538X/06/$08.00+0     doi:10.1128/JVI.00957-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1{triangledown}

Morgan Hakki,2,4 Emily E. Marshall,3 Katherine L. De Niro,1 and Adam P. Geballe1,2,3,4*

Divisions of Human Biology,1 Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109,2 Department of Microbiology,3 Division of Allergy and Infectious Diseases, University of Washington, Seattle, Washington 981154

Received 10 May 2006/ Accepted 11 September 2006

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2{alpha}) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VV{Delta}E3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2{alpha} kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VV{Delta}E3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VV{Delta}E3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VV{Delta}E3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VV{Delta}E3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2{alpha}.

* Corresponding author. Mailing address: Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., MS C2-023, Seattle, WA 98109. Phone: (206) 667-5122. Fax: (206) 667-6523. E-mail: ageballe{at}fhcrc.org.

{triangledown} Published ahead of print on 20 September 2006.

Journal of Virology, December 2006, p. 11817-11826, Vol. 80, No. 23
0022-538X/06/$08.00+0     doi:10.1128/JVI.00957-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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