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Journal of Virology, June 2005, p. 7311-7318, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7311-7318.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Double-Stranded RNA Binding by Human Cytomegalovirus pTRS1

Morgan Hakki and Adam P. Geballe*

Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, and Departments of Microbiology and Medicine, University of Washington, Seattle, Washington 98115

Received 19 November 2004/ Accepted 28 February 2005

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes rescue replication of vaccinia virus (VV) that has a deletion of the double-stranded RNA binding protein gene E3L (VV{Delta}E3L). Like E3L, these HCMV genes block the activation of key interferon-induced, double-stranded RNA (dsRNA)-activated antiviral pathways. We investigated the hypothesis that the products of these HCMV genes act by binding to dsRNA. pTRS1 expressed by cell-free translation or by infection of mammalian cells with HCMV or recombinant VV bound to dsRNA. Competition experiments revealed that pTRS1 preferentially bound to dsRNA compared to double-stranded DNA or single-stranded RNA. 5'- and 3'-end deletion analyses mapped the TRS1 dsRNA-binding domain to amino acids 74 through 248, a region of identity to pIRS1 that contains no homology to known dsRNA-binding proteins. Deletion of the majority of this region ({Delta}86-246) completely abrogated dsRNA binding. To determine the role of the dsRNA-binding domain in the rescue of VV{Delta}E3L replication, wild-type or deletion mutants of TRS1 were transfected into HeLa cells, which were then infected with VV{Delta}E3L. While full-length TRS1 rescued VV{Delta}E3L replication, deletion mutants affecting a carboxy-terminal region of TRS1 that is not required for dsRNA binding failed to rescue VV{Delta}E3L. Analyses of stable cell lines revealed that the carboxy-terminal domain is necessary to prevent the shutoff of protein synthesis and the phosphorylation of eIF2{alpha} after VV{Delta}E3L infection. Thus, pTRS1 contains an unconventional dsRNA-binding domain at its amino terminus, but a second function involving the carboxy terminus is also required for countering host cell antiviral responses.

* Corresponding author. Mailing address: Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, MS C2-023, Seattle, WA 98109-1024. Phone: (206) 667-5122. Fax: (206) 667-6523. E-mail: ageballe{at}fhcrc.org.

Journal of Virology, June 2005, p. 7311-7318, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7311-7318.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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