Double-stranded RNA binding by human cytomegalovirus pTRS1.
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 (VVDeltaE3L). 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 (Delta86-246) completely abrogated dsRNA binding. To determine the role of the dsRNA-binding domain in the rescue of VVDeltaE3L replication, wild-type or deletion mutants of TRS1 were transfected into HeLa cells, which were then infected with VVDeltaE3L. While full-length TRS1 rescued VVDeltaE3L replication, deletion mutants affecting a carboxy-terminal region of TRS1 that is not required for dsRNA binding failed to rescue VVDeltaE3L. Analyses of stable cell lines revealed that the carboxy-terminal domain is necessary to prevent the shutoff of protein synthesis and the phosphorylation of eIF2alpha after VVDeltaE3L 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.
Hakki
Morgan
M
Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, MS C2-023, Seattle, WA 98109-1024, USA.
Geballe
Adam P
AP
D000818:Animals; D003587:Cytomegalovirus / Q000378:metabolism / Q000472:pathogenicity*; D006367:HeLa Cells; D006801:Humans; D012330:RNA, Double-Stranded / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
C081374:E3L protein, Vaccinia virus; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; C072216:TRS1 protein, Human herpesvirus 5; D014764:Viral Proteins