Characterization of RNA Determinants Recognized by the Arginine- and Proline-Rich Region of Us11, a Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation

Abstract

The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I ⅐ C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I ⅐ C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I ⅐ C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2 hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine-and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA.

Computational approaches toward the design of pools for the in vitro selection of complex aptamers

“…Analysis of existing aptamers has revealed that the majority of oligonucleotide sequences generated from random in vitro selection experiments have simple structures with low degrees of complexity and that highly complex structures are far less abundant (Khoo et al 2002;Zinnen et al 2002;Laserson et al 2004). This lack of structural diversity in random pools may explain why complex structure motifs such as high-order junctions are rare in known synthetic aptamers.…”

Herpes simplex virus type 1 virion-derived US11 inhibits type 1 interferon-induced protein kinase R phosphorylation

“…Recently, researchers have reported that degradation of nuclear IFI16 by ICP0 inhibits IRF3 signaling, and that ICP0 interacts with PML and induces its degradation (15)(16)(17). In addition, that US11 reportedly inhibits phosphorylation of PKR by binding to dsRNA, the protein activator of the interferoninduced protein kinase (PACT) and PKR (18)(19)(20)(21)(22)(23)(24). Recently, researchers have reported that US11 can also competitively inhibit activation of OAS by binding to dsRNA (25).…”

To replicate or not to replicate: achieving selective oncolytic virus replication in cancer cells through translational control

“…Significantly, the Us12 promoter is active at IE times and directs the synthesis of Us11 mRNA, which is normally produced very late in the viral life cycle, as an IE mRNA. Thus, by altering the temporal pattern of Us11 expression, the suppressor mutation enables g 1 34.5 mutants to replicate efficiently in what were previously nonpermissive cells (Mohr and Gluzman, 1996;Mulvey et al, 1999;Poppers et al, 2000;Khoo et al, 2002). The considerable surprise was that although the suppressor mutant was capable of restored growth in cells that failed to support the replication of the g 1 34.5 parent virus, it essentially remained as neuroattenuated as the parental g 1 34.5 mutant at the doses examined (up to 2 Â 10 7 PFU).…”

“…Ensuing genetic studies revealed that HSV-1 actually encodes multiple functions to control eIF2a phosphorylation, as the dsRNA binding protein specified by the g 2 or 'true late' Us11 gene prevents PKR activation (Mohr and Gluzman, 1996;Mulvey et al, 1999;Poppers et al, 2000;Khoo et al, 2002;Peters et al, 2002). Analysis of a panel of g 1 34.5 and Us11 mutants established that both Us11 and g 1 34.5 gene products act at different times in the productive growth cycle to regulate eIF2a phosphorylation in infected cells ( Figure 6; Mulvey et al, 2003).…”