During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating the decay of all mRNAs, it helps redirect the cell from host to viral gene expression and facilitates the sequential expression of different classes of viral genes. While it is clear that Vhs induces mRNA degradation, it is uncertain whether it is itself an RNase or somehow activates a cellular enzyme. This question was addressed by using a combination of genetic and biochemical approaches. The Vhs homologues of alphaherpesviruses share sequence similarities with a family of mammalian, yeast, bacterial, and phage nucleases. To test the functional significance of these similarities, Vhs was mutated to alter residues corresponding to amino acids known to be critical to the nuclease activity of cellular homologues. In every instance, mutations that inactivated the nuclease activity of cellular homologues also abolished Vhs activity. Recent experiments showed that Vhs interacts with the cellular translation initiation factor eIF4H. In this study, the coexpression of Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein in bacteria resulted in the formation of a complex of the proteins. The wild-type Vhs/GST-eIF4H complex was isolated and shown to have RNase activity. In contrast, Vhs mutations that altered key residues in the nuclease motif abolished the nuclease activity of the recombinant Vhs/GST-eIF4H complex. The results provide genetic and biochemical evidence that Vhs is an RNase, either alone or as a complex with eIF4H.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with a variety of malignancies including nasopharyngeal carcinoma. The EBV-encoded latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced B-lymphocyte transformation and has been shown to transform rodent fibroblasts. LMP1 contains two signaling domains, the carboxy-terminal activating region 1 and 2 (CTAR1 and CTAR2), by which NF-jB, phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase, and c-Jun N-terminal kinase are activated. In this study, the role of CTAR1 and CTAR2 in LMP1-mediated transformation of rodent fibroblasts was analysed. CTAR1 was found to be necessary for rodent fibroblast transformation, whereas CTAR2 was dispensable. The activation of the PI3K pathway in Rat-1 cells by LMP1 and LMP1-CTAR1 in transformed cells resulted in phosphorylated Akt and phosphorylated glycogen synthase kinase 3b. The role of PI3K and NF-jB activation in LMP1-mediated transformation was further analysed using the chemical inhibitors LY294002 and BAY 11-7085. LY294002 inhibited CTAR1-induced focus formation and anchorage-independent growth, whereas BAY 11-7085 did not inhibit focus formation or anchorage-independent growth. Similar studies in human fibroblasts confirmed that LMP1-CTAR1 also mediates aberrant growth, phosphorylation of Akt, and decreased levels of p27. These findings indicate that LMP1-mediated rodent fibroblast transformation is dependent upon activation of PI3K and Akt and is independent of activation of NF-jB.

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5 end of mRNAs prior to the 3 end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using the Saccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathione S-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.

During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.During lytic infections with herpes simplex virus (HSV), viral and cellular gene expression is regulated through a complex interplay of transcriptional and posttranscriptional controls (68). Of the posttranscriptional mechanisms, one of the best characterized is the degradation of host and viral mRNAs by the HSV virion host shutoff (Vhs) protein (UL41) (15,59). Vhs is an endoribonuclease that is a minor structural component of HSV virions (12,15,61,72,92). Following uncoating, copies of Vhs from the infecting virions destabilize host mRNAs in the cytoplasm (19,70,81). This, together with the inhibition of pre-mRNA splicing by the HSV immediate-early protein ICP27 (24, 25), contributes to an overall decrease in host protein synthesis. Following the onset of viral transcription, Vhs accelerates the turnover of viral mRNAs from all kinetic classes (38,53,54,81). In this role, it helps determine viral mRNA levels and facilitates sequential expression of different classes of viral genes (38,53,54,59).While mutations that inactivate Vhs have a modest effect upon virus growth in cell culture (39, 60, 61), they have a striking impact upon HSV virulence in animals (3,22,31,32,40,52,74,75,(78)(79)(80). The mechanisms by which Vhs affects pathogenesis are unclear, but they appear to involve effects upon both the adaptive and innate immune responses to HSV infection (73). Vhs impedes antigen presentation by major histocompatibility complex cl...

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