The Splicing History of an mRNA Affects Its Level of Translation and Sensitivity to Cleavage by the Virion Host Shutoff Endonuclease during Herpes Simplex Virus Infections.

During lytic herpes simplex virus (HSV) infections, the virion host shutoff (Vhs) (UL41) endoribonuclease degrades many cellular and viral mRNAs. In uninfected cells, spliced mRNAs emerge into the cytoplasm bound by exon junction complexes (EJCs) and are translated several times more efficiently than unspliced mRNAs that have the same sequence but lack EJCs. Notably, most cellular mRNAs are spliced, whereas most HSV mRNAs are not. To examine the effect of splicing on gene expression during HSV infection, cells were transfected with plasmids harboring an unspliced renilla luciferase (RLuc) reporter mRNA or RLuc constructs with introns near the 5' or 3' end of the gene. After splicing of intron-containing transcripts, all three RLuc mRNAs had the same primary sequence. Upon infection in the presence of actinomycin D, spliced mRNAs were much less sensitive to degradation by copies of Vhs from infecting virions than were unspliced mRNAs. During productive infections (in the absence of drugs), RLuc was expressed at substantially higher levels from spliced than from unspliced mRNAs. Interestingly, the stimulatory effect of splicing on RLuc expression was significantly greater in infected than in uninfected cells. The translational stimulatory effect of an intron during HSV-1 infections could be replicated by artificially tethering various EJC components to an unspliced RLuc transcript. Thus, the splicing history of an mRNA, and the consequent presence or absence of EJCs, affects its level of translation and sensitivity to Vhs cleavage during lytic HSV infections. IMPORTANCE: Most mammalian mRNAs are spliced. In contrast, of the more than 80 mRNAs harbored by herpes simplex virus 1 (HSV-1), only 5 are spliced. In addition, synthesis of the immediate early protein ICP27 causes partial inhibition of pre-mRNA splicing, with the resultant accumulation of both spliced and unspliced versions of some mRNAs in the cytoplasm. A common perception is that HSV-1 infection necessarily inhibits the expression of spliced mRNAs. In contrast, this study demonstrates two instances in which pre-mRNA splicing actually enhances the synthesis of proteins from mRNAs during HSV-1 infections. Specifically, splicing stabilized an mRNA against degradation by copies of the Vhs endoribonuclease from infecting virions and greatly enhanced the amount of protein synthesized from spliced mRNAs at late times after infection. The data suggest that splicing, and the resultant presence of exon junction complexes on an mRNA, may play an important role in gene expression during HSV-1 infections.

mRNA decay during herpes simplex virus (HSV) infections: mutations that affect translation of an mRNA influence the sites at which it is cleaved by the HSV virion host shutoff (Vhs) protein.

During lytic infections, the herpes simplex virus (HSV) virion host shutoff (Vhs) endoribonuclease degrades many host and viral mRNAs. Within infected cells it cuts mRNAs at preferred sites, including some in regions of translation initiation. Vhs binds the translation initiation factors eIF4H, eIF4AI, and eIF4AII, suggesting that its mRNA degradative function is somehow linked to translation. To explore how Vhs is targeted to preferred sites, we examined the in vitro degradation of a target mRNA in rabbit reticulocyte lysates containing in vitro-translated Vhs. Vhs caused rapid degradation of mRNAs beginning with cleavages at sites in the first 250 nucleotides, including a number near the start codon and in the 5' untranslated region. Ligation of the ends to form a circular mRNA inhibited Vhs cleavage at the same sites at which it cuts capped linear molecules. This was not due to an inability to cut any circular RNA, since Vhs cuts circular mRNAs containing an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) at the same sites as linear molecules with the IRES. Cutting linear mRNAs at preferred sites was augmented by the presence of a 5' cap. Moreover, mutations that altered the 5' proximal AUG abolished Vhs cleavage at nearby sites, while mutations that changed sequences surrounding the AUG to improve their match to the Kozak consensus sequence enhanced Vhs cutting near the start codon. The results indicate that mutations in an mRNA that affect its translation affect the sites at which it is cut by Vhs and suggest that Vhs is directed to its preferred cut sites during translation initiation.

The virion host shutoff endonuclease (UL41) of herpes simplex virus interacts with the cellular cap-binding complex eIF4F.

The herpes simplex virus Vhs endonuclease degrades host and viral mRNAs. Isolated Vhs cuts any RNA at many sites. Yet, within cells, it targets mRNAs and cuts at preferred sites, including regions of translation initiation. Previous studies have shown that Vhs binds the translation factors eIF4A and eIF4H. Here, we show that Vhs binds the cap-binding complex eIF4F. Association with eIF4F correlated with the ability of Vhs to bind eIF4A but not eIF4H. All Vhs proteins that degrade mRNAs associated with eIF4F. However, simply tethering an active endonuclease to eIF4F is not sufficient to degrade mRNAs. Binding to eIF4H may also be required.

Evidence for translational regulation by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff protein (vhs) encoded by gene UL41 is an mRNA-specific RNase that triggers accelerated degradation of host and viral mRNAs in infected cells. We report here that vhs is also able to modulate reporter gene expression without greatly altering the levels of the target mRNA in transient-transfection assays conducted in HeLa cells. We monitored the effects of vhs on a panel of bicistronic reporter constructs bearing a variety of internal ribosome entry sites (IRESs) located between two test cistrons. As expected, vhs inhibited the expression of the 5' cistrons of all of these constructs; however, the response of the 3' cistron varied with the IRES: expression driven from the wild-type EMCV IRES was strongly suppressed, while expression controlled by a mutant EMCV IRES and the cellular ApaF1, BiP, and DAP5 IRES elements was strongly activated. In addition, several HSV type 1 (HSV-1) 5' untranslated region (5' UTR) sequences also served as positive vhs response elements in this assay. IRES activation was also observed in 293 and HepG2 cells, but no such response was observed in Vero cells. Mutational analysis has yet to uncouple the ability of vhs to activate 3' cistron expression from its shutoff activity. Remarkably, repression of 5' cistron expression could be observed under conditions where the levels of the reporter RNA were not correspondingly reduced. These data provide strong evidence that vhs can modulate gene expression at the level of translation and that it is able to activate cap-independent translation through specific cis-acting elements.

Small interfering RNAs that deplete the cellular translation factor eIF4H impede mRNA degradation by the virion host shutoff protein of herpes simplex virus.

The herpes simplex virus (HSV) virion host shutoff (Vhs) protein is an endoribonuclease that accelerates decay of many host and viral mRNAs. Purified Vhs does not distinguish mRNAs from nonmessenger RNAs and cuts target RNAs at many sites, yet within infected cells it is targeted to mRNAs and cleaves those mRNAs at preferred sites including, for some, regions of translation initiation. This targeting may result in part from Vhs binding to the translation initiation factor eIF4H; in particular, several mutations in Vhs that abrogate its binding to eIF4H also abolish its mRNA-degradative activity, even though the mutant proteins retain endonuclease activity. To further investigate the role of eIF4H in Vhs activity, HeLa cells were depleted of eIF4H or other proteins by transfection with small interfering RNAs (siRNAs) 48 h prior to infection or mock infection in the presence of actinomycin D. Cellular mRNA levels were then assayed 5 h after infection. In cells transfected with an siRNA for the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase, wild-type HSV infection reduced beta-actin mRNA levels to between 20 and 30% of those in mock-infected cells, indicative of a normal Vhs activity. In contrast, in cells transfected with any of three eIF4H siRNAs, beta-actin mRNA levels were indistinguishable in infected and mock-infected cells, suggesting that eIF4H depletion impeded Vhs-mediated degradation. Depletion of the related factor eIF4B did not affect Vhs activity. The data suggest that eIF4H binding is required for Vhs-induced degradation of many mRNAs, perhaps by targeting Vhs to mRNAs and to preferred sites within mRNAs.

Virus-encoded endonucleases: expected and novel functions.

Endonucleases catalyze critical steps in the processing, function, and turnover of many cellular RNAs. It is, therefore, not surprising that a number of viruses encode endonucleases that play important roles in viral gene expression. The virion host shutoff (Vhs) endonuclease of herpes simplex virus, the SOX protein of Kaposi Sarcoma Herpesvirus (KSHV), and the influenza virus PB1 endonuclease have well-characterized functions that stem from their abilities to cleave RNA. Vhs accelerates turnover of many cellular and viral mRNAs, redirecting the cell from host to viral gene expression, counteracting key elements of the innate immune response, and facilitating sequential expression of different classes of viral genes. SOX reduces synthesis of many host proteins during the lytic phase of KSHV infections. PB1 is a component of the influenza RNA polymerase that snatches capped oligonucleotides from cellular pre-mRNAs to serve as primers during viral mRNA synthesis. However, all three proteins have important second functions. Vhs stimulates translation of the 3' cistron of bicistronic mRNAs that have selected cellular internal ribosome entry sites, and stimulates polysome loading and translation of selected viral mRNAs at late times during productive infections. SOX has an alkaline exonuclease activity that is important for processing and maturation of newly synthesized copies of the KSHV genome. The influenza RNA polymerase binds the cap and 5' region of viral mRNAs and recruits eIF4G and other factors to viral mRNAs, allowing them to be translated under conditions of reduced eIF4E functionality. This review will discuss the novel and expected functions of these viral endonucleases.

Packaging of the virion host shutoff (Vhs) protein of herpes simplex virus: two forms of the Vhs polypeptide are associated with intranuclear B and C capsids, but only one is associated with enveloped virions.

The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.

mRNA decay during herpes simplex virus (HSV) infections: protein-protein interactions involving the HSV virion host shutoff protein and translation factors eIF4H and eIF4A.

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.

mRNA degradation by the virion host shutoff (Vhs) protein of herpes simplex virus: genetic and biochemical evidence that Vhs is a nuclease.

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.