Tristetraprolin Recruits the Herpes Simplex Virion Host Shutoff RNase to AU-Rich Elements in Stress Response mRNAs To Enable Their Cleavage.

UNLABELLED: Herpes simplex viruses (HSV) package and bring into cells an RNase designated virion host shutoff (VHS) RNase. In infected cells, the VHS RNase targets primarily stress response mRNAs characterized by the presence of AU-rich elements in their 3' untranslated regions (UTRs). In uninfected cells, these RNAs are sequestered in exosomes or P bodies by host proteins that bind to the AU-rich elements. In infected cells, the AU-rich RNAs are deadenylated and cleaved close to the AU-rich elements, leading to long-term persistence of nontranslatable RNAs consisting of the 5' portions of the cleavage products. The host proteins that bind to the AU-rich elements are either resident in cells (e.g., TIA-1) or induced (e.g., tristetraprolin). Earlier, this laboratory reported that tristetraprolin binds VHS RNase. To test the hypothesis that tristetraprolin directs VHS RNase to the AU-rich elements, we mapped the domains of VHS and tristetraprolin required for their interactions. We report that VHS binds to the domain of tristetraprolin that enables its interaction with RNA. A single amino acid substitution in that domain abolished the interaction with RNA but did not block the binding to VHS RNase. In transfected cells, the mutant but not the wild-type tristetraprolin precluded the degradation of the AU-rich RNAs by VHS RNase. We conclude that TTP mediates the cleavage of the 3' UTRs of stress response mRNAs by recruiting the VHS RNase to the AU-rich elements. IMPORTANCE: The primary host response to HSV infection is the synthesis of stress response mRNAs characterized by the presence of AU-rich elements in their 3' UTRs. These mRNAs are the targets of the virion host shutoff (VHS) RNase. The VHS RNase binds both to mRNA cap structure and to tristetraprolin, an inducible host protein that sequesters AU-rich mRNAs in exosomes or P bodies. Here we show that tristetraprolin recruits VHS RNase to the AU-rich elements and enables the degradation of the stress response mRNAs.

Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the UL47 tegument protein.

Herpes simplex virus 1 (HSV-1) encodes an endoribonuclease that is responsible for the shutoff of host protein synthesis [virion host shutoff (VHS)-RNase]. The VHS-RNase released into cells during infection targets differentially four classes of mRNAs. Thus, (a) VHS-RNase degrades stable cellular mRNAs and α (immediate early) viral mRNAs; (b) it stabilizes host stress response mRNAs after deadenylation and subsequent cleavage near the adenylate-uridylate (AU)-rich elements; (c) it does not effectively degrade viral ß or γ mRNAs; and (d) it selectively spares from degradation a small number of cellular mRNAs. Current evidence suggests that several viral and at least one host protein (tristetraprolin) regulate its activity. Thus, virion protein (VP) 16 and VP22 neutralize the RNase activity at late times after infection. By binding to AU-rich elements via its interaction with tristetraprolin, the RNase deadenylates and cleaves the mRNAs in proximity to the AU-rich elements. In this report we show that another virion protein, UL47, brought into the cell during infection, attenuates the VHS-RNase activity with respect to stable host and viral α mRNAs and effectively blocks the degradation of ß and γ mRNAs, but it has no effect on the processing of AU-rich mRNAs. The properties of UL47 suggest that it, along with the α protein infected cell protein 27, attenuates degradation of mRNAs by the VHS-RNase through interaction with the enzyme in polyribosomes. Mutants lacking both VHS-RNase and UL47 overexpress α genes and delay the expression of ß and γ genes, suggesting that overexpression of α genes inhibits the downstream expression of early and late genes.

The herpes simplex virus host shutoff RNase degrades cellular and viral mRNAs made before infection but not viral mRNA made after infection.

A herpes simplex virus tegument protein brought into the cell during infection and designated the virion host shutoff protein (VHS) is an endoribonuclease that degrades mRNA. The prevailing view for many years has been that the VHS-RNase does not discriminate between cellular and viral RNAs and that the viruses prevail because the accumulation of viral transcripts outpaces their degradation. Here we report the following. (i) The degradation of viral mRNA made during infection of Vero or HEp-2 cells proceeds at a much-reduced rate compared to that of cellular mRNA. In effect, viral mRNAs are largely stable, whereas cellular mRNAs are rapidly degraded or, in the case of AU-rich mRNA, cleaved and rendered dysfunctional. (ii) In contrast to viral mRNAs made after infection, viral mRNAs expressed by plasmids transfected into cells prior to infection are degraded after infection at a rate comparable to that of cellular mRNAs. Moreover, the mRNA encoded by the transfected plasmid is hyperadenylated in the infected cell. Hyperadenylation but not degradation of mRNAs is blocked by actinomycin D. The results indicate that VHS-mRNA discriminates between viral and cellular mRNA but only in the context of infection and that discrimination is not based on the sequence of the mRNA but most likely on one or more viral factors expressed in the infected cell.

The nuclear-cytoplasmic shuttling of virion host shutoff RNase is enabled by pUL47 and an embedded nuclear export signal and defines the sites of degradation of AU-rich and stable cellular mRNAs.

The herpes simplex virus host shutoff RNase (VHS-RNase) is the major early block of host responses to infection. VHS-RNase is introduced into cells during infection and selectively degrades stable mRNAs made before infection and the normally short-lived AU-rich stress response mRNAs induced by sensors of innate immunity. Through its interactions with pUL47, another tegument protein, it spares from degradation viral mRNAs. Analyses of embedded motifs revealed that VHS-RNase contains a nuclear export signal (NES) but not a nuclear localization signal. To reconcile the potential nuclear localization with earlier studies showing that VHS-RNase degrades mRNAs in polyribosomes, we constructed a mutant in which NES was ablated. Comparison of the mutant and wild-type VHS-RNases revealed the following. (i) On infection, VHS-RNase is transported to the nucleus, but only the wild-type protein shuttles between the nucleus and cytoplasm. (ii) Both VHS-RNases localized in the cytoplasm following transfection. On cotransfection with pUL47, a fraction of VHS-RNase was translocated to the nucleus, suggesting that pUL47 may enable nuclear localization of VHS-RNase. (iii) In infected cells, VHS-RNase lacking NES degraded the short-lived AU-rich mRNAs but not the stable mRNAs. In transfected cells, both wild-type and NES mutant VHS-RNases effectively degraded cellular mRNAs. Our results suggest that the stable mRNAs are degraded in the cytoplasm, whereas the AU-rich mRNAs may be degraded in both cellular compartments. The selective sparing of viral mRNAs may take place during the nuclear phase in the course of interaction of pUL47, VHS-RNase, and nascent viral mRNAs.

The virion host shutoff RNase plays a key role in blocking the activation of protein kinase R in cells infected with herpes simplex virus 1.

Earlier studies have shown that active MEK blocks the activation of protein kinase R (PKR), a component of antiviral innate immune responses. In this report we show that the herpes simplex virus 1 virion host shutoff (VHS) RNase protein and MEK (mitogen-activated protein kinase kinase) act cooperatively in blocking the activation of PKR. This conclusion is based on the following. (i) In contrast to viral gene expression in the parental cell line or a cell line expressing a constitutively active MEK, the replication of a VHS mutant is particularly impaired in cells expressing dominant negative MEK. In this cell line PKR is activated by phosphorylation, and the accumulation of several viral proteins is delayed. (ii) In transfected cells, wild-type VHS blocked the activation of PKR, whereas PKR was activated in cells transfected with a mutant VHS or with plasmids encoding the VHS RNase and VP16 and VP22, the two viral proteins that neutralize the RNase activity of VHS. The results suggest that early in infection the VHS RNase degrades RNAs that activate PKR. Coupled with published data, the results suggest that inhibition of activation of PKR or its effect on viral replication is staged early in infection by VHS, postsynthesis of VP16 and VP22 by the γ(1)34.5 protein, and very late in infection by the U(S)11 protein.

The virion-packaged endoribonuclease of herpes simplex virus 1 cleaves mRNA in polyribosomes.

The virion host shutoff protein product of the U(L)41 gene of herpes simplex virus 1 is an endoribonuclease that selectively degrades mRNAs during the first hours after infection. Specifically, in contrast to the events in uninfected cells or cells infected with a mutant lacking the RNase, in wild-type virus-infected cells mRNA of housekeeping genes exemplified by GAPDH is degraded rapidly, whereas mRNAs containing AU elements are cleaved and the 5' cleavage product of these RNAs persists for many hours. We report that in wild-type virus-infected cells there was a rapid increase in the number and size of processing bodies (P-bodies). These P-bodies were also preset in cycloheximide (CHX)-treated cells but not in either treated or untreated uninfected cells or cells infected with the RNase minus mutant. Additional studies revealed that polyribosomes extracted from cytoplasm of wild-type virus-infected cells treated with CHX and displayed in sucrose gradients contained ribosome-loaded, truncated AU-rich mRNAs lacking the 3' UTR and poly(A) tails. The results suggest that the virion RNase is bound to polyribosomes by virtue of the reported association with translation machinery and cleaves the RNAs 5' to the AU elements. In contrast to the slow degradation of the of the residual 5' domain, the 3' UTR of the AU-rich mRNA and the GAPDH mRNA are rapidly degraded in wild-type virus-infected cells.

Role of herpes simplex virus ICP27 in the degradation of mRNA by virion host shutoff RNase.

The virion host shutoff (VHS) RNase tegument protein released into cells by infecting virus has two effects. Preexisting stable mRNAs (e.g., GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) are rapidly degraded. Stress response RNAs containing AU-rich elements (AREs) in the 3' untranslated region (3'UTR) are deadenylated and cleaved, but the cleavage products persist for hours, in contrast to the short half-lives of ARE-containing mRNAs in uninfected cells. At late times, the VHS RNase is neutralized by the viral structural proteins VP16 and VP22. A recent study (J. A. Corcoran, W. L. Hsu, and J. R. Smiley, J. Virol. 80:9720-9729, 2006) reported that, at relatively late times after infection, ARE RNAs are rapidly degraded in cells infected with DeltaICP27 mutant virus and concluded that ICP27 "stabilizes" ARE mRNAs. We report the following. (i) The rates of degradation of ARE mRNA at early times (3 h) after infection with the wild type or the DeltaICP27 mutant virus are virtually identical, and hence ICP27 plays no role in this process. (ii) In noncomplementing cells, VHS RNase or VP22 is not synthesized. Therefore, the only VHS that is active is brought into cells by the DeltaICP27 mutant. (ii) The VHS RNase brought into the cells by the DeltaICP27 virus is reduced in potency relative to that of wild-type virus. Hence the rapid degradation of ARE mRNAs noted in DeltaICP27 mutant-infected cells at late times is similar to that taking place in mock-infected or in DeltaVHS RNase mutant-virus-infected cells and does not by itself support the hypothesis that ICP27 stabilizes ARE mRNAs. (iii) Concurrently, we present the first evidence that VHS RNase interacts with ICP27 most likely when bound to cap- and poly(A)-binding proteins, respectively.

Fibroblast Growth Factor-2 and the HIV-1 Tat Protein Synergize in Promoting Bcl-2 Expression and Preventing Endothelial Cell Apoptosis: Implications for the Pathogenesis of AIDS-Associated Kaposi's Sarcoma.

Kaposi's sarcoma (KS) is a vascular tumor frequently occurring in Human Immunodeficiency Virus- (HIV-) 1-infected individuals. Our previous work indicated that the angiogenic fibroblast growth factor (FGF)-2 and the Tat protein of HIV-1, both expressed in KS lesions of HIV-infected patients, synergize at inducing angioproliferative, KS-like lesions in mice. Here we show that the development of angioproliferative lesions promoted in mice by combined Tat and FGF-2 associates with an increase in the levels of expression of the antiapoptotic Bcl-2 protein. Upregulation of Bcl-2 expression by combined FGF-2 and Tat occurs also in vitro, and this protects human primary endothelial cells from programmed cell death. As Bcl-2 is expressed in human KS lesions in a fashion paralleling the progression of the disease, these findings suggest a molecular mechanism by which Tat and FGF-2 cooperate in KS maintenance and progression in HIV-infected individuals.

Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for accumulation of mRNA.

The virion host shutoff (vhs) protein encoded by the U(L)41 gene of herpes simplex virus 1 is an endoribonuclease. The enzyme is introduced into the cell during unpackaging of the virion upon entry and selectively degrades mRNA for several hours. The RNase activity ceases after the onset of synthesis of late (gamma) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the U(L)41 gene. However, vhs does accumulate in cells cotransfected with plasmids expressing two other tegument proteins, VP16 and VP22. vhs does not directly interact with VP22 but, instead, binds VP22 only in the presence of VP16. In contrast to these findings, the amounts of vhs mRNA accumulating in the cells transfected solely with vhs are not significantly different from those detected in cells coexpressing vhs, VP16, and VP22. We conclude from these studies that the steady state of vhs mRNA, reflecting synthesis and turnover of mRNA, is not affected by the interaction of vhs protein with VP16 with VP22. A model is proposed in which the vhs protein may function to sequester mRNAs in compartments inaccessible to the cellular translational machinery and that VP16 and VP22 rescue the mRNAs by interacting with the vhs protein.

Replication-competent herpes simplex virus 1 isolates selected from cells transfected with a bacterial artificial chromosome DNA lacking only the UL49 gene vary with respect to the defect in the UL41 gene encoding host shutoff RNase.

To generate a null U(L)49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U(L)49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-DeltaU(L)49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the U(L)41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-U(L)49R DNA (R-U(L)49) accumulated a full-length vhs protein, indicating that in the parental BAC-DeltaU(L)49 DNA, the U(L)41 gene was intact. We conclude that expression of the vhs protein in the absence of U(L)49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of U(L)49, to be neutralized.

The virion host shutoff protein (UL41) of herpes simplex virus 1 is an endoribonuclease with a substrate specificity similar to that of RNase A.

Earlier, our laboratory reported that purified glutathione S-transferase-virion host shutoff (GST-vhs) protein exhibited endoribonucleolytic activity in in vitro assays using as substrates in vitro-transcribed regions of IEX-1 mRNA. Here, we report that studies of the cleavage patterns of synthetic RNA oligonucleotides defined the activity of GST-vhs as being similar to that of RNase A. Thus, GST-vhs cleaved the RNA at the 3' end of single-stranded cytidine and uridine residues. Since the GST-mvhs nuclease-defective mutant protein failed to cleave the synthetic RNAs, the results unambiguously attribute the activity to vhs.

The U(L)41 protein of herpes simplex virus 1 degrades RNA by endonucleolytic cleavage in absence of other cellular or viral proteins.

The herpes simplex virus 1 ORF U(L)41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or beta-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3' domain disappears rapidly, a large 5' domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: (i) a GST-vhs protein produced in Escherichia coli, solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST-vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. (ii) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3' untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. (iii) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.

The UL41 protein of herpes simplex virus mediates selective stabilization or degradation of cellular mRNAs.

The U(L)41 protein of herpes simplex virus 1 has been reported to mediate the degradation of both viral and cellular mRNAs. Extensive studies on beta-actin and some viral mRNAs were consonant with this conclusion. In earlier studies, we reported that the U(L)41-dependent degradation of cellular mRNAs up-regulated after infection was selective. One class of the up-regulated mRNAs, exemplified by the stress-inducible immediate-early 1 mRNA, is deadenylated, 3' to 5' degraded and is not translated. Another class of up-regulated mRNAs, exemplified by GADD45beta, does not undergo this pattern of degradation and is translated. A puzzling feature of the earlier results is that the amounts of up-regulated mRNAs accumulating in the cytoplasm of DeltaU(L)41 mutant virus-infected cells was lower than in WT virus-infected cells, a contradiction, inasmuch as if the rates of accumulation were identical and degradation of the mRNAs were higher in WT virus-infected cells, the steady-state levels should have been higher in DeltaU(L)41 mutant virus-infected cells. In this report, we show that in DeltaU(L)41 mutant virus-infected cells, the rates of degradation of the stress-inducible immediate-early response gene 1 and other up-regulated mRNAs are approximately the same as those observed in mock-infected cells and are faster than in WT virus-infected cells. This is contrary to the observed U(L)41-dependent degradation of beta-actin and other mRNAs. The U(L)41 protein thus mediates two functions, i.e., it mediates rapid degradation of some mRNAs exemplified by beta-actin and stabilizes or delays the degradation of other mRNAs exemplified by GADD45beta, tristetraprolin, etc. A model unifying both activities of the U(L)41 protein is presented.

Herpes simplex virus 1 induces cytoplasmic accumulation of TIA-1/TIAR and both synthesis and cytoplasmic accumulation of tristetraprolin, two cellular proteins that bind and destabilize AU-rich RNAs.

Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the U(L)41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3'-to-5' degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking U(L)41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking U(L)41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

The patterns of accumulation of cellular RNAs in cells infected with a wild-type and a mutant herpes simplex virus 1 lacking the virion host shutoff gene.

Cellular RNA extracted from quiescent human foreskin fibroblasts harvested at 1, 3, 7, or 12 h after infection was profiled on Affymetrix HG-U95Av2 arrays designed to detect 12,626 unique human transcripts. We also profiled RNA extracted from cells harvested at 1 and 7 h after infection with a mutant lacking the gene (DeltaU(L)41) encoding a protein (vhs) brought into cells by the virus and responsible for nonselective degradation of RNA early in infection. We report the following: (i) of the 12 tested genes, up-regulated at least 3-fold relative to the values of mock infected cells, 9 were confirmed by real-time PCR. The microchip assays analyses indicate that there were 475 genes up-regulated > or =3-fold. The up-regulated genes were clustered into 15 groups with respect to temporal pattern of transcript accumulation, and classified into 20 groups on the basis of their function. The preponderance of cellular genes up-regulated early in infection play a predominant role in transcription, whereas those up-regulated at later times respond to intracellular stress or concern themselves with the cell cycle and apoptosis. (ii) The number of genes up-regulated early in infection was higher in cells infected with the DeltaU(L)41 mutant. Conversely, more genes were down-regulated late in infection with wild-type virus than with mutant viruses. Both observations are compatible with the known function of the U(L)41 gene product early in infection and with degradation of cellular RNAs in the absence of replenishment by de novo transcription of cellular genes.

Activation of NF-kappaB in cells productively infected with HSV-1 depends on activated protein kinase R and plays no apparent role in blocking apoptosis.

Microarray data reported elsewhere indicated that herpes simplex virus 1 induces the up-regulation of nuclear factor kappaB (NF-kappaB)-regulated genes, including that of its inhibitor, IkappaBalpha, consistent with the reports that wild-type virus induces the activation of NF-kappaB. In this report we show that activation of NF-kappaB in infected cells is linked to the activation of protein kinase R (PKR). Specifically: (i) PKR is activated in infected cells although the effects of the activated enzyme on protein synthesis are negated by the viral gene gamma134.5, which encodes a protein phosphatase 1alpha accessory factor that enables the dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. NF-kappaB is activated in wild-type murine embryonic fibroblasts but not in related PKR-null cells. (ii) In cells infected with a replication-competent Deltagamma134.5 mutant (R5104), but carrying a US11 gene expressed early in infection, eukaryotic translation initiation factor 2alpha is not phosphorylated, and in in vitro assays, PKR bound to the US11 protein is not phosphorylated on subsequent addition of double-stranded RNA. Here we report that this mutant does not activate PKR, has no effect on the accumulation of IkappaBalpha, and does not cause the translocation of NF-kappaB in infected cells. (iii) One hypothesis advanced for the activation of NF-kappaB is that it blocks apoptosis induced by viral gene products. The replication-competent R5104 mutant does not induce the programmed cell's death. We conclude that in herpes simplex virus 1-infected cells, activation of NF-kappaB depends on activation of PKR and that NF-kappaB is not required to block apoptosis in productively infected cells.

The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific.

In cells infected with herpes simplex virus 1, the RNA encoded by the stress-inducible immediate early response gene IEX-1 was up-regulated immediately after infection. However, the accumulated RNA was degraded 3'-5', and the protein was detectable only at very early times after infection. The degradation was dependent on the U(L)41 gene encoding the virion host shutoff (vhs) protein and resulted in the accumulation of truncated RNA containing the 5'-end portion of the transcript. IEX-1 contains an AU-rich element (ARE) in its 3'-untranslated domains known to regulate negatively the RNA lifespan. To examine the role of ARE in signaling the degradation, we compared the stability of several RNAs up-regulated during infection to WT virus. These were ARE-containing RNAs encoding IEX-1, c-fos, and IkappaBalpha and the non-ARE-containing RNAs GADD45beta and tristetraprolin. We report that the ARE-containing RNAs exemplified by IEX-1 RNA are deadenylated and cleaved in the ARE within the 3' UTR in a U(L)41-dependent manner. In contrast, Northern blot hybridizations and analyses of poly(A) tails revealed no evidence of degradation of GADD45beta RNA. GADD45beta protein was detected in WT virus-infected cells. These results indicate that the degradation of RNAs and the mechanism by which cellular RNAs are degraded are selective and may be sequence specific. The persistence of partially degraded ARE-containing RNAs may reflect specific targeting of the vhs proteins to the ARE and the modification of the RNA degradation machinery of the cell induced by the presence of the vhs protein.

Cells lacking NF-kappaB or in which NF-kappaB is not activated vary with respect to ability to sustain herpes simplex virus 1 replication and are not susceptible to apoptosis induced by a replication-incompetent mutant virus.

Earlier we reported that NF-kappaB is activated by protein kinase R (PKR) in herpes simplex virus 1-infected cells. Here we report that in PKR(-/-) cells the yields of wild-type virus are 10-fold higher than in PKR(+/+) cells. In cells lacking NF-kappaB p50 (nfkb1), p65 (relA), or both p50 and p65, the yields of virus were reduced 10-fold. Neither wild-type nor mutant cells undergo apoptosis following infection with wild-type virus. Whereas PKR(+/+) and NF-kappaB(+/+) control cell lines undergo apoptosis induced by the d120 (Deltaalpha4) mutant of HSV-1, the mutant PKR(-/-) and NF-kappaB(-/-) cell lines were resistant. The evidence suggests that the stress-induced apoptosis resulting from d120 infection requires activation of NF-kappaB and that this proapoptotic pathway is blocked in cells in which NF-kappaB is not activated or absent. Activation of NF-kappaB in the course of viral infection may have dual roles of attempting to curtain viral replication by rendering the cell susceptible to apoptosis induced by the virus and by inducing the synthesis of proteins that enhance viral replication.

The stress-inducible immediate-early responsive gene IEX-1 is activated in cells infected with herpes simplex virus 1, but several viral mechanisms, including 3' degradation of its RNA, preclude expression of the gene.

The accumulation of cellular transcripts from cells infected with herpes simplex virus 1 (HSV-1) as measured with the aid of Affymetrix microchips has been reported elsewhere. Among these transcripts were genes that respond to stress and that could have a noxious effect on viral replication. We have selected the stress-inducible cellular gene encoding the immediate-early response protein IEX-1 to verify and determine the significance of the accumulation of these transcripts in infected cells. We report that we verified the increase in accumulation of IEX-1 transcripts after infection by Northern analyses and real-time PCR. These transcripts reach peak levels between 3 and 7 h after infection and decrease thereafter. However, IEX-1 protein was detected in cells 1 h after infection but not at later intervals. Studies designed to elucidate the failure of IEX-1 protein to be synthesized revealed the following points. (i) IEX-1 RNA transported to the cytoplasm after 1 h of infection consisted of at least two populations, a partially degraded population and a population consisting of unspliced IEX-1 RNA. Neither of these RNAs could translate the authentic IEX-1 protein. (ii) The partially degraded IEX-1 RNA was not detected in the cytoplasm of cells infected with a mutant virus lacking the U(L)41 gene encoding the virion host shutoff protein (vhs). Although degradation of RNA mediated by vhs was reported to be 5' to 3', the partially degraded IEX-1 RNA lacked the 3' sequences rather than the 5' sequences. (iii) The unspliced pre-RNA form containing the IEX-1 intron sequences was detected in the cytoplasm of cell infected with wild-type virus but not in those infected with a mutant lacking the alpha27 gene encoding the infected cell protein No. 27. (iv) Overexpression of IEX-1 protein by transduction of the gene prior to infection with 1 PFU of HSV-1 per cell had no effect on the accumulation of late genes and virus yield. We conclude that the failure of IEX-1 to express its protein reflects the numerous mechanisms by which the virus thwarts the cells from expressing its genes after infection.

Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection.

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (i) (32)P-labeled riboprobe generated by in vitro transcription of the U(S)8.5 ORF bound three proteins identified as the products of U(S)11, U(L)47, and U(L)49 (VP22) genes. (ii) Viral RNA was bound to U(L)47 or U(S)11 proteins immune precipitated from cells transduced with baculoviruses expressing U(L)47 or U(S)11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (iii) Virions were purified from cells transduced with a baculovirus encoding a U(S)8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the U(S)8-12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in approximately 1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent-U(S)8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (i) three virion proteins are capable of binding RNA; (ii) the packaged RNA can be expressed in newly infected cells; and (iii) the U(L)47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.