Brunella Taddeo

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) 32P-labeled riboprobe generated by in vitro transcription of the US8.5 ORF bound three proteins identified as the products of US11, UL47, and UL49 (VP22) genes. (ii) Viral RNA was bound to UL47 or US11 proteins immune precipitated from cells transduced with baculoviruses expressing UL47 or US11 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 US8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the US8–12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in ≈1% of infected cells. (iv) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent–US8.5 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 UL47 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.

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 (ΔUL41) 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 ≥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 ΔUL41 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 UL41 gene product early in infection and with degradation of cellular RNAs in the absence of replenishment by de novo transcription of cellular genes.

RNAs Extracted from Herpes Simplex Virus 1 Virions: Apparent Selectivity of Viral but Not Cellular RNAs Packaged in Virions

ABSTRACT Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, we investigated the presence and identity of RNAs from purified virions of herpes simple virus 1. To facilitate these studies, we designed primers for all known open reading frames (ORFs) and also constructed cDNA arrays containing probes designed to detect all known transcripts. In the first series of experiments, labeled DNA made by reverse transcription of poly(A)+ RNA extracted from infected HEp-2 or rabbit skin cells hybridized to all but two of the probes in the cDNA array. A similar analysis of the RNA extracted from purified extracellular virions derived from infected HEp-2 cells hybridized to probes representing 24 of the ORFs. In the second series of analyses, we reverse transcribed and amplified by PCR RNAs from purified intracellular or extracellular virions derived from infected HEp-2 or Vero cell lines. The positive RNAs were retested by PCR with and without prior reverse transcription to ensure that the samples tested were free of contaminating DNA. The results were as follows. (i) Only a fraction of viral ORF transcripts were represented in virion RNA, and only nine RNAs (UL10, UL34/UL35, UL36, UL42, UL48, UL51, US1/US1.5, US8.5, and US10/US11) were positive in all RT PCR assays. Of these, seven were positive by hybridization to cDNA arrays. (ii) RNA extracted from cells infected with a mutant virus lacking the US8 to US12 genes yielded results similar to those described above, indicating that US11, a known RNA binding protein, does not play a role in packaging RNA in virions. (iii) Cellular RNAs detected in virions were representative of the abundant cellular RNAs. Last, RNA extracted from virions was translated in vitro and the translation products were reacted with antibody to αTIF (VIP16). The immune precipitate contained a labeled protein with the apparent molcular weight of αTIF, indicating that at least one mRNA packaged in virions was intact and capable of being translated. The basis for the apparent selectivity in the packaging of the viral RNAs packaged in virions is unknown.

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

The UL41 protein of herpes simplex virus 1 has been reported to mediate the degradation of both viral and cellular mRNAs. Extensive studies on β-actin and some viral mRNAs were consonant with this conclusion. In earlier studies, we reported that the UL41-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 GADD45β, 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 ΔUL41 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 ΔUL41 mutant virus-infected cells. In this report, we show that in ΔUL41 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 UL41-dependent degradation of β-actin and other mRNAs. The UL41 protein thus mediates two functions, i.e., it mediates rapid degradation of some mRNAs exemplified by β-actin and stabilizes or delays the degradation of other mRNAs exemplified by GADD45β, tristetraprolin, etc. A model unifying both activities of the UL41 protein is presented.

The Virion Host Shutoff Protein (UL41) of Herpes Simplex Virus 1 Is an Endoribonuclease with a Substrate Specificity Similar to That of RNase A

ABSTRACT 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.

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.

ABSTRACT 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 UL41 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 UL41 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 UL41, 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.

Activation of NF-κB 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 κB (NF-κB)-regulated genes, including that of its inhibitor, IκBα, consistent with the reports that wild-type virus induces the activation of NF-κB. In this report we show that activation of NF-κB 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 γ134.5, which encodes a protein phosphatase 1α accessory factor that enables the dephosphorylation of the α subunit of eukaryotic translation initiation factor 2. NF-κB is activated in wild-type murine embryonic fibroblasts but not in related PKR-null cells. (ii) In cells infected with a replication-competent Δγ134.5 mutant (R5104), but carrying a US11 gene expressed early in infection, eukaryotic translation initiation factor 2α 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 IκBα, and does not cause the translocation of NF-κB in infected cells. (iii) One hypothesis advanced for the activation of NF-κB is that it blocks apoptosis induced by viral gene products. The replication-competent R5104 mutant does not induce the programmed cells death. We conclude that in herpes simplex virus 1-infected cells, activation of NF-κB depends on activation of PKR and that NF-κB is not required to block apoptosis in productively infected cells.

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 UL41 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 (γ) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the UL41 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.

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

ABSTRACT 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 UL41 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 α27 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.

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

Significance Herpes simplex virus encodes an RNase linked to the shutoff of host gene expression early in infection (virion host shutoff-RNase), and recent studies showed that its activity is highly selective: it targets stable host mRNAs and α (immediate early) mRNAs but spares other viral mRNAs. Here we report that RNase’s function early in infections is regulated by a viral protein designated UL47. UL47 is packed in virions; it binds the RNase and attenuates the degradation of all viral mRNAs but has no effect on the processing of the stress response mRNAs targeted by the viral RNase. 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 β 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 β 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 β and γ genes, suggesting that overexpression of α genes inhibits the downstream expression of early and late genes.