scholarly journals Role of Herpes Simplex Virus ICP27 in the Degradation of mRNA by Virion Host Shutoff RNase

2010 ◽  
Vol 84 (19) ◽  
pp. 10182-10190 ◽  
Author(s):  
Brunella Taddeo ◽  
Weiran Zhang ◽  
Bernard Roizman
Keyword(s):  
Mutant Virus ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Tegument Protein ◽  
Wild Type ◽  
Virion Host Shutoff ◽  
Infected Cells ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT 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 ΔICP27 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 ΔICP27 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 ΔICP27 mutant. (ii) The VHS RNase brought into the cells by the ΔICP27 virus is reduced in potency relative to that of wild-type virus. Hence the rapid degradation of ARE mRNAs noted in ΔICP27 mutant-infected cells at late times is similar to that taking place in mock-infected or in ΔVHS 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.

scholarly journals 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

2004 ◽  
Vol 78 (16) ◽  
pp. 8582-8592 ◽  
Author(s):  
Audrey Esclatine ◽  
Brunella Taddeo ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
Simplex Virus

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.

scholarly journals Role of the VP16-Binding Domain of vhs in Viral Growth, Host Shutoff Activity, and Pathogenesis

2004 ◽  
Vol 78 (24) ◽  
pp. 13562-13572 ◽  
Author(s):  
Stephanie S. Strand ◽  
David A. Leib
Keyword(s):  
Actinomycin D ◽  
Rna Degradation ◽  
Binding Domain ◽  
Wild Type Virus ◽  
Wild Type ◽  
Virion Host Shutoff ◽  
Significant Difference ◽  
Host Shutoff ◽  
Amino Acid Binding ◽  
Simplex Virus

ABSTRACT The virion host shutoff (vhs) protein of herpes simplex virus type 1 causes the degradation of host and viral mRNA immediately upon infection of permissive cells. vhs can interact with VP16 through a 20-amino-acid binding domain, and viruses containing a deletion of this VP16-binding domain of vhs (Δ20) and a corresponding marker rescue (Δ20R) were constructed and characterized. Transient-transfection assays showed that this domain was dispensable for vhs activity. The Δ20 recombinant virus, however, was unable to induce mRNA degradation in the presence of actinomycin D, while degradation induced by Δ20R was equivalent to that for wild-type virus. Δ20, Δ20R, and KOS caused comparable RNA degradation in the absence of actinomycin D. Western blot analysis of infected cells indicated that comparable levels of vhs were expressed by Δ20, Δ20R, and KOS, and there was only a modest reduction of vhs packaging in Δ20. Immunoprecipitation of protein from cells infected with Δ20 and Δ20R showed equivalent coprecipitation of vhs and VP16. Pathogenesis studies with Δ20 showed a significant decrease in replication in the corneas, trigeminal ganglia, and brains, as well as a significant reduction in clinical disease and lethality, but no significant difference in the establishment of, or reactivation from, latency compared to results with KOS and Δ20R. These results suggest that the previously described VP16-binding domain is not required for vhs packaging or for binding to VP16. It is required, however, for RNA degradation activity of tegument-derived vhs and wild-type replication and virulence in mice.

scholarly journals Role of Cellular Phosphatase cdc25C in Herpes Simplex Virus 1 Replication

2008 ◽  
Vol 82 (9) ◽  
pp. 4527-4532 ◽  
Author(s):  
Benjamin A. Smith-Donald ◽  
Lizette O. Durand ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cyclin B1 ◽  
Wild Type Virus ◽  
Physical Interaction ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT Earlier studies have shown that in herpes simplex virus 1-infected cells, ICP22 upregulates the accumulation of a subset of γ2 proteins exemplified by the products of the UL38, UL41, and US11 genes. The ICP22-dependent process involves degradation of cyclins A and B1, the stabilization and activation of cdc2, physical interaction of activated cdc2 with the UL42 DNA synthesis processivity factor, and recruitment and phosphorylation of topoisomerase IIα by the cdc2/UL42 complex. Activation of cdc2, the first step in the process, is a key function of the mitotic phosphatase cdc25C. To define the role of cdc25C, we probed some features of the ICP22-dependent pathway of upregulation of γ2 genes in cdc25C−/− cells and in cdc25C+/+ cells derived from sibling mice. We report that cyclin B1 turned over in cdc25C+/+ or cdc25C−/− cells at the same rate, that cdc2 increased in amount, and that US11 and UL38 proteins and infectious virus accumulated in smaller amounts than in wild-type infected cells. The reduction in UL38 protein accumulation and virus was greater in cdc25C−/− cells infected with virus lacking ICP22 than in cells infected with wild-type virus. We conclude that cdc25C phosphatase plays a role in viral replication and that this role extends beyond its function of activating cdc2 for initiation of the ICP22-dependent cascade for upregulation of γ2 gene expression.

scholarly journals Herpes Simplex Virus 1 Precludes Replenishment of the Short-Lived Receptor of Tumor Necrosis Factor Alpha by Virion Host Shutoff-Dependent Degradation of Its mRNA

2006 ◽  
Vol 80 (15) ◽  
pp. 7756-7759 ◽  
Author(s):  
Li Liang ◽  
Bernard Roizman
Keyword(s):  
Tumor Necrosis ◽  
Wild Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Necrosis Factor Alpha ◽  
Virion Host Shutoff ◽  
Infected Cells ◽  
Factor Alpha ◽  
Simplex Virus ◽  
Necrosis Factor

ABSTRACT Cell function is tightly regulated by surface receptors. Earlier reports showed that herpes simplex virus 1 regulates by diverse mechanisms the presentation of antigenic peptides, downregulates the signaling pathways associated with receptor tyrosine kinases, and posttranslationally modifies members of the Src family of protein kinases. Here we report that the receptor for tumor necrosis factor alpha (TNF-R1) rapidly disappears from both the cell surface and total cell lysates in cells infected with wild-type virus or a variety of mutants but not in cells infected with the mutant ΔUL41, which lacks the UL41 gene, the virion host shutoff gene. The half-life of TNF-R1 appears to be less than 30 min in both mock-infected and infected cells. The disappearance of TNF-R1 correlates with the disappearance of cytoplasmic TNF-R1 mRNA in wild-type-virus-infected cells. The results suggest that by degrading the TNFR1 mRNA, the virus precludes the replenishment of naturally decaying TNF-R1.

scholarly journals Expression of Gamma Interferon-Dependent Genes Is Blocked Independently by Virion Host Shutoff RNase and by US3 Protein Kinase

2008 ◽  
Vol 82 (10) ◽  
pp. 4688-4696 ◽  
Author(s):  
Li Liang ◽  
Bernard Roizman
Keyword(s):  
Protein Kinase ◽  
Gamma Interferon ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Virion Host Shutoff ◽  
Gene Transcripts ◽  
Infected Cells ◽  
Ifn Γ ◽  
Host Shutoff ◽  
In Cells

ABSTRACT Gamma interferon receptor α (IFN-γRα) is stable but posttranslationally modified in herpes simplex virus 1(F) [HSV-1(F)]-infected cells. Studies with antibody directed to the phosphorylation site indicate that IFN-γRα is phosphorylated by the US3 kinase. The modification is abolished in cells infected with ΔUS3, ΔUL13, or Δ(US3/UL13) mutant virus. Transcripts of the IFN-γ-dependent genes do not accumulate in cells transduced with the US3 protein kinase and treated with IFN-γ. In contrast, the accumulation of IFN-γ-dependent gene transcripts is suppressed in cells infected with the wild-type virus, in cells infected with the ΔUS3 mutant virus, and to a lesser extent in the ΔUL41 virus-infected cells. The accumulation of IFN-γ-dependent gene transcripts in ΔUL41-infected cells could be due at least in part to a significant delay and reduction in the accumulation of the US3 protein. The results suggest that the expression of IFN-γ-dependent genes is blocked independently by the degradation of IFN-γ-dependent gene transcripts—a function of the virion host shutoff RNase—and by posttranslational modification of the IFN-γRα protein.

scholarly journals Interwoven Roles of Cyclin D3 and cdk4 Recruited by ICP0 and ICP4 in the Expression of Herpes Simplex Virus Genes

2010 ◽  
Vol 84 (19) ◽  
pp. 9709-9717 ◽  
Author(s):  
Maria Kalamvoki ◽  
Bernard Roizman
Keyword(s):  
Cyclin D3 ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Nuclear Retention ◽  
Enhanced Expression ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
Simplex Virus ◽  
Cdk4 Inhibitor

ABSTRACT Elsewhere this laboratory reported that (i) ICP0 interacts with cyclin D3 but not D1 or D2. The 3 cyclins independently partially rescue ΔICP0 mutants. (ii) Interaction with cyclin D3 is required for the switch from nuclear to cytoplasmic accumulation of ICP0. (iii) In infected cells cdk4 is activated whereas cdk2 is not. Inhibition of cdk4 results in nuclear retention of ICP0. Overexpression of cyclin D3 reverses the effect of the inhibitor. Here we report the following. (i) cdk4 interacts with ICP0, ICP4, and possibly with ICP8. This interaction is required to recruit cdk4 initially to ND10 and later to the viral replication compartments. (ii) cdk4 inhibitor I reduced or delayed the transcription and ultimately translation of mRNAs of ICP4, ICP27, or ICP8 and to a lesser extent that of the ICP0 gene in wild-type virus-infected cells. (iii) Overexpression of cyclin D3 resulted in a more rapid transcription of these genes. In the presence of inhibitor, the rates of accumulation of the products of these genes resemble those of wild-type virus in the absence of inhibitor. (iv) Overexpression of cyclin D3 also results in mobilization of cdk6 in nuclei of infected cells. We conclude that ICP0 encodes a function that enhances the recruitment of cyclin D3 to ND10 structures to activate cdk4 and that ICP0 along with other viral proteins recruits cdk4 to ND10 structures and ultimately to replication compartments for enhanced expression of viral genes and viral DNA synthesis.

scholarly journals Replication-Competent Controlled Herpes Simplex Virus

2015 ◽  
Vol 89 (20) ◽  
pp. 10668-10679 ◽  
Author(s):  
David C. Bloom ◽  
Joyce Feller ◽  
Peterjon McAnany ◽  
Nuria Vilaboa ◽  
Richard Voellmy
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Traditional Approach ◽  
Wild Type Virus ◽  
Infection Model ◽  
Wild Type ◽  
Infected Cells ◽  
Simplex Virus ◽  
Gene Switch ◽  
Hsv 1

ABSTRACTWe present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model.IMPORTANCEThe alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety.

scholarly journals A Null Mutation in the Gene Encoding the Herpes Simplex Virus Type 1 UL37 Polypeptide Abrogates Virus Maturation

2001 ◽  
Vol 75 (21) ◽  
pp. 10259-10271 ◽  
Author(s):  
Prashant Desai ◽  
Gerry L. Sexton ◽  
J. Michael McCaffery ◽  
Stanley Person
Keyword(s):  
Herpes Simplex ◽  
Gene Product ◽  
Vero Cells ◽  
Mutant Virus ◽  
Null Mutation ◽  
Wild Type ◽  
Gene Encoding ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated KΔUL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus KΔUL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, KΔUL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in KΔUL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of KΔUL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of KΔUL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.

scholarly journals Role of Cyclin D3 in the Biology of Herpes Simplex Virus 1 ICP0

2001 ◽  
Vol 75 (4) ◽  
pp. 1888-1898 ◽  
Author(s):  
Charles Van Sant ◽  
Pascal Lopez ◽  
Sunil J. Advani ◽  
Bernard Roizman
Keyword(s):  
Cyclin D1 ◽  
Binding Site ◽  
Mutant Virus ◽  
Cyclin D3 ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Cyclin D ◽  
Wild Type ◽  
Human Embryonic Lung

ABSTRACT Earlier reports from this laboratory have shown that the promiscuous transactivator infected-cell protein 0 (ICP0) binds and stabilizes cyclin D3, that the binding site maps to aspartic acid 199 (D199), and that replacement of D199 with alanine abolishes binding and reduces the capacity of the mutant virus to replicate in quiescent cells or to cause mortality in mice infected by a peripheral site. The objective of this report was to investigate the role of cyclin D3 in the biology of ICP0. We report the following results. (i) Wild-type ICP0 activates cyclin D-dependent kinase 4 (cdk4) and stabilizes cyclin D1 although ICP0 does not interact with this cyclin. (ii) The D199A mutant virus (R7914) does not activate cdk4 or stabilize cyclin D1, and neither the wild-type nor the mutant virus activates cdk2. (iii) Early in infection of human embryonic lung (HEL) fibroblasts both wild-type and D199A mutant ICP0s colocalize with PML, and in these cells the ND10 nuclear structures are dispersed. Whereas wild-type ICP0 is transported to the cytoplasm between 3 and 9 h. after infection, ICPO containing the D199A substitution remains quantitatively in the nucleus. (iv) To examine the interaction of ICP0 with cyclin D3, we used a previously described mutant carrying a wild-type ICP0 but expressing cyclin D3 (R7801) and in addition constructed a virus (R7916) that was identical except that it carried the D199A-substituted ICP0. Early in infection with R7801, ICP0 colocalized with cyclin D3 in structures similar to those containing PML. At 3 h after infection, ICP0 was translocated to the cytoplasm whereas cyclin D3 remained in the nucleus. The translocation of ICP0 to the cytoplasm was accelerated in cells expressing cyclin D3 compared with that of ICP0 expressed by wild-type virus. In contrast, ICP0 carrying the D199A substitution remained in the nucleus and did not colocalize with cyclin D3. These studies suggest the following conclusions. (i) ICP0 brings to the vicinity of ND10 cyclin D3 and, in consequence, an activated cdk4. The metabolic events occurring at or near that structure and involving cyclin D3 cause the translocation of ICP0 to the cytoplasm. (ii) In the absence of the cyclin D3 binding site in ICP0, cyclin D3 is not brought to ND10, cyclin D is not stabilized, and the function responsible for the translocation of ICP0 is not expressed, and in quiescent HEL fibroblasts the yields of virus are reduced.

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