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 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 Enhanced Pathogenesis of an Attenuated Herpes Simplex Virus for Mice Lacking Stat1

2008 ◽  
Vol 82 (12) ◽  
pp. 6052-6055 ◽  
Author(s):  
Tracy Jo Pasieka ◽  
Betty Lu ◽  
David A. Leib
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Wild Type Virus ◽  
Growth Defect ◽  
Type Virus ◽  
Interferon Signaling ◽  
Wild Type ◽  
Virion Host Shutoff ◽  
Simplex Virus

ABSTRACT Mice lacking the Stat1 interferon signaling gene were infected with herpes simplex virus type 1 (HSV-1) or an attenuated recombinant lacking virion host shutoff (Δvhs). Δvhs virus-infected Stat1−/− mice showed levels of replication equivalent to that of the wild-type virus-infected control mice but reduced relative to wild-type virus-infected Stat1−/− mice. Stat1 deficiency relieves the immunomodulatory deficiency of Δvhs virus, but not its inherent growth defect. Also Vhs is dispensable for reactivation.

scholarly journals Pathogenesis of Herpes Simplex Virus Type 2 Virion Host Shutoff (vhs) Mutants

2002 ◽  
Vol 76 (5) ◽  
pp. 2054-2061 ◽  
Author(s):  
Tracy J. Smith ◽  
Lynda A. Morrison ◽  
David A. Leib
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Rna Degradation ◽  
Host Protein ◽  
Trigeminal Ganglia ◽  
Virion Host Shutoff ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT During lytic infection, the virion host shutoff (vhs) protein mediates the rapid degradation of mRNA and the shutoff of host protein synthesis. In vivo, herpes simplex virus type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. Homologs of vhs exist in all of the neurotropic herpesviruses, and the goal of this study was to determine the virulence of HSV-2 mutants lacking vhs. Two HSV-2 recombinants were used in this study: 333-vhsB, which has a lacZ cassette inserted into the N terminus of vhs, and 333d41, which has a 939-bp deletion in vhs. As expected, both 333-vhsB and 333d41 failed to induce the cellular RNA degradation characteristic of HSV. Corneal, vaginal, and intracerebral routes of infection were used to study pathogenesis. Both viruses grew to significantly lower titers in the corneas, trigeminal ganglia, vaginas, dorsal root ganglia, spinal cords, and brains of mice than wild-type and rescue viruses, with a correspondingly reduced induction of disease. Both viruses, however, reactivated efficiently from explanted trigeminal ganglia, showing that vhs is dispensable for reactivation. The lethality of 333d41 following peripheral infection of mice, however, was significantly higher than that of 333-vhsB, suggesting that some of the attenuation of 333-vhsB may be due to the presence of a lacZ cassette in the vhs locus. Taken together, these data show that vhs represents an important determinant of HSV-2 pathogenesis and have implications for the design of HSV-2 recombinants and vaccines.

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 Herpes Simplex Virus Virion Host Shutoff (vhs) Activity Alters Periocular Disease in Mice

2000 ◽  
Vol 74 (8) ◽  
pp. 3598-3604 ◽  
Author(s):  
Tracy J. Smith ◽  
Cathleen E. Ackland-Berglund ◽  
David A. Leib
Keyword(s):  
Cell Culture ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Degradation ◽  
Host Protein ◽  
Trigeminal Ganglia ◽  
Virion Host Shutoff ◽  
Host Shutoff ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role ofvhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fastvhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.

scholarly journals Effect of Deletion of the Ribonucleotide Reductase Gene in Wild Type and Virion Associated Host Shutoff (vhs-1) Mutant Herpes Simplex Virus-1 on Viral Proliferation and Infected-Carcinoma Cell Cultures Growth

2020 ◽  
Author(s):  
Pnina Schlesinger ◽  
Niza Frenkel
Keyword(s):  
Glioblastoma Multiforme ◽  
Cell Cultures ◽  
Ribonucleotide Reductase ◽  
Viral Vector ◽  
Green Fluorescence Protein ◽  
Vero Cells ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Host Shutoff ◽  
Simplex Virus

Glioblastoma multiforme is the most prevalent and deadliest form of glioma and brain cancer, with a very poor prognosis. In an effort to develop an oncolytic viral vector for the treatment of Glioblastoma multiforme, we replaced the UL39 and UL40 genes encoding ribonucleotide reductase (RR) with green fluorescence protein and luciferase genes in wild type KOS and in the virion host shutoff mutant vhs-1, resulting in strains KOS-RR and Vhs-RR, respectively. KOS-RR and Vhs-RR caused death of infected U87 Glioblastoma multiforme cell cultures within one day after infection, whereas KOS and vhs-1-infected cells were more viable. All four viral strains caused apoptotic DNA laddering in infected H1299 lung cancer cells, while only Vhs-RR caused apoptosis in U87 cell cultures. Vhs-RR gave higher yields on U87 than on Vero cells, while it barely proliferated on non-dividing Goiter cells. These results indicate that Vhs-RR proliferates well in actively growing U87 Glioblastoma multiforme cells, causing their death in a mechanism involving apoptosis, while sparing non-dividing cells. Therefore, Vhs-RR is a promising candidate for oncolytic treatment of brain tumor malignancies.

scholarly journals Virucidal Activity of a GT-Rich Oligonucleotide against Herpes Simplex Virus Mediated by Glycoprotein B

2006 ◽  
Vol 80 (10) ◽  
pp. 4740-4747 ◽  
Author(s):  
Benjamin Shogan ◽  
Lori Kruse ◽  
Gilbert B. Mulamba ◽  
André Hu ◽  
Donald M. Coen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Resistant Mutant ◽  
Glycoprotein B ◽  
Wild Type Virus ◽  
Type Virus ◽  
Virucidal Activity ◽  
Wild Type ◽  
Phosphorothioate Oligonucleotide ◽  
Simplex Virus

ABSTRACT We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in glycoprotein B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhibited attachment of virus to cells at 4°C; however, the resistant mutant did not exhibit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhibiting attachment. Similarly, ISIS 5652 was able to inhibit entry of preattached virions into cells at 37°C, but the mutant did not exhibit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incubation of wild-type virus with 1 to 2 μM ISIS 5652 at 37°C led to a time-dependent, irreversible loss of infectivity (virucidal activity). No virucidal activity was detected at 4°C or with an unrelated oligonucleotide at 37°C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhibited substantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.

scholarly journals Point Mutations in Herpes Simplex Virus Type 1 oriL, but Not in oriS, Reduce Pathogenesis during Acute Infection of Mice and Impair Reactivation from Latency

2006 ◽  
Vol 80 (1) ◽  
pp. 440-450 ◽  
Author(s):  
John W. Balliet ◽  
Priscilla A. Schaffer
Keyword(s):  
Herpes Simplex ◽  
Point Mutations ◽  
Tear Film ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT In vitro studies of herpes simplex virus type 1 (HSV-1) viruses containing mutations in core sequences of the viral origins of DNA replication, oriL and oriS, that eliminate the ability of these origins to initiate viral-DNA synthesis have demonstrated little or no effect on viral replication in cultured cells, leading to the conclusion that the two types of origins are functionally redundant. It remains unclear, therefore, why origins that appear to be redundant are maintained evolutionarily in HSV-1 and other neurotropic alphaherpesviruses. To test the hypothesis that oriL and oriS have distinct functions in the HSV-1 life cycle in vivo, we determined the in vivo phenotypes of two mutant viruses, DoriL-ILR and DoriS-I, containing point mutations in oriL and oriS site I, respectively, that eliminate origin DNA initiation function. Following corneal inoculation of mice, tear film titers of DoriS-I were reduced relative to wild-type virus. In all other tests, however, DoriS-I behaved like wild-type virus. In contrast, titers of DoriL-ILR in tear film, trigeminal ganglia (TG), and hindbrain were reduced and mice infected with DoriL-ILR exhibited greatly reduced mortality relative to wild-type virus. In the TG explant and TG cell culture models of reactivation, DoriL-ILR reactivated with delayed kinetics and, in the latter model, with reduced efficiency relative to wild-type virus. Rescuant viruses DoriL-ILR-R and DoriS-I-R behaved like wild-type virus in all tests. These findings demonstrate that functional differences exist between oriL and oriS and reveal a prominent role for oriL in HSV-1 pathogenesis.

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