scholarly journals Herpes Simplex Virus Type 1 Immediate-Early Protein Vmw110 Induces the Proteasome-Dependent Degradation of the Catalytic Subunit of DNA-Dependent Protein Kinase

1999 ◽  
Vol 73 (1) ◽  
pp. 650-657 ◽  
Author(s):  
Jane Parkinson ◽  
Susan P. Lees-Miller ◽  
Roger D. Everett
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Herpes Simplex Virus Type ◽  
Dependent Protein Kinase ◽  
Early Protein ◽  
Dependent Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) infection causes the active degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and this process is reliant on the expression of the HSV-1 immediate-early protein Vmw110. In this study we investigated in more detail the mechanism by which the degradation occurs, the domains of Vmw110 which are required, and whether Vmw110 is by itself sufficient for the effect. We found that proteasome inhibitors prevented the degradation of DNA-PKcs, indicating the involvement of a proteasome pathway. Furthermore, the continued activity of DNA-PK during infection in the presence of these inhibitors indicated that Vmw110 does not directly alter the enzyme activity of DNA-PKcs prior to its degradation in a normal infection. Indeed, Vmw110 was found to bind to neither the catalytic nor Ku subunits of DNA-PK. Using mutant Vmw110 viruses we show that the RING finger domain of Vmw110 is essential for the induced degradation of DNA-PKcs but that the ability of Vmw110 to bind to a cellular ubiquitin-specific protease (HAUSP) is not required. When expressed in the absence of other viral proteins, Vmw110 was sufficient to cause the degradation of DNA-PKcs, indicating that the effect on the stability of DNA-PKcs was a direct consequence of Vmw110 activity and not an indirect Vmw110-dependent effect of virus infection. Finally, the Vmw110-induced degradation of DNA-PKcs and loss in DNA-PK activity appears to be beneficial to HSV-1 infection, as virus replication was more efficient in cells lacking DNA-PKcs, especially at low multiplicities of infection.

scholarly journals Origin of Expression of the Herpes Simplex Virus Type 1 Protein US1.5

2009 ◽  
Vol 83 (18) ◽  
pp. 9183-9194 ◽  
Author(s):  
J. Jason Bowman ◽  
Priscilla A. Schaffer
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Expression Patterns ◽  
Full Length ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT ICP22, an immediate-early protein of herpes simplex virus type 1 (HSV-1), is required for viral replication in nonpermissive cell types and for expression of a class of late viral proteins which includes glycoprotein C. An understanding of the mechanism of ICP22 function has been complicated by the coexpression of the full-length protein with an in-frame, C-terminus-specific protein, US1.5. In this report, we confirm that the US1.5 protein is a bona fide translation product since it is detected during infections with three laboratory strains and two low-passage clinical isolates of HSV-1. To clarify the expression patterns of the ICP22 and US1.5 proteins, we examined their synthesis from plasmids in transient expression assays. Because previous studies had identified two different US1.5 translational start sites, we attempted to determine which is correct by studying the effects of a series of deletion, nonsense, and methionine substitutions on US1.5 expression. First, amino acids 90 to 420 encoded by the ICP22 open reading frame (ORF) migrated at the mobility of US1.5 in sodium dodecyl sulfate-polyacrylamide gels. Second, introduction of a stop codon downstream of M90 ablated expression of both ICP22 and US1.5. Finally, mutation of M90 to alanine (M90A) allowed expression of full-length ICP22 while dramatically reducing expression of US1.5. Levels of US1.5 but not ICP22 protein expression were also reduced in cells infected with an M90A mutant virus. Thus, we conclude that expression of IC22 and that of US1.5 can occur independently of each other and that US1.5 translation initiates at M90 of the ICP22 ORF.

scholarly journals RNA-Dependent Protein Kinase Is Required for Alpha-1 Interferon Transgene-Induced Resistance to Genital Herpes Simplex Virus Type 2

2005 ◽  
Vol 79 (14) ◽  
pp. 9341-9345 ◽  
Author(s):  
Daniel J. J. Carr ◽  
Lisa Tomanek ◽  
Robert H. Silverman ◽  
Iain L. Campbell ◽  
Bryan R. G. Williams
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Genital Herpes ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Simplex Virus

ABSTRACT We investigated the mechanism of resistance to genital herpes simplex virus type 2 (HSV-2) infection in mice transfected with the murine alpha-1 interferon (IFN-α1) transgene. In situ transfection of mice with the IFN-α1 transgene resulted in an elevation in an IFN-responsive gene, RNA-dependent protein kinase (PKR), but not 2′,5′-oligoadenylate synthetases (OAS), in vaginal tissue. Coupled with the finding that mice lacking a functional PKR pathway were no longer resistant to genital HSV-2 infection following transfection with the IFN-α1 transgene in comparison to wild-type mice or mice lacking a functional OAS pathway, these results suggest that PKR is the dominant antiviral pathway activated by the IFN-α1 transgene.

scholarly journals The lack of RNA-dependent protein kinase enhances susceptibility of mice to genital herpes simplex virus type 2 infection

Immunology ◽  
2006 ◽  
Vol 0 (0) ◽  
pp. 060606080407004-???
Author(s):  
Daniel J. J. Carr ◽  
Todd Wuest ◽  
Lisa Tomanek ◽  
Robert H. Silverman ◽  
Bryan R. G. Williams
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Genital Herpes ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Simplex Virus

Cross-reactivity of the anti-PML antibody PG-M3 with the herpes simplex virus type 1 immediate early protein ICP4

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1773-1777 ◽  
Author(s):  
Stefanie L. Boulware ◽  
Peter C. Weber
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Cross Reactivity ◽  
Immediate Early ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

The PML protein is one of the components of ND10, nuclear matrix-associated structures which undergo rapid disintegration at the onset of herpes simplex virus type 1 (HSV-1) infection. This disruption event has been frequently visualized in immunofluorescence assays using the anti-PML mouse monoclonal antibody PG-M3. This antibody was surprisingly found to also stain nuclear virus replication compartments when employed at higher concentrations. This was shown to be due to an unexpected cross-reactivity of the PG-M3 antibody with the HSV-1 immediate early protein ICP4, a known component of replication compartments. The sequences of ICP4 recognized by PG-M3 were found to map to the extreme amino-terminal end of the protein, which includes a 21 amino acid segment that is partially homologous to the peptide of PML that was used to make PG-M3. These results suggest that PG-M3 may no longer represent an appropriate antibody for use in visualizing the fate of PML and ND10 during HSV-1 infection.

scholarly journals Transactivation of Herpes Simplex Virus Type 1 Immediate-Early Gene Expression by Virion-Associated Factors Is Blocked by an Inhibitor of Cyclin-Dependent Protein Kinases

1999 ◽  
Vol 73 (10) ◽  
pp. 8843-8847 ◽  
Author(s):  
Robert Jordan ◽  
Luis Schang ◽  
Priscilla A. Schaffer
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Immediate Early ◽  
Dependent Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Initiation of productive infection by human herpes simplex virus type 1 (HSV-1) requires cell cycle-dependent protein kinase (cdk) activity. Treatment of cells with inhibitors of cdks blocks HSV-1 replication and prevents accumulation of viral transcripts, including immediate-early (IE) transcripts (26). Inhibition of IE transcript accumulation suggests that virion proteins, such as VP16, require functional cdks to activate viral transcription. In this report, we show that a cdk inhibitor, Roscovitine, blocks VP16-dependent IE gene expression. In the presence of Roscovitine, the level of virion-induced activation of a transfected reporter gene (the gene encoding chloramphenicol acetyltransferase) linked to the promoter-regulatory region of the ICP0 gene was reduced 40-fold relative to that of untreated samples. Roscovitine had little effect on the interaction of VP16 with VP16-responsive DNA sequences as measured by electrophoretic mobility shift assays. These data indicate that VP16-dependent activation of IE gene expression requires functional cdks and that this requirement is independent of the ability of VP16 to bind to DNA.

scholarly journals Live Covisualization of Competing Adeno-Associated Virus and Herpes Simplex Virus Type 1 DNA Replication: Molecular Mechanisms of Interaction

2007 ◽  
Vol 81 (9) ◽  
pp. 4732-4743 ◽  
Author(s):  
Daniel L. Glauser ◽  
Regina Strasser ◽  
Andrea S. Laimbacher ◽  
Okay Saydam ◽  
Nathalie Clément ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT We performed live cell visualization assays to directly assess the interaction between competing adeno-associated virus (AAV) and herpes simplex virus type 1 (HSV-1) DNA replication. Our studies reveal the formation of separate AAV and HSV-1 replication compartments and the inhibition of HSV-1 replication compartment formation in the presence of AAV. AAV Rep is recruited into AAV replication compartments but not into those of HSV-1, while the single-stranded DNA-binding protein HSV-1 ICP8 is recruited into both AAV and HSV-1 replication compartments, although with differential staining patterns. Slot blot analysis of coinfected cells revealed a dose-dependent inhibition of HSV-1 DNA replication by wild-type AAV but not by rep-negative recombinant AAV. Consistent with this, Western blot analysis indicated that wild-type AAV affects the levels of the HSV-1 immediate-early protein ICP4 and the early protein ICP8 only modestly but strongly inhibits the accumulation of the late proteins VP16 and gC. Furthermore, we demonstrate that the presence of Rep in the absence of AAV DNA replication is sufficient for the inhibition of HSV-1. In particular, Rep68/78 proteins severely inhibit the formation of mature HSV-1 replication compartments and lead to the accumulation of ICP8 at sites of cellular DNA synthesis, a phenomenon previously observed in the presence of viral polymerase inhibitors. Taken together, our results suggest that AAV and HSV-1 replicate in separate compartments and that AAV Rep inhibits HSV-1 at the level of DNA replication.

scholarly journals Phosphorylation of Structural Components Promotes Dissociation of the Herpes Simplex Virus Type 1 Tegument

1998 ◽  
Vol 72 (9) ◽  
pp. 7108-7114 ◽  
Author(s):  
Ewan E. Morrison ◽  
Yi-Fen Wang ◽  
David M. Meredith
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Structural Components ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.

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