Infected Cell Protein No. 22 Is Subject to Proteolytic Cleavage by Caspases Activated by a Mutant That Induces Apoptosis

AbstractEarlier reports have shown that the d120 mutant of herpes simplex virus 1 lacking both copies of the gene encoding the infected cells protein No. 4 (ICP4) induces apoptosis in a variety of cell lines. The programmed cell death induced by this mutant is blocked by overexpression of Bcl-2 or by transduction of infected cells with the gene encoding the viral US3 protein kinase. HEp-2 cells infected with the d120 mutant express predominantly α proteins. Studies on these proteins revealed the accumulation of a Mr 37,500 protein that reacted with antibody directed against the carboxyl-terminal domain of ICP22. We report that the Mr 37,500 protein is a product of the proteolytic cleavage of ICP22 by a caspase activated by the d120 mutant. Thus the accumulation of the Mr 37,500 protein was blocked in cells transduced with the US3 protein kinase, in cells overexpressing Bcl-2, or in infected cells treated with the general caspase inhibitor zVAD-fmk. Exposure of ICP22 made in wild-type virus-infected cells to caspase 3 yielded two polypeptides, of which one could not be differentiated from the Mr 37,500 protein with respect to electrophoretic mobility. We conclude that the cellular apoptotic response targets at least one viral protein for destruction

UL13 Protein Kinase of Herpes Simplex Virus 1 Complexes with Glycoprotein E and Mediates the Phosphorylation of the Viral Fc Receptor: Glycoproteins E and I

AbstractHerpes simplex virus 1 encodes a Fc receptor consisting of glycoproteins E (gE) and I (gI) and two protein kinases specified by UL13 and US3, respectively. We report the following: (i) Antibody to UL13 formed immune complexes containing gE and gI in addition to UL13 protein. Immune complexes formed by monoclonal antibody to gE, but not those formed by monoclonal antibody to gI, also contained the UL13 protein. This association may reflect direct interaction between gE and UL13 inasmuch as IgG in preimmune rabbit serum and an antiserum made against another viral protein which does not react with the UL13 protein directly also bound gE and UL13. (ii) In cells infected with the wild-type virus, gE formed two sharp bands and a diffuse, slower migrating band. The slower sharp band was undetectable, and the diffuse slower migrating forms of gE were diminished in lysates of cells infected with a mutant virus lacking the UL13 gene (ΔUL13). (iii) Both gE and gI were labeled with32Pi in cells infected with wild-type or the ΔUL13 virus, but the labeling was significantly stronger in cells infected with the wild-type virus than in those infected with the ΔUL13 virus. (iv) In anin vitroprotein kinase assay, UL13 immunoprecipitated from cells infected with wild-type virus labeled gE in the presence of [γ-32P]ATP. This activity was absent in precipitates from cells infected with ΔUL13 virus. The labeled gE comigrated with the slower, sharp band of gE. (v) gI present in the UL13 immune complex was also phosphorylated in thein vitrokinase assay. (vi) The cytoplasmic domain of gE contains recognition sequences for phosphorylation by casein kinase II (CKII). Exogenous CKII phosphorylated gE in immune complexes from lysates of cells infected with the ΔUL13 mutant or in immune complexes from lysates of cells infected with wild-type virus that had been heated to inactivate all endogenous kinase activity including that of UL13. In both instances, CKII phosphorylated gE in both the slow and fast migrating sharp bands. We conclude that UL13 physically associates with gE and mediates the phosphorylation of gE and gI. UL13 may also be a determinant in posttranslational processing of gE

Antisense RNA directed to the human papillomavirus type 16 E7 mRNA from herpes simplex virus type 1 derived vectors is expressed in CaSki cells and downregulates E7 mRNA

<p>Abstract</p> <p>Background</p> <p>Human papillomavirus (HPV) infection is known to be the most important etiologic factor of cervical cancer. There is no HPV specific therapy available for treatment of invasive squamous cell carcinoma of the cervix and its precursor lesions. The present study elucidates the potential to use herpes simplex virus (HSV) derived vectors for expression of antisense RNA to HPV -16 E7 oncogene.</p> <p>Results</p> <p>We have constructed replication competent, nonneuroinvasive HSV-1 vectors, deleted of the γ₁34.5 gene. The vectors express RNA antisense to the first 100 nucleotides of the HPV-16 E7 gene. We assayed the ability of the antisense E7 vectors R5225 (<it>tk</it>-) and R5226 (<it>tk+</it>), to produce antisense RNA, as well as the consequent effects on E7 mRNA and protein levels in HPV-16 positive CaSki cells. Anti-E7 RNA was expressed by both constructs in a dose-dependent manner. Expression of HPV-16 E7 mRNA was downregulated effectively in CaSki cells infected with the <it>tk- </it>recombinant R5225 or with R5226. The <it>tk+ </it>recombinant R5226 was effective in downregulating E7 protein expression.</p> <p>Conclusion</p> <p>We have shown that anti-E7 RNA expressed from an HSV vector could efficiently downregulate HPV-16 E7 mRNA and E7 protein expression in CaSki cells. We conclude that HSV vectors may become a useful tool for gene therapy of HPV infections.</p

Role of ICP0 in the Strategy of Conquest of the Host Cell by Herpes Simplex Virus 1

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