The studies described in this application for continuing support of a long term project center on the functions of the infected cell protein No. 22 (ICP22), a regulatory protein, in the biology and pathogenesis of herpes simplex viruses (HSV) infections. Our studies relevant to this proposal and its aims are as follows: (i) the 420-residue ICP22 is encoded by the a22 gene. The a22 open reading frame also encodes a second protein, Us1.5, consisting of the C-terminal 273 residues of ICP22. (ii) Mutants lacking the coding sequences of ICP22 or Us1.5 proteins are unable to replicate in mice but do replicate in cell culture in a cell type-dependent manner. (iii) No phenotype other than ability to replicate in mice has been mapped to the N-terminal domain of ICP22. Mutants lacking the coding sequence of Us 11 are attenuated and in culture accumulate grossly reduced levels of a subset of late viral proteins exemplified by Us11, UL41 and UL38. The objective of the studies proposed in this application is to continue studies of three functions mapped to the Us1.5 domain of the a22 gene. Specifically (i) In wild-type virus-infected cells, the accumulation of ICP22-dependent subset of late proteins is dependent on activation of the mitotic kinase cdc2, degradation of cdc2 kinase partners, cyclins A and B, the acquisition of a new partner, the UL42 DNA polymerase processivity factor, and finally, the ICP22-dependent binding and modification of topoisomerase IIa by the cdc2-UL42 protein complex. In uninfected cells cdc2 is normally activated by the cdc25C phosphatase, which is also activated in wild-type virus infected cells but not in ?a22-mutant virus-infected cells. Cdc2 is not activated in cdc25c-/- (knockout) cells.
AIM 1 of this study is to determine whether HSV-1 activates cdc2 via an interaction of cdc25C-phosphatase with ICP22 and the UL13 protein kinase. The objective of AIM 2 is to investigate the role of the interaction of UL42 and topoisornerase IIa. (ii) ICP22 and UL13 mediate an aberrant phosphorylation of RNA POL II. We found that ICP22 interacts with cdk9, a T1 cyclin-dependent kinase involved in transcriptional activation. We have also shown that the C-terminal domain of RNA POL II is phosphorylated by cdk9 precipitates from wild type but nor from ?a22 mutant virus-infected cells. The objective of AIM 3 is to determine the role of ICP22 and cdk9 in the modification of RNA POL II. Lastly, (iii) our earlier studies have identified a cellular protein (p60) that binds both ICP22 and ICP0. A central question addressed in AIM 4 is the role of this protein in the biology of HSV-1. In all instances a key question to be resolved is the contribution of each of these functions of ICP22 to the biology of the virus in the experimental animal host.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA083939-08
Application #
7163507
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Daschner, Phillip J
Project Start
2000-01-01
Project End
2009-12-31
Budget Start
2007-01-01
Budget End
2007-12-31
Support Year
8
Fiscal Year
2007
Total Cost
$491,187
Indirect Cost
Name
University of Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Jovasevic, Vladimir; Liang, Li; Roizman, Bernard (2008) Proteolytic cleavage of VP1-2 is required for release of herpes simplex virus 1 DNA into the nucleus. J Virol 82:3311-9
Smith-Donald, Benjamin A; Durand, Lizette O; Roizman, Bernard (2008) Role of cellular phosphatase cdc25C in herpes simplex virus 1 replication. J Virol 82:4527-32
Kalamvoki, Maria; Qu, Jianguo; Roizman, Bernard (2008) Translocation and colocalization of ICP4 and ICP0 in cells infected with herpes simplex virus 1 mutants lacking glycoprotein E, glycoprotein I, or the virion host shutoff product of the UL41 gene. J Virol 82:1701-13
Smith-Donald, Benjamin A; Roizman, Bernard (2008) The interaction of herpes simplex virus 1 regulatory protein ICP22 with the cdc25C phosphatase is enabled in vitro by viral protein kinases US3 and UL13. J Virol 82:4533-43
Durand, Lizette Olga; Roizman, Bernard (2008) Role of cdk9 in the optimization of expression of the genes regulated by ICP22 of herpes simplex virus 1. J Virol 82:10591-9
Poon, Alice P W; Roizman, Bernard (2007) Mapping of key functions of the herpes simplex virus 1 U(S)3 protein kinase: the U(S)3 protein can form functional heteromultimeric structures derived from overlapping truncated polypeptides. J Virol 81:1980-9
Kalamvoki, Maria; Roizman, Bernard (2007) Bcl-2 blocks accretion or depletion of stored calcium but has no effect on the redistribution of IP3 receptor I mediated by glycoprotein E of herpes simplex virus 1. J Virol 81:6316-25
Sciortino, Maria Teresa; Taddeo, Brunella; Giuffre-Cuculletto, Maria et al. (2007) Replication-competent herpes simplex virus 1 isolates selected from cells transfected with a bacterial artificial chromosome DNA lacking only the UL49 gene vary with respect to the defect in the UL41 gene encoding host shutoff RNase. J Virol 81:10924-32
Benetti, Luca; Roizman, Bernard (2007) In transduced cells, the US3 protein kinase of herpes simplex virus 1 precludes activation and induction of apoptosis by transfected procaspase 3. J Virol 81:10242-8
Zhou, Guoying; Roizman, Bernard (2007) Separation of receptor-binding and profusogenic domains of glycoprotein D of herpes simplex virus 1 into distinct interacting proteins. Proc Natl Acad Sci U S A 104:4142-6

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