This application is a continuation and replacement of a grant to study the mechanisms by which herpes simplex viruses (HSV) thwart attempts of the host cell to commit suicide (apoptosis) to block viral replication. The discovery that the US3 kinase blocks apoptosis induced by defective viruses, activated pro-apoptotic genes, or exogenous agents (e.g. sorbitol) was followed by the findings that (a) HSV encodes two isomers of US3 designated US3 and US3.5 that only the full- length US3 kinases, and not the US3.5 kinase initiating at methionine 77 blocks apoptosis, (b) US3 is more efficient than US3.5 in enabling the release of capsids from infected cells, (c) both US3 and US3.5 phosphorylate histone deacetylases 1 and 2 (HDAC-1/2) and enable the expression of genes introduced into cells by co-transduction (helper function). In the absence of the kinase, the virus is totally avirulent in mice. The present application proposes a broader investigation of this unique regulatory viral kinase. The objective of AIM 1 is to define the functions of post-translational modifications of the US3 protein kinase. The US3 protein is extensively posttranslationally modified by auto-phosphorylation, by UL13 viral kinase and by cellular kinases. In addition there is evidence that it aggregates to form higher order structures. The objective of this aim is to elucidate the role of posttranslational modifications in the various functions of the US3 kinase by appropriate mapping and mutagenesis of the sites. The objective of AIM 2 is to map the domains encoding key functions of the US3 protein kinase and identify the interacting proteins for each of the functions. We found that the anti-apoptotic functions and the modification of the nuclear envelope to enable release of capsids from nuclei map in the N- terminal domain whereas the kinase activity, the phosphorylation of HDAC-1/2 and functions designed to enable viral gene expression map C-terminal with respect to residue 182. The objective of these studies is to map the domains responsible for anti-apoptotic activity and egress of capsids from nuclei and to identify and investigate the role of viral or cellular proteins that interact with these domains. The objective of AIM 3 is to verify that the helper function of the US3 protein kinase is due to the phosphorylation of HDAC1/2 and not to the phosphorylation of some unknown proteins. The objective of AIM 4 is to define the role of the various functions of the HSV-1 US3 kinase in neuronal transport and pathogenesis of HSV-1 infections. The US3 mutants are totally avirulent and are impaired in neuronal transport. We want to determine the contributions of the various functions of the US3 protein kinase enumerated above to the ability of HSV to cause morbidity and mortality in experimental animals systems. In essence, the US3 kinases turn out to be key enzymes that not only block the attempts of the host cell to thwart viral replication but they also play a key role in neuroinvasiveness and replication of the virus. The mechanisms by which viruses conquer human cells are key objectives of virology today.

Public Health Relevance

The US3 protein kinase is an essential protein that performs two sets of function: it blocks key host responses to infection (blocks apoptosis, blocks silencing of DNA by host proteins, and inactivates interferon 3 receptor) and modifies viral and cellular proteins for efficient replication and spread of virus. The proposed studies will elucidate the mechanisms by which US3 performs these functions, contribute to our understanding of apoptosis and silencing of DNA, and pinpoint the functions that account for the pathogenicity of herpes simplex virus 1. ? ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Virology - B Study Section (VIRB)
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Daschner, Phillip J
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University of Chicago
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Sciortino, Maria Teresa; Parisi, Tiziana; Siracusano, Gabriel et al. (2013) The virion host shutoff RNase plays a key role in blocking the activation of protein kinase R in cells infected with herpes simplex virus 1. J Virol 87:3271-6
Wang, Xiaojia; Patenode, Caroline; Roizman, Bernard (2011) US3 protein kinase of HSV-1 cycles between the cytoplasm and nucleus and interacts with programmed cell death protein 4 (PDCD4) to block apoptosis. Proc Natl Acad Sci U S A 108:14632-6
Jovasevic, Vladimir; Roizman, Bernard (2010) The novel HSV-1 US5-1 RNA is transcribed off a domain encoding US5, US4, US3, US2 and alpha22. Virol J 7:103
Liang, Li; Roizman, Bernard (2008) Expression of gamma interferon-dependent genes is blocked independently by virion host shutoff RNase and by US3 protein kinase. J Virol 82:4688-96
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
Yang, Kui; Poon, Alice P W; Roizman, Bernard et al. (2008) Temperature-sensitive mutations in the putative herpes simplex virus type 1 terminase subunits pUL15 and pUL33 preclude viral DNA cleavage/packaging and interaction with pUL28 at the nonpermissive temperature. J Virol 82:487-94
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
Taddeo, Brunella; Sciortino, Maria Teresa; Zhang, Weiran et al. (2007) Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for accumulation of mRNA. Proc Natl Acad Sci U S A 104:12163-8

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