The long-term objective of these studies is to elucidate the mechanisms that mediate cleavage of intranuclear concatameric herpes simplex virus (HSV) DNA, and insertion of cleaved genomes into preformed capsids. This process is the target of several novel antiviral drugs and understanding the process may lead to modified drugs with broader specificities. The packaging reaction can be viewed as an interaction of two components of a molecular motor. One part receives the DNA (the procapsid), and the second part, the terminase, processes concatameric DNA and drives it into the capsid. Studies focus on members of a protein complex comprised of pULl5, pUL28 and pUL33 that we have identified in infected cells and that have been shown to be required for DNA packaging and are capsid components. The first specific aim is to identify and characterize the ATPase responsible for DNA cleavage and packaging. UL15 is the strongest candidate for fulfilling such a role, although proteins that interact with UL15 may also be involved. Purified proteins will be tested for ATPase activity and mutants lacking the activity will be tested for the ability to support viral replication. The second specific aim seeks to identify and characterize DNA binding relevant to DNA packaging. The UL28 protein has been shown in vitro to bind Pac1 DNA (a sequence that is necessary for formation of the terminus of the short component of viral DNA). Efforts will be made to determine if this occurs in vivo. Efforts will also seek to identify proteins that associate with Pac2, a sequence required to initiate the packaging reaction at the terminus of the long component of viral DNA. To determine if the in vitro activities are relevant in vivo, mutants lacking DNA binding activities will be tested for their ability to support viral DNA cleavage/packaging and replication. The third specific aim will seek to determine the morphology of the molecular motor that drives DNA packaging and thus, will provide considerable information about how the machine functions. Evidence supports the possibility that the UL6 protein (pUL6) forms the pore into which DNA is inserted and that pUL15 may bind this pore. Efforts are described to determine the localization in capsids of components of the UL15, UL28, and UL33. At least pUL15 is hypothesized to interact with pUL6 to mediate docking of the terminase with the procapsid, so should be present on the external surface of the capsid as revealed by electron microscopic immunogold analysis. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Experimental Virology Study Section (EVR)
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Basavappa, Ravi
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Cornell University
Schools of Veterinary Medicine
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Yang, Kui; Wills, Elizabeth G; Baines, Joel D (2012) Release of the herpes simplex virus 1 protease by self cleavage is required for proper conformation of the portal vertex. Virology 429:63-73
Mbong, Ekaette F; Woodley, Lucille; Frost, Elizabeth et al. (2012) Deletion of UL21 causes a delay in the early stages of the herpes simplex virus 1 replication cycle. J Virol 86:7003-7
Johnson, David C; Baines, Joel D (2011) Herpesviruses remodel host membranes for virus egress. Nat Rev Microbiol 9:382-94
Yang, Kui; Baines, Joel D (2009) Tryptophan residues in the portal protein of herpes simplex virus 1 critical to the interaction with scaffold proteins and incorporation of the portal into capsids. J Virol 83:11726-33
Scholtes, Luella; Baines, Joel D (2009) Effects of major capsid proteins, capsid assembly, and DNA cleavage/packaging on the pUL17/pUL25 complex of herpes simplex virus 1. J Virol 83:12725-37
Duffy, Carol; Mbong, Ekaette F; Baines, Joel D (2009) VP22 of herpes simplex virus 1 promotes protein synthesis at late times in infection and accumulation of a subset of viral mRNAs at early times in infection. J Virol 83:1009-17
Yang, Kui; Baines, Joel D (2008) Domain within herpes simplex virus 1 scaffold proteins required for interaction with portal protein in infected cells and incorporation of the portal vertex into capsids. J Virol 82:5021-30
Yang, Kui; Homa, Fred; Baines, Joel D (2007) Putative terminase subunits of herpes simplex virus 1 form a complex in the cytoplasm and interact with portal protein in the nucleus. J Virol 81:6419-33
Wills, Elizabeth; Scholtes, Luella; Baines, Joel D (2006) Herpes simplex virus 1 DNA packaging proteins encoded by UL6, UL15, UL17, UL28, and UL33 are located on the external surface of the viral capsid. J Virol 80:10894-9
Duffy, Carol; Lavail, Jennifer H; Tauscher, Andrew N et al. (2006) Characterization of a UL49-null mutant: VP22 of herpes simplex virus type 1 facilitates viral spread in cultured cells and the mouse cornea. J Virol 80:8664-75

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