The long term objective of this research is to understand the molecular basis of cytomegalovirus (CMV) replication and use that information to develop new ways to diagnose and treat CMV-related diseases of man. This will be accomplished by identifying and studying the synthesis, structure, and function of specific viral proteins that are essential for virus replication. In this application for renewed support, two general sets of studies are proposed. One represents a continuation of ongoing work to learn more about proteins that are already under investigation. The other will pursue leads generated during the last grant period to better define two new sets of proteins. A major emphasis is placed on learning more about a """"""""scaffoldinglike"""""""" protein, referred to as the assembly protein and suspected to have a key role in capsid formation and possibly DNA-packaging. Experiments are outlined to identify, characterize, and genetically map the protease responsible for cleaving the precursor form of this protein. Chemical mutagenesis of the cloned assembly protein gene will be used in an attempt to generate temperature-sensitive mutants that will aid functional studies. Four proteins of the virion tegument will also be investigated. Two of these, referred to as the basic phosphoprotein and the lower matrix protein, appear to interact directly with proteins of the capsid and envelope. The nature of these associations will be explored by chemical, immunological, and genetic approaches. Two other tegument proteins, referred to as the high molecular weight protein and the 115K protein, cosediment as a possible heterodimer and are speculated to be related to the herpes simplex DNA cleavage/packaging complex. Experiments based on chemical dissociation, rate-velocity sedimentation, band-shift assays, and DNA-affinity chromatography are described to characterize the complex and determine whether it has properties of a cleavage/packaging enzyme. Studies of a more exploratory nature will try to identify the proteins encoded by three newly discovered HCMV genes that are homologous to cellular G protein-coupled receptors, and determine by recombinant DNA methods whether they are required for growth of the virus in cell culture.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology Study Section (VR)
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Johns Hopkins University
Schools of Medicine
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Tullman, Jennifer A; Harmon, Mary-Elizabeth; Delannoy, Michael et al. (2014) Recovery of an HMWP/hmwBP (pUL48/pUL47) complex from virions of human cytomegalovirus: subunit interactions, oligomer composition, and deubiquitylase activity. J Virol 88:8256-67
Fernandes, Steve M; Brignole, Edward J; Taori, Kanchan et al. (2011) Cytomegalovirus capsid protease: biological substrates are cleaved more efficiently by full-length enzyme (pUL80a) than by the catalytic domain (assemblin). J Virol 85:3526-34
Margulies, Barry J; Gibson, Wade (2007) The chemokine receptor homologue encoded by US27 of human cytomegalovirus is heavily glycosylated and is present in infected human foreskin fibroblasts and enveloped virus particles. Virus Res 123:57-71
Loveland, Amy N; Nguyen, Nang L; Brignole, Edward J et al. (2007) The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production. J Virol 81:620-8
Wang, Jianlei; Loveland, Amy N; Kattenhorn, Lisa M et al. (2006) High-molecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable. J Virol 80:6003-12
McCartney, Stephen A; Brignole, Edward J; Kolegraff, Keli N et al. (2005) Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a. J Biol Chem 280:33206-12
Loveland, Amy N; Chan, Chee-Kai; Brignole, Edward J et al. (2005) Cleavage of human cytomegalovirus protease pUL80a at internal and cryptic sites is not essential but enhances infectivity. J Virol 79:12961-8
Chan, Chee-Kai; Brignole, Edward J; Gibson, Wade (2002) Cytomegalovirus assemblin (pUL80a): cleavage at internal site not essential for virus growth; proteinase absent from virions. J Virol 76:8667-74
Baxter, M K; Gibson, W (2001) Cytomegalovirus basic phosphoprotein (pUL32) binds to capsids in vitro through its amino one-third. J Virol 75:6865-73
Plafker, S M; Woods, A S; Gibson, W (1999) Phosphorylation of simian cytomegalovirus assembly protein precursor (pAPNG.5) and proteinase precursor (pAPNG1): multiple attachment sites identified, including two adjacent serines in a casein kinase II consensus sequence. J Virol 73:9053-62

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