Human cytomegalovirus (HCMV), a member of the herpesvirus group, is capable of causing serious medical problems that affect all age groups. This virus is one of the principal pathogens in patients with AIDS and has been implicated as a potential cofactor for the development or progression of the disease. The multiple pathogenic effects of HCMV are likely manifested through a complex interplay of viral gene products and induced and repressed cellular gene functions. HCMV immediate early and early gene expression clearly play a central role in this scheme. The research in my laboratory has been directed towards molecular analysis of HCMV genome organization and gene expression. Specific focus has been on the regulation of expression of three representative early transcription units including: the 2.7 kb and 1.2 kb RNA transcripts encoded by the repeat bonding the long unique segment of the genome; and the 2.2 kb family of RNA transcripts containing the cell-related sequences encoded by HCMV strain AD 169 EcoRI fragments R and d. In this renewal application, we propose to continue our studies on the cis-acting sequences and the trans-acting factors (both viral and cellular) necessary for control of these three early genes. For these studies, we will couple site-directed mutagenesis and functional in vivo transient expression assays with DNA-protein binding assays. To elucidate the means by which specific HCMV immediate early gene products (particularly those expressed by the IE1 and IE2 regions) activate transcription of the early genes, we propose to overexpress the proteins, purify them to homogeneity, and analyze their biochemical interaction with the early promoter elements as well as with the basic RNA polymerase II transcription machinery. With the use of in vitro transcription assays, we will analyze the effect of the IE proteins on: the overall transcription reaction: transcription initiation: commitment of the transcription complex to the DNA template; and the assembly and transcription of nucleosome templates. The long range goals of this research are to define at the molecular level the mechanisms which operate to control HCMV gene expression, thus providing the basis for understanding the process by which this virus causes disease. The knowledge is essential for the development of effective strategies for prevention and treatment.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA034729-12
Application #
2088743
Study Section
Experimental Virology Study Section (EVR)
Project Start
1983-05-01
Project End
1996-07-31
Budget Start
1995-01-01
Budget End
1996-07-31
Support Year
12
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Clark, Elizabeth; Spector, Deborah H (2015) Studies on the Contribution of Human Cytomegalovirus UL21a and UL97 to Viral Growth and Inactivation of the Anaphase-Promoting Complex/Cyclosome (APC/C) E3 Ubiquitin Ligase Reveal a Unique Cellular Mechanism for Downmodulation of the APC/C Subunits APC1, J Virol 89:6928-39
Strang, Blair L; Bender, Brian J; Sharma, Mayuri et al. (2012) A mutation deleting sequences encoding the amino terminus of human cytomegalovirus UL84 impairs interaction with UL44 and capsid localization. J Virol 86:11066-77
DuRose, Jenny B; Li, Julie; Chien, Shu et al. (2012) Infection of vascular endothelial cells with human cytomegalovirus under fluid shear stress reveals preferential entry and spread of virus in flow conditions simulating atheroprone regions of the artery. J Virol 86:13745-55
Burgdorf, Sarah W; Clark, Charles L; Burgdorf, James R et al. (2011) Mutation of glutamine to arginine at position 548 of IE2 86 in human cytomegalovirus leads to decreased expression of IE2 40, IE2 60, UL83, and UL84 and increased transcription of US8-9 and US29-32. J Virol 85:11098-110
Tran, Karen; Mahr, Jeffrey A; Spector, Deborah H (2010) Proteasome subunits relocalize during human cytomegalovirus infection, and proteasome activity is necessary for efficient viral gene transcription. J Virol 84:3079-93
Tran, Karen; Kamil, Jeremy P; Coen, Donald M et al. (2010) Inactivation and disassembly of the anaphase-promoting complex during human cytomegalovirus infection is associated with degradation of the APC5 and APC4 subunits and does not require UL97-mediated phosphorylation of Cdh1. J Virol 84:10832-43
Sanders, Rebecca L; Spector, Deborah H (2010) Human cytomegalovirus IE2 86 and IE2 40 proteins differentially regulate UL84 protein expression posttranscriptionally in the absence of other viral gene products. J Virol 84:5158-70
Kapasi, Anokhi J; Clark, Charles L; Tran, Karen et al. (2009) Recruitment of cdk9 to the immediate-early viral transcriptosomes during human cytomegalovirus infection requires efficient binding to cyclin T1, a threshold level of IE2 86, and active transcription. J Virol 83:5904-17
Kapasi, Anokhi J; Spector, Deborah H (2008) Inhibition of the cyclin-dependent kinases at the beginning of human cytomegalovirus infection specifically alters the levels and localization of the RNA polymerase II carboxyl-terminal domain kinases cdk9 and cdk7 at the viral transcriptosome. J Virol 82:394-407
Sanders, Rebecca L; Clark, Charles L; Morello, Christopher S et al. (2008) Development of cell lines that provide tightly controlled temporal translation of the human cytomegalovirus IE2 proteins for complementation and functional analyses of growth-impaired and nonviable IE2 mutant viruses. J Virol 82:7059-77

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