Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and poses a serious health threat to immunocompromised individuals, particularly those with AIDS. This virus appears to affect the host cell metabolism in ways that mimic processes involved in cell activation, but the molecular basis of these effects and their relationship to viral replication have yet to be elucidated. We have found that the HCMV infection markedly affects key components of the cell cycle, leading to cell cycle arrest. Early after infection, HCMV induced elevated levels of cyclins E and B and their associated kinase activities, p53, and hyperphosphorylated Rb. However, the synthesis of cyclin A was inhibited, and only at very late times in the infection was there any increase in the levels of protein and kinase activity. Our preliminary studies indicate that p53, but not Rb, becomes sequestered in viral replication centers in the nucleus. We also have evidence which shows that both the enhanced expression of cyclin E and the inhibition of cyclin A are due to effects at the level of transcription, and we hypothesize that the E2F/DP family of transcription factors are involved. In contrast, the increased levels of cyclin B appear to be due to lack of cell-cycle mediated degradation. Preliminary data also indicate that during the infection there is a distinct redistribution of the proteasomes and their overall level increases. Thus, it appears that the HCMV infection has altered the transcription, subcellular localization, and stability of key cell cycle regulatory proteins. The goal of the proposed studies is to decipher the molecular basis of these cell cycle perturbations. The approach is to couple in vivo analyses with in vitro biochemical and molecular assays to achieve the following specific aims: 1) identification of the cis-acting sequences and trans-acting factors responsible for the HCMV-mediated effects on the transcription of the cyclin E and cyclin A genes; 2) determination of the role of ubiquitination and the proteasomes in mediating the effects of the HCMV infection on the stability of cell cycle regulatory proteins; and 3) analysis of the functional properties of the HCMV 1E2 86 and IE1 72 gene products with respect to dysregulation of the cell cycle. These studies are important not only to advance our knowledge of how the complex interactions of viral and host functions relate to viral replication and pathogenesis but also to help elucidate the general mechanisms that operate to control the cell cycle.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA073490-05
Application #
6475905
Study Section
Virology Study Section (VR)
Program Officer
Daschner, Phillip J
Project Start
1997-12-20
Project End
2002-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
5
Fiscal Year
2002
Total Cost
$296,821
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
Spector, Deborah H (2015) Human cytomegalovirus riding the cell cycle. Med Microbiol Immunol 204:409-19
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
Tran, Karen; Mahr, Jeffrey A; Choi, Jiwon et al. (2008) Accumulation of substrates of the anaphase-promoting complex (APC) during human cytomegalovirus infection is associated with the phosphorylation of Cdh1 and the dissociation and relocalization of APC subunits. J Virol 82:529-37
Sanders, Rebecca L; Del Rosario, Christia J; White, Elizabeth A et al. (2008) Internal deletions of IE2 86 and loss of the late IE2 60 and IE2 40 proteins encoded by human cytomegalovirus affect the levels of UL84 protein but not the amount of UL84 mRNA or the loading and distribution of the mRNA on polysomes. J Virol 82:11383-97
Sanchez, Veronica; Mahr, Jeffrey A; Orazio, Nicole I et al. (2007) Nuclear export of the human cytomegalovirus tegument protein pp65 requires cyclin-dependent kinase activity and the Crm1 exporter. J Virol 81:11730-6

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