SV40 large tumor antigen (T antigen) is a potent viral oncoprotein which is capable of a large array of biochemical functions. These functions affect DNA replication, cell cycle control and the control of transcription. Much of our previous work has dealt with the transcriptional activation function of T antigen using the SV40 late promoter as a model system. However, T antigen is a promiscuous activator of many heterologous promoters. This may account, at least in part, for the dramatic alterations in cellular gene expression seen during transformation and lytic infection. Our analyses of the transcriptional activation mechanisms mediated by T antigen have shown that the promoter structure necessary for activation is relatively simple: a TATA or initiator element with an upstream binding site for a transcription factor. There is a great deal of variability allowed in these promoters, which may explain T antigen's promiscuous activation. Our data suggest that the late promoter is made up of a number of these more simple promoters. Using simple promoter constructions we have found that the overlapping binding sites for octamer and TEF transcription factors, form a specific upstream region through which T antigen mediates transcriptional control of the late promoter. Activation is mediated through TEF, while octamer factor binding mediates negative regulation of late promoter activity. Evidence suggests that octamer and/or TEF may be modified in T antigen containing cells. However, the number of different promoters activated by T antigen indicates that it may also affect many other factors. The utilization of synthetic, simple promoters allows us to determine promoter structures required for activation by T antigen. Therefore, in Aim 1 we propose to utilize well defined promoters to identify: 1) the simplest promoter necessary for activation by T antigen; 2) the requirements for most efficient activation, e.g. the factors, the spacing requirements, preferences between TATA and initiator elements; 3) the variability in the promoter elements that can be used; 4) the effects of multiplicity of homologous and heterologous elements, i.e. systematically building more complex promoters. The findings of these studies provide us with different defined promoter elements and structures which we are utilizing, in Aim 2, for the determination of the mechanism by which T antigen activates transcription. With limited and well defined elements in the promoters we have specific factors to examine in order to determine, for example, whether factors are modified or induced, whether T antigen binds them or whether a cellular protein they interact with has been affected by T antigen. The SV40 late leader region has also been implicated in late promoter function and RNA stability. Therefore, in Aim 3, we will include this region in our studies, examining it for elements involved in transcriptional control and RNA stability.
In Aim 4, we propose a complementary approach to our studies of T antigen transcriptional activation. We have expressed T antigen in Saccharomyces cerevisiae and have found that it mediates transcriptional activation an affects cell cycle control. Therefore, we will utilize the genetics of yeast as an alternate system to determine the mechanisms by which T antigen affects transcriptional control and the cell cycle.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA028379-12
Application #
3168108
Study Section
Virology Study Section (VR)
Project Start
1980-06-01
Project End
1995-12-31
Budget Start
1992-01-01
Budget End
1992-12-31
Support Year
12
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Clippinger, Amy J; Maguire, Tobi G; Alwine, James C (2011) The changing role of mTOR kinase in the maintenance of protein synthesis during human cytomegalovirus infection. J Virol 85:3930-9
Yu, Yongjun; Clippinger, Amy J; Pierciey Jr, Francis J et al. (2011) Viruses and metabolism: alterations of glucose and glutamine metabolism mediated by human cytomegalovirus. Adv Virus Res 80:49-67
Yu, Yongjun; Maguire, Tobi G; Alwine, James C (2011) Human cytomegalovirus activates glucose transporter 4 expression to increase glucose uptake during infection. J Virol 85:1573-80
Buchkovich, Nicholas J; Maguire, Tobi G; Alwine, James C (2010) Role of the endoplasmic reticulum chaperone BiP, SUN domain proteins, and dynein in altering nuclear morphology during human cytomegalovirus infection. J Virol 84:7005-17
Chambers, Jeremy W; Maguire, Tobi G; Alwine, James C (2010) Glutamine metabolism is essential for human cytomegalovirus infection. J Virol 84:1867-73
Buchkovich, Nicholas J; Yu, Yongjun; Pierciey Jr, Francis J et al. (2010) Human cytomegalovirus induces the endoplasmic reticulum chaperone BiP through increased transcription and activation of translation by using the BiP internal ribosome entry site. J Virol 84:11479-86
Buchkovich, Nicholas J; Maguire, Tobi G; Paton, Adrienne W et al. (2009) The endoplasmic reticulum chaperone BiP/GRP78 is important in the structure and function of the human cytomegalovirus assembly compartment. J Virol 83:11421-8
Yu, Yongjun; Alwine, James C (2008) Interaction between simian virus 40 large T antigen and insulin receptor substrate 1 is disrupted by the K1 mutation, resulting in the loss of large T antigen-mediated phosphorylation of Akt. J Virol 82:4521-6
Alwine, J C (2008) Modulation of host cell stress responses by human cytomegalovirus. Curr Top Microbiol Immunol 325:263-79
Buchkovich, Nicholas J; Yu, Yongjun; Zampieri, Carisa A et al. (2008) The TORrid affairs of viruses: effects of mammalian DNA viruses on the PI3K-Akt-mTOR signalling pathway. Nat Rev Microbiol 6:266-75

Showing the most recent 10 out of 45 publications