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Associate Professor, Neurology Telephone: 215-204-0607 Fax: 215-204-0679 Email: sawaya@temple.edu
Department of Neurology
Molecular biology of HIV-1 in CNS, role of viral regulatory proteins including Tat and Vpr in the control of HIV-1 transcription and replication, development and use of dominant negative factors for blocking HIV-1 replication; identification of mechanisms that regulate MCP-1 gene expression and the effect of Tat and Vpr on this chemokine.
Research Summary The human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, infects a variety of human cells including brain cells. This infection can be divided into three stages: immediate early (before synthesis of viral proteins), early (after the synthesis of Tat), and late infection (after the synthesis of Vpr proteins). At the immediate early stage of infection, the viral proteins are not synthesized, and therefore, the virus uses a number of cellular factors, which trigger viral transcription. C/EBP and Smad proteins are among the transcription factors identified by our laboratory as a part of many players used by the virus at this stage.
At the early and late stages, the viral regulatory proteins Tat and Vpr are synthesized and active. Tat interacts with the cellular complex p-TEFb in order to enhance viral elongation. A part of our studies is to identify the cellular proteins used by Tat and/or Vpr to induce viral gene expression. We have demonstrated that p21WAF1 and TCF-4 proteins play a certain role during these stages. We have observed a physical interaction between Vpr and p21WAF1. As a result of this interaction, p21 loses its nuclear localization and remains in the cytoplasm which prevents its activity on control of the cell cycle. The interaction between Tat and TCF-4, a member of the Wnt pathway, helps the phosphorylation of Sp1 by DNA-PK. We have shown that TCF-4 inhibits the phosphorylation of Sp1 by DNA-PK as well as prevents Sp1-DNA binding. On the other hand, we have been able to show the formation of a ternary complex between Tat-TCF-4 and p300, which is important for Tat acetylation and plays a major role during elongation.
Another series of studies has focused on the identification of the mechanism involved in the regulation of cdk9 gene expression. Cdk9, in addition to cyclin T1, forms the p-TEFb complex. The main role of cdk9 is the phosphorylation of holoenzyme polymerase II. We have shown that the tumor suppressor protein p53 plays an important role in the activation of the cdk9 promoter as well as has the ability to prevent cdk9 from phosphorylating pol II. We also have been able to demonstrate that cdk9 phosphorylates p53, triggers its ubiquitination by mdm2, hand helps its degradation. This cellular event prevents p53 from causing cell cycle arrest and preventing viral replication. We have been trying to use adno-p53 in order to target HIV-1 infected cells to stop viral regulation.
The de-regulated expression of cytokines and chemokines is a common event seen during the course of HIV-1 infection and the progression of AIDS. MCP-1 is one of these proteins because of its level which is increased in patients with AIDS encephalitis and HIV-1 associated dementia (HAD) patients. MCP-1 is a chemokine that recruits monocytes and lymphocytes to areas of inflammation, a feature which is seen in AIDS brain with CNS disorders. While the mechanism responsible for the increased expression of MCP-1 in HAD remains to be investigated, results from our laboratory and others have demonstrated that the HIV-1 regulatory protein, Tat, has the ability to stimulate transcription of the MCP-1 gene in human astrocytes. We also been investigating the role of C/EBPb and its partners in the basal transcription of MCP-1 in astrocytes and microglial cells. We will assess the effect of Tat on the activity of C/EBPb on its communication with CHOP and c-myb, and determine the impact of various signaling pathways including TNFa and TGFb on C/EBPb-mediated regulation of MCP-1 in cells expressing Tat as well as in cells infected with HIV-1.
Recently, we have shifted some of our research focus to study the regulation of HHV8 immediate early gene expression, ORF-50 and its downstream gene target pK8 alpha. Extracellular Tat binds specific cell surface proteins, affecting immune response inhibition and stimulating Kaposi's sarcoma (KS) tumor cell growth. In addition, Tat has the ability to activate several promoters independently of TAR. Both of these functions are central to AIDS pathogenesis and morbidity. We have shown that Tat uses several cellular proteins to achieve these functions and to activate the ORF-50 promoter of HHV8. Therefore, understanding how Tat interacts with those factors will be critical in developing effective therapies aimed at suppressing the ability of HIV-1 to compromise and preventing KS in AIDS patients. Our goal is to discover how Tat binds different cellular factors, and the molecular basis for this multiple specificity. Finally, we and others have shown the implication of Vpr in causing DNA damage. Several mechanisms were proposed for this function including the interaction between Vpr and HHR23A, a protein involved in DNA repair. We have identified the region within Vpr responsible for causing DNA damage and we have been able to show the mechanism used by Vpr to fulfill this function. This mechanism is made possible through the interaction of Vpr with HHR23A and the release of S5A, a member of the proteosome complexes. Our goal is to use this Vpr function to suppress tumors and/or prevent HIV-1 replication.
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Bassel E. Sawaya, PhD