HIV-1 infection is often associated with central nervous system (CNS) dysfunction. Detection of HIV-l in microglia/macrophages, as well as in astrocytes of brains from AIDS patients led us to believe that these cells have an important role in inducing CNS disease directly by accommodating virus gene expression and replication, and indirectly by releasing viral and cellular factors. Expression and replication of the HIV- 1 genome in CNS and non CNS cells is regulated by a series of cell-derived transcription factors which exhibit unique ability to interact directly or indirectly with various DNA sequences located in the viral control region spanning the long terminal repeat (LTR). In addition, viral-derived regulatory factors such as Tat and Vpr which are produced at immediate early and late phases of lytic infection, respectively, often play a pivotal role in enhancing transcriptional activity of the LTR. In particular, extensive study on Tat protein revealed that cooperative interaction of this viral protein with several cellular factors including cyclin T and its partner, cdk9, and the LTR sequence called TAR, which is positioned between +19 to +43 with respect to the transcription start site at +1, is critical for the transactivation of the viral genome by Tat in a variety of lymphoid cells. However, our earlier results showed that in astrocytic cells, TAR is dispensable as Tat has the ability to communicate with the alternative regulatory sequence, i.e. KB motif which is positioned between -104 to -81, and its binding factors, the NFkB p50/p65, and facilitates HIV-1 replication in these cells. Our most current results showed that Tat can also interact with the viral late regulator protein Vpr and that may include participation of several other important cellular factors such as p300 and p21, all of which exert their regulatory action through the KB motif of the LTR. Thus, it is evident that a delicate regulatory mechanism, which involves the cooperative physical and functional interaction of the viral and cellular proteins may modulate HIV- 1 gene expression and replication in the infected cells. In this research project we will focus our attention on microglial and astrocytic cell infection and by employing molecular biology, genetics, and virological approaches. We will: i) investigate the interaction of Tat and Vpr in these cells during the course of infection; ii) study the involvement of cellular proteins such as p300 and p21 alone and/or in context of Tat and Vpr via NFKB in the modulation of HIV-l replication; and iii) create and utilize dominant regulatory proteins based on Vpr and Tat to investigate the mechanism of viral gene regulation by these proteins in microglia and astrocytes. In parallel, clinical samples from AIDS patients with neurological disorders will be utilized to evaluate the expression and interaction of relevant viral and cellular factors. The outcome from these molecular studies will generate important and unique information, which could be translated into devising and utilizing an effective strategy for the prevention and treatment of HIV-l induced CNS injury in AIDS.
