This Core facility is designed to provide sophisticated molecular techniques for the evaluation of HIV-1 replication in various central nervous system (CNS)-derived cells and tissues. A number of techniques developed in this laboratory will be brought to bear in the understanding of HIV-1 expression in primary cells and cell-lines of CNS origin. Firstly, an in situ polymerase chain reaction (IS-PCR) will be utilized to evaluated HIV-1 proviral copies within an infected cell. Using this technique, a single copy of HIV-1 provirus can be detected within intact cells. As such, this technique allows the sensitive detection of HIV-1 provirus at the single cell level. The morphological characteristics can be accessed to determine cell-type. As well, recent data suggests that this technique can be utilized in association with HIV-1. Various primers and probes can be used in this technique to determine the patterns of HIV-1 reverse transcription, as it has been determined that reverse transcription occurs with varying efficiencies in different cell- types. As such, using specific primer/probes combinations, IS-PCR will be utilized to determine blocks to reverse transcription in various CNS- derived cells. In addition, standard in situ hybridization can be performed. In this methodology, without the amplification steps, the major positive signal represents HIV-1 specific RNA. As such, comparing IS-PCR to standard in situ hybridization, one can determine latently infected from efficiently productive cells infected with HIV-1. In addition, a quantitative reverse transcriptase-initiated PCR technique, developed in this laboratory, will also be utilized to evaluate HIV-1 replication in various CNS-derived cells. Utilizing in vitro transcribed RNA standards and specific primer pairs, this technique allows the quantitative assessment of various species of HIV-1 specific RNA. As many cells latently-infected with HIV-1 have been demonstrated to have a predominance of multiply-spliced, as compared to unspliced HIV-1 specific RNA, this technique will be useful in determining patterns of HIV-1 replication in various neurological cell-types and tissues. In particular, this methodology will be useful in properly assessing the patterns and locations in the viral life-cycle which lead to restricted replication in astrocytes, oligodendrocyte, and microglial cells. As such, we propose a Core facility to study HIV-1 replication in various CNS-derived cell-types. This Core facility will allow efficient use of these powerful molecular techniques in the various proposed projects to elucidate the pathogenesis of HI-1 encephalopathy.
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