Project VI: Reverse Transcriptase Complex (Aim III) Project Leader: Nicolas Sluis-Cremer Reverse transcription of the viral single-stranded (+) RNA genome into double-stranded DNA is an essential step in the human immunodeficiency virus (HIV) life-cycle. Although recombinant purified viral reverse transcriptase (RT) can accomplish the DNA polymerization, strand-transfer, strand displacement, and ribonuclease H activities in vitro, in newly infected cells, the reverse transcription reaction is accomplished by RT molecules that are present within a reverse transcription complex (RTC) that includes other viral and cellular factors207'208. In this regard, RTCs have been shown to contain, in addition to RT, viral RNA, tRNALysS (which serves as a primer for reverse transcription), newly synthesized viral DNA, matrix, nucleocapsid, protease, Vpr, and integrase207. Several host factors such as HMGI, barrier to autointegration factor, and lens epithelium-derived growth factor/p75 may also be associated with the RTC207. Surprisingly, however, no host cell factors have been identified that specifically interact with RT. Given that in all living organisms the essential function of DNA replication is a complex process that is carried out by a network of proteins which work together to rapidly and accurately duplicate the genetic information of the cell268, it seems unlikely that reverse transcription of the viral RNA into double-stranded DNA by HIV RT does not also involve host cell proteins. DNA synthesomes have been shown to consist of polymerases and "accessory" enzymes and proteins such as DNA processivity factors, replication factors, helicases, endonucleases, ligases, glycosylases, methyltransferases, kinases, and DNA binding proteins (e.g. histones and assembly factors) that physically interact with the polymerases to regulate the spatial and temporal events involved in replication . In that regard, it is of interest to note, that two separate studies implicated RT phosphorylation by protein kinases and that this phosphorylation event may regulate RT activity, drugresistance, and metabolism '27[unreadable]. These putative biological interactions have not yet been reconstituted in vitro. Therefore, the goal of this exploratory project is to identify host cell factors that interact with HIV-1 RT and regulate the reverse transcription process, using both computational and experimental approaches. These studies will provide unique and novel insights into basic events that precede viral DNA integration.