In the next 5-year phase of this R37 grant, we will continue our investigations in 2 of the three aims originally proposed. The third Aim, is a new aim and outgrowth of the current trends In HIV research of Investigating the role of host factors in specific viral functions. In Aim 1, we will continue the wori< on the RT determinants of dNTP selection and expand into determinants of mispair extension fidelity. We will further expand from our novel observation that the K65 residue plays a critical role in forcing the insertion of incorrect basepairs due to the interaction between the lysine side chain and the y phosphate of incoming dNTP. We will use two novel ways of improving the fidelity of HIV-1 RT variants in which we will either use dNTP analogs that do not have a negative charge on the y phosphate or remove the positive charge on the amino acid side chain by using unnatural amino acid residues. We believe that these two methods afford powerful means of testing the pivotal importance of this interaction to HIV-1 RT accuracy. We have also made a novel observation that certain amino acid substitutions and certain sequences in template-primer dnve the RT molecule to an unproductive, -2 position with respect to the templating base position. We will further explore and delineate the various conditions that facilitate a higher fidelity by displacing RT. In Aim 2, we will continue our previous work on RT-IN complexes by co-crystallizing HIV-1 RT-IN complexes in collaboration with Dr. Steve Almo. When the structural information becomes available, we can carry out site-directed mutagenesis to disrupt residues in the interaction interface and carry out genetic protein-protein interaction assays and virological assays to determine the significance of the interaction. In Aim 3, we will Investigate the role of host factors in HIV reverse transcription. We will examine the genes Implicated in reverse trancription by shRNA screens by Konig et al. The specific role of selected genes in reverse transcription will be confirmed by eliminating possible effects on entry and/or uncoating. First, we will develop an interactome of each hit via co-1.P. from both infected and uninfected cells to understand the pathways involved. Second, we will silence other members of the complexes thus identified for role in reverse transcription. Third, we will test whether the silencing of the 'hit's or their interacting partners will affect only HIV or also the related lenti- and retroviruses including SIVcpz, SIVmac, HIV-2, MuLV and RSV - to understand the wider significance of the proteins thus identified to retroviruses.