| Several drugs target the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT); however, the evolution of resistant viruses has reduced the efficacy of these therapies. The development of new drugs for inhibiting RT, despite inherent difficulties, depends on a better understanding RT function within infectious HIV-1 particles. First, mutations in RT/Gag-Pol have pleiotropic effects on virus replication, including impairment in virion assembly and maturation. Second, since both RT subunits (p51 and p66) are derived from the same coding region and since the relative arrangement of their subdomains differs markedly, mutations are structurally and functionally nonequivalent between the two subunits. Both of these characteristics have hindered detailed structure-function analysis of RT and, in particular, its individual subunits in the context of infectious virions and human target cells.; This dissertation describes a subunit-specific molecular analysis of RT in infectious virions that uses a novel, biologically relevant approach developed during the course of this research endeavor. The specific approaches, alternative strategies, and pitfalls that shaped this viable approach to analyzing RT are discussed. We demonstrate the ability of this novel approach to analyze anti-RT drugs in a subunit-specific manner, which is significant since it provides new avenues to analyze and understand drug resistance and viral fitness. By analyzing subunit-specific function with infectious particles, we demonstrate that p66 is alone sufficient for the polymerase catalytic function of RT. Furthermore, we found that residues involved in p66-mediated catalysis are catalytically nonfunctional in p51 but rather are structurally important in p51 for an active RT heterodimer in virions and infected cells. Finally, we examine the contributions of amino acid residues in the "tryptophan-repeat motif", a structural motif at the dimer interface composed of the connection subdomains of the two subunits, to RT subunit interaction and function and identify specific residues that are important for subunit interface interactions and virus replication.; This dissertation research provides important new insights into the role of p51 and p66 in HIV-1 reverse transcription. We anticipate that future research based upon this dissertation work will help further elucidate determinants that mediate p51/p66 subunit interactions and will have direct relevance in structure-based drug design targeting RT heterodimerization. |
