STRUCTURE/FUNCTION RELATIONSHIP IN HIV1 RT

描述(改编自申请人的摘要)：本提案的目标是 定义HIV-1RT的生化和结构方面。这将涉及到 氨基酸残基和蛋白质结构域的鉴定 逆转录。新近解决的HIV-1RT-AS的三维晶体结构 酶-DNA-dNTP三元复合体表明，许多氨基酸可能是 参与特定的酶促步骤。这些残留物将由 定点突变，突变体的生化性质将被 其特征是确定它们在催化中的功能作用。诱变 将包括沿DNA结合轨迹的氨基酸来确定影响 对这些残基进行RNase-H和链转移反应。删除/插入 P51亚单位的点突变将决定它们在催化中的作用 以及便于将P66装载到模板底漆上。的影响 这些变异对病毒复制和传染性的影响将被确定。这些 研究将有助于确定氨基酸的侧链取向 催化域。氨基酸功能意义的赋值 RT的3D结构将促进更好地理解 RT催化DNA合成的机制，抗RT药物的抑制作用， 以及耐药的结构性基础。具体目标是：1)。至 确定p51在逆转录中的功能作用，2)。要确定 3)特定氨基酸在RT催化中的功能作用。要确定 RT突变对HIV-1复制/传染性的体内影响，3)。至 确定突变热点与体系结构之间的相关性 HIV-1基因组RNA

DESCRIPTION (Adapted from applicant's abstract): The goal of this proposal is to define biochemical and structural aspects of HIV-1 RT. This will involve identification of amino acid residues, and protein domains that are involved in reverse transcription. The recently solved 3D crystal structure of HIV-1 RT as an enzyme-DNA-dNTP ternary complex suggests a number of amino acids that may be involved in specific enzymatic steps. These residues will be modified by site-directed mutagenesis, and biochemical properties of the mutants will be characterized to determine their functional roles in catalysis. Mutagenesis will include amino acids along the DNA binding track to determine the influence of these residues on RNAse-H and strand transfer reactions. Deletion/insertion and point mutations in the p51 subunit will determine their roles in catalysis and in facilitating loading of p66 onto the template primer. The effect of these mutations on viral replication and infectivity will be determined. These studies will help to define the side chain orientation of amino acids in the catalytic domain. The assignment of functional significance to amino acids within the 3D structure of RT will promote better understanding of the mechanisms of RT catalyzed DNA synthesis, inhibitory action of anti-RT drugs, and structural basis for drug resistance. The specific aims are: 1). to determine the functional role of p51 in reverse transcription, 2). to determine the functional role of specific amino acids in RT catalysis, 3). to determine the in vivo effect of RT mutations on HIV-1 replication/infectivity, 3). to determine the correlation between mutational hotspots and the architecture of HIV-1 genomic RNA