The long-term objectives of this research are to construct and charaterize RNA-dependent DNA-polymerase mutants of the human immunodeficiency virus (HIV). Mutations will be introduced into cloned HIV DNA sing three different approaches. First, linker insertions will be made within the DNA polymerase coding domain. Second, deletions will be made at the site of silent insertions. Third, random point mutations will be made by chemical modification of viral DNA. DNA polymerase activity will be assayed after expression of the mutated DNA polymerase gene in bacteria. Following this initial screening of DNA polymerase mutants, selected mutants will br recombined into an appropirate expression vector, and the mutant will be expressed in animal cells. Chemical mutagenesis will be targeted to the DNA polymerase domain of an infectious HIV proviral DNA clone and the mutated HIV DNA will be introduced in cells susceptable to HIV infection. Drug-resistant DNA polymerase mutants will be selected for their ability to grow in the presence of the DNA polymerase inhibitor A509U. We will also examine the genetic basis of drug-resistant HIV mutants isolated in a clinical setting. Using these approaches we will develop a detailed genetic map of the HIV DNA polymerase domain, a map that relates the primary structure of the enzyme to its catalytic function.
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