STRUCTURE-BASED MUTAGENESIS OF THE CATALYTIC DOMAIN OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE

Two different crystal structures of the human immunodeficiency virus t ype 1 (HIV-1) integrase (IN) catalytic domain were analyzed for intera ctions at the enzyme active site, Gln-62 and Glu-92 interact with acti ve-site residue Asp-64, and Lys-136 interacts with active-site residue Asp-116 across a dimer interface, Conservative and nonconservative su bstitutions were introduced at these positions to probe the roles of t hese interactions in HIV-1 integration, Purified mutant proteins were assayed for in vitro 3' processing, DNA strand transfer, and disintegr ation activities, and HIV-1 mutants were assayed for virion protein co mposition, reverse transcription, and infectivities in human cell line s. Each of the mutant IN proteins displayed wild-type disintegration a ctivity, indicating that none of the interactions is essential for cat alysis, Mutants carrying Gin or Ala for Glu-92 displayed wild-type act ivities, but substituting Lys for Glu-92 reduced in vitro 3' processin g and DNA strand transfer activities 5- to 10-fold and yielded a repli cation-defective IN active-site mutant viral phenotype. Substituting G lu for Gln-62 reduced in vitro 3' processing and DNA strand transfer a ctivities 5- to 10-fold without grossly affecting viral replication ki netics, suggesting that HIV-1 can replicate in T-cell lines with less than the wild-type level of IN activity, The relationship between IN s olubility and HIV-I replication was also investigated, We previously s howed that substituting Lys for Phe-185 dramatically increased the sol ubility of recombinant IN but caused an HIV-1 particle assembly defect . Mutants carrying His at this position displayed increased solubility and wild-type replication kinetics, showing that increased IN solubil ity per se is not detrimental to virus growth.