Selection of amino acid substitutions restoring activity of HIV-1 integrase mutated in its catalytic site using the yeast Saccharomyces cerevisiae

The integration of proviral DNA into the genome of the host cell is an esse ntial step in the replication of retroviruses. This reaction is catalyzed b y a viral-encoded enzyme, the integrase (IN). We have previously shown that human immunodeficiency virus type 1 (HIV-1) IN causes a lethal effect when expressed in yeast cells. This system, called yeast lethal assay, was used as a tool to study IN activity in a cellular context. The yeast lethal ass ay allowed the selection and characterization of mutations affecting both t he lethal phenotype and the in vitro IN activities. IN mutants were produced by random PCR mutagenesis in an IN gene bearing th e inactivating D116A mutation in the catalytic site. The corresponding D116 A substituted IN does not lead to lethality in yeast. Subsequent selection of mutants able to restore the lethal effect of IN was carried out using th e yeast lethal assay. We isolated three mutants presenting a restored pheno type. The mutated IN genes were sequenced and the corresponding proteins we re purified to characterize their in vitro activities. The three mutants pr esented restoration of the in vitro strand transfer activity, while 3' proc essing was only partially restored. The three mutants differ from D116A IN by at least one amino acid substitut ion located in the N-terminal domain of the protein, outside of the active site. These new mutated HIV-1 INs may therefore allow a better understandin g of the N-terminal domain function in the integration reaction. In additio n, these results support our hypothesis that explains the lethal effect as a consequence of the nuclear damage caused by wildtype IN in yeast cells. T hese data also indicate that the yeast lethal assay can be used as a tool t o study the retroviral integration mechanism in a cellular context and to s elect specific inhibitors. (C) 2000 Academic Press.