Genetic analyses of human immunodeficiency virus type 1 integrase mutants

Efficient human immunodeficiency virus type 1 (HIV-1) replication is dependent on integrase (IN)-mediated recombination of the viral cDNA into a host chromosome. Previous reports however indicated that IN-defective viruses could replicate in a cell-type dependent manner: permissive cells supported replication of IN-defective HIV-1 and nonpermissive cells failed to support replication. Unintegrated DNA, which can be transcriptionally active, is transiently elevated in cells infected with IN-defective HIV-1, suggesting a potential role in its replication, however unintegrated DNA is typically lost from cells during division. Preserving or amplifying unintegrated DNA could in theory permit replication in nonpermissive cells. Incorporation of the simian virus 40 (SV40) episome amplification cassette into IN-defective HIV-l yielded a chimeric virus capable of replicating in previously nonpermissive Jurkat T-cells and monocyte-derived macrophages. This provided a means by which antivirals could be screened for specificity against IN: agents specific to IN would not block HIV-1/SV40 chimera replication while nonspecific antivirals would inhibit HIV-1 and chimera replication. Although IN is sufficient to catalyze the insertion of viral DNA into target DNA in vitro, IN likely participates in multiple interactions to facilitate virus replication. These include IN multimerization, reverse transcription, host and viral DNA binding, HIV-1 nuclear import, and integration. Previous studies identified IN residues important for nuclear localization. We found that substitutions within putative IN nuclear localization signals (NLSs) did not affect HIV-1 nuclear localization, but instead primarily affected integration and cDNA synthesis. Though the NLS mutant viruses were replication-defective, their INs retained catalytic activity suggesting the loss of an interaction between IN and unidentified factors) essential for post nuclear entry chromosomal targeting of HIV-l. The C-terminal domain of HIV-1 IN contains residues that contribute to DNA binding and multimerization in vitro assays, but the roles of these residues in virus replication were for the most part untested. Substitutions of these residues yielded replication-defective viruses with pleiotropic defects in cDNA synthesis and virion release. One mutant, HIV-1E246K, was notably defective for Pr55Gag polyprotein processing. Thus we uncovered potential new targets for antiviral drug design against IN and a method by which antiviral agent specificity can be determined.