REVERSION OF A HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE MUTANT AT A 2ND SITE RESTORES ENZYME FUNCTION AND VIRUS INFECTIVITY

The integration of a DNA copy of the retroviral RNA genome into the ho st cell genome is essential for viral replication. The virion-associat ed integrase protein, encoded by the 3' end of the viral pol gene, is required for integration, Stable virus-producing T-cell lines were est ablished for replication-defective human immunodeficiency virus type 1 carrying single amino acid substitutions at conserved residues in the catalytic domain of integrase, Phenotypically reverted virus was dete cted 12 weeks after transfection with the integrase mutant carrying th e P-109-->S mutation (P109S). Unlike the defective P109S virus, the re vertant virus (designated P109S(R)) grew in CD4(+) SupT1 cells, In add ition to the Ser substitution at Pro-109, P109S(R) had a second substi tution of Ala for Thr at position 125 in integrase. Site-directed muta genesis was used to show that the P109S T125A genotype was responsible for the P109S(R) replication phenotype. The T125A substitution also r escued the in vitro enzyme activities of recombinant P109S integrase p rotein. P109S integrase did not display detectable 3' processing or DN A strand transfer activity, although 5 to 10% of wild-type disintegrat ion activity was detected, P109S T125A integrase displayed nearly wild -type levels of 3' processing, DNA strand transfer, and disintegration activities, confirming that T125A is a second-site intragenic suppres sor of P109S. P109S integrase ran as a large aggregate on a size exclu sion column, whereas wild-type integrase ran as a monomer and P109S T1 25A integrase ran as a mixed population, Pro-109 and Thr-125 are not i mmediately adjacent in the crystal structure of the integrase catalyti c domain, We suggest that the T125A substitution restores integrase fu nction by stabilizing a structural alteration(s) induced by the P109S mutation.