Reversion of a human immunodeficiency virus type 1 matrix mutation affecting Gag membrane binding, endogenous reverse transcriptase activity, and virus infectivity

We previously characterized mutations in the human immunodeficiency virus t ype 1 matrix (MA) protein that displayed reduced infectivity in single-roun d assays, defects in the stable synthesis of viral DNA In infected cells, a nd impaired endogenous reverse transcriptase activity. The mutants, which c ontained substitutions in a highly conserved Leu at MA amino acid 20, also increased binding of Gag to membrane. To elucidate further the role of MA i n the virus replication cycle, we have characterized a viral revertant of a n amino acid 20 mutant (20LK), The revertant virus, which replicates with e ssentially wild-type kinetics in H9 cells, contains second-site compensator y changes at MA amino acids 73 (E-->K) and 82 (A-->T), while retaining the original 20LK mutation. Single-cycle infectivity assays, performed with luc iferase-expressing viruses, show that the 20LK/73EK/82AT triple mutant disp lays markedly improved infectivity relative to the original 20LK mutant. Th e stable synthesis of viral DNA in infected cells is also significantly inc reased compared with that of 20LK DNA, Furthermore, activity of revertant v irions in endogenous reverse transcriptase assays is restored to near-wild- type-levels. Interestingly, although 20LK/73EK/82AT reverses the defects in replication kinetics, postentry events, and endogenous reverse transcripta se activity induced by the 20LK mutation, the reversion does not affect the 20LK-imposed increase in Gag membrane binding. Mutants containing single a nd double amino acid substitutions were constructed, and their growth kinet ics were examined. Only virus containing all three changes (20LK/73EK/82AT) grew with significantly accelerated kinetics; 73EK, 73EK/82AT, and 20LK/82 AT mutants displayed pronounced defects in virus particle production. Viral core-like complexes were isolated by sucrose density gradient centrifugati on of detergent-treated virions. Intriguingly, the protein composition of w ild-type and mutant detergent-resistant complexes differed markedly. In wil d-type and 20LK; complexes, MA was removed following detergent solubilizati on of the viral membrane. In contrast, in revertant preparations, the major ity of MA cosedimented with the detergent-resistant complex. These results suggest that the 20LK/73EK/82AT mutations induced a significant alteration in MA-MA or Il ZA-core interactions.