Correlation of Recombinant Integrase Activity and Functional Preintegration Complex Formation during Acute Infection by Replication-Defective Integrase Mutant Human Immunodeficiency Virus

Abstract

Previous studies characterized two types of replication-defective human immunodeficiency virus type 1 (HIV-1) integrase mutants: class I, which are specifically blocked at the integration step, and class II, which harbor additional virion production and/or reverse transcription defects. Class I mutant enzymes supported little if any metal ion-dependent 3=-processing and DNA strand transfer activities in vitro, whereas class II enzymes displayed partial or full catalytic function in studies with simplified assay designs, suggesting that defective interaction(s) with heterologous integrase binding proteins might underlie the class II mutant viral phenotype. To address this hypothesis, class I and II mutant enzymes were interrogated under expanded sets of in vitro conditions. The majority failed to catalyze the concerted integration of two viral DNA ends into target DNA, highlighting defective integrase function as the root cause of most class II in addition to all class I mutant virus infection defects. One mutant protein, K264E, in contrast, could support the wild-type level of concerted integration activity. After accounting for its inherent reverse transcription defect, HIV-1 K264E moreover formed preintegration complexes that supported the efficient integration of endogenous viral DNA in vitro and normal levels and sequences of 2-long terminal repeat-containing circle junctions during acute infection. K264E integrase furthermore efficiently interacted in vitro with two heterologous binding partners, LEDGF/p75 and reverse transcriptase. Our results underscore the physiological relevance of concerted integration assays for tests of integrase mutant function and suggest that the K264E mutation disrupts an interaction with an intranuclear integrase binding partner that is important for HIV-1 integration.

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

“…5D). Thus our and published (72,73) findings collectively argue for the importance of this IN segment for HIV-1 integration and call for further more detailed genetic analysis of these substitutions during HIV-1 infection. Because the IN R262E/R263E/K264E mutant is defective for interactions with TNPO3, Importin-␣3, and viral DNA, the main focus of future genetic studies should be to carefully delineate pleotropic affects arising from these substitutions.…”

“…Furthermore, a more recent report has uncovered the significance of Lys-264 for the IN interaction with an undefined nuclear import factor and HIV-1 integration (73). Although the K264E substitution fully impaired IN CTD binding with TNPO3 (Fig.…”

Multimode, Cooperative Mechanism of Action of Allosteric HIV-1 Integrase Inhibitors

“…The concentration of HIV-1 NL4-3 in the supernatant of plasmid pNL4 -3-transfected HEK293T cells was determined using a radionuclide-based exogenous assay for reverse transcriptase (RT) activity (27), and SupT1 cells (4 ϫ 10 4 per well of a 96-well plate) were infected with 5 ϫ 10 5 32 P counts per minute in 200 l. The effective concentration of compound required to inhibit 50% (EC 50 ) of HIV-1 replication was determined after 5 days using the WST-1 assay (Roche Applied Science) to quantify cell viability. Control compounds raltegravir and saquinavir were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.…”

Interaction between Reverse Transcriptase and Integrase Is Required for Reverse Transcription during HIV-1 Replication

“…Depending on their effects on viral processes, IN mutants are grouped as either class I or class II (20)(21)(22)(23). Class I IN mutants are enzymatically inactive in catalyz-ing the 3= end processing and strand transfer, and viruses harboring such mutations are specifically blocked at the integration step.…”