Sequential Deletion of the Integrase (Gag-Pol) Carboxyl Terminus Reveals Distinct Phenotypic Classes of Defective HIV-1

Abstract

A requisite step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is the insertion of the viral genome into that of the host cell, a process catalyzed by the 288-amino-acid (32-kDa) viral integrase (IN). IN recognizes and cleaves the ends of reverse-transcribed viral DNA and directs its insertion into the chromosomal DNA of the target cell. IN function, however, is not limited to integration, as the protein is required for other aspects of viral replication, including assembly, virion maturation, and reverse transcription. Previous studies demonstrated that IN is comprised of three domains: the N-terminal domain (NTD), catalytic core domain (CCD), and C-terminal domain (CTD). Whereas the CCD is mainly responsible for providing the structural framework for catalysis, the roles of the other two domains remain enigmatic. This study aimed to elucidate the primary and subsidiary roles that the CTD has in protein function. To this end, we generated and tested a nested set of IN C-terminal deletion mutants in measurable assays of virologic function. We discovered that removal of up to 15 residues (IN 273) resulted in incremental diminution of enzymatic function and infectivity and that removal of the next three residues resulted in a loss of infectivity. However, replication competency was surprisingly reestablished with one further truncation, corresponding to IN 269 and coinciding with partial restoration of integration activity, but it was lost permanently for all truncations extending N terminal to this position. Our analyses of these replication-competent and -incompetent truncation mutants suggest potential roles for the IN CTD in precursor protein processing, reverse transcription, integration, and IN multimerization.The defining hallmarks of retroviruses are reverse transcription of the viral genomic information as encoded in polyadenylated RNA and the subsequent integration of the copied DNA genome into that of a host cell. The latter is an essential and irreversible event which is mediated by the catalytic activities of the viral integrase protein (IN), the recent target of successful chemotherapeutic intervention against HIV-1 infection (1). HIV-1 IN is a 288-amino-acid, 32-kDa protein that is cleaved from the C terminus of the Gag-Pol polyprotein (Pr160 Gag-Pol ) via viral proteolytic activity. The biochemical mechanisms that lead to retroviral integration, which have been extensively studied in vitro, are defined by two catalytically related and sequentially dependent steps (18) which may be distinguished by their respective sensitivities to current inhibitors of IN function (39). Following the completion of reverse transcription of the viral RNA into its DNA copy, IN removes two nucleotides from the 3Ј end of each strand of the viral DNA. This step, termed 3Ј processing, generates a chemically reactive 3Ј-hydroxyl group (CA OH -3Ј) at the 3Ј ends of the DNA molecule, effectively activating the termini for the subsequent reaction (strand transfer). This enzymatic step is the target of all I...

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

“…Besides integration and reverse transcription, other steps include nuclear import of preintegration complexes (26,(35)(36)(37), polyprotein processing, assembly, and maturation (22, 31, 38-40, 65, 74). Since IN is synthesized and packaged into the immature virion as part of the Gag-Pol polyprotein, many alterations in virion morphology and defects at the late steps of replication may be caused by the effect of IN mutations on the Gag-Pol precursor protein (22,38,40,65,74). However, certain IN mutations, such as His12Ala and Phe185Ala substitutions and deletion of the C-terminal 22 residues, specifically impair reverse transcription with no apparent effects on the GagPol polyprotein and other steps in the life cycle (29,30,33).…”

The HIV-1 Integrase Mutant R263A/K264A Is 2-fold Defective for TRN-SR2 Binding and Viral Nuclear Import

“…However, because IN is an essential component of the nucleoprotein complex in the budding virus and of the reverse transcription complex, IN mutations can result in pleiotropic effects extending well beyond the integration step. It is well established that some IN mutants negatively affect reverse transcription (44,45). Of the five amino acids identified as hot spots for interaction with TRN-SR2 (see Fig.…”

Identification of Residues in the C-terminal Domain of HIV-1 Integrase That Mediate Binding to the Transportin-SR2 Protein

“…K186Q, R228A, R262A/R263A, R262A/K264A, K266A, and R269A) were shown to affect reverse transcription as well (51,52,56). Unfortunately, all mutants identified in this study are known to display reduced reverse transcription during replication (51,52,56), confounding the study of their role in the nuclear import of HIV in cellular replication assays. Still, insight into the hot spots that define the protein-protein interaction between IN and TRN-SR2 is necessary to guide future drug discovery efforts targeting the nuclear entry step of replication.…”

“…The interface of this interaction has been mapped to three crucial amino acids (K258A, W243E, and V250E) (55), but IN mutations outside of this interface (e.g. K186Q, R228A, R262A/R263A, R262A/K264A, K266A, and R269A) were shown to affect reverse transcription as well (51,52,56). Unfortunately, all mutants identified in this study are known to display reduced reverse transcription during replication (51,52,56), confounding the study of their role in the nuclear import of HIV in cellular replication assays.…”

“…Multiple IN mutations were inserted into viruses and tested for their impact on viral replication. IN mutants affecting HIV nuclear import were assayed for reduced a two-LTR circle formation (51,52). Despite these extensive studies, so far no single NLS was unambiguously identified as the determinant of HIV nuclear import.…”