Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase

Abstract

The catalytic core domain (CCD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) harbors the enzyme active site and binds viral and chromosomal DNA during integration. Thirty-five CCD mutant viruses were constructed, paying particular attention to conserved residues in the Phe 139 -Gln 146 flexible loop and abutting Ser 147 -Val 165 amphipathic alpha helix that were implicated from previous in vitro work as important for DNA binding. Defective viruses were typed as class I mutants (specifically blocked at integration) or pleiotropic class II mutants (additional particle assembly and/or reverse transcription defects). Whereas HIV-1 P145A and HIV-1 Q146K grew like the wild type, HIV-1 N144K and HIV-1 Q148L were class I mutants, reinforcing previous results that Gln-148 is important for DNA binding and uncovering for the first time an important role for Asn-144 in integration. HIV-1 Q62K , HIV-1 H67E , HIV-1 N120K , and HIV-1 N155K were also class I mutants, supporting findings that Gln-62 and Asn-120 interact with viral and target DNA, respectively, and suggesting similar integration-specific roles for His-67 and Asn-155. Although results from complementation analyses established that IN functions as a multimer, the interplay between active-site and CCD DNA binding functions was unknown. By using Vpr-IN complementation, we determined that the CCD protomer that catalyzes integration also preferentially binds to viral and target DNA. We additionally characterized E138K as an intramolecular suppressor of Gln-62 mutant virus and IN. The results of these analyses highlight conserved CCD residues that are important for HIV-1 replication and integration and define the relationship between DNA binding and catalysis that occurs during integration in vivo.An essential step in the retroviral life cycle is the integration of the cDNA made by reverse transcription into a cell chromosome. Integration is mediated by the viral integrase (IN) protein, a specialized DNA recombinase that catalyzes two distinct endonucleolytic reactions during the early phase of infection. The first reaction, 3Ј processing, occurs shortly after reverse transcription is completed. During 3Ј processing, human immunodeficiency virus type 1 (HIV-1) IN removes a GT dinucleotide from each end of linear viral cDNA. IN performs its second reaction, DNA strand transfer, after finding a suitable chromosome target for integration. During DNA strand transfer, IN uses the oxygen moieties at the processed 3Ј ends to cut the target DNA at two 5Ј phosphates that are separated by 5 bp, which concomitantly joins the 3Ј ends to the chromosome. The resulting DNA recombination intermediate, with viral 5Ј ends unattached to the chromosome, is likely repaired by cellular enzymes (see references 16 and 34 for reviews).Partial proteolysis (24), deletion mutagenesis (9, 70), and functional complementation (23, 69) analyses revealed that HIV-1 IN is comprised of three domains, the N-terminal domain, catalytic core domain (CCD), and C-terminal domain (CTD) (reviewe...

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

“…Lee and Robinson (63) have analyzed the HIV-1 IN mutant S153Y and demonstrated that this mutation causes only a small delay in virus replication. The S153R, studied in this report, has been analyzed recently in the context of the virus system in cells and found to have little or no detectable affect on viral replication (68). In contrast to position 153, the substitution at position 72 (V72W) changed to a larger extent the specificity for 3Ј processing from the HIV-1 to the RSV LTR (Fig.…”

Integrase-defective lentiviral vectors: progress and applications

“…12,62 The N-terminal domain contains an HH-CC zinc-finger binding domain that primarily binds viral DNA 5,63,64 and facilitates required IN multimerization. 65,66 The C-terminal domain is the least conserved retroviral IN domain 5,67,68 and binds nonspecific target DNA. 63,69 The IN core domain contains the D-D35E catalytic amino-acid triad motif, comprised of amino-acids D64, D116 and E152 in HIV.…”

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.…”