Genetic analyses of DNA-binding mutants in the catalytic core domain of human immunodeficiency virus type 1 integrase.
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.
Lu
Richard
R
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney St. Boston, MA 02115, USA.
Limón
Ana
A
Ghory
Hina Z
HZ
Engelman
Alan
A
D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D019427:HIV Integrase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D014779:Virus Replication
D004247:DNA; D019427:HIV Integrase