Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase
Ghory, Hina Z.
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CitationLu, R., A. Limon, H. Z. Ghory, and A. Engelman. 2005. “Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase.” Journal of Virology 79 (4): 2493–2505. https://doi.org/10.1128/jvi.79.4.2493-2505.2005.
AbstractThe 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 11 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.
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