Structure-based mutagenesis of the catalytic domain of human immunodeficiency virus type 1 integrase.
Two different crystal structures of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalytic domain were analyzed for interactions at the enzyme active site. Gln-62 and Glu-92 interact with active-site residue Asp-64, and Lys-136 interacts with active-site residue Asp-116 across a dimer interface. Conservative and nonconservative substitutions were introduced at these positions to probe the roles of these interactions in HIV-1 integration. Purified mutant proteins were assayed for in vitro 3' processing, DNA strand transfer, and disintegration activities, and HIV-1 mutants were assayed for virion protein composition, reverse transcription, and infectivities in human cell lines. Each of the mutant IN proteins displayed wild-type disintegration activity, indicating that none of the interactions is essential for catalysis. Mutants carrying Gln or Ala for Glu-92 displayed wild-type activities, but substituting Lys for Glu-92 reduced in vitro 3' processing and DNA strand transfer activities 5- to 10-fold and yielded a replication-defective IN active-site mutant viral phenotype. Substituting Glu for Gln-62 reduced in vitro 3' processing and DNA strand transfer activities 5- to 10-fold without grossly affecting viral replication kinetics, suggesting that HIV-1 can replicate in T-cell lines with less than the wild-type level of IN activity. The relationship between IN solubility and HIV-1 replication was also investigated. We previously showed that substituting Lys for Phe-185 dramatically increased the solubility of recombinant IN but caused an HIV-1 particle assembly defect. Mutants carrying His at this position displayed increased solubility and wild-type replication kinetics, showing that increased IN solubility per se is not detrimental to virus growth.
Engelman
A
A
Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Liu
Y
Y
Chen
H
H
Farzan
M
M
Dyda
F
F
D004279:DNA, Viral / Q000096:biosynthesis; D015497:HIV-1 / Q000737:chemistry / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000737:chemistry / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D012995:Solubility; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000032:analysis; D014771:Virion / Q000737:chemistry; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; D019426:Integrases