title: Genetic analyses of conserved residues in the carboxyl-terminal domain of human immunodeficiency virus type 1 integrase.
abstract: Results of in vitro assays identified residues in the C-terminal domain (CTD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) important for IN-IN and IN-DNA interactions, but the potential roles of these residues in virus replication were mostly unknown. Sixteen CTD residues were targeted here, generating 24 mutant viruses. Replication-defective mutants were typed as class I (blocked at integration) or class II (additional reverse transcription and/or assembly defects). Most defective viruses (15 of 17) displayed reverse transcription defects. In contrast, replication-defective HIV-1(E246K) synthesized near-normal cDNA levels but processing of Pr55(gag) was largely inhibited in virus-producing cells. Because single-round HIV-1(E246K.Luc(R-)) transduced cells at approximately 8% of the wild-type level, we concluded that the late-stage processing defect contributed significantly to the overall replication defect of HIV-1(E246K). Results of complementation assays revealed that the CTD could function in trans to the catalytic core domain (CCD) in in vitro assays, and we since determined that certain class I and class II mutants defined a novel genetic complementation group that functioned in cells independently of IN domain boundaries. Seven of eight novel Vpr-IN mutant proteins efficiently trans-complemented class I active-site mutant virus, demonstrating catalytically active CTD mutant proteins during infection. Because most of these mutants inefficiently complemented a class II CCD mutant virus, the majority of CTD mutants were likely more defective for interactions with cellular and/or viral components that affected reverse transcription and/or preintegration trafficking than the catalytic activity of the IN enzyme.
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Lu
authors.forename: Hina Z
authors.initials: HZ
authors.lastname: Ghory
authors.forename: Alan
authors.initials: A
authors.lastname: Engelman
mesh_terms: D002478:Cells, Cultured; D004279:DNA, Viral / Q000096:biosynthesis; D016336:Gene Products, vpr / Q000502:physiology; D005816:Genetic Complementation Test; D019427:HIV Integrase / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D014779:Virus Replication; D054325:vpr Gene Products, Human Immunodeficiency Virus
chemical_list: D004279:DNA, Viral; D016336:Gene Products, vpr; D054325:vpr Gene Products, Human Immunodeficiency Virus; D019427:HIV Integrase