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Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase

Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency... Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase Richard Lu , Hina Z. Ghory , and Alan Engelman * Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 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 g ag 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Virology American Society For Microbiology

Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase

Journal of Virology , Volume 79 (16): 10356 – Aug 15, 2005

Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase

Journal of Virology , Volume 79 (16): 10356 – Aug 15, 2005

Abstract

Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase Richard Lu , Hina Z. Ghory , and Alan Engelman * Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 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 g ag 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.

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References (90)

Publisher
American Society For Microbiology
Copyright
Copyright © 2005 by the American society for Microbiology.
ISSN
0022-538X
eISSN
1098-5514
DOI
10.1128/JVI.79.16.10356-10368.2005
pmid
16051828
Publisher site
See Article on Publisher Site

Abstract

Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase Richard Lu , Hina Z. Ghory , and Alan Engelman * Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 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 g ag 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.

Journal

Journal of VirologyAmerican Society For Microbiology

Published: Aug 15, 2005

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