title: Engineered hyperactive integrase for concerted HIV-1 DNA integration.
abstract: The DNA cutting and joining reactions of HIV-1 integration are catalyzed by integrase (IN), a viral protein that functions as a tetramer bridging the two viral DNA ends (intasome). Two major obstacles for biochemical and structural studies of HIV-1 intasomes are 1) the low efficiency of assembly with oligonucleotide DNA substrates, and 2) the non-specific aggregation of both intasomes and free IN in the reaction mixture. By fusing IN with a small non-specific DNA binding protein, Sulfolobus solfataricus chromosomal protein Sso7d (PDB: 1BNZ), we have engineered a highly soluble and hyperactive IN. Unlike wild-type IN, it efficiently catalyzes intasome assembly and concerted integration with oligonucleotide DNA substrates. The fusion IN protein also functions to integrate viral reverse transcripts during HIV-infection. The hyperactive HIV-1 IN may assist in facilitating future biochemical and structural studies of HIV-1 intasomes. Understanding the mechanistic basis of the Sso7d-IN fusion protein could provide insight into the factors that have hindered biophysical studies of wild-type HIV-1 IN and intasomes.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Kellie A
authors.initials: KA
authors.lastname: Jurado
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Shiqiang
authors.initials: S
authors.lastname: Lin
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Alan
authors.initials: A
authors.lastname: Engelman
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Craigie
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology; D019427:HIV Integrase / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000235:genetics* / Q000378:metabolism; D016662:Virus Integration
chemical_list: D019843:Archaeal Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C093228:Sso7d protein, Sulfolobus; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1