title: Structural and sequencing analysis of local target DNA recognition by MLV integrase.
abstract: Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection.
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Sriram
authors.initials: S
authors.lastname: Aiyer
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium (NESG), Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Paolo
authors.initials: P
authors.lastname: Rossi
authors.affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Nirav
authors.initials: N
authors.lastname: Malani
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway, NJ 08854, USA.
authors.forename: William M
authors.initials: WM
authors.lastname: Schneider
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway, NJ 08854, USA.
authors.forename: Ashwin
authors.initials: A
authors.lastname: Chandar
authors.affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium (NESG), Rutgers University, Piscataway, NJ 08854, USA Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA roth@rwjms.rutgers.edu.
authors.forename: Monica J
authors.initials: MJ
authors.lastname: Roth
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D004247:DNA; D019426:Integrases