Article

Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

Louisiana State University Health Sciences Center, United States of America
PLoS ONE (Impact Factor: 3.23). 12/2011; 6(12):e27751. DOI: 10.1371/journal.pone.0027751
Source: PubMed

ABSTRACT We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins.
Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate.
Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.

Download full-text

Full-text

Available from: Mark D Andrake, Aug 24, 2015
0 Followers
 · 
195 Views
  • Source
    • ", 2002 , 2003 , 2004 ) and others ( Bojja et al . , 2011 ; Peletskaya et al. , 2011 ). Our analysis was conducted with the support of the above- mentioned HIV- 1 intasome model ( Krishnan et al . "
    [Show abstract] [Hide abstract]
    ABSTRACT: A functional study of mutants of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) was conducted with the support of a recently proposed HIV-1 intasome model. Firstly, we investigated the predicted position of the C-terminal domain (CTD) and the flexibility of the alpha-6 helix by mutating the residue Ile-203. This had no impact on the 3'-processing reaction but reduced the strand transfer reaction and the formation of tetramers. Secondly, the residues Ile-141 of the catalytic loop and Glu-246 of the CTD are predicted to bind the Td-3 base of the viral DNA maintaining it in a "flipped out" position and stabilizing the catalytic core domain (CCD)-CTD interface. Our data showed that the Ile-141/Td-3 interaction was important for the strand transfer activity and the oligomerization of IN. Interestingly, mutating the Glu-246 residue by an alanine enhanced half- and full-site integrations, suggesting that this residue may not be optimized for integration.
    Virology 03/2013; 439(2). DOI:10.1016/j.virol.2013.02.001 · 3.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 3' DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves as a viral DNA binding surface for RSV IN.
    PLoS ONE 02/2013; 8(2):e56892. DOI:10.1371/journal.pone.0056892 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We determined conditions to produce mg quantities of highly soluble Rous sarcoma virus (RSV) synaptic complex that is kinetically trapped by HIV strand transfer inhibitors (STIs). Concerted integration catalyzed by RSV integrase (IN) is effectively inhibited by HIV STIs. Optimized assembly of the RSV synaptic complex required IN, a gain-of-function 3' OH recessed U3 oligonucleotide, and an STI under specific conditions to maintain solubility of the trapped synaptic complex at 4°C. A C-terminal truncated IN (1-269 residues) produced a homogeneous population of trapped synaptic complex that eluted at ~151,000 Da upon Superdex 200 size exclusion chromatography (SEC). Approximately 90% of input IN and DNA are incorporated into the trapped synaptic complex using either the C-terminally truncated IN or wild type IN (1-286 residues). No STI is present in the SEC running buffer suggesting the STI-trapped synaptic complex is kinetically stabilized. The yield of trapped synaptic complex correlates with the dissociative half-life of the STI observed with HIV IN-DNA complexes. Dolutegravir, MK-2048 and MK-0536 are equally effective while Raltegravir is ~70% as effective. Without an STI present in the assembly mixture, no trapped synaptic complex was observed. Fluorescence and mass spectroscopy analyses demonstrated that the STI remain associated with the trapped complex. SEC-MALS analyses demonstrated that wild type IN and the C-terminal IN truncation are dimers that acted as precursors to the tetramer. The purified STI-trapped synaptic complex contained a tetramer as shown by cross-linking studies. Structural studies of this 3-domain RSV IN in complex with viral DNA may be feasible.
    Journal of Biological Chemistry 05/2014; 289(28). DOI:10.1074/jbc.M114.573311 · 4.57 Impact Factor