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.

Sites of retroviral DNA integration: From basic research to clinical applications

“…The extent of central tDNA flexibility was moreover inversely proportional to TSD length. The examined viruses harbored a neutral, compact amino acid at the position analogous to Ala188 in PFV, and the polarity of the amino acid side chain correlated with the positioning of base preference significance relative to the points of vDNA insertion – a finding that was since confirmed by analyzing the behavior of mutants of the non-polar Pro187 side-chain in Mo-MLV IN (Aiyer et al, 2015). Taken together these studies imply that, though retroviral INs have structurally evolved to target unique nucleotide signatures, the common functional purpose of integration site base preferences may be to generate strand transfer-facilitating central tDNA distortion within the TCC.…”

“…This finding was in line with prior (Appa et al, 2001, Harper et al, 2001, Nowak et al, 2009) and subsequent (Demeulemeester et al, 2014) studies that implicated Ser119 in the process of HIV-1 tDNA site selection. The analogous residue in Mo-MLV IN, Pro187, plays the same role as Ala188 in PFV IN and Ser119 in HIV-1 in determining tDNA base selectivity (Aiyer et al, 2015). …”

“…Also intriguing is that chromatin density in the regions surrounding integration sites can negatively modulate the DNA strand transfer activity of some retroviral INs, while having the opposite effect on other proteins (Taganov et al, 2004, Lesbats et al, 2011, Benleulmi et al, 2015). It nonetheless appears that, regardless of viral preferences for chromatin density in the region surrounding the average integration site, distorted phosphodiester bonds are generally favored by IN at the points of integration (Maertens et al, 2010, Serrao et al, 2014, Serrao et al, 2015, Aiyer et al, 2015), and nucleosomes provide for natural sources of DNA distortion. The mechanism of how nucleosome structure could accommodate the natural propensity to integrate into flexible tDNA sequences was recently revealed through the cryo-electron microscopy structure of the PFV intasome in complex with a bound nucleosome (Maskell et al, 2015).…”

Retroviral DNA Integration

“…Initially identified by limited proteolysis of HIV-1 IN, 108 the domains were characterized in isolation using X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy (Figure 2a). 147−153 The NTD folds into a compact three-helical bundle stabilized by coordination of a Zn 2+ ion by side chains of His and Cys residues comprising the invariant HHCC motif. 147,150 Often misidentified as a “zinc finger domain”, the NTD is most closely related to helix–turn–helix DNA binding domains.…”

Structure of the Brd4 ET domain bound to a C-terminal motif from γ-retroviral integrases reveals a conserved mechanism of interaction

“…1B, yellow) (12,23), and NMR studies of the MLV IN C-terminal domain revealed that this region is unstructured, suggesting a "fly-casting" mechanism for BET protein binding (23). Modifications to this region compromise MLV IN binding to BET proteins and alter MLV integration patterns (12,(23)(24)(25). In addition, the sequence of the ET domain is highly conserved (Fig.…”