FRET analysis reveals distinct conformations of IN tetramers in the presence of viral DNA or LEDGF/p75

Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Research (Impact Factor: 9.11). 07/2011; 39(20):9009-22. DOI: 10.1093/nar/gkr581
Source: PubMed


A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN-viral DNA and IN-LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN-viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN-LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.

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    • "In vitro concerted integration assays allow reproduction of the integration process as observed in vivo . This assay has allowed biochemical and biophysical analyses of the HIV integration as well as to dissect the nucleoprotein complexes intermediates in HIV integration ( Grandgenett et al . , 2009 ; Kessl et al . , 2011 ; Li and Craigie , 2009 ; Pandey et al . , 2011 ) . The donor DNA ( Fig . 3A ) was mixed with a suitable plasmid as acceptor DNA ( pBSK - Zeo ) and the recombinant IN enzyme . Products of the integration reaction ( Fig . 3B ) arise from : ( i ) full - site integration ( also called two - ended or concerted integration ( Moreau et al . ,"
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    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.
    Full-text · Article · Mar 2013 · Virology
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    • "Several groups have described similar 2-(quinolin-3-yl)acetic acid derivatives that inhibit LEDGF/p75-IN binding in vitro and integration during the acute phase of HIV-1 infection (50–53). Initially dubbed LEDGINs (LEDGF/p75-IN inhibitors), it has recently become apparent that these compounds inhibit the assembly of IN on viral DNA (51–53), a step that in all likelihood precedes the binding of LEDGF/p75 to IN during HIV-1 infection (61,62). Accordingly, the drugs inhibit IN catalytic function in vitro in a LEDGF/p75 independent manner (51–53). "
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    ABSTRACT: The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.
    Full-text · Article · Oct 2012 · Nucleic Acids Research
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    • "It remains to be seen whether PFV structures can aid in the elucidation of non-catalytic site INIs, given major differences that may exist distal from the IN active site. For instance, PFV integrase does not interact with LEDGF [87]; as such, models based on PFV may not be able to help in the design of IN-LEDGF inhibitors. Further insights into integration based on PFV structures are discussed in other reviews [88,89]. "
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    ABSTRACT: Integration of the viral genome into host cell chromatin is a pivotal and unique step in the replication cycle of retroviruses, including HIV. Inhibiting HIV replication by specifically blocking the viral integrase enzyme that mediates this step is an obvious and attractive therapeutic strategy. After concerted efforts, the first viable integrase inhibitors were developed in the early 2000s, ultimately leading to the clinical licensure of the first integrase strand transfer inhibitor, raltegravir. Similarly structured compounds and derivative second generation integrase strand transfer inhibitors, such as elvitegravir and dolutegravir, are now in various stages of clinical development. Furthermore, other mechanisms aimed at the inhibition of viral integration are being explored in numerous preclinical studies, which include inhibition of 3' processing and chromatin targeting. The development of new clinically useful compounds will be aided by the characterization of the retroviral intasome crystal structure. This review considers the history of the clinical development of HIV integrase inhibitors, the development of antiviral drug resistance and the need for new antiviral compounds.
    Full-text · Article · Apr 2012 · BMC Medicine
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