Dolutegravir Interactions with HIV-1 Integrase-DNA: Structural Rationale for Drug Resistance and Dissociation Kinetics

<div><p>Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.</p> </div

Structural model of Q148R HIV-1 integrase with U5 LTR DNA.

<p>The side chain of residue Q148R was modeled interacting with the side chain of E152 and in this conformation the residue may interfere with the binding of elvitegravir. Molecular representations and coloring schemes as described in Figure 2.</p

Structural models of HIV-1 integrase with U5 LTR DNA and (A, B) raltegravir, (C) elvitegravir or (D) dolutegravir.

<p>For raltegravir, the terminal 3′ adenylate is depicted in 2 distinct conformations: panel 2A shows the published conformer and panel 2B shows an alternative conformer that is also consistent with the observed electron density. Raltegravir, elvitegravir and dolutegravir are in stick representation with carbon, nitrogen, oxygen, fluorine and chlorine atoms colored gray, blue, red, cyan and green, respectively. A select subset of amino acids and nucleotides is depicted and labeled with residue type and number (numbering schemes as listed in Figure S1); all residues are in stick representation with carbon atoms colored by secondary structure/chain and nitrogen and oxygen atoms colored blue and red, respectively. The Mg²⁺ ions are represented as small yellow spheres with coordinate bonds to the inhibitors depicted as dashed yellow lines.</p

2D structures of (A) dolutegravir, (B) raltegravir and (C) elvitegravir.

<p>Red ovals encircle the oxygen atoms that chelate the divalent metal cations in the active site; green ovals encircle the halobenzyl groups; and blue boxes encircle the approximate regions of the scaffolds that can accommodate positive charge after chelation of the metals. The purple circles at (B) encircle raltegravir’s gem-dimethyl (small circle) and oxadiazole groups, and the purple oval at (C) encircles elvitegravir’s 1-hydroxymethyl-2-methylpropyl group.</p

Structural models of (A) Q148H/G140S and (B) N155H HIV-1 integrase with U5 LTR DNA and dolutegravir.

<p>(A) The Q148H/G140S mutations are predicted to disrupt the structure of the flexible active-site loop, displacing the 3₁₀ helix away from the DDE motif and weakening the H-bond interaction between the backbone CO of Q148H and the backbone NH of E152. (B) The N155H mutation is predicted to disrupt the structure of the α4 helix, widen the base of the catalytic pocket, alter the placement of at least the Mg²⁺ ion coordinated to residues D64 and E152 and alter the conformation of the terminal 3′ adenosine forming part of the pocket. Molecular representations and coloring schemes are described in Figure 2.</p

Phosphorylation of the HIV-1 capsid by MELK triggers uncoating to promote viral cDNA synthesis

<div><p>Regulation of capsid disassembly is crucial for efficient HIV-1 cDNA synthesis after entry, yet host factors involved in this process remain largely unknown. Here, we employ genetic screening of human T-cells to identify maternal embryonic leucine zipper kinase (MELK) as a host factor required for optimal uncoating of the HIV-1 core to promote viral cDNA synthesis. Depletion of MELK inhibited HIV-1 cDNA synthesis with a concomitant delay of capsid disassembly. MELK phosphorylated Ser-149 of the capsid in the multimerized HIV-1 core, and a mutant virus carrying a phosphorylation-mimetic amino-acid substitution of Ser-149 underwent premature capsid disassembly and earlier HIV-1 cDNA synthesis, and eventually failed to enter the nucleus. Moreover, a small-molecule MELK inhibitor reduced the efficiency of HIV-1 replication in peripheral blood mononuclear cells in a dose-dependent manner. These results reveal a previously unrecognized mechanism of HIV-1 capsid disassembly and implicate MELK as a potential target for anti-HIV therapy.</p></div

MELK phosphorylates Ser-149 of HIV-1 CA.

<p><b>(A)</b><i>In vitro</i> luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated GST fusion proteins in the presence or absence of OTSSP167 (100 nM). Phosphorylation of the proteins was monitored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006441#ppat.1006441.g003" target="_blank">Fig 3B</a>. Error bars indicate the standard deviations calculated from five independent experiments. <b>(B)</b> Immunoblotting to detect Ser-149 phosphorylation by MELK. Envelope-stripped cores prepared as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006441#ppat.1006441.g002" target="_blank">Fig 2D</a> were incubated with recombinant MELK for the indicated times. Aliquots of each reaction sample were processed for immunoblotting using anti-phospho-S149-CA (CA-S149p), anti-p24 (CA), anti-p17 (MA), anti-gp120 (Env) or anti-MELK (MELK) (upper 5 panels). Envelope-stripped cores were incubated with recombinant MELK in the presence of 100 nM or 500 nM of OTSSP167 [panel: OTSSP167 (100 nM or 500 nM)]. The samples were also treated with (+) or without (-) 100 U of calf intestine alkaline phosphatase (CIAP) and immunoblotted (lower 2 panels) with CA-S149p (panel: CA-S149p) or with anti-p24 antibody [panel: CA (reprobed)]. Experiments were performed three times and one representative set of data is shown. <b>(C)</b> Non-T or MELK-KD-2 MT4C5 cells were infected with VSV-G-pseudotyped HIV-1 or VSV-G-pseudotyped HIV-1 CA-S149A for 8 h. The proteasome inhibitor MG132 (2 μM) was added 5 h after infection to prevent the degradation of CA proteins dissociated from the viral core in the cytoplasm [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006441#ppat.1006441.ref048" target="_blank">48</a>]. Cell lysates were separated by SDS-PAGE containing Manganese(II)-Phos-tag (Mn²⁺-phos-tag) or SDS-PAGE without Mn²⁺-phos-tag (Normal), and analyzed by immunoblotting with anti-p24 antibody (CA) or anti-alpha-tubulin antibody (α-tubulin). Cell lysates were incubated for 60 min at 37°C without (lanes 2 and 3) or with (lanes 4 and 5) calf intestine alkaline phosphatase (CIAP). “non-pCA” indicates the position of CA dephosphorylated by CIAP and “pCA” indicates phosphorylated CA. Experiments were performed at least three times and one representative set of data is shown. <b>(D)</b> Non-T or MELK-KD-2 MT4C5 cells were infected with VSV-G-pseudotyped HIV-1 CA-S149A for 8 h. Cell lysates were separated as in <b>(C)</b> and analyzed by immunoblotting with anti-p24 antibody (CA) or anti-alpha-tubulin antibody (α-tubulin). Similar results were obtained in three independent experiments and a representative result is shown. Statistical significance was determined by two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test <b>(A)</b>. ns, not significant (<i>P</i>>0.05); *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Phosphorylation of Ser-149 in CA regulates CA disassembly and viral cDNA synthesis in human T cells.

<p><b>(A-C)</b> Quantitative DNA-PCR analyses of viral cDNA metabolism after HIV-1 infection of MT4C5 cells. Total DNA was extracted from non-target shRNA (Non-T) or MELK-depleted (MELK-KD-2) MT4C5 cells at the indicated time points (2, 4, 8 and 24 h) after infection with wild-type or the indicated mutants of HIV-1 and analyzed for the amounts of the <i>R/U5</i> region as early viral cDNA <b>(A)</b>, the <i>env</i> region as late viral cDNA <b>(B)</b> and the 2-LTR circle form <b>(C)</b>. Experiments were performed at least three times and error bars are standard deviations calculated from three independent experiments. The ratios of each viral cDNA level to beta-globin DNA level are given. <b>(D)</b> Fate-of-capsid assays with non-target shRNA (Non-T) or MELK-KD-2 MT4C5 cells infected with NL4-3 or its S149E CA mutant for 8 h in the presence or absence of 10 μM MG132 (MG132 [+] or MG132 [–]). HIV-1 stock inactivated by incubation at 65°C for 30 min was used as a negative control (HI control). Cell lysates were prepared and analyzed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006441#ppat.1006441.g002" target="_blank">Fig 2I</a>. Aliquots of input, fraction #1 and #3 were processed for immunoblotting with anti-p24 antibody (CA). The amount of CA in each fraction in the absence of MG132 was quantified by HIV-1 p24 ELISA (MG132 [–], lower panels). Experiments were performed five times and one representative set of data is shown. <b>(E)</b> Percentages of pelletable CA (fraction #3) within total CA in the absence of MG132 were calculated based on the p24 ELISA data shown in <b>Fig 4D</b>. Total CA denotes the sum of the amount of p24 antigen which was calculated based on the p24 ELISA data of fractions #1, #2, and #3. Error bars represent the standard deviations calculated from five independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test <b>(D)</b>, or unpaired two-tailed Student’s <i>t</i> test <b>(E)</b>. ns, not significant (<i>P</i>>0.05); *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Depletion of MELK delays HIV-1 CA disassembly.

<p><b>(A)</b> Non-T or MELK-KD-2 MT4C5 cells were mock-infected or infected with 100 or 500 ng of p24-measured amounts of NL4-3 virions containing BlaM-Vpr, based on the measured amount of p24, in the presence or absence of AMD3100 (100 nM). They were then analyzed in the fusion assay by flow cytometry using a violet laser to excite CCF2. Each experiment was performed in triplicate, repeated three times and one set of representative data is shown. <b>(B)</b> Relative numbers of BlaM⁺ MELK-KD-2 MT4C5 cells are shown as percentages (%) of Non-T MT4C5 cells with standard deviations calculated from three independent experiments. <b>(C)</b> Virion-associated viral RNA was quantified by quantitative RT-PCR 2 h after infection of Non-T or MELK-KD-2 MT4C5 cells with wild-type HIV-1. Error bars indicate the standard deviations calculated from five independent experiments. (<b>D)</b> Immunoblot analysis of envelope-stripped HIV-1 cores. Concentrated virions were subjected to step-gradient centrifugation in the absence (-) or presence (+) of 0.1% of Triton-X100. <b>(E)</b> Electron micrographs showing envelope-stripped cores of HIV-1. TEM images of a negatively stained envelope-stripped core of HIV-1 prepared from HIV-1_NL4-3 virions. Bars indicate 50 nm. <b>(F)</b> Immunoblot analyses showing MELK bound to envelope-stripped cores of HIV-1. HeLa cells were transfected with pCAG-OSF (lane 1) or increasing amounts of pCAG-OSF-GFP (lanes 2 to 3), pCAG-OSF-MELK (lanes 4 to 5), pCAG-FOS2-rhT5α (lanes 6 to 7) or pCAG-OSF-CypA (lanes 8 to 9). Purified OSF- or FOS2-tagged proteins were incubated with envelope-stripped cores and complex formation was assessed. Masses of molecular weight standards are indicated on the left. Arrows indicate the position of MELK in the gel. <b>(G)</b> Schematic diagram of the fate-of-capsid assay. <b>(H)</b> Forced expression of rhesus Trim5α (rhT5α) inhibits HIV-1 replication in human T cells. Cell lysates were prepared from MT4C5 cells transduced with empty lentivirus (vector-control) or lentivirus for C-terminally HA-tagged rhesus Trim5α expression (rhT5α-HA) and processed for immunoblotting with anti-HA (HA) and anti-alpha-tubulin (α-tubulin) antibodies. Experiments were performed at least three times and one representative set of data is shown (upper panels). Vector-control and rhT5α-HA cells were infected with VSV-G-env-pseudotyped NL4-3luc. Relative luciferase activity is shown as a percentage (%) of the RLU of vector-control cells with standard deviations calculated from five independent experiments (lower panel). <b>(I)</b> Effect of MELK depletion on the fate of the HIV-1 CA in MT4C5 cells. Non-T, MELK-KD-2, MT4C5 cells expressing rhT5α (rhT5α), or Non-T cells treated with 10 μM nevirapine (Non-T + NVP) were infected with wild-type HIV-1 for 8 h in the presence or absence of 10 μM MG132 (MG132 [+] or MG132 [–]). HIV-1 stock inactivated by incubation at 65°C for 30 min was used as a negative control (HI control). Cell lysates were subjected to 20%–60% step-gradient centrifugation and three fractions were collected from the top (fraction #1), middle (fraction #2) and interface between the 20% and 60% sucrose layers (fraction #3). Aliquots of each fraction were processed for immunoblotting with anti-p24 antibody (CA) (upper panel). Experiments were performed at least five times and one representative set of data is shown. The amount of CA in each fraction in the absence of MG132 was quantified by HIV-1 p24 ELISA (lower panel). Error bars indicate the standard deviations calculated from five independent experiments. <b>(J)</b> Percentage of the pelletable CA (fraction #3) within total CA in the absence of MG132 was calculated based on the p24 ELISA data shown in Fig 2H. Total CA denotes the sum of the amount of p24 antigen which was calculated based on the p24 ELISA data of fractions #1, #2, and #3. Error bars represent the standard deviations calculated from five independent experiments. Statistical significance was determined by unpaired two-tailed Student’s <i>t</i> test <b>(B</b>, <b>C</b>, <b>H</b> and <b>J)</b>, or one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test <b>(I)</b>. ns, not significant (<i>P</i>>0.05); *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Catalytic activity of MELK is required for HIV-1 infection.

<p><b>(A)</b> Effect of exogenous wt or T167A MELK on single-round HIV-1 infection. VSV-G-pseudotyped NL4-3luc was used to infect parental MT4C5 (white bar: lane 1), Non-T (gray bars: lanes 2 and 9) and MELK-KD-1 (dark gray bars: lanes 3 to 8) cells transduced with control vector (lane 4), wild<b>-</b>type MELK (lanes 5 and 6) or catalytically inactive T167A MELK mutant (lanes 7 and 8) (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006441#ppat.1006441.s009" target="_blank">S7 Fig</a>). Two independent MELK-KD-1 cell pools expressing wild<b>-</b>type MELK (lanes 5 and 6) or T167A MELK mutant (lanes 7 and 8) were used. Error bars indicate the standard deviations calculated from five independent experiments. <b>(B)</b> <i>In vitro</i> luminescent kinase assay with recombinant active MELK (10 or 100 ng) and increasing amounts of ZIPtide, a substrate for MELK (upper panel). Phosphorylation of the substrate was monitored as the amount of ADP produced during the kinase reaction. Effect of OTSSP167, a MELK kinase inhibitor, on <i>in vitro</i> MELK kinase activity (lower panel). Error bars indicate the standard deviations calculated from three independent experiments. <b>(C)</b> <i>In vitro</i> luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated GST fusion proteins in the presence or absence of OTSSP167 (100 nM). Mean values from five independent experiments are shown. Error bars indicate the standard deviations calculated from five independent experiments. <b>(D)</b> <i>In vitro</i> luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated substrates in the presence or absence of OTSSP167 (100 nM). Phosphorylation of proteins was monitored as in <b>(C)</b>. Error bars indicate the standard deviations calculated from five independent experiments. <b>(E)</b> List of fifteen different peptides containing serine or threonine residues in HIV-1 CA. <b>(F)</b> <i>In vitro</i> luminescent kinase assay with recombinant active MELK and increasing amounts of each peptide shown in <b>(E)</b>. Phosphorylation of the peptides was monitored as in <b>(B)</b>. Experiments were performed at least three times and error bars are standard deviations calculated from three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test <b>(A)</b>, or two-way ANOVA with Tukey’s multiple comparison test <b>(C</b> and <b>D)</b>. ns, not significant (<i>P</i>>0.05); *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p