Báo cáo khoa học: Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41 ppt

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Retrocyclin RC-101 overcomes cationic mutations on theheptad repeat 2 region of HIV-1 gp41Christopher A. Fuhrman1, Andrew D. Warren1, Alan J. Waring2, Stephen M. Dutz3,Shantanu Sharma3, Robert I. Lehrer2, Amy L. Cole1and Alexander M. Cole11 Molecular Biology & Microbiology, Biomolecular Science Center, Burnett College of Biomedical Sciences at University of Central Florida,Orlando, FL, USA2 Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA3 Department of Chemistry and Center for Macromolecular Modeling & Materials Design, California State Polytechnic University, Pomona,CA, USADefensins are effector molecules of the innate immunesystem, which protect humans and other animals froma wide range of pathogens, including bacteria, fungiand viruses [1]. There are three major defensinfamilies (a, b and h). They are classified based ontheir b-sheet conformation, cationic charge andunique disulfide bond pattern [2]. a- and b-defensinsarose from a common pre-mammalian protein [3],whereas h-defensins evolved directly from a-defensins[2,4]. h-defensins are formed by the fusion of twotruncated a-defensin nonapeptides by a yet to be iden-tified mechanism, to form an octadecapeptide thatcontains three intramolecular disulfide bonds, and ismacrocyclic through fusion of the N- and C-termini.Fully translated and processed h-defensins were origi-nally isolated from the leukocytes of rhesus monkeys,and intact h-defensin genes exist in Old Worldmonkeys, orangutans and a lesser ape species [4,5].Humans, gorillas, bonobos and chimpanzees retainmultiple-mutated, but largely intact h-defensin genes.Humans express h-defensin mRNA in a variety ofcells and tissues, and their lack of h-defensin peptideexpression is due to a conserved stop codon in thesignal sequence that prevents translation. A search ofthe human genome revealed five h-defensin pseudoge-nes clustered on chromosome 8 near the other a- andKeywordsAUTODOCK; defensin; HIV-1; innate immunity;retrocyclinCorrespondenceA. M. Cole, Department. of MolecularBiology & Microbiology, Burnett School ofBiomedical Sciences, University of CentralFlorida, 4000 Central Florida Boulevard,Building 20, Room 236, Orlando, FL 32816,USAFax: +1 407 823 3635Tel: +1 407 823 3633E-mail: acole@mail.ucf.edu(Received 8 August 2007, revised 24 Octo-ber 2007, accepted 25 October 2007)doi:10.1111/j.1742-4658.2007.06165.xRetrocyclin RC-101, a h-defensin with lectin-like properties, potently inhib-its infection by many HIV-1 subtypes by binding to the heptad repeat 2(HR2) region of glycoprotein 41 (gp41) and preventing six-helix bundle for-mation. In the present study, we used in silico computational explorationto identify residues of HR2 that interacted with RC-101, and then analyzedthe HIV-1 sequence database at Los Alamos National Laboratory (NewMexico, USA) for residue variations in the heptad repeat 1 (HR1) andHR2 segments that could plausibly impart in vivo resistance. DockingRC-101 to gp41 peptides in silico confirmed its strong preference for HR2over HR1, and implicated residues crucial for its ability to bind HR2. Wemutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses,and subjected them to single-round replication assays in the presence of1.25–10 lgÆmL)1RC-101. Apart from one mutant that was partially resis-tant to RC-101, the other pseudotyped viruses with single-site cationicmutations in HR2 manifested absent or impaired infectivity or retainedwild-type susceptibility to RC-101. Overall, these data suggest that mostmutations capable of rendering HIV-1 resistant to RC-101 will also exertdeleterious effects on the ability of HIV-1 to initiate infections – an inter-esting and novel property for a potential topical microbicide.Abbreviations6HB, six helix bundle; gp41, glycoprotein of 41 kDa; HR1, heptad repeat 1; HR2, heptad repeat 2.FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6477b-defensin genes, and one additional h-defensin genethat had translocated to chromosome 1 [5].Utilizing genetic information present in the pseudo-gene, we recreated h-defensins using solid-phase synthe-sis, and tested for antimicrobial activity [6,7]. Theputative wild-type h-defensin, called retrocyclin orRC-100, exhibited modest activity against severalGram-positive and Gram-negative bacteria, yetpotently prevented both X4 and R5 HIV-1 replicationin CD4+ peripheral blood mononuclear cells [6]. Anumber of RC-100 analogues have been developed thatare effective in preventing HIV-1 infection, includingthe highly active analogue RC-101 [8,9]. RC-101 differsfrom ‘wild-type’ retrocyclin-1 by a single arginine-to-lysine mutation on one of the b-turns. It is nonhemolyt-ic for human red blood cells, and noncytotoxic againstseveral human cell lines at concentrations up to500 lgÆmL)1[6,10]. Importantly, RC-101 preventedinfection at low to submicromolar concentrations, andwas active against 27 clinical HIV-1 isolates from fivedifferent clades [8,11].Retrocyclins act prior to viral entry into host cells bydisrupting the function of glycoprotein 41 (gp41) ofHIV-1 [12,13]. Retrocyclin prevented formation of thesix-helix bundle (6HB) of in vitro synthesized heptadrepeat 1 (HR1) and heptad repeat 2 (HR2) regions ofgp41 [12]. During 100 days of serial passaging, HIV-1strain BaL developed three cationic mutations in thepresence of sublethal concentrations of RC-101 [14]. Ofthe three mutations, one was found in gp120 and oneeach in the HR1 and HR2 regions of gp41, and allthree mutations converted a polar or anionic residue toa cationic residue [14]. In addition, the cationic muta-tion in HR2 ablated a commonly glycosylated aspara-gine residue. Loss of glycosylated residues in gp41 canreduce the fusion ability of the virus, and alter theshape of discontinuous epitopes [15,16], and thus thedependence of viral replication on the presence ofRC-101 is not surprising. The cationic mutations andloss of glycosylated residues suggest an attempt by thevirus to repel the cationic lectin, RC-101 [14,17].As all nonsynonymous mutations in RC-101-exposed HIV-1 were cationic mutations [14], andbecause HR2 is the principal target of retrocyclins [12],we decided to study how other mutations that lead toan increase in net positive charge in HR2 would affectviral resistance to retrocyclins [18]. Computationalanalysis of gp41 revealed a region of low amino aciddiversity in the HR1-binding region of HR2 that wasfavored by RC-101 in our docking model. Cationicmutations revealed only one mutation in HR2 thatoffered partial resistance to RC-101; all other muta-tions showed poor infection, did not infect, or wereinhibited by RC-101 in a manner similar to the wild-type JR.FL pseudotype.ResultsVariability of amino acids in HR1 and HR2corresponds to the structural role in the 6HBconformationIn order to measure the susceptibility of the envelopegene to mutation and identify viable escape mutants,we analyzed over 900 HIV-1 group M envelope proteinsequences from the HIV sequence database at Los Ala-mos National Laboratory (NM, USA). The aminoacid diversity indices of HR1 and HR2 are distinctlydissimilar (Fig. 1A). The majority of sites (28 of 36)on HR1 are monomorphic and do not readily change,whereas the majority of sites (21 of 34) on HR2 arehighly pliable and change readily between viral strains.Of the eight non-monomorphic sites found on HR1,six are externally exposed to the environment in the6HB conformation.To visualize the sequence variation as a function ofbiochemical structure, we mapped the amino aciddiversity values to the 3D model of HR2 (Fig. 1B).The externally exposed regions of HR2 in the 6HBshow a high amount of amino acid diversity, while theHR1-binding domain on HR2 is predominantly mono-morphic. Yamaguchi-Kabata et al. [20] found that dis-continuous epitopes in the a-helices of gp120 wereunder putative positive selection. By contrast, themonomorphic sites of HR2 suggest a region undervery little selection. Alternatively, the regions exposedin the 6HB conformation may be under strong puta-tive positive selection. The long-term potency ofRC-101 against HIV-1 BaL could be attributed tointeraction with discontinuous epitopes of the mono-morphic residues of HR2.Because all three known nonsynonymous, RC-101-evasive mutations were cationic residues [14], we choseto measure the isoelectric points of the heptad repeatregions of all group M sequences as a marker ofcharge diversity. The isoelectric points of the heptadrepeats illustrate the ability of HIV-1 to alter its regio-nal charge in vivo. While the amino acid compositionof HR2 is highly variable, its isoelectric range is acidicand significantly restricted: 96% of the isoelectricpoints range between 3.89 and 4.66. In contrast, HR1is highly monomorphic but covers a wide range of iso-electric points (Fig. 1C). In line with having only eightnon-monomorphic sites, the isoelectric points showa strong inclination to cluster around certain values:8.49 (n ¼ 30), 9.99 (n ¼ 52), 10.29 (n ¼ 33), 10.83RC-101 overcomes cationic mutations in HIV-1 gp41 C. A. Fuhrman et al.6478 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS(n ¼ 569), 11 (n ¼ 140), 11.71 (n ¼ 58) and 12.01(n ¼ 12). Sequences with higher isoelectric points havea greater number of cationic mutations with feweranionic residues; the converse is true for HR1sequences with more acidic isoelectric points (data notshown). The isoelectric range of HR1 is over twice thatof HR2, suggesting a greater in vivo variation in elec-trostatic density.The change in free energy upon binding ofRC-101 to HR2 is consistently higher than theenergy of binding to HR1While charge interaction plays an important role inRC-101 viral inhibition, it is not known which residuesplay an important role in binding. Because RC-101still binds gp41 in the absence of linked sugar mole-cules, we can reasonably exclude the sugar moietiesfrom having a direct interaction with RC-101 [12,14].The molecular docking program autodock [28,29]was used to determine the affinity of RC-101 for HR1,HR2 and the dimer (HR1 + HR2). Previous dockingprocedures using the protein models of HR1 and HR2focused on docking small molecules to the helices [36].In contrast, RC-101 contains a large number of flexibleside chains and flexible side groups. Consequently, ourdocking procedures did not identify just one residuethat can be considered the principal docking site ofRC-101, but a number of RC-101 binding conforma-tions. Docking of RC-101 to HR1 alone did not resultin a strong binding energy [Fig. 2]. Conversely, theminimum energy of binding to HR2 is predominantlylower than values for small molecule inhibitors previ-ously docked to this model [Fig. 2] [36].Anionic-to-cationic mutations on HR2 wereunable to elicit appreciable resistance to RC-101We created HIV-1 env molecular clones to identifymutations that alter HIV-1 susceptibility to RC-101.An expression vector containing env from JR.FL,an R5 pseudotype, was subjected to site-directedmutagenesis to create mutant clones. Because RC-101ABCFig. 1. The ‘a’ and ‘d’ heptamers of HR2 are predominantly mono-morphic. (A) The amino acid diversity index of HR1 and HR2 wascalculated for 913 group M HIV-1 viruses. All amino acids for whichthe index value is below the dotted line (0.05) are consideredmonomorphic. (B) The diversity indices were mapped to the 3Dstructure of HR2 (N-terminus at the top). Monomorphic residues,more red in color, are found in the HR1-binding region of HR2.Highly diverse residues, more white in color, are exposed to theexternal environment in the 6HB. (C) Isoelectric points for HR1 andHR2 were obtained by inputting the group M sequences into thepI ⁄ MWtool of EXPASY. The range of isoelectric points for each axishas been restricted in order to clearly visualize the isoelectric pointsof the majority of HR1 and HR2 molecules.C. A. Fuhrman et al. RC-101 overcomes cationic mutations in HIV-1 gp41FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6479viral entry inhibition is glycan-independent and chargealteration is a common mechanism for microbialevasion of antimicrobial peptides [12,14,37,38], weindividually mutated each negatively charged aminoacid to a positively charged lysine or arginine(Fig. 3A). After alteration of the env gene, the wild-type stock (nonmutated) or mutant JR.FL env cloneswere then used to create pseudotyped single-cycleHIV-1 luciferase reporter viruses, and RC-101 activityagainst each viral clone was measured. Of the 10JR.FL variants, five showed scant ability to infectHOS-CD4-CCR5 cells (Fig. 3B). For all five low- ornoninfectious variants, the mutation was located onthe region of HR2 that is externally exposed in the6HB conformation (heptamers b, c, e, f and g). Of thefive pseudotyped variants that effectively entered HOS-CD4-CCR5 cells, only the pseudotype with a lysine atamino acid position 648 showed partial resistance toRC-101 (Fig. 3C,D). Residue 648, part of the ‘g’heptamer, is located in the central region of the helix,and, based on our modeling simulations, is a potentialbinding site for the positive residues on RC-101. TheseABCDFig. 3. Single-site anionic-to-cationic mutations revealed only one partially resistant variant. The JR.FL env molecular clone was mutatedusing site-directed mutagenesis based on the HR2 sequences shown in (A). Pseudotyped viruses were then used to infect HOS-CD4-CCR5cells. (B) Pseudotypes that infected HOS cells very little or not at all in the absence of RC-101. (C) Pseudotypes that caused infection in amanner similar to the wild-type JR.FL molecular clone. The percentage inhibition was calculated relative to normal infectious virus. (D) Allthe pseudotypes were inhibited similarly to wild-type, except for E648K (P ¼ 0.05). In (A), ‘Hept.’ indicates heptamer location (‘a’–‘g’), asshown in Fig. 4(C). In (B)–(D), error bars represent the SEM (n ¼ 4).Fig. 2. RC-101 forms stronger intermolecular bonds with HR2 thanwith HR1. Four in silico docking experiments revealed a signifi-cantly lower DG for RC-101 upon binding HR2 than HR1(P ¼ 0.0005). The DG upon binding is also referred to as the finaldocked energy. Error bars represent the SEM.RC-101 overcomes cationic mutations in HIV-1 gp41 C. A. Fuhrman et al.6480 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBSdata suggest that the ability of the virus to form the6HB was significantly decreased and ⁄ or the mutantslost the ability to properly form the gp41 pre-fusioncomplex.RC-101 binds to the HR1-binding regions of HR2Figure 4A shows backbone renderings of five RC-101molecules docked to HR2, representing the five mostenergetically favorable dockings in a single dockingsimulation. Ligands binding to HR1 were nonspecific,as evidenced by the highly dispersed RC-101 mole-cules. In contrast, RC-101 repeatedly bound to HR2in the same region. Examination of a helical represen-tation of HR2 (Fig. 4B) shows that the backbone ofRC-101 covers the ‘a’ and ‘d’ heptamers, and thelong flexible side chains of RC-101 extend out andinteract with heptamer locations ‘e’ and ‘g’. Interest-ingly, these heptamer positions are areas of lowamino acid diversity (Fig. 1B) that coincide with theregion that binds HR1 upon 6HB formation. Thestrong affinity of RC-101 for HR2 prevents the inter-action of HR1 and HR2, formation of the 6HB, andsubsequent fusion of the host and viral membranes(Fig. 4C), as supported by recent in vitro studies[12,14].N & C TerminalHR2HR1Side ViewABDECFig. 4. RC-101 preferentially docks to the HR1-binding domain of HR2. (A) The top five docked RC-101 molecules for a representative dock-ing, as measured by the final docked energy, are shown as gray loops near the a-helix to which they were docked. The RC-101 mole-cules docked to HR1 are much more dispersed than the RC-101 molecules docked to HR2. The color of each residue of HR1 and HR2 in (A)correlates with the heptamer designation shown in (B). (C) RC-101 binds to the HR1-binding region of HR2. An interaction in this region the-oretically prevents formation of the 6-helix bundle. RC-101 is shown by both (D) cartoon and (E) structural representations.C. A. Fuhrman et al. RC-101 overcomes cationic mutations in HIV-1 gp41FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6481Anionic, polar and hydrophobic residues of HR2create a preferred binding site for RC-101The computer programs ligplot and hbplus wereused to identify specific interactions between theligand, RC-101 and HR2 based on proximity andatomic angles. We quantified the number of interac-tions per HR2 residue for the lowest (best) 25% of thedocked RC-101 molecules, based on the final dockedenergy for each docking experiment comprising 200iterations of the Lamarckian genetic algorithm(Fig. 5). This allowed us to isolate regions and residuesof ligand–macromolecule interaction. The applicationsidentified two sets of molecular interactions betweenRC-101 and HR2: hydrogen bonds at residues Ser649,Gln653 and Asn656, and hydrophobic or nonhydro-gen-bonded contacts at residues Tyr638, Ile642 andLeu645 (Fig. 5, asterisks). Five of the six residues arelocated in the ‘a’ and ‘d’ heptamer regions of HR2,which bind HR1 upon 6HB formation. The sixth resi-due, Gln653, is located on the ‘e’ heptamer. Four ofthe residues are monomorphic, and the remaining tworesidues have reasonably low amino acid diversity val-ues. autodock consistently bound RC-101 to a loca-tion with low amino acid diversity that has animportant role in 6HB formation.Mutation of residues in the HR1-binding domainof HR2 resulted in viruses that were notreplication-competent or not resistant to RC-101Based on the above study, we created mutant pseudo-typed JR.FL env clones that contained a cationic muta-tion at each of the six residues observed to interact withRC-101 in silico. In addition, we mutated two residueson the 6HB-exposed portion of HR2 (heptamers ‘f’and ‘c’) as negative controls (Fig. 6A). Both of thesecontrol pseudotypes infected HOS-CD4-CCR5 cellsand remained sensitive to RC-101. Four mutants werenoninfectious even in the absence of RC-101 (Fig. 6B).All noninfectious JR.FL mutants were located on hep-tamers that indirectly or directly interacted with HR1[27,39]. Of the JR.FL mutants that did infect HOS-CD4-CCR5 cells, none were resistant to RC-101.DiscussionThe envelope protein of HIV-1 is under many kineticrestraints for proper functionality. First, the short timebetween gp120–CD4 interaction and 6HB formationlimits the time that 6HB inhibitors have to act [40,41].The strong net negative charge of HR2 and net posi-tive charge of RC-101 create a strong electrostaticattraction that probably promotes binding. This is evi-dent in the marked difference observed between thenonspecific binding of RC-101 to HR1 and the specificbinding to HR2 seen in this work. RC-101 bindsreversibly but with high affinity to glycoproteins andassociates with the cellular lipids and proteins involvedin host–viral fusion [17,42]. This lectin-like bindingplaces RC-101 in the most advantageous location toaffect 6HB formation.As a response to opposing host and environmentalfactors, HIV-1 employs a number of counter-measures,including a ‘glycan shield’ and the error-prone natureof its reverse transcriptase. Alterations in the glycanFig. 5. RC-101 dockings prefer both thepolar and hydrophobic residues on HR2.Four docking experiments were completed,each involving 200 repetitions of theLamarckian genetic algorithm. The best25% docked RC-101 molecules (n ¼ 50)from each docking experiment were ana-lyzed for intermolecular interactions (hydro-gen bonding and hydrophobic contacts), andtabulated per HR2 residue. Asterisks indi-cate the six residues of HR2 that had thegreatest number of interactions with RC-101, and which were mutated for in vitroinfection assays (Fig. 6). Error bars repre-sent the SEM.RC-101 overcomes cationic mutations in HIV-1 gp41 C. A. Fuhrman et al.6482 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBSshield affect access to binding sites [43]. In addition,the 6HB formed in solution with the synthetic N36peptide and a glycosylated C34 peptide was less com-pact than its nonglycosylated counterpart [16]. Thissuggests variation in the interhelical distance, and apossible change in the discontinuous epitopes targetedby site-specific antibodies [44]. In our analysis of HIV-1 protein sequences, we observed many non-monomor-phic sites with variation primarily within an aminoacid chemical grouping (e.g. Ile M Leu), further alter-ing possible binding epitopes. The question remains asto whether HIV-1 mutations that confer partial resis-tance against RC-101 change the binding site of RC-101 or alter access to the binding site. Both scenariosare possible.In attempting to evade RC-101 inhibition, HIV-1developed three cat ionic mutations, one of whichremoved a glycosylated residue but caused the virus toremain dependent on RC-101 for infectivity [14]. Anio-nic-to-cationic mutations in the HR2 region resulted in anormal infectious mutant only 50% of the time, with allmutants susceptible to RC-101. This suggests that thenegative charge on HR2 may be important for maintain-ing the normal replication efficiency of HIV-1, possiblyby stabilizing its interaction with HR1 during 6HB for-mation. Although mutations that alter the negativecharge of HR2 may impair RC-101 binding, they mayalso have the untoward effect (for the virus) of preventingits ability to mediate the fusion process and infect cells.The virus’s inability to become fully resistant to RC-101 is further illustrated by an extension of our previ-ous work [14]. Passaging the virus from days 100–140in the presence of 10–20 lgÆmL)1RC-101 neitherinduced additional mutations nor increased its resis-tance (data not shown). Collectively, our data indicatethat it is unlikely that HIV-1 can mount further resis-tance to RC-101: aside from one partially resistantvirus, mutant viruses either remained infectious butsensitive to RC-101, or suffered from a significant lossof fusion efficiency.The predominant problem with current HIV-1 treat-ments is the eventual emergence of fully resistantABCDFig. 6. Mutation of residues in the HR1-binding region of HR2 led to noninfectious or non-RC-101-resistant mutants. The JR.FL env mole-cular clone was mutated using site-directed mutagenesis based on the HR2 sequences shown in (A). Pseudotyped viruses were then usedto infect HOS-CD4-CCR5 cells. Cationic mutations of RC-101-interacting residues revealed nonviable mutations (B) or normally inhibitedmutant pseudotypes (C). (D) All normally infectious mutants were inhibited similarly to the wild-type. In (A), ‘Hept.’ indicates heptamerlocation (‘a’–‘g’), as shown in Fig. 4(C). In (B)–(D), error bars represent the SEM (n ¼ 4).C. A. Fuhrman et al. RC-101 overcomes cationic mutations in HIV-1 gp41FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6483mutants that are then transmitted to new hosts. Thesame problem is theoretically possible for widely usedtopical microbicides. Our work has shown that theability of HIV-1 to generate escape mutants againstRC-101 is limited, and thus RC-101 holds great poten-tial as an anti-HIV-1 microbicide because it remainseffective against the virus.Experimental proceduresComputational analysis of the variationin HR1 and HR2Aligned envelope protein sequences of 913 unique HIV-1group M viruses were obtained from the HIV sequencedatabase at the Los Alamos National Laboratory (http://www.hiv.lanl.gov), which has been curated by Los AlamosNational Laboratory scientific staff for duplicate sequencesfrom the same source. HIV-1 group M represents a groupof viral isolates that diverged in humans and originatedfrom one chimpanzee-to-human transmission event, andis the most common group found in humans [19].The amino acid diversity index was calculated asDaa¼P20i¼1xiÀÁ2ÀP20i¼1x2i, where x is the proportion ofthe ith amino acid of the 20 standard amino acids at thatlocation [20]. The value is similar to that obtained for genediversity in population genetics [21]. An amino acid with adiversity index less than 0.05 is considered monomorphic[20]. The residues corresponding to HR1 and HR2 werespliced out of the sequence file and used for evaluation inthe expasy pI ⁄ Mwtool to determine the isoelectric point[22–24].Preparation of HR1, HR2 and retrocyclin structuremodelsThree separate structural representations were required forbio-computational experimentation: the HR1 and HR2regions of JR.FL and the h-defensin RC-101. In the contextof computational data, the HR1 ⁄ HR2 nomenclature refersonly to the N36 and C34 peptides, respectively. Three-dimensional structural models of the HR1 and HR2 regionsof JR.FL were generated using the swiss-model proteinhomology web server based on the HIV-1 gp41 core struc-ture (Protein Data Bank accession number 1AIK) pub-lished previously [25–27]. The structure for RC-101 wascreated using the mutagenesis function of the pymol molec-ular graphics system, based on the structure of retrocyclin-2(Protein Data Bank accession number 2ATG). Two in silicomutations were performed to create RC-101: the secondarginine to a glycine and the fourth arginine to a lysine [9].The backbone atoms for both mutated residues remainedstationary. There was no need to minimize the rotationalbond energy of the mutated bonds, as all carbon–carbon orcarbon–nitrogen bonds were deemed ‘rotatable’ in thedocking procedure.Computational modeling of RC-101 binding‘Grid’ and ‘Docking’ parameter files for all RC-101 doc-kings to dimer and comparative monomer macromoleculeswere prepared using autodocktools (ADT) and accompa-nying scripts, and then run with autodock 3.0 and auto-grid 3.0 [28,29]. The grid parameters were the same for allthree macromolecules. The numbers of points in the x, yand z direction were 76, 76 and 126, respectively. The gridspacing value was 0.4527 A˚. Finally, the grid center wasdefined as the x,y,z coordinate (17.449, 13.8, 5.67). All otherautogrid parameters remained at their default values. Theligand, RC-101, was prepared using ADT according to theautodock manual [28]. For each macromolecule (HR1,HR2, HR1 + HR2) and ligand (RC-101), hydrogen posi-tions were reassigned, nonpolar hydrogens were merged,and Kollman united charges were assigned to each residue.The genetic algorithm variables of population size, maxi-mum number of energy evaluations and the maximumnumber of generations were increased to 200, 2 · 106and2 · 105, respectively, using the methods described by Hete-nyi et al. [30] as a general guideline. Lower values wereused because the protein model contained substantially lesssolvent-exposed surface area and contained less than halfthe average number of residues tested in previous blind-docking studies [30–32]. For each docking simulation, thegenetic algorithm was run 200 times to return 200 possibleRC-101 docked conformations. autodock reports thechange in free energy upon binding for each conformation.An approximate threshold range (– 9 to )11 kcalÆmol)1)separates nonspecific interactions from prominent inter-molecular bonds. Each docking simulation was executedfour times, and the quantitative measures of all four dock-ing simulations were averaged and the SEM calculated.Defining hydrogen and nonhydrogen bondsTables of hydrogen bonds and nonhydrogen bonds weregenerated using ligplot in conjunction with hbplus[33,34]. The best 25% of the docked RC-101 molecules(n ¼50), according to the final docked energy, were tabu-lated, and the mean number of bonds per residue for fourindependent docking executions was reported by the pro-gram, together with the SEM.Preparation of peptideThe 18-amino-acid peptide RC-101 was synthesized as pre-viously described [4,6,35] with the sequence: cyclic-GIC-RCICGKGICRCICGR. After each step, the peptide wassubjected to MALDI-TOF mass spectrometry to assessRC-101 overcomes cationic mutations in HIV-1 gp41 C. A. Fuhrman et al.6484 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBShomogeneity (typically approximately 95%), and to confirmthat the observed mass agreed with the theoretical mass.Peptide concentrations were determined by quantitativepeptide analysis.Cell cultureHOS-CD4-CCR5 cells (N. R. Landau, Salk Institute forBiological Studies, La Jolla, CA, USA), which allow entryof R5 HIV-1, were acquired from the National Institutes ofHealth AIDS Research and Reference Reagent Program(Germantown, MD, USA). HOS cells were grown inDMEM supplemented with penicillin, streptomycin, 10%fetal bovine serum, 1 lgÆmL)1puromycin, and mycophenol-ic acid selection medium. 293T cells were grown in DMEMwith penicillin, streptomycin and 10% fetal bovine serum.HIV-1 plasmid constructs and viral entry assayThe expression vectors pNL-LucR–E–and JR.FL env weregifts from N. R. Landau. JR.FL is an R5 strain of HIV-1. Intotal, 18 JR.FL env mutants were constructed. Nine glutamicacids on the HR2 of HIV-1 gp41 were mutated to lysines.Amino acids 632, 634, 641, 647, 648, 654, 657 and 659 weremutated from Glu (GAA) to Lys (AAA). Amino acid 636was mutated from Asp (GAC) to Arg (CGC). For the secondset of mutagenesis studies, polar or hydrophobic residueswere mutated to an arginine or lysine: Tyr638 (TAC) to Arg(CGC), Ser640 (AGC) to Arg (AGG), Ile642 (ATA) to Lys(AAA), Leu645 (CTA) to Arg (CGA), Ser649 (TCG) toLys (CGC), Asn651 (AAC) to Lys (AAA), Gln653 (CAA)to Lys (AAA), and Asn656 (AAT) to Lys (AAA).Each mutation was created from the wild-type JR.FL envplasmid using the QuikChange multi site-directed mutagen-esis kit (Stratagene, La Jolla, CA, USA), verified bysequencing (University of Central Florida BiomolecularScience Center Genomics Core Laboratory, Orlando, FL,USA) and compared to the published JR.FL wild-typesequence (accession number U63632). Subsequently, HIV-1single-cycle (replication-incompetent) luciferase reporterviruses were produced by cotransfecting 293T cells with10 lg each of pNL-LucR–E–and one of the JR.FL envclones. Virus-containing clarified supernatants were col-lected after 48 h by centrifugation at 1000 g for 10 min,filtered though a 0.45 lm filter and stored at )80 °Cinaliquots until needed. One aliquot was used to quantifypropagated pseudovirus by p24 ELISA (Perkin Elmer, Wal-tham, MA, USA). Another aliquot was used to ensure theintegrity of the envelope gene. Viral RNA was isolatedfrom the JR.FL pseudotypes (viral RNA mini kit; Qiagen,Valencia, CA, USA). A cDNA library was created fromthe isolated viral RNA using the iScript Select cDNA syn-thesis kit (Bio-Rad, Hercules, CA, USA). Then a 666 bpenvelope region containing HR1 and HR2 was PCR-ampli-fied, and the DNA was separated using a 1.5% agarose gel.The sense primer used was 5¢-CTGTGTTCCTTGGGTTCTTGG-3¢, and the antisense primer was 5¢-CTCCACCTTCTTCTTCGATTCC-3¢. To measure the infectious abil-ity of the JR.FL pseudotypes, HOS-CD4-CCR5 cells(5 · 103per well; 96-well plate) were infected with 50 ngp24 per well of virus in the presence or the absence ofRC-101 (0, 1.25, 2.5, 5 or 10 lgÆmL)1), and luciferaseactivity was measured 2 days later.AcknowledgementsThis work was supported by grants from the NationalInstitutes of Health: AI052017, AI065430 andAI060753 (to AMC) and AI056921 (to RIL), and fromthe National Science Foundation: EIA-0321333 (toSS). We thank Martin Kline (UCF) for his excellenttechnical help and Dr G. M. Morris (The ScippsResearch Institute) for autodock support. We arealso grateful to the National Biomedical ComputationResource and Dr W. Li (NBCR) for the use of theircomputer grid cluster. SS thanks ProfessorP. W. Mobley (California State Polytechnic University)for insightful discussions of the computational results,and CAF thanks Dr C. 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During 100 days of serial passaging, HIV-1 strain BaL developed three cationic mutations in the presence of
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Xem thêm: Báo cáo khoa học: Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41 ppt, Báo cáo khoa học: Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41 ppt, Báo cáo khoa học: Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41 ppt