Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat region of HIV-1 gp41 Christopher A Fuhrman1, Andrew D Warren1, Alan J Waring2, Stephen M Dutz3, Shantanu Sharma3, Robert I Lehrer2, Amy L Cole1 and Alexander M Cole1 Molecular Biology & Microbiology, Biomolecular Science Center, Burnett College of Biomedical Sciences at University of Central Florida, Orlando, FL, USA Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Department of Chemistry and Center for Macromolecular Modeling & Materials Design, California State Polytechnic University, Pomona, CA, USA Keywords AUTODOCK; defensin; HIV-1; innate immunity; retrocyclin Correspondence A M Cole, Department of Molecular Biology & Microbiology, Burnett School of Biomedical Sciences, University of Central Florida, 4000 Central Florida Boulevard, Building 20, Room 236, Orlando, FL 32816, USA Fax: +1 407 823 3635 Tel: +1 407 823 3633 E-mail: acole@mail.ucf.edu (Received August 2007, revised 24 October 2007, accepted 25 October 2007) doi:10.1111/j.1742-4658.2007.06165.x Retrocyclin RC-101, a h-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat (HR2) region of glycoprotein 41 (gp41) and preventing six-helix bundle formation In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101, and then analyzed the HIV-1 sequence database at Los Alamos National Laboratory (New Mexico, USA) for residue variations in the heptad repeat (HR1) and HR2 segments that could plausibly impart in vivo resistance Docking RC-101 to gp41 peptides in silico confirmed its strong preference for HR2 over HR1, and implicated residues crucial for its ability to bind HR2 We mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses, and subjected them to single-round replication assays in the presence of 1.25–10 lgỈmL)1 RC-101 Apart from one mutant that was partially resistant to RC-101, the other pseudotyped viruses with single-site cationic mutations in HR2 manifested absent or impaired infectivity or retained wild-type susceptibility to RC-101 Overall, these data suggest that most mutations capable of rendering HIV-1 resistant to RC-101 will also exert deleterious effects on the ability of HIV-1 to initiate infections – an interesting and novel property for a potential topical microbicide Defensins are effector molecules of the innate immune system, which protect humans and other animals from a wide range of pathogens, including bacteria, fungi and viruses [1] There are three major defensin families (a, b and h) They are classified based on their b-sheet conformation, cationic charge and unique disulfide bond pattern [2] a- and b-defensins arose 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 two truncated a-defensin nonapeptides by a yet to be identified mechanism, to form an octadecapeptide that contains three intramolecular disulfide bonds, and is macrocyclic through fusion of the N- and C-termini Fully translated and processed h-defensins were originally isolated from the leukocytes of rhesus monkeys, and intact h-defensin genes exist in Old World monkeys, orangutans and a lesser ape species [4,5] Humans, gorillas, bonobos and chimpanzees retain multiple-mutated, but largely intact h-defensin genes Humans express h-defensin mRNA in a variety of cells and tissues, and their lack of h-defensin peptide expression is due to a conserved stop codon in the signal sequence that prevents translation A search of the human genome revealed five h-defensin pseudogenes clustered on chromosome near the other a- and Abbreviations 6HB, six helix bundle; gp41, glycoprotein of 41 kDa; HR1, heptad repeat 1; HR2, heptad repeat FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6477 RC-101 overcomes cationic mutations in HIV-1 gp41 C A Fuhrman et al b-defensin genes, and one additional h-defensin gene that had translocated to chromosome [5] Utilizing genetic information present in the pseudogene, we recreated h-defensins using solid-phase synthesis, and tested for antimicrobial activity [6,7] The putative wild-type h-defensin, called retrocyclin or RC-100, exhibited modest activity against several Gram-positive and Gram-negative bacteria, yet potently prevented both X4 and R5 HIV-1 replication in CD4+ peripheral blood mononuclear cells [6] A number of RC-100 analogues have been developed that are effective in preventing HIV-1 infection, including the highly active analogue RC-101 [8,9] RC-101 differs from ‘wild-type’ retrocyclin-1 by a single arginine-tolysine mutation on one of the b-turns It is nonhemolytic for human red blood cells, and noncytotoxic against several human cell lines at concentrations up to 500 lgỈmL)1 [6,10] Importantly, RC-101 prevented infection at low to submicromolar concentrations, and was active against 27 clinical HIV-1 isolates from five different clades [8,11] Retrocyclins act prior to viral entry into host cells by disrupting the function of glycoprotein 41 (gp41) of HIV-1 [12,13] Retrocyclin prevented formation of the six-helix bundle (6HB) of in vitro synthesized heptad repeat (HR1) and heptad repeat (HR2) regions of gp41 [12] During 100 days of serial passaging, HIV-1 strain BaL developed three cationic mutations in the presence of sublethal concentrations of RC-101 [14] Of the three mutations, one was found in gp120 and one each in the HR1 and HR2 regions of gp41, and all three mutations converted a polar or anionic residue to a cationic residue [14] In addition, the cationic mutation in HR2 ablated a commonly glycosylated asparagine residue Loss of glycosylated residues in gp41 can reduce the fusion ability of the virus, and alter the shape of discontinuous epitopes [15,16], and thus the dependence of viral replication on the presence of RC-101 is not surprising The cationic mutations and loss of glycosylated residues suggest an attempt by the virus to repel the cationic lectin, RC-101 [14,17] As all nonsynonymous mutations in RC-101exposed HIV-1 were cationic mutations [14], and because HR2 is the principal target of retrocyclins [12], we decided to study how other mutations that lead to an increase in net positive charge in HR2 would affect viral resistance to retrocyclins [18] Computational analysis of gp41 revealed a region of low amino acid diversity in the HR1-binding region of HR2 that was favored by RC-101 in our docking model Cationic mutations revealed only one mutation in HR2 that offered partial resistance to RC-101; all other mutations showed poor infection, did not infect, or were 6478 inhibited by RC-101 in a manner similar to the wildtype JR.FL pseudotype Results Variability of amino acids in HR1 and HR2 corresponds to the structural role in the 6HB conformation In order to measure the susceptibility of the envelope gene to mutation and identify viable escape mutants, we analyzed over 900 HIV-1 group M envelope protein sequences from the HIV sequence database at Los Alamos National Laboratory (NM, USA) The amino acid diversity indices of HR1 and HR2 are distinctly dissimilar (Fig 1A) The majority of sites (28 of 36) on HR1 are monomorphic and not readily change, whereas the majority of sites (21 of 34) on HR2 are highly pliable and change readily between viral strains Of the eight non-monomorphic sites found on HR1, six are externally exposed to the environment in the 6HB conformation To visualize the sequence variation as a function of biochemical structure, we mapped the amino acid diversity values to the 3D model of HR2 (Fig 1B) The externally exposed regions of HR2 in the 6HB show a high amount of amino acid diversity, while the HR1-binding domain on HR2 is predominantly monomorphic Yamaguchi-Kabata et al [20] found that discontinuous epitopes in the a-helices of gp120 were under putative positive selection By contrast, the monomorphic sites of HR2 suggest a region under very little selection Alternatively, the regions exposed in the 6HB conformation may be under strong putative positive selection The long-term potency of RC-101 against HIV-1 BaL could be attributed to interaction with discontinuous epitopes of the monomorphic residues of HR2 Because all three known nonsynonymous, RC-101evasive mutations were cationic residues [14], we chose to measure the isoelectric points of the heptad repeat regions of all group M sequences as a marker of charge diversity The isoelectric points of the heptad repeats illustrate the ability of HIV-1 to alter its regional charge in vivo While the amino acid composition of HR2 is highly variable, its isoelectric range is acidic and significantly restricted: 96% of the isoelectric points range between 3.89 and 4.66 In contrast, HR1 is highly monomorphic but covers a wide range of isoelectric points (Fig 1C) In line with having only eight non-monomorphic sites, the isoelectric points show a strong inclination to cluster around certain values: 8.49 (n ¼ 30), 9.99 (n ¼ 52), 10.29 (n ¼ 33), 10.83 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS C A Fuhrman et al Fig The ‘a’ and ‘d’ heptamers of HR2 are predominantly monomorphic (A) The amino acid diversity index of HR1 and HR2 was calculated for 913 group M HIV-1 viruses All amino acids for which the index value is below the dotted line (0.05) are considered monomorphic (B) The diversity indices were mapped to the 3D structure 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 the external environment in the 6HB (C) Isoelectric points for HR1 and HR2 were obtained by inputting the group M sequences into the pI ⁄ MW tool of EXPASY The range of isoelectric points for each axis has been restricted in order to clearly visualize the isoelectric points of the majority of HR1 and HR2 molecules (n ¼ 569), 11 (n ¼ 140), 11.71 (n ¼ 58) and 12.01 (n ¼ 12) Sequences with higher isoelectric points have a greater number of cationic mutations with fewer anionic residues; the converse is true for HR1 sequences with more acidic isoelectric points (data not shown) The isoelectric range of HR1 is over twice that of HR2, suggesting a greater in vivo variation in electrostatic density RC-101 overcomes cationic mutations in HIV-1 gp41 A B The change in free energy upon binding of RC-101 to HR2 is consistently higher than the energy of binding to HR1 While charge interaction plays an important role in RC-101 viral inhibition, it is not known which residues play an important role in binding Because RC-101 still binds gp41 in the absence of linked sugar molecules, we can reasonably exclude the sugar moieties from 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 docking procedures using the protein models of HR1 and HR2 focused on docking small molecules to the helices [36] In contrast, RC-101 contains a large number of flexible side chains and flexible side groups Consequently, our docking procedures did not identify just one residue that can be considered the principal docking site of RC-101, but a number of RC-101 binding conformations Docking of RC-101 to HR1 alone did not result in a strong binding energy [Fig 2] Conversely, the minimum energy of binding to HR2 is predominantly lower than values for small molecule inhibitors previously docked to this model [Fig 2] [36] Anionic-to-cationic mutations on HR2 were unable to elicit appreciable resistance to RC-101 We created HIV-1 env molecular clones to identify mutations that alter HIV-1 susceptibility to RC-101 C An expression vector containing env from JR.FL, an R5 pseudotype, was subjected to site-directed mutagenesis to create mutant clones Because RC-101 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6479 RC-101 overcomes cationic mutations in HIV-1 gp41 C A Fuhrman et al Fig RC-101 forms stronger intermolecular bonds with HR2 than with HR1 Four in silico docking experiments revealed a significantly lower DG for RC-101 upon binding HR2 than HR1 (P ¼ 0.0005) The DG upon binding is also referred to as the final docked energy Error bars represent the SEM viral entry inhibition is glycan-independent and charge alteration is a common mechanism for microbial evasion of antimicrobial peptides [12,14,37,38], we individually mutated each negatively charged amino acid to a positively charged lysine or arginine (Fig 3A) After alteration of the env gene, the wildtype stock (nonmutated) or mutant JR.FL env clones were then used to create pseudotyped single-cycle HIV-1 luciferase reporter viruses, and RC-101 activity against each viral clone was measured Of the 10 JR.FL variants, five showed scant ability to infect HOS-CD4-CCR5 cells (Fig 3B) For all five low- or noninfectious variants, the mutation was located on the region of HR2 that is externally exposed in the 6HB conformation (heptamers b, c, e, f and g) Of the five pseudotyped variants that effectively entered HOSCD4-CCR5 cells, only the pseudotype with a lysine at amino acid position 648 showed partial resistance to RC-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 potential binding site for the positive residues on RC-101 These A B C D Fig Single-site anionic-to-cationic mutations revealed only one partially resistant variant The JR.FL env molecular clone was mutated using site-directed mutagenesis based on the HR2 sequences shown in (A) Pseudotyped viruses were then used to infect HOS-CD4-CCR5 cells (B) Pseudotypes that infected HOS cells very little or not at all in the absence of RC-101 (C) Pseudotypes that caused infection in a manner similar to the wild-type JR.FL molecular clone The percentage inhibition was calculated relative to normal infectious virus (D) All the pseudotypes were inhibited similarly to wild-type, except for E648K (P ¼ 0.05) In (A), ‘Hept.’ indicates heptamer location (‘a’–‘g’), as shown in Fig 4(C) In (B)–(D), error bars represent the SEM (n ¼ 4) 6480 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS C A Fuhrman et al RC-101 overcomes cationic mutations in HIV-1 gp41 data suggest that the ability of the virus to form the 6HB was significantly decreased and ⁄ or the mutants lost the ability to properly form the gp41 pre-fusion complex in the same region Examination of a helical representation of HR2 (Fig 4B) shows that the backbone of RC-101 covers the ‘a’ and ‘d’ heptamers, and the long flexible side chains of RC-101 extend out and interact with heptamer locations ‘e’ and ‘g’ Interestingly, these heptamer positions are areas of low amino acid diversity (Fig 1B) that coincide with the region that binds HR1 upon 6HB formation The strong affinity of RC-101 for HR2 prevents the interaction of HR1 and HR2, formation of the 6HB, and subsequent fusion of the host and viral membranes (Fig 4C), as supported by recent in vitro studies [12,14] RC-101 binds to the HR1-binding regions of HR2 Figure 4A shows backbone renderings of five RC-101 molecules docked to HR2, representing the five most energetically favorable dockings in a single docking simulation Ligands binding to HR1 were nonspecific, as evidenced by the highly dispersed RC-101 molecules In contrast, RC-101 repeatedly bound to HR2 A N & C Terminal Side View HR2 HR1 B D C E Fig RC-101 preferentially docks to the HR1-binding domain of HR2 (A) The top five docked RC-101 molecules for a representative docking, as measured by the final docked energy, are shown as gray loops near the a-helix to which they were docked The RC-101 molecules 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 theoretically prevents formation of the 6-helix bundle RC-101 is shown by both (D) cartoon and (E) structural representations FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6481 RC-101 overcomes cationic mutations in HIV-1 gp41 C A Fuhrman et al Anionic, polar and hydrophobic residues of HR2 create a preferred binding site for RC-101 The computer programs ligplot and hbplus were used to identify specific interactions between the ligand, RC-101 and HR2 based on proximity and atomic angles We quantified the number of interactions per HR2 residue for the lowest (best) 25% of the docked RC-101 molecules, based on the final docked energy for each docking experiment comprising 200 iterations of the Lamarckian genetic algorithm (Fig 5) This allowed us to isolate regions and residues of ligand–macromolecule interaction The applications identified two sets of molecular interactions between RC-101 and HR2: hydrogen bonds at residues Ser649, Gln653 and Asn656, and hydrophobic or nonhydrogen-bonded contacts at residues Tyr638, Ile642 and Leu645 (Fig 5, asterisks) Five of the six residues are located in the ‘a’ and ‘d’ heptamer regions of HR2, which bind HR1 upon 6HB formation The sixth residue, Gln653, is located on the ‘e’ heptamer Four of the residues are monomorphic, and the remaining two residues have reasonably low amino acid diversity values autodock consistently bound RC-101 to a location with low amino acid diversity that has an important role in 6HB formation Mutation of residues in the HR1-binding domain of HR2 resulted in viruses that were not replication-competent or not resistant to RC-101 Based on the above study, we created mutant pseudotyped JR.FL env clones that contained a cationic muta- tion at each of the six residues observed to interact with RC-101 in silico In addition, we mutated two residues on the 6HB-exposed portion of HR2 (heptamers ‘f’ and ‘c’) as negative controls (Fig 6A) Both of these control pseudotypes infected HOS-CD4-CCR5 cells and remained sensitive to RC-101 Four mutants were noninfectious even in the absence of RC-101 (Fig 6B) All noninfectious JR.FL mutants were located on heptamers that indirectly or directly interacted with HR1 [27,39] Of the JR.FL mutants that did infect HOSCD4-CCR5 cells, none were resistant to RC-101 Discussion The envelope protein of HIV-1 is under many kinetic restraints for proper functionality First, the short time between gp120–CD4 interaction and 6HB formation limits the time that 6HB inhibitors have to act [40,41] The strong net negative charge of HR2 and net positive charge of RC-101 create a strong electrostatic attraction that probably promotes binding This is evident in the marked difference observed between the nonspecific binding of RC-101 to HR1 and the specific binding to HR2 seen in this work RC-101 binds reversibly but with high affinity to glycoproteins and associates with the cellular lipids and proteins involved in host–viral fusion [17,42] This lectin-like binding places RC-101 in the most advantageous location to affect 6HB formation As a response to opposing host and environmental factors, HIV-1 employs a number of counter-measures, including a ‘glycan shield’ and the error-prone nature of its reverse transcriptase Alterations in the glycan Fig RC-101 dockings prefer both the polar and hydrophobic residues on HR2 Four docking experiments were completed, each involving 200 repetitions of the Lamarckian genetic algorithm The best 25% docked RC-101 molecules (n ¼ 50) from each docking experiment were analyzed for intermolecular interactions (hydrogen bonding and hydrophobic contacts), and tabulated per HR2 residue Asterisks indicate the six residues of HR2 that had the greatest number of interactions with RC101, and which were mutated for in vitro infection assays (Fig 6) Error bars represent the SEM 6482 FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS C A Fuhrman et al RC-101 overcomes cationic mutations in HIV-1 gp41 A B C D Fig Mutation of residues in the HR1-binding region of HR2 led to noninfectious or non-RC-101-resistant mutants The JR.FL env molecular clone was mutated using site-directed mutagenesis based on the HR2 sequences shown in (A) Pseudotyped viruses were then used to infect HOS-CD4-CCR5 cells Cationic mutations of RC-101-interacting residues revealed nonviable mutations (B) or normally inhibited mutant pseudotypes (C) (D) All normally infectious mutants were inhibited similarly to the wild-type In (A), ‘Hept.’ indicates heptamer location (‘a’–‘g’), as shown in Fig 4(C) In (B)–(D), error bars represent the SEM (n ¼ 4) shield affect access to binding sites [43] In addition, the 6HB formed in solution with the synthetic N36 peptide and a glycosylated C34 peptide was less compact than its nonglycosylated counterpart [16] This suggests variation in the interhelical distance, and a possible change in the discontinuous epitopes targeted by site-specific antibodies [44] In our analysis of HIV1 protein sequences, we observed many non-monomorphic sites with variation primarily within an amino acid chemical grouping (e.g Ile M Leu), further altering possible binding epitopes The question remains as to whether HIV-1 mutations that confer partial resistance against RC-101 change the binding site of RC101 or alter access to the binding site Both scenarios are possible In attempting to evade RC-101 inhibition, HIV-1 developed three cationic mutations, one of which removed a glycosylated residue but caused the virus to remain dependent on RC-101 for infectivity [14] Anionic-to-cationic mutations in the HR2 region resulted in a normal infectious mutant only 50% of the time, with all mutants susceptible to RC-101 This suggests that the negative charge on HR2 may be important for maintaining the normal replication efficiency of HIV-1, possibly by stabilizing its interaction with HR1 during 6HB formation Although mutations that alter the negative charge of HR2 may impair RC-101 binding, they may also have the untoward effect (for the virus) of preventing its ability to mediate the fusion process and infect cells The virus’s inability to become fully resistant to RC101 is further illustrated by an extension of our previous work [14] Passaging the virus from days 100–140 in the presence of 10–20 lgỈmL)1 RC-101 neither induced additional mutations nor increased its resistance (data not shown) Collectively, our data indicate that it is unlikely that HIV-1 can mount further resistance to RC-101: aside from one partially resistant virus, mutant viruses either remained infectious but sensitive to RC-101, or suffered from a significant loss of fusion efficiency The predominant problem with current HIV-1 treatments is the eventual emergence of fully resistant FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS 6483 RC-101 overcomes cationic mutations in HIV-1 gp41 C A Fuhrman et al mutants that are then transmitted to new hosts The same problem is theoretically possible for widely used topical microbicides Our work has shown that the ability of HIV-1 to generate escape mutants against RC-101 is limited, and thus RC-101 holds great potential as an anti-HIV-1 microbicide because it remains effective against the virus Experimental procedures Computational analysis of the variation in HR1 and HR2 Aligned envelope protein sequences of 913 unique HIV-1 group M viruses were obtained from the HIV sequence database at the Los Alamos National Laboratory (http:// www.hiv.lanl.gov), which has been curated by Los Alamos National Laboratory scientific staff for duplicate sequences from the same source HIV-1 group M represents a group of viral isolates that diverged in humans and originated from one chimpanzee-to-human transmission event, and is the most common group found in humans [19] The amino acid diversity index was calculated as ÀP20 Á2 P20 À i¼1 xi , where x is the proportion of Daa ¼ i¼1 xi the ith amino acid of the 20 standard amino acids at that location [20] The value is similar to that obtained for gene diversity in population genetics [21] An amino acid with a diversity index less than 0.05 is considered monomorphic [20] The residues corresponding to HR1 and HR2 were spliced out of the sequence file and used for evaluation in the expasy pI ⁄ Mw tool to determine the isoelectric point [22–24] Preparation of HR1, HR2 and retrocyclin structure models Three separate structural representations were required for bio-computational experimentation: the HR1 and HR2 regions of JR.FL and the h-defensin RC-101 In the context of computational data, the HR1 ⁄ HR2 nomenclature refers only to the N36 and C34 peptides, respectively Threedimensional structural models of the HR1 and HR2 regions of JR.FL were generated using the swiss-model protein homology web server based on the HIV-1 gp41 core structure (Protein Data Bank accession number 1AIK) published previously [25–27] The structure for RC-101 was created using the mutagenesis function of the pymol molecular graphics system, based on the structure of retrocyclin-2 (Protein Data Bank accession number 2ATG) Two in silico mutations were performed to create RC-101: the second arginine to a glycine and the fourth arginine to a lysine [9] The backbone atoms for both mutated residues remained stationary There was no need to minimize the rotational bond energy of the mutated bonds, as all carbon–carbon or 6484 carbon–nitrogen bonds were deemed ‘rotatable’ in the docking procedure Computational modeling of RC-101 binding ‘Grid’ and ‘Docking’ parameter files for all RC-101 dockings to dimer and comparative monomer macromolecules were prepared using autodocktools (ADT) and accompanying scripts, and then run with autodock 3.0 and autogrid 3.0 [28,29] The grid parameters were the same for all three macromolecules The numbers of points in the x, y and z direction were 76, 76 and 126, respectively The grid ˚ spacing value was 0.4527 A Finally, the grid center was defined as the x,y,z coordinate (17.449, 13.8, 5.67) All other autogrid parameters remained at their default values The ligand, RC-101, was prepared using ADT according to the autodock manual [28] For each macromolecule (HR1, HR2, HR1 + HR2) and ligand (RC-101), hydrogen positions were reassigned, nonpolar hydrogens were merged, and Kollman united charges were assigned to each residue The genetic algorithm variables of population size, maximum number of energy evaluations and the maximum number of generations were increased to 200, · 106 and · 105, respectively, using the methods described by Hetenyi et al [30] as a general guideline Lower values were used because the protein model contained substantially less solvent-exposed surface area and contained less than half the average number of residues tested in previous blinddocking studies [30–32] For each docking simulation, the genetic algorithm was run 200 times to return 200 possible RC-101 docked conformations autodock reports the change in free energy upon binding for each conformation An approximate threshold range (– to )11 kcalỈmol)1) separates nonspecific interactions from prominent intermolecular bonds Each docking simulation was executed four times, and the quantitative measures of all four docking simulations were averaged and the SEM calculated Defining hydrogen and nonhydrogen bonds Tables of hydrogen bonds and nonhydrogen bonds were generated 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 tabulated, and the mean number of bonds per residue for four independent docking executions was reported by the program, together with the SEM Preparation of peptide The 18-amino-acid peptide RC-101 was synthesized as previously described [4,6,35] with the sequence: cyclic-GICRCICGKGICRCICGR After each step, the peptide was subjected to MALDI-TOF mass spectrometry to assess FEBS Journal 274 (2007) 6477–6487 ª 2007 The Authors Journal compilation ª 2007 FEBS C A Fuhrman et al homogeneity (typically approximately 95%), and to confirm that the observed mass agreed with the theoretical mass Peptide concentrations were determined by quantitative peptide analysis Cell culture HOS-CD4-CCR5 cells (N R Landau, Salk Institute for Biological Studies, La Jolla, CA, USA), which allow entry of R5 HIV-1, were acquired from the National Institutes of Health AIDS Research and Reference Reagent Program (Germantown, MD, USA) HOS cells were grown in DMEM supplemented with penicillin, streptomycin, 10% fetal bovine serum, lgỈmL)1 puromycin, and mycophenolic acid selection medium 293T cells were grown in DMEM with penicillin, streptomycin and 10% fetal bovine serum HIV-1 plasmid constructs and viral entry assay The expression vectors pNL-LucR–E– and JR.FL env were gifts from N R Landau JR.FL is an R5 strain of HIV-1 In total, 18 JR.FL env mutants were constructed Nine glutamic acids on the HR2 of HIV-1 gp41 were mutated to lysines Amino acids 632, 634, 641, 647, 648, 654, 657 and 659 were mutated from Glu (GAA) to Lys (AAA) Amino acid 636 was mutated from Asp (GAC) to Arg (CGC) For the second set of mutagenesis studies, polar or hydrophobic residues were 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) to Lys (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 env plasmid using the QuikChange multi site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), verified by sequencing (University of Central Florida Biomolecular Science Center Genomics Core Laboratory, Orlando, FL, USA) and compared to the published JR.FL wild-type sequence (accession number U63632) Subsequently, HIV-1 single-cycle (replication-incompetent) luciferase reporter viruses were produced by cotransfecting 293T cells with 10 lg each of pNL-LucR–E– and one of the JR.FL env clones Virus-containing clarified supernatants were collected after 48 h by centrifugation at 1000 g for 10 min, filtered though a 0.45 lm filter and stored at )80 °C in aliquots until needed One aliquot was used to quantify propagated pseudovirus by p24 ELISA (Perkin Elmer, Waltham, MA, USA) Another aliquot was used to ensure the integrity of the envelope gene Viral RNA was isolated from the JR.FL pseudotypes (viral RNA mini kit; Qiagen, Valencia, CA, USA) A cDNA library was created from the isolated viral RNA using the iScript Select cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) Then a 666 bp envelope region containing HR1 and HR2 was PCR-amplified, and the DNA was separated using a 1.5% agarose gel RC-101 overcomes cationic mutations in HIV-1 gp41 The sense primer used was 5¢-CTGTGTTCCTTGGG TTCTTGG-3¢, and the antisense primer was 5¢-CTCCACC TTCTTCTTCGATTCC-3¢ To measure the infectious ability of the JR.FL pseudotypes, HOS-CD4-CCR5 cells (5 · 103 per well; 96-well plate) were infected with 50 ng p24 per well of virus in the presence or the absence of RC-101 (0, 1.25, 2.5, or 10 lgỈmL)1), and luciferase activity was measured days later Acknowledgements This work was supported by grants from the National Institutes of Health: AI052017, AI065430 and AI060753 (to AMC) and AI056921 (to RIL), and from the National Science Foundation: EIA-0321333 (to SS) We thank Martin Kline (UCF) for his excellent technical help and Dr G M Morris (The Scipps Research Institute) for autodock support We are also grateful to the National Biomedical Computation 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