Title: Detailed Mechanistic Insights into HIV-1 Sensitivity to Three Generations of Fusion Inhibitors
Abstract: Peptides based on the second heptad repeat (HR2) of viral class I fusion proteins are effective inhibitors of virus entry. One such fusion inhibitor has been approved for treatment of human immunodeficiency virus-1 (T20, enfuvirtide). Resistance to T20 usually maps to the peptide binding site in HR1. To better understand fusion inhibitor potency and resistance, we combined virological, computational, and biophysical experiments with comprehensive mutational analyses and tested resistance to T20 and second and third generation inhibitors (T1249 and T2635). We found that most amino acid substitutions caused resistance to the first generation peptide T20. Only charged amino acids caused resistance to T1249, and none caused resistance to T2635. Depending on the drug, we can distinguish four mechanisms of drug resistance: reduced contact, steric obstruction, electrostatic repulsion, and electrostatic attraction. Implications for the design of novel antiviral peptide inhibitors are discussed. Peptides based on the second heptad repeat (HR2) of viral class I fusion proteins are effective inhibitors of virus entry. One such fusion inhibitor has been approved for treatment of human immunodeficiency virus-1 (T20, enfuvirtide). Resistance to T20 usually maps to the peptide binding site in HR1. To better understand fusion inhibitor potency and resistance, we combined virological, computational, and biophysical experiments with comprehensive mutational analyses and tested resistance to T20 and second and third generation inhibitors (T1249 and T2635). We found that most amino acid substitutions caused resistance to the first generation peptide T20. Only charged amino acids caused resistance to T1249, and none caused resistance to T2635. Depending on the drug, we can distinguish four mechanisms of drug resistance: reduced contact, steric obstruction, electrostatic repulsion, and electrostatic attraction. Implications for the design of novel antiviral peptide inhibitors are discussed. The HIV-1 envelope glycoprotein complex (Env), 3The abbreviations used are: Envenvelope glycoprotein complexHIV-1human immunodeficiency virus type 1HRheptad repeatWTwild type. 3The abbreviations used are: Envenvelope glycoprotein complexHIV-1human immunodeficiency virus type 1HRheptad repeatWTwild type. a class I viral fusion protein, is responsible for viral attachment to CD4+ target T cells and subsequent fusion of viral and cellular membranes resulting in release of the viral core in the cell. Other examples of viruses using class I fusion proteins are Coronaviridae (severe acute respiratory syndrome virus), Paramyxoviridae (Newcastle disease virus, human respiratory syncytial virus, Nipah virus, Hendra virus), and Orthomyxoviridae (influenza virus), some of which cause fatal diseases in humans (1Harrison S.C. Nat. Struct. Mol. Biol. 2008; 15: 690-698Crossref PubMed Scopus (919) Google Scholar, 2Kielian M. Rey F.A. Nat. Rev. Microbiol. 2006; 4: 67-76Crossref PubMed Scopus (432) Google Scholar, 3Melikyan G.B. Retrovirology. 2008; 5: 111Crossref PubMed Scopus (144) Google Scholar). The entry process of these viruses is an attractive target for therapeutic intervention.The functional trimeric Env spike on HIV-1 virions consists of three gp120 and three gp41 molecules that are the products of cleavage of the precursor gp160 by cellular proteases such as furin (4Decroly E. Vandenbranden M. Ruysschaert J.M. Cogniaux J. Jacob G.S. Howard S.C. Marshall G. Kompelli A. Basak A. Jean F. J. Biol. Chem. 1994; 269: 12240-12247Abstract Full Text PDF PubMed Google Scholar, 5Hallenberger S. Bosch V. Angliker H. Shaw E. Klenk H.D. Garten W. Nature. 1992; 360: 358-361Crossref PubMed Scopus (476) Google Scholar). The gp120 surface subunits are responsible for binding to the cellular receptors, whereas the gp41 subunits anchor the complex in the viral membrane and mediate the fusion of viral and cellular membranes. Env undergoes several conformational changes that culminate in membrane fusion. The gp120 subunit binds the CD4 receptor, resulting in creation and/or exposure of the binding site for a coreceptor, usually CCR5 or CXCR4 (6Trkola A. Purtscher M. Muster T. Ballaun C. Buchacher A. Sullivan N. Srinivasan K. Sodroski J. Moore J.P. Katinger H. J. Virol. 1996; 70: 1100-1108Crossref PubMed Google Scholar, 7Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1081) Google Scholar). Two α-helical leucine zipper-like motifs, heptad repeat 1 (HR1) and heptad repeat 2 (HR2), located in the extracellular part of gp41, play a major role in the following conformational changes. Binding of the receptors to gp120 induces formation of the pre-hairpin intermediate of gp41 in which HR1 is exposed and the N-terminal fusion peptide is inserted into the target cell membrane (1Harrison S.C. Nat. Struct. Mol. Biol. 2008; 15: 690-698Crossref PubMed Scopus (919) Google Scholar, 8Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1829) Google Scholar, 9Cladera J. Martin I. Ruysschaert J.M. O'Shea P. J. Biol. Chem. 1999; 274: 29951-29959Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 10Jacobs A. Garg H. Viard M. Raviv Y. Puri A. Blumenthal R. Vaccine. 2008; 26: 3026-3035Crossref PubMed Scopus (18) Google Scholar, 11Jones P.L. Korte T. Blumenthal R. J. Biol. Chem. 1998; 273: 404-409Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 12Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1457) Google Scholar). Subsequently, three HR1 and three HR2 domains assemble into a highly stable six-helix bundle structure that juxtaposes the viral and cellular membranes for the membrane merger. Other viruses with class I viral fusion proteins use similar HR1-HR2-mediated membrane fusion for target cell entry.Peptides based on the HR domains of class I viral fusion proteins have proven to be efficient inhibitors of virus entry for a broad range of viruses (13Bosch B.J. Martina B.E. Van Der Zee R. Lepault J. Haijema B.J. Versluis C. Heck A.J. De Groot R. Osterhaus A.D. Rottier P.J. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 8455-8460Crossref PubMed Scopus (288) Google Scholar, 14Ou W. Silver J. J. Virol. 2005; 79: 4782-4792Crossref PubMed Scopus (9) Google Scholar, 15Porotto M. Carta P. Deng Y. Kellogg G.E. Whitt M. Lu M. Mungall B.A. Moscona A. J. Virol. 2007; 81: 10567-10574Crossref PubMed Scopus (58) Google Scholar, 16Wang E. Sun X. Qian Y. Zhao L. Tien P. Gao G.F. Biochem. Biophys. Res. Commun. 2003; 302: 469-475Crossref PubMed Scopus (50) Google Scholar, 17Zhu J. Jiang X. Liu Y. Tien P. Gao G.F. J. Mol. Biol. 2005; 354: 601-613Crossref PubMed Scopus (13) Google Scholar). The HIV-1 fusion inhibitor T20 (enfuvirtide (Fuzeon)) has been approved for clinical use. T20 mimics HR2 and can bind to HR1, thereby preventing the formation of the six-helix bundle (Fig. 1) (18Wild C. Greenwell T. Matthews T. AIDS Res. Hum. Retroviruses. 1993; 9: 1051-1053Crossref PubMed Scopus (371) Google Scholar, 19Wild C. Greenwell T. Shugars D. Rimsky-Clarke L. Matthews T. AIDS Res. Hum. Retroviruses. 1995; 11: 323-325Crossref PubMed Scopus (103) Google Scholar, 20Wild C. Oas T. McDanal C. Bolognesi D. Matthews T. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 10537-10541Crossref PubMed Scopus (479) Google Scholar, 21Wild C.T. Shugars D.C. Greenwell T.K. McDanal C.B. Matthews T.J. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 9770-9774Crossref PubMed Scopus (880) Google Scholar). T1249 is a second-generation fusion inhibitor with improved antiviral potency compared with the first-generation peptide T20 (22Chinnadurai R. Münch J. Kirchhoff F. AIDS. 2005; 19: 1401-1405Crossref PubMed Scopus (29) Google Scholar, 23Eron J.J. Gulick R.M. Bartlett J.A. Merigan T. Arduino R. Kilby J.M. Yangco B. Diers A. Drobnes C. DeMasi R. Greenberg M. Melby T. Raskino C. Rusnak P. Zhang Y. Spence R. Miralles G.D. J. Infect. Dis. 2004; 189: 1075-1083Crossref PubMed Scopus (124) Google Scholar, 24Lalezari J.P. Bellos N.C. Sathasivam K. Richmond G.J. Cohen C.J. Myers Jr., R.A. Henry D.H. Raskino C. Melby T. Murchison H. Zhang Y. Spence R. Greenberg M.L. Demasi R.A. Miralles G.D. J. Infect. Dis. 2005; 191: 1155-1163Crossref PubMed Scopus (90) Google Scholar, 25Menzo S. Castagna A. Monachetti A. Hasson H. Danise A. Carini E. Bagnarelli P. Lazzarin A. Clementi M. New Microbiol. 2004; 27: 51-61PubMed Google Scholar). Recently, a series of more potent third-generation fusion inhibitors were designed (26He Y. Xiao Y. Song H. Liang Q. Ju D. Chen X. Lu H. Jing W. Jiang S. Zhang L. J. Biol. Chem. 2008; 283: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Dwyer J.J. Wilson K.L. Davison D.K. Freel S.A. Seedorff J.E. Wring S.A. Tvermoes N.A. Matthews T.J. Greenberg M.L. Delmedico M.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12772-12777Crossref PubMed Scopus (219) Google Scholar). These include T2635, which has an improved helical structure that increases stability and activity against both wild type (WT) HIV-1 and fusion inhibitor resistant variants.Both the in vitro and in vivo selection of resistance has been described for T20 (28Aquaro S. D'Arrigo R. Svicher V. Perri G.D. Caputo S.L. Visco-Comandini U. Santoro M. Bertoli A. Mazzotta F. Bonora S. Tozzi V. Bellagamba R. Zaccarelli M. Narciso P. Antinori A. Perno C.F. J. Antimicrob. Chemother. 2006; 58: 714-722Crossref PubMed Scopus (68) Google Scholar, 29Melby T. Sista P. DeMasi R. Kirkland T. Roberts N. Salgo M. Heilek-Snyder G. Cammack N. Matthews T.J. Greenberg M.L. AIDS Res. Hum. Retroviruses. 2006; 22: 375-385Crossref PubMed Scopus (62) Google Scholar, 30Mink M. Mosier S.M. Janumpalli S. Davison D. Jin L. Melby T. Sista P. Erickson J. Lambert D. Stanfield-Oakley S.A. Salgo M. Cammack N. Matthews T. Greenberg M.L. J. Virol. 2005; 79: 12447-12454Crossref PubMed Scopus (111) Google Scholar, 31Rimsky L.T. Shugars D.C. Matthews T.J. J. Virol. 1998; 72: 986-993Crossref PubMed Google Scholar, 32Wei X. Decker J.M. Liu H. Zhang Z. Arani R.B. Kilby J.M. Saag M.S. Wu X. Shaw G.M. Kappes J.C. Antimicrob. Agents Chemother. 2002; 46: 1896-1905Crossref PubMed Scopus (1326) Google Scholar, 33Baldwin C.E. Sanders R.W. Deng Y. Jurriaans S. Lange J.M. Lu M. Berkhout B. J. Virol. 2004; 78: 12428-12437Crossref PubMed Scopus (129) Google Scholar) and T1249 (23Eron J.J. Gulick R.M. Bartlett J.A. Merigan T. Arduino R. Kilby J.M. Yangco B. Diers A. Drobnes C. DeMasi R. Greenberg M. Melby T. Raskino C. Rusnak P. Zhang Y. Spence R. Miralles G.D. J. Infect. Dis. 2004; 189: 1075-1083Crossref PubMed Scopus (124) Google Scholar, 34Chinnadurai R. Rajan D. Münch J. Kirchhoff F. J. Virol. 2007; 81: 6563-6572Crossref PubMed Scopus (40) Google Scholar, 35Eggink D. Baldwin C.E. Deng Y. Langedijk J.P. Lu M. Sanders R.W. Berkhout B. J. Virol. 2008; 82: 6678-6688Crossref PubMed Scopus (72) Google Scholar, 36Melby T. Demasi R. Cammack N. Miralles G.D. Greenberg M.L. AIDS Res. Hum. Retroviruses. 2007; 23: 1366-1373Crossref PubMed Scopus (31) Google Scholar). Resistance is often caused by mutations in the HR1 binding site of the fusion inhibitor. In particular, substitutions at positions 36 (G36D/M/S), 38 (V38A/W/M/E), and 43 (N43D/K) of gp41 can cause resistance. Strikingly, substitutions at position 38 can cause resistance to both T20 and T1249, but distinct amino acid substitutions are required. At position 38 only charged amino acids (V38E/R/K) cause resistance to T1249 (35Eggink D. Baldwin C.E. Deng Y. Langedijk J.P. Lu M. Sanders R.W. Berkhout B. J. Virol. 2008; 82: 6678-6688Crossref PubMed Scopus (72) Google Scholar). Surprisingly, none of the known T20 and T1249 resistance mutations at position 38 affect the susceptibility to the third generation inhibitor T2635.We hypothesized that the use of HIV-1 as a model system could provide a more detailed understanding of resistance to fusion inhibitors. We, therefore, analyzed the effect of all 20 amino acids at resistance hotspot 38 on Env function, viral fitness, biochemical properties of gp41, and resistance to the fusion inhibitors. From the results we can propose four resistance mechanisms that differ in the way the drug-target interaction is affected at the molecular level. Furthermore, we can deduce general principles on the mechanisms of resistance against fusion inhibitors and the requirements for effective antiviral drugs.DISCUSSIONMutation of position 38 in HIV-1 gp41 can induce resistance to T20 and T1249, both in vivo and in vitro. Here, we have determined the impact of all 20 amino acids at this gp41 position for the sensitivity against three generations of peptide fusion inhibitors: T20, T1249, and T2635. In addition, we have collected biophysical and computational data on these gp41 variants. Combined, these data provide detailed insight into the underlying mechanisms of fusion inhibitor resistance.Estimated Binding Energy Versus DockingWe found a weak correlation between increased T20 resistance and decreased binding energy of the T20-HR1 complex. Thus, the binding energy can only in part explain the resistance profiles of the 38 variants. For T1249 and T2635 the resistance cannot readily be explained by the variations in binding energy. It is, therefore, likely that residue 38 does not only contribute to the binding energy but is important for the initial docking of the peptides onto HR1. Indeed, it has been described that the LLSGIV stretch (residues 32–38 of gp41) is a critical docking site for T20 (66Trivedi V.D. Cheng S.F. Wu C.W. Karthikeyan R. Chen C.J. Chang D.K. Protein Eng. 2003; 16: 311-317Crossref PubMed Scopus (43) Google Scholar), and this may also explain the critical role of position 38 in resistance development.If residue 38 is essential for binding/docking of T20, one would expect it to be crucial for the HR2 interaction as well as T20 and HR2 are similar in sequence. Indeed, we found a correlation between T20-HR1 binding energy and six-helix bundle stability (p = 0.0042), and we observed dramatic effects on six-helix bundle formation and infectivity with some substitutions. However, it should be noted that HR2 may be less dependent on such an initial docking event as it is already tethered to HR1 via the gp41 loop domain. Therefore, resistance mutations may have a more dramatic impact on the T20-HR1 interaction than on the HR2-HR1 interaction, and this is exactly a scenario that viruses would prefer for the selection of drug resistance mutations.The increase in total binding energy of T2635-HR1 and to a lesser extent T1249-HR1 may explain why these drugs are less dependent on residue 38. The relative contribution of this single residue to the total binding energy is decreased. Another issue that may be taken into account is the level of structure of the drugs in solution. T20 exists predominantly as a random coil (∼12% helix content) and needs the docking onto the HR1 coiled coil to acquire an α-helical structure, whereas T1249 is ∼50% helical in solution. T2635 was specifically designed to be more stable (∼75% helix content and a Tm of 86 °C in complex with HR1 compared with 59 °C for T1249 and <5 °C for T20) and, therefore, may be less dependent on the initial docking event to obtain its structure (27Dwyer J.J. Wilson K.L. Davison D.K. Freel S.A. Seedorff J.E. Wring S.A. Tvermoes N.A. Matthews T.J. Greenberg M.L. Delmedico M.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12772-12777Crossref PubMed Scopus (219) Google Scholar, 67Martins Do Canto A.M. Palace Carvalho A.J. Prates Ramalho J.P. Loura L.M. J. Pept. Sci. 2008; 14: 442-447Crossref PubMed Scopus (19) Google Scholar).Because the binding site of T1249 and T2635 is shifted compared with that of T20, their binding and/or docking may be more dependent on a hydrophobic pocket which is located near the C-terminal end of the HR1 coiled coil (68Liu S. Jing W. Cheung B. Lu H. Sun J. Yan X. Niu J. Farmar J. Wu S. Jiang S. J. Biol. Chem. 2007; 282: 9612-9620Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The pocket binding domain, which is absent in T20, may contribute to the enhanced binding energy for T1249 and T2635 observed in our in silico analysis. We note that the binding of a lipid binding domain at the C terminus of T20 is absent in our analysis (68Liu S. Jing W. Cheung B. Lu H. Sun J. Yan X. Niu J. Farmar J. Wu S. Jiang S. J. Biol. Chem. 2007; 282: 9612-9620Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 69Caffrey M. Cai M. Kaufman J. Stahl S.J. Wingfield P.T. Covell D.G. Gronenborn A.M. Clore G.M. EMBO J. 1998; 17: 4572-4584Crossref PubMed Scopus (368) Google Scholar, 70Caffrey M. Cai M. Kaufman J. Stahl S.J. Wingfield P.T. Gronenborn A.M. Clore G.M. J. Mol. Biol. 1997; 271: 819-826Crossref PubMed Scopus (36) Google Scholar). This may constitute an underestimation of the binding energy for T20.Mechanisms of ResistanceFrom our results, we can deduce four well defined mechanisms of resistance to fusion inhibitors (excluding the resistance of V38P that probably causes a kink in HR1) (Fig. 6).Reduced ContactSmall amino acids such as Ala/Gly/Ser at position 38 are smaller than the WT Val, creating a “gap” in the binding site for T20. This reduces the number of contacts between HR1 and T20, resulting in a lower binding energy and resistance (Fig. 6). Obviously, the same should be the case for the HR2-HR1 interaction. Indeed we observed dramatically reduced six-helix bundle stability despite the fact that A/G did not alter six-helix bundle formation itself (as indicated for example by the high α-helix content).Steric ObstructionThe opposite effect is induced by large residues Phe/His/Met/Trp/Tyr that create a “bulge,” which sterically hinders the attachment of T20 to HR1. Although these larger residues can be accommodated quite well in the six-helix bundle complex (as indicated by high helix content, considerable six-helix bundle stability, relatively high infectivity, and large binding energies for T20), they probably affect the initial docking of T20 onto HR1.These two types of resistance mechanisms suggest that there is a correlation between the size of an amino acid at position 38 and resistance to T20. We indeed observed such a trend (data not shown), although it was not significant. Both small and large amino acid caused resistance to T20, whereas intermediate sized amino acids displayed T20 sensitivity (Val/Ile/Leu). Several amino acids did not fit this trend. In particular, the charged amino acids behaved differently, which calls for alternative resistance mechanisms.Electrostatic RepulsionA third type of resistance proposed for negatively charged residues is electrostatic repulsion (Asp/Glu). This is facilitated by the juxtaposition of a negatively charged residue (Glu-146) in HR2, T20, and T1249. The presence of a negative charge at position 38 caused a decrease in α-helical content, six-helix bundle stability, and T20-HR1 binding energy. Despite the fact that amino acids like Asp and especially Glu, are well accommodated in α-helices (60Chou P.Y. Fasman G.D. Biochemistry. 1974; 13: 211-222Crossref PubMed Scopus (1826) Google Scholar, 61Levitt M. Biochemistry. 1978; 17: 4277-4285Crossref PubMed Scopus (540) Google Scholar), a large decrease in helical content is observed. This suggests that these amino acids do not disturb the formation of the HR1 helix but affect the HR2 helix. This explains the high level of resistance to T20 and the resistance to T1249. The structural design of the T2635 peptide reveals why this peptide is not repulsed by the negative HR1 charge. In the design of T2635, this Glu-146 residue is specifically placed in a helix stabilizing salt bridge, resulting in a net neutral charge in that area of the peptide (Fig. 5C). To directly test this scenario, we paired the negative charge of T1249 in an intramolecular salt bridge (T1249mut), which reduced the resistance profile of the V38D/E mutants dramatically.Electrostatic AttractionInterestingly, positive charges also resulted in resistance to both T20 and T1249. We do not have an entirely satisfying explanation for this, but the potential formation of a salt bridge between Arg/Lys and Glu-146 may not be accommodated in a configuration that is compatible with the proper T20-HR1 or HR2-HR1 packing (as confirmed by a reduced helicity and stability of the six-helix bundle). The introduction of a positive charge affects the electrostatic potential and might result in improper steering of the components when docking. Although a salt bridge might form, this is probably not compatible with an optimal HR1-peptide interface.Other common T20 and T1249 resistance mutations identified in vitro and in vivo are G36D/E and N43D/K, representing changes from neutral to negative and positive charges (29Melby T. Sista P. DeMasi R. Kirkland T. Roberts N. Salgo M. Heilek-Snyder G. Cammack N. Matthews T.J. Greenberg M.L. AIDS Res. Hum. Retroviruses. 2006; 22: 375-385Crossref PubMed Scopus (62) Google Scholar, 30Mink M. Mosier S.M. Janumpalli S. Davison D. Jin L. Melby T. Sista P. Erickson J. Lambert D. Stanfield-Oakley S.A. Salgo M. Cammack N. Matthews T. Greenberg M.L. J. Virol. 2005; 79: 12447-12454Crossref PubMed Scopus (111) Google Scholar, 35Eggink D. Baldwin C.E. Deng Y. Langedijk J.P. Lu M. Sanders R.W. Berkhout B. J. Virol. 2008; 82: 6678-6688Crossref PubMed Scopus (72) Google Scholar, 71Greenberg M.L. Cammack N. J. Antimicrob. Chemother. 2004; 54: 333-340Crossref PubMed Scopus (208) Google Scholar, 72Sista P.R. Melby T. Davison D. Jin L. Mosier S. Mink M. Nelson E.L. DeMasi R. Cammack N. Salgo M.P. Matthews T.J. Greenberg M.L. AIDS. 2004; 18: 1787-1794Crossref PubMed Scopus (142) Google Scholar, 73Xu L. Pozniak A. Wildfire A. Stanfield-Oakley S.A. Mosier S.M. Ratcliffe D. Workman J. Joall A. Myers R. Smit E. Cane P.A. Greenberg M.L. Pillay D. Antimicrob. Agents Chemother. 2005; 49: 1113-1119Crossref PubMed Scopus (155) Google Scholar) that may influence the HR1-peptide interaction in a similar fashion as V38D/E/K/R. Residue 43 in HR1 is in close contact with residues Glu-137 in HR2 or peptide inhibitors (74Bai X. Wilson K.L. Seedorff J.E. Ahrens D. Green J. Davison D.K. Jin L. Stanfield-Oakley S.A. Mosier S.M. Melby T.E. Cammack N. Wang Z. Greenberg M.L. Dwyer J.J. Biochemistry. 2008; 47: 6662-6670Crossref PubMed Scopus (30) Google Scholar). We studied the binding energies of G36D, V38D, and N43D HR1 variants with the three generations of peptide inhibitors in silico (supplemental Fig. S3 and Table S1). G36D, V38D, and N43D caused a decrease in binding energy of T20 corresponding with the observed resistance (29Melby T. Sista P. DeMasi R. Kirkland T. Roberts N. Salgo M. Heilek-Snyder G. Cammack N. Matthews T.J. Greenberg M.L. AIDS Res. Hum. Retroviruses. 2006; 22: 375-385Crossref PubMed Scopus (62) Google Scholar, 30Mink M. Mosier S.M. Janumpalli S. Davison D. Jin L. Melby T. Sista P. Erickson J. Lambert D. Stanfield-Oakley S.A. Salgo M. Cammack N. Matthews T. Greenberg M.L. J. Virol. 2005; 79: 12447-12454Crossref PubMed Scopus (111) Google Scholar, 32Wei X. Decker J.M. Liu H. Zhang Z. Arani R.B. Kilby J.M. Saag M.S. Wu X. Shaw G.M. Kappes J.C. Antimicrob. Agents Chemother. 2002; 46: 1896-1905Crossref PubMed Scopus (1326) Google Scholar). N43D, but not G36D and V38D, caused a decrease in binding energy of T1249, confirming that binding energy is not the only determinant for sensitivity to T1249. All three HR1 variants displayed decreased binding energy with T2635, but the differences are relatively small when the total binding energy is taken into account. Thus, the resistance mechanisms we describe are likely to apply to other positions than 38 as well.Implications for the Design of Novel Antiviral Fusion InhibitorsWe can make two important recommendations for the design of novel peptide fusion inhibitors. First, we give indications for the relative minimal binding energy that is required for potent inhibition and that may prevent easy viral escape by a single mutation in the binding site. To prevent a single substitution in the binding site from causing resistance, the binding energy for the peptide-target interaction, which can be computed from the six-helix bundle structure, should be high such that a single mutation only causes a relatively small change in the overall binding energy. We cannot give absolute values as our calculated binding energies are only an approximation and different methods might lead to different estimates. An additional advantage of such a mutation-tolerant mechanism of inhibition is that more natural virus variants will display sensitivity to the drug. The accumulation of multiple resistance mutations may provide resistance but will require more time (75Keulen W. Boucher C. Berkhout B. Antiviral Res. 1996; 31: 45-57Crossref PubMed Scopus (58) Google Scholar) and will likely have a more dramatic effect on viral fitness.Second, our data suggest that the presence of exposed charges on the peptide at the drug-target interface, unless involved in an intramolecular salt bridge, are not desirable as it provides the virus with an easy possibility to generate the most powerful mechanism of resistance; that is, electrostatic repulsion. These findings may guide the design of novel fusion inhibitors targeting viruses with class I fusion proteins. The HIV-1 envelope glycoprotein complex (Env), 3The abbreviations used are: Envenvelope glycoprotein complexHIV-1human immunodeficiency virus type 1HRheptad repeatWTwild type. 3The abbreviations used are: Envenvelope glycoprotein complexHIV-1human immunodeficiency virus type 1HRheptad repeatWTwild type. a class I viral fusion protein, is responsible for viral attachment to CD4+ target T cells and subsequent fusion of viral and cellular membranes resulting in release of the viral core in the cell. Other examples of viruses using class I fusion proteins are Coronaviridae (severe acute respiratory syndrome virus), Paramyxoviridae (Newcastle disease virus, human respiratory syncytial virus, Nipah virus, Hendra virus), and Orthomyxoviridae (influenza virus), some of which cause fatal diseases in humans (1Harrison S.C. Nat. Struct. Mol. Biol. 2008; 15: 690-698Crossref PubMed Scopus (919) Google Scholar, 2Kielian M. Rey F.A. Nat. Rev. Microbiol. 2006; 4: 67-76Crossref PubMed Scopus (432) Google Scholar, 3Melikyan G.B. Retrovirology. 2008; 5: 111Crossref PubMed Scopus (144) Google Scholar). The entry process of these viruses is an attractive target for therapeutic intervention. envelope glycoprotein complex human immunodeficiency virus type 1 heptad repeat wild type. envelope glycoprotein complex human immunodeficiency virus type 1 heptad repeat wild type. The functional trimeric Env spike on HIV-1 virions consists of three gp120 and three gp41 molecules that are the products of cleavage of the precursor gp160 by cellular proteases such as furin (4Decroly E. Vandenbranden M. Ruysschaert J.M. Cogniaux J. Jacob G.S. Howard S.C. Marshall G. Kompelli A. Basak A. Jean F. J. Biol. Chem. 1994; 269: 12240-12247Abstract Full Text PDF PubMed Google Scholar, 5Hallenberger S. Bosch V. Angliker H. Shaw E. Klenk H.D. Garten W. Nature. 1992; 360: 358-361Crossref PubMed Scopus (476) Google Scholar). The gp120 surface subunits are responsible for binding to the cellular receptors, whereas the gp41 subunits anchor the complex in the viral membrane and mediate the fusion of viral and cellular membranes. Env undergoes several conformational changes that culminate in membrane fusion. The gp120 subunit binds the CD4 receptor, resulting in creation and/or exposure of the binding site for a coreceptor, usually CCR5 or CXCR4 (6Trkola A. Purtscher M. Muster T. Ballaun C. Buchacher A. Sullivan N. Srinivasan K. Sodroski J. Moore J.P. Katinger H. J. Virol. 1996; 70: 1100-1108Crossref PubMed Google Scholar, 7Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1081) Google Scholar). Two α-helical leucine zipper-like motifs, heptad repeat 1 (HR1) and heptad repeat 2 (HR2), located in the extracellular part of gp41, play a major role in the following conformational changes. Binding of the receptors to gp120 induces formation of the pre-hairpin intermediate of gp41 in which HR1 is exposed and the N-terminal fusion peptide is inserted into the target cell membrane (1Harrison S.C. Nat. Struct. Mol. Biol. 2008; 15: 690-698Crossref PubMed Scopus (919) Google Scholar, 8Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1829) Google Scholar, 9Cladera J. Martin I. Ruysschaert J.M. O'Shea P. J. Biol. Chem. 1999; 274: 29951-29959Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 10Jacobs A. Garg H. Viard M. Raviv Y. Puri A. Blumenthal R. Vaccine. 2008; 26: 3026-3035Crossref PubMed Scopus (18) Google Scholar, 11Jones P.L. Korte T. Blumenthal R. J. Biol. Chem. 1998; 273: 404-409Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 12Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1457) Google Scholar). Subsequently, three HR1 and three HR2 domains assemble into a highly stable six-helix bundle structure that juxtaposes the viral and cellular membranes for the membrane merger. Other viruses with class I viral fusion proteins use similar HR1-HR2-mediated membrane fusion for target cell entry. Peptides based on the HR domains of class I viral fusion proteins have proven to be efficient inhibitors of virus entry for a broad range of viruses (13Bosch B.J. Martina B.E. Van Der Zee R. Lepault J. Haijema B.J. Verslu