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• W2088341183 abstract "Sifuvirtide, a novel fusion inhibitor against human immunodeficiency virus type I (HIV-1), which is more potent than enfuvirtide (T20) in cell culture, is currently under clinical investigation for the treatment of HIV-1 infection. We now report that in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide has led to several specific mutations in the gp41 region that had not been previously reported. Many of these substitutions were confined to the N-terminal heptad repeat region at positions 37, 38, 41, and 43, either singly or in combination. A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat region was also found. Site-directed mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses. Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide. Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic utility of sifuvirtide. Sifuvirtide, a novel fusion inhibitor against human immunodeficiency virus type I (HIV-1), which is more potent than enfuvirtide (T20) in cell culture, is currently under clinical investigation for the treatment of HIV-1 infection. We now report that in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide has led to several specific mutations in the gp41 region that had not been previously reported. Many of these substitutions were confined to the N-terminal heptad repeat region at positions 37, 38, 41, and 43, either singly or in combination. A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat region was also found. Site-directed mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses. Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide. Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic utility of sifuvirtide. The envelope glycoprotein (Env) of HIV-1 is critical in mediating viral entry into the target cells, and it represents a major target for the development of novel antiretroviral therapeutics. The entry process starts with the binding of gp120 to a cellular receptor, CD4, and subsequently with a chemokine receptor, CCR5 or CXCR4, on the surface of the target cells (1Melikyan G.B. Retrovirology. 2008; 5: 111Crossref PubMed Scopus (144) Google Scholar). These interactions trigger a cascade of conformational changes that lead to the formation of a prehairpin intermediate of gp41 in which the hydrophobic N-terminal heptad repeat (NHR) 2The abbreviations used are: NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; 6-HB, six-helix bundle. is exposed and allows the fusion peptides to insert into the target cell membrane (1Melikyan G.B. Retrovirology. 2008; 5: 111Crossref PubMed Scopus (144) Google Scholar, 2Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar). This transient gp41 intermediate then refolds into a stabilized trimer of hairpins, also called the six-helix bundle (6-HB) structure, which brings the viral envelope and the target cell membrane into close proximity, thus facilitating the completion of the fusion process (1Melikyan G.B. Retrovirology. 2008; 5: 111Crossref PubMed Scopus (144) Google Scholar, 2Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar, 3Root M.J. Kay M.S. Kim P.S. Science. 2001; 291: 884-888Crossref PubMed Scopus (381) Google Scholar, 4Jiang S. Debnath A.K. Biochem. Biophys. Res. Commun. 2000; 269: 641-646Crossref PubMed Scopus (53) Google Scholar). Structure and function studies indicate that the 6-HB core consists of a parallel trimeric coiled-coil of NHR with the C-terminal heptad repeat (CHR) wrapped on the outside in an antiparallel fashion (5Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar, 6Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1460) Google Scholar, 7Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12303-12308Crossref PubMed Scopus (519) Google Scholar). Similar features have been found in the fusion-mediating subunits of other viruses with class I membrane fusion proteins (8Rapaport D. Ovadia M. Shai Y. EMBO J. 1995; 14: 5524-5531Crossref PubMed Scopus (163) Google Scholar, 9Blacklow S.C. Lu M. Kim P.S. Biochemistry. 1995; 34: 14955-14962Crossref PubMed Scopus (126) Google Scholar, 10Lombardi S. Massi C. Indino E. La Rosa C. Mazzetti P. Falcone M.L. Rovero P. Fissi A. Pieroni O. Bandecchi P. Esposito F. Tozzini F. Bendinelli M. Garzelli C. Virology. 1996; 220: 274-284Crossref PubMed Scopus (39) Google Scholar, 11Wang E. Sun X. Qian Y. Zhao L. Tien P. 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Chem. 2004; 279: 20836-20849Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 17Xu Y. Lou Z. Liu Y. Pang H. Tien P. Gao G.F. Rao Z. J. Biol. Chem. 2004; 279: 49414-49419Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Inhibitors capable of disrupting the entry process hold great promise for improved efficacy of clinical antiviral therapeutics. Several entry inhibitors have been developed, and two of these have been approved for clinical use, such as a peptide-based inhibitor enfuvirtide (T20) and a small molecule inhibitor Maraviroc against HIV-1 co-receptor CCR5 (18Dorr P. Westby M. Dobbs S. Griffin P. Irvine B. Macartney M. Mori J. Rickett G. Smith-Burchnell C. Napier C. Webster R. Armour D. Price D. Stammen B. Wood A. Perros M. Antimicrob. Agents Chemother. 2005; 49: 4721-4732Crossref PubMed Scopus (963) Google Scholar, 19Fätkenheuer G. Pozniak A.L. Johnson M.A. Plettenberg A. Staszewski S. Hoepelman A.I. Saag M.S. Goebel F.D. Rockstroh J.K. Dezube B.J. Jenkins T.M. Medhurst C. Sullivan J.F. Ridgway C. Abel S. James I.T. Youle M. van der Ryst E. Nat. Med. 2005; 11: 1170-1172Crossref PubMed Scopus (451) Google Scholar, 20Kromdijk W. Huitema A.D. Mulder J.W. Expert Opin. Pharmacother. 2010; 11: 1215-1223Crossref PubMed Scopus (27) Google Scholar). In addition, several other entry inhibitors are being developed. These include the co-receptor antagonist small molecule inhibitor vicriviroc, the anti-CCR5 monoclonal antibody (21Trkola A. Ketas T.J. Nagashima K.A. Zhao L. Cilliers T. Morris L. Moore J.P. Maddon P.J. Olson W.C. J. Virol. 2001; 75: 579-588Crossref PubMed Scopus (226) Google Scholar, 22Jacobson J.M. Saag M.S. Thompson M.A. Fischl M.A. Liporace R. Reichman R.C. Redfield R.R. Fichtenbaum C.J. Zingman B.S. Patel M.C. Murga J.D. Pemrick S.M. D'Ambrosio P. Michael M. Kroger H. Ly H. Rotshteyn Y. Buice R. Morris S.A. Stavola J.J. Maddon P.J. Kremer A.B. Olson W.C. J. Infect. Dis. 2008; 198: 1345-1352Crossref PubMed Scopus (97) Google Scholar), the anti-human CD4 monoclonal antibody ibalizumab (TNX-355) (23Kuritzkes D.R. Jacobson J. Powderly W.G. Godofsky E. DeJesus E. Haas F. Reimann K.A. Larson J.L. Yarbough P.O. Curt V. Shanahan Jr., W.R. J. Infect. Dis. 2004; 189: 286-291Crossref PubMed Scopus (176) Google Scholar), and new generations of peptide-based and small molecule inhibitors targeting the fusion process (24Dwyer 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, 25Liu K. Lu H. Hou L. Qi Z. Teixeira C. Barbault F. Fan B.T. Liu S. Jiang S. Xie L. J. Med. Chem. 2008; 51: 7843-7854Crossref PubMed Scopus (119) Google Scholar, 26Katritzky A.R. Tala S.R. Lu H. Vakulenko A.V. Chen Q.Y. Sivapackiam J. Pandya K. Jiang S. Debnath A.K. J. Med. Chem. 2009; 52: 7631-7639Crossref PubMed Scopus (104) Google Scholar, 27Pan C. Cai L. Lu H. Qi Z. Jiang S. J. Virol. 2009; 83: 7862-7872Crossref PubMed Scopus (52) Google Scholar, 28Chen X. Lu L. Qi Z. Lu H. Wang J. Yu X. Chen Y. Jiang S. J. Biol. Chem. 2010; 285: 25506-25515Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We have previously characterized a new generation peptide-based fusion inhibitor known as sifuvirtide, which has an improved stability, pharmacokinetics, and antiviral potency, as compared with enfuvirtide (29He 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). In this report, we describe the in vitro selection and characterization of HIV-1 variants with relative resistance to sifuvirtide. We derived resistant variants of HIV-1 NL4-3 by serial passage in the presence of increasing concentrations of sifuvirtide. By sequencing analysis of resistant variants, we have identified several novel specific mutations in the NHR and CHR regions in gp41 associated with observed resistant phenotypes. Site-directed mutagenesis studies have further identified the critical amino acid substitutions in conferring the resistant genotypes to sifuvirtide and cross-resistance to enfuvirtide. Finally, biophysical and structural analyses of these substitutions have revealed several potential mechanisms against sifuvirtide. These results may help to predict potential patterns of resistance in vivo and may facilitate the further clinical development and therapeutic utility of sifuvirtide. MT4 cells were seeded at 3 × 105/ml in RPMI 1640 medium containing 10% fetal bovine serum on a 96-well plate. Serial dilutions of wild-type virus, molecular clone NL4-3, were added and followed by incubation at 37 °C with 5% CO2 for 5 days. The concentration of sifuvirtide required to inhibit 50% viral infection was calculated based on the cytopathic effect. For selection of sifuvirtide-resistant virus, we initially used 7.8 ng/ml sifuvirtide for wild-type virus, which can inhibit virus replication by about 90%. Cells were incubated at 37 °C with 5% CO2 until extensive cytopathic effect was observed, and supernatants were used for the next passage in MT4 cells with 1.5–2-fold increasing concentrations of sifuvirtide. During the course of selection, wild-type virus was used without sifuvirtide as a parallel control. After 15 passages, the virus isolates were able to grow at a sifuvirtide concentration up to 5 μg/ml. Cells were pelleted by centrifugation, and DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen). The full-length gp41 gene was amplified from cellular DNA with primers (forward primer, 5′-TGGAGGAGGCGATATGAGGG-3′; reverse primer, 5′-GATAGTAGGAGGCTTGGTAG-3′) using the Platinum® TaqDNA polymerase high fidelity PCR system (Invitrogen). The PCR products were analyzed on 1% agarose gel and then purified by the QIAquick gel extraction kit (Qiagen). PCR products were sequenced and analyzed by BioEdit software (available on the World Wide Web). Plasmid pNL4-3 containing the full-length genome of HIV-1 (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health) was digested with restriction endonucleases NheI and BamHI to release the fragment containing the full-length gp41 and cloned into the vector pVAX1/lacZ (Invitrogen). Mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene) verified by direct sequencing, and the fragments containing mutations were cloned back to original NL4-3. The virus stock for wild type and mutant viruses containing various mutations were produced by transfecting 293T cells using FuGENE®6 transfection reagent (Roche Applied Science). The supernatants containing infectious wild-type and mutant viruses were harvested after a 48-h incubation. The concentration of p24 of each virus was measured by an HIV-1 antigen kit (Vironostika, Netherlands) and stored at −80 °C until use. We used TZM-bl cells (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health) to study viral infectivity. These cells are genetically engineered to stably express high levels of CD4 and HIV-1 co-receptors CCR5 and CXCR4 and also to contain the luciferase and β-galactosidase genes under the control of the HIV-1 long terminal repeat promoter. Approximately 3 × 104 cells/well were plated on a 96-well plate in Dulbecco's modified Eagle's medium containing 10% fetal bovine and penicillin/streptomycin and then incubated at 37 °C with 5% CO2. Serial dilution of sifuvirtide or enfuvirtide was added to the cells. One nanogram of p24 equivalent viral stock was added, and each concentration of sifuvirtide or enfuvirtide was assayed in triplicate. Forty-eight hours postinfection, 50 μl of supernatants were discarded, and 50 μl of Bright Glo reagent (Promega) was added to the each well. After a 5-min incubation at room temperature to allow cell lysis, 100 μl of cell lysate was transferred to 96-well black solid plates for measurements of luminescence using a Berthold Centro LB 960 luminometer (Berthold Technologies). Nonlinear regression curves were generated, and 50% inhibitory concentration (IC50) was calculated using Prism software version 4.0 (GraphPad Software). Sifuvirtide peptide was kindly provided by Dr. Genfa Zhou (FusoGen Pharmaceuticals, Inc., Tianjin, China). C34, N36, and mutant N peptides were synthesized by a standard solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) method using a ThuraMed TETRAS synthesizer (Louisville, KY). All peptides were acetylated at the N termini and amidated at the C termini. The peptides were purified to homogeneity (>95% purity) by HPLC (Agilent 1200) and further analyzed by laser desorption mass spectrometry (Waters, Milford, MA). The concentration of peptides was determined by UV absorbance and a theoretically calculated molar extinction coefficient ϵ (280 nm) of 5500 mol/liter−1 cm−1 and 1490 mol/liter−1 cm−1 based on the number of tryptophan residues and tyrosine residues (all of the tested peptides contain Trp and/or Tyr). Native PAGE was carried out to determine the 6-HB formation between the C and N peptides as described previously (30Liu S. Zhao Q. Jiang S. Peptides. 2003; 24: 1303-1313Crossref PubMed Scopus (69) Google Scholar). The ability of various mutant N peptides to form 6-HB with C34 was measured by an ELISA (31Jiang S. Lin K. Zhang L. Debnath A.K. J. Virol. Methods. 1999; 80: 85-96Crossref PubMed Scopus (110) Google Scholar) with modification. The secondary structure of the peptides and their mixtures was analyzed by CD spectroscopy as described previously (32Liu 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, 33Zhu Y. Lu L. Xu L. Yang H. Jiang S. Chen Y.H. J. Virol. 2010; 84: 9359-9368Crossref PubMed Scopus (18) Google Scholar). The structural modeling of interaction between mutant N peptides (including Q41K, Q41H, Q41R, N43K, I37T, and V38A) and C peptide (C34 or mutant N126K) was based on the trimeric structure recently obtained from HIV-1 HXB2 (Protein Data Bank code 2X7R) (34Buzon V. Natrajan G. Schibli D. Campelo F. Kozlov M.M. Weissenhorn W. PLoS Pathog. 2010; 6: e1000880Crossref PubMed Scopus (203) Google Scholar). The updated structure contains both fusion peptide and membrane-proximal external regions of gp41 and represents the most complete example of interaction between the N peptide and C peptide (34Buzon V. Natrajan G. Schibli D. Campelo F. Kozlov M.M. Weissenhorn W. PLoS Pathog. 2010; 6: e1000880Crossref PubMed Scopus (203) Google Scholar). The 6-HB for each mutant peptide was constructed through symmetry operation in alignment mode using the automated protein modeling program on the SWISSMODEL protein-modeling server (35Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Crossref PubMed Scopus (899) Google Scholar). HIV-1 isolates resistant to sifuvirtide were generated and selected by serial passage of molecular clone HIV-1 NL4-3 through MT-4 cells with increasing concentrations of sifuvirtide. A total of 12 independent cultures were conducted with a starting concentration of sifuvirtide at 7.8 ng/ml, which is sufficient to inhibit about 90% of virus replication. About 1 ng of p24 equivalent of HIV-1 NL4-3 was added to the MT-4 cells 1 h before the addition of sifuvirtide. Half of the culture medium was replaced every other day, and sifuvirtide with appropriate concentration was maintained in the culture medium throughout the passage process. Cells were routinely monitored for cytopathic effect. Supernatant in which the cytopathic effect was present was collected and continued to culture with a 2-fold higher concentration of sifuvirtide. After 9–15 passages, all 12 HIV-1 isolates continued to replicate in the presence of sifuvirtide up to 5 μg/ml, although variability existed among different isolates on the detectable levels of cytopathic effect. The marked increase in sifuvirtide concentration suggests the presence of highly resistant variants in the viral culture. To study the genetic changes associated with resistance to sifuvirtide, proviral DNAs from infected cells of all 12 independent cultures at the last passages were subjected to PCR to amplify the entire gp41 region. As shown in the upper panel of Fig. 1, several amino acid substitutions were identified in the NHR region, some of which have been identified previously against C34, SC34, and SC34-EK (36Izumi K. Kodama E. Shimura K. Sakagami Y. Watanabe K. Ito S. Watabe T. Terakawa Y. Nishikawa H. Sarafianos S.G. Kitaura K. Oishi S. Fujii N. Matsuoka M. J. Biol. Chem. 2009; 284: 4914-4920Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 37Shimura K. Nameki D. Kajiwara K. Watanabe K. Sakagami Y. Oishi S. Fujii N. Matsuoka M. Sarafianos S.G. Kodama E.N. J. Biol. Chem. 2010; 285: 39471-39480Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), whereas some or a combination thereof have not been reported previously. No substitutions were found in the pocket-binding domain despite the ubiquitous presence of sifuvirtide (Fig. 1, top). These substitutions are located at positions 37, 38, 41, and 43, either singly or in combination. Notably, all substitutions at position 37 involve the change of isoleucine to threonine (I37T, 6 of 12) (1a, 1b, 1c, 2c, 3b, and 4b), whereas the substitution at position 43 produces the change from asparagine to lysine (N43K, 5 of 12) (1b, 1c, 2c, 3a, and 4a). Substitutions at position 38 are more variable, from valine to either methionine (V38M, 2 of 12) or alanine (V38A, 1 of 12). Substitutions at position 41 are unique in that many of the mutants bear positively charged residues, such as arginine (Q41R, 2 of 12), lysine (Q41K, 2 of 12), or histidine (Q41H, 1 of 12), which have only been reported a few times among over 2229 standard HIV-1 sequences in the data base (available on the World Wide Web). Furthermore, analysis of combinational substitutions revealed that double mutations at positions 37 and 43 (I37T/N43K) are the most frequent, having been identified in three (1b, 1c, and 2c) of the 12 independent cultures (Fig. 1). The paired substitutions have so far not been reported in naturally infected or treated patients or in any in vitro culture resistant to other peptide-based fusion inhibitors against HIV-1. Triple mutations were also identified in two cultures (3b and 4a), both of which share the positively charged residues at position 41 (Fig. 1). Sporadic mutations at positions 71 and 113 involving alanine to threonine (A71T) and asparagine to aspartic acid (N113D), respectively, were identified only for clone 1c (data now shown). Last, a downstream substitution from asparagine to lysine at position 126 within the CHR region was also observed in five of the 12 cultures (1a, 1c, 2c, 3b, 3c, and 4a) (data not shown). Codon changes at the nucleotide level leading to the non-synonymous substitutions shown in Fig. 1 are listed in boldface italic type in Table 1. No preferences were found toward either transition or transversion.TABLE 1Amino acid substitutions and corresponding codon changes in 12 independent culturesCulture numberAmino acid substitution(s)Codon change1aI37T/N126KATA→ACA/AAT→AAA1bI37T/N43KATA→ACA/AAT→AAA1cI37T/N43K/A71T/N113D/N126KATA→ACA/AAT→AAA/GCT→ACT/AAT→GAT/AAT→AAG2aV38MGTG→ATG2bQ41HCAG→CAC2cI37T/N43K/N126KATA→ACA/AAT→AAA/AAT→AAG3aN43KAAT→AAA3bI37T/V38A/ Q41R/N126KATA→ACA/GTG→GCG/CAG→CGG/AAT→AAA3cQ41R/N126KCAG→CGG/AAT→AAA4aV38M/Q41K/N43K/N126KGTG→ATG/CAG→AAG/AAT→AAA/AAT→AAA4bI37TATA→ACA4cQ41KCAG→AAG Open table in a new tab To study which mutation(s) play the dominant role in conferring observed resistance to sifuvirtide, we have introduced each individual mutation, as well as double and multiple mutations, into the NHR region of gp41 and further cloned into the backbone of wild-type molecular clone pNL4-3. All 12 mutant clones identified in the culture (1a–4c), as well as some additional single mutants, were constructed to study the relative contribution of individual mutations to the overall resistance. A total of 22 mutant clones were constructed, and their sequences were confirmed by sequencing before generating the viral stock for infection analysis (Table 2). Fig. 2 shows the drug sensitivity data to sifuvirtide of nine representative mutant clones. It is evident that among all of the single mutants, there is a clear trend of increase in the level of resistance from Q41H, V38M, I37T, V38A, N43K, Q41R, and Q41K relative to the wild-type NL4-3 (Table 2 and Fig. 2). Substitutions at position 41 result in dramatic differences in resistance, with Q41K the strongest, Q41R intermediate, and Q41H the least resistant (Fig. 2 and Table 2). For the double mutants, both I37T/N43K and I37T/V38A demonstrated significant increases in resistance (Fig. 2 and Table 2, middle). However, substitution N43K clearly contributes more than V38A in the context of I37T background. Last, triple and multiple substitutions have clear added benefit to the viruses upon challenge by sifuvirtide. Most notable is the substitution N126K, which elevates the resistance level dramatically in the context of I37T/N43K, whereas the impact of sporadic substitutions, such as A71T and N113D, is rather minimal (Table 2, bottom). It should be noted that mutants with substitutions identical to culture 3b and 4a and other clones bearing substitutions in addition to that at position 41 (Q41K or Q41R) resulted in defective viruses, which failed to infect the target cells despite multiple attempts (Table 2, bottom).TABLE 2Resistance of mutant viruses to sifuvirtide and T20SifuvirtideEnfuvirtideMutant virusesIC50 (S.D.)ResistanceIC50 (S.D.)Resistancenm-foldnm-foldNL4–32.87 (0.36)1.00229.44 (30.11)1.00I37T15.31 (0.38)5.331677.75 (379.55)7.31V38A18.17 (1.28)6.333520.67 (481.12)15.34V38M9.22 (1.53)3.211549.89 (111.69)6.76Q41K114.92 (5.67)40.043889.44 (1711.46)16.95Q41H5.28 (3.32)1.84800.22 (444.04)3.49Q41R80.96 (9.71)28.214093.93 (715.51)17.84N43K27.61 (1.67)9.621462.02 (262.25)6.37A71T5.71 (1.02)1.99301.57 (24.27)1.31N113D1.95 (1.43)0.68298.2 (112.13)1.30N126K10.83 (1.47)3.77471.24 (57.3)2.05I37T/V38A126.59 (30.98)44.108301.35 (2246.29)36.18I37T/N43K277.85 (25.96)96.802182.47 (696.63)9.51I37T/N43K/A71T248.61 (2.58)86.622973.93 (417.3)12.96I37T/N43K/N126K439.18 (30.05)153.011916.4 (937.08)8.35I37T/N43K/N113D256.51 (29.3)89.374629.89 (372.13)20.18I37T/N43K/A71T/N126K419.97 (125.79)146.322014.38 (339.55)8.78I37T/N43K/A71T/N113D/N126K590.06 (114.53)205.584807.87 (189.89)20.95V38A/Q41RV38M/Q41KI37T/V38A/Q41RV38M/Q41K/N43KI37T/V38A/Q41R/N126K Open table in a new tab Taken together, our results suggest that resistance to sifuvirtide is most likely a result of multiple different substitutions, either alone or in combination, leading to different resistance mechanisms. Single substitution at position 41 (Q41K or Q41R) and double substitutions I37T/N43K or I37T/V38A in combination with N126K are probably the key mutations responsible for observed increases in resistant phenotype. A cross-resistance study was further conducted against the clinically approved peptide-based inhibitor enfuvirtide. All mutant clones, together with wild-type molecular clone NL4-3, were subjected to serial concentrations of either enfuvirtide or sifuvirtide in the cell supernatant. Their sensitivity to inhibitions by either enfuvirtide or sifuvirtide and -fold increases relative to wild-type NL4-3 are summarized in Table 2. In terms of absolute molar concentration, sifuvirtide is close to 80-fold more potent than enfuvirtide in inhibiting wild-type NL4-3, consistent with our previous observation (29He 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). For cross-resistance analysis, we focused more on the -fold increases and investigated whether the same substitutions resulted in different resistance variation against sifuvirtide and enfuvirtide. For all mutants with single substitutions, there is a good correlation between -fold increases for sifuvirtide and enfuvirtide. The most evident are the Q41K and Q41R substitutions, which result in double-digit increases toward both sifuvirtide (40.04 and 28.21) and enfuvirtide (16.95 and 17.84) (Table 2, top). Q41H substitution, despite being at the same location, had only minimal impact on cross-resistance (1.84 versus 3.49) (Table 2, top). Substitutions at position 37, 38, or 43 share a similar level of cross-resistance at the single-digit level, although the V38A mutant is probably more resistant to enfuvirtide than to sifuvirtide (15.34 versus 6.33), whereas the N43K mutant seems to be more resistant to sifuvirtide (9.62 versus 6.37) (Table 2, top). Downstream substitutions, such as A71T and N113D, have negligible impact on overall resistance, whereas minor increases in cross-resistance were observed for the mutant with N126K substitution (3.77 versus 2.05) (Table 2, top). For mutants with double substitutions, I37T/V38A resulted in similar level of increase in resistance to sifuvirtide and cross-resistance to enfuvirtide (44.10 versus 36.18) (Table 2, middle), which is quite comparable with resistance conferred by single substitutions Q41K and Q41R (see above). Substitution I37T/N43K, however, preferentially shows more resistance to sifuvirtide than cross-resistance to enfuvirtide, and the difference reached over more than 10-fold (96.80 versus 9.51) (Table 2, middle). For mutants with triple substitutions, it is clear that the additional downstream substitution of N126K in the context of I37T/N43K has led to three-digit increases in resistance against sifuvirtide but only minimal cross-resistance to enfuvirtide (153.01 versus 8.35) (Table 2, bottom). Although additional single A71T or N113D substitution had no added resistance to sifuvirtide, there was a trend toward more cross-resistance to enfuvirtide (Table 2, bottom). Finally, increases in resistance for mutants with four or more substitutions are quite comparable with those of triple mutants, although both a slightly higher resistance and cross-resistance were noticed for the mutant with I37T/N43K/A71T/N113D/N126K substitutions in combination (Table 2, bottom). To measure the infectivity of mutant clones, we have performed an infection assay in the absence of sifuvirtide or other inhibitors using the TZM-bl reporter cell line. One nanogram of p24 equivalent of each mutant virus, together with wild-type NL4-3, was used to infect the target cells, and luciferase activity was measured 2 days after infection. Infectivity of NL4-3 was normalized to 100%, and the relative infectivity of other mutants was calculated accordingly. For mutants with single substitutions, I37T resulted in an ∼50% increase in infectivity, whereas Q41H remained similar to that of the wild-type NL4-3 (Fig. 3). In contrast, other mutant viruses revealed variable and diminished infectivity. Some substitutions, such as Q41K, Q41R, and V38M, severely" @default.
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• W2088341183 title "In Vitro Selection and Characterization of HIV-1 Variants with Increased Resistance to Sifuvirtide, a Novel HIV-1 Fusion Inhibitor" @default.
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