Molecular mechanism of HIV-1 resistance to sifuvirtide,a clinical trial–approved membrane fusion inhibitorReceived for publication, April 18, 2018, and in revised form, June 13, 2018 Published, Papers in Press, June 21, 2018, DOI 10.1074/jbc.RA118.003538

Danwei Yu‡§1, Xiaohui Ding‡§1, Zixuan Liu‡§1, Xiyuan Wu‡§1, Yuanmei Zhu‡§, Huanmian Wei‡§, Huihui Chong‡§,Sheng Cui‡, and Yuxian He‡§2

From the ‡MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and the §Center for AIDS Research,Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China

Edited by Charles E. Samuel

Host cell infection with HIV-1 requires fusion of viral and cellmembranes. Sifuvirtide (SFT) is a peptide-based HIV-1 fusioninhibitor approved for phase III clinical trials in China. Here, wefocused on characterizing HIV-1 variants highly resistant toSFT to gain insight into the molecular resistance mechanism.Three primary substitutions (V38A, A47I, and Q52R) located atthe inhibitor-binding site of HIV-1’s envelope protein (Env) andone secondary substitution (N126K) located at the C-terminalheptad repeat region of the viral protein gp41, which is part ofthe envelope, conferred high SFT resistance and cross-resis-tance to the anti-HIV-1 drug T20 and the template peptide C34.Interestingly, SFT’s resistance profile could be dramaticallyimproved with an M–T hook structure–modified SFT (MTSFT)and with short-peptide inhibitors that mainly target the gp41pocket (2P23 and its lipid derivative LP-19). We found that theV38A and Q52R substitutions reduce the binding stabilities ofSFT, C34, and MTSFT, but they had no effect on the binding of2P23 and LP-19; in sharp contrast, the A47I substitutionenhanced fusion inhibitor binding. Furthermore, the primaryresistance substitutions impaired Env-mediated membranefusion and cell entry and changed the conformation of the gp41core structure. Importantly, whereas the V38A and Q52R sub-stitutions disrupted the N-terminal helix of gp41, a single A47Isubstitution greatly enhanced its thermostability. Takentogether, our results provide crucial structural insights into themechanism of HIV-1 resistance to gp41-dependent fusioninhibitors, which may inform the development of additionalanti-HIV drugs.

Infection of HIV type 1 (HIV-1) requires fusion between viraland target cell membranes (1). In a generally accepted model,the viral envelope (Env) surface glycoprotein gp120 bindssequentially to cell receptor CD4 and a coreceptor (CCR5 orCXCR4), which triggers huge conformational changes in theEnv complex. The transmembrane glycoprotein gp41 isreleased from the gp120 constraint and undergoes a structural

rearrangement to activate its fusogenic activity. First, the N-ter-minal fusion peptide of gp41 inserts into the cell membrane,resulting in an extended, membrane-bridging prehairpin struc-ture; then its C-terminal heptad repeat (CHR)3 packs in anantiparallel manner into the internal hydrophobic grooves cre-ated by trimeric N-terminal heptad repeat (NHR) coiled coils,resulting in a stable six-helix bundle (6-HB) conformation (Fig.1). Thus, the viral and cellular membranes are driven togetherfor bilayer fusion and viral entry. Notably, the crystal structureof 6-HB reveals a deep pocket at the C-terminal site of NHRhelices, which is occupied by hydrophobic residues from thepocket-binding domain (PBD) of the CHR helix (2–4). A bodyof data demonstrate that the deep pocket is essential for thestability of 6-HB core and the fusogenic activity of gp41, beingsuggested as an ideal target site for developing anti-HIV agents(5–7).

Synthetic peptides derived from the NHR and CHR of gp41can competitively bind to the prehairpin intermediate of gp41to prevent the formation of viral 6-HB structure, thereby inhib-iting HIV-1 Env-mediated cell fusion and entry (8 –10). As amilestone, T20 (enfuvirtide/fuzeon), a native CHR-derivedpeptide with 36 amino acids, was approved for clinical use in2003, generating the first member of a new class of anti-HIVdrugs: HIV entry inhibitors (11, 12). T20 has demonstratedeffectiveness in combination therapy of HIV-1 infection; how-ever, it requires frequent high dosages (90 mg, twice daily) andreadily acquires drug resistance. The resistance mutations toT20 are primarily mapped to the inhibitor-binding sites on theNHR region of gp41, especially within the amino acid Gly36–Leu45 stretch (13–18). To develop new fusion inhibitors withimproved pharmaceutical profiles, the CHR-derived peptideC34 has been widely used as a design template, because itsN-terminal sequence contains a PBD that critically determinesthe anti-HIV activity of inhibitors (8, 9). Prominent C34 deriv-atives involve sifuvirtide (SFT) (19), T2635 (20), SC34EK (21),and CP32M (22). By modifying the PBD sequence with theM–T hook structure, short-peptide inhibitors, such as HP23and 2P23, have been recently developed, which mainly targetthe gp41 pocket site and possess potent anti-HIV activity (23,24). The M–T hook–modified SFT, termed MTSFT, alsoThis work was supported by Natural Science Foundation of China Grants

81630061 and 81473255 and CAMS Innovation Fund for Medical SciencesGrant 2017-I2M-1-014. The authors declare that they have no conflicts ofinterest with the contents of this article.

This article contains Figs. S1–S4.1 These authors contributed equally to this work.2 To whom correspondence should be addressed. Tel.: 8610-67870275; Fax:

8610-67870275; E-mail: yhe@ipbcams.ac.cn.

3 The abbreviations used are: CHR, C-terminal heptad repeat; NHR, N-terminalheptad repeat; 6-HB, six-helix bundle; PBD, pocket-binding domain; SFT,sifuvirtide; MTSFT, M–T hook structure–modified SFT; SIV, simian immuno-deficiency virus; DSP, dual split protein; N-PAGE, native PAGE.

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showed greatly increased anti-HIV activity and genetic barrierto inducing drug resistance (25).

Among the newly designed fusion inhibitor peptides, SFTwas engineered on the basis of a C34-based gp41 core structure,in which the charged glutamic acid and lysine residues wereintroduced into the solvent-accessible site while the residuesresponsible for the NHR binding were maintained unchanged(Fig. 1). Further, a serine residue was added to the N terminus ofSFT to increase its helical stability, and Glu119 was substitutedby threonine to enhance its binding with the NHR pocket site.We previously determined the crystal structure of SFT boundto a target mimic peptide, which verified an electronically con-strained �-helical peptide with significantly improved bindingstability (26). As anticipated, SFT showed greatly increasedactivities in inhibiting both diverse subtypes of WT and T20-resistant strains (19, 26). In the phase I clinical trials, SFTdemonstrated its good safety and pharmacokinetic profiles,especially with a dramatically prolonged half-life (19). In anadvanced stage, SFT has been evaluated in clinical phase II trialsand approved for clinical phase II trials in China, and thus it will

hopefully become the next fusion inhibitor for clinical applica-tion. In this study, we focused on characterizing SFT-inducedHIV-1 variants to gain new insights into the genetic pathwaysand mechanisms of HIV-1 resistance to newly developed fusioninhibitors. Several new mutations were identified as conferringhigh resistance to SFT and cross-resistance to T20 and C34.The mechanisms underlying the resistance were further char-acterized from multiple aspects, including the binding stabilityof inhibitors, the functionality of viral Env glycoproteins, theconformation of gp41 core structure, and the structuralchanges of the NHR helices. The data have provided criticalinformation for the structure–function relationship of HIV-1Env and gp41-dependent fusion inhibitors and will definitelyaid in the development of new anti-HIV drugs.

Results

Identification of SFT-induced resistance mutations

To characterize the structure–activity relationship of newlydesigned HIV-1 fusion inhibitors, we previously performed the

Figure 1. Schematic illustration of HIV-1 gp41 and peptides. A, functional domains of gp41 and its peptide derivatives. FP, fusion peptide; TM, transmem-brane domain; CT, cytoplasmic tail. The gp41 numbering of HIV-1HXB2 is used. The sequences corresponding to the T20-resistant site and the pocket-formingsite are marked in purple and blue, respectively, on the NHR-derived peptide N36, with the SFT-induced mutations highlighted in red. The sequences corre-sponding to the PBD are marked in deep red on the CHR-derived peptides, whereas the position and sequence of the M–T hook structure are shown in green.The negatively and positively charged residues introduced for potential formation of salt bridges in SFT are indicated with solid black lines. B, the interactionbetween the NHR and CHR of gp41. In the current model, the CHR sequence of gp41 packs back to the NHR sequence to form a hairpin-like structure. Threehairpins associate with each other to form a 6-HB conformation. The dashed lines between the NHR and CHR indicate the interaction between the residueslocated at the e, g and a, d positions in the NHR and CHR, respectively. The NHR- and CHR-derived peptides are depicted as lines to express their sequences andbinding sites.

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in vitro selection of escape HIV-1 variants to SFT, in which thedrug concentration was raised from 0.2 to 9,600 nM after 38generations of virus passage over 9 months (25). To identify thegenetic pathway of SFT-induced resistance, the entire envgenes of resistant viruses were amplified by PCR and cloned forDNA sequencing. As shown in Fig. 2, two Env clones (M1 andM2) carry a single V38A mutation; one Env clone (M3) carries asingle A47I mutation; 12 Env clones (M4 –M15) carry V38A/A47I/N126K mutations, and three Env clones (M16 –M18)carry V38A/A47I/Q52R/N126K quaternary mutations. Therefore,four mutant viruses were finally identified from the pool ofSFT-resistant HIV-1 variants. The V38A, A47I, and Q52Rlocated within the inhibitor-binding site of gp41 mightdetermine the resistance as primary mutations, whereas theN126K in the CHR served as a secondary mutation. Afteranalyzing all of the cloned Env sequences, no consistent sub-stitutions that apparently caused the resistance were identi-fied in the other sites of gp41 or in gp120 sequence (Fig. S1),and we thus focused on the mutations on the NHR and CHRsequences of gp41.

Resistance profile of HIV-1 mutants to SFT

To identify the mutations responsible for SFT-induced resis-tance, we generated a panel of HIV-1NL4-3 Env mutants carry-ing the amino acid substitutions either singly or in combination(Table 1). The corresponding pseudoviruses were then pro-duced and used in a single cycle infection assay to determine theinhibitory activity of SFT. Among the single mutations, V38A,A47I, and Q52R conferred 7.93-, 4.58-, and 39.49-fold resis-tance to SFT, respectively, whereas N126K had minor effect onthe inducer (1.7-fold). Apparently, the mutant viruses carrying

the combined mutations resulted in dramatically increased-fold changes in resistance to SFT, as shown by the mutantswith V38A/A47I (41.61-fold), V38A/A47I/N126K (81.2-fold),V38A/A47I/Q52R (1107.19-fold), and V38A/A47I/Q52R/N126K (1316.01-fold). Therefore, the mutant virus with V38A/A47I/Q52R/N126K mutations might finally dominate the resis-tance phenotype to SFT.

Cross-resistance of HIV-1 mutants to the drug T20 and thetemplate C34

We next analyzed the cross-resistance profiles of SFT-in-duced mutants to the drug T20 and the template peptide C34.As shown in Table 1, the single mutants V38A and Q52R alsomediated high level resistance to T20 and C34. Specifically,whereas V38A conferred 26.5-fold resistance to T20 and 12.79-fold resistance to C34, Q52R rendered 17.55-fold resistance toT20 and 34.98-fold resistance to C34. Interestingly, whereas thevirus with A47I had slight resistance to C34 (2.15-fold), itexhibited markedly increased susceptibility to T20. Among thevariants with multiple mutations, V38A/A47I, V38A/A47I/N126K, V38A/A47I/Q52R, and V38A/A47I/Q52R/N126Kshowed 13.6-, 17.85-, 31.05-, and 35.39-fold resistance to T20,respectively, and 9.58-, 17.77-, 70.97-, and 121.85-fold resis-tance to C34, respectively. Therefore, these results suggestedthat SFT-resistant mutants also displayed high cross-resistanceto the first-generation fusion inhibitors T20 and C34.

Cross-resistance of HIV-1 mutants to newly designed fusioninhibitors that contain the M–T hook structure

We previously demonstrated that adding an M–T hookstructure to the CHR-derived peptide fusion inhibitors

Figure 2. Sequence characterization of SFT-induced mutations. The amino acid sequences in the NHR and CHR of WT and mutant HIV-1NL4-3 gp41 arealigned. The positions of selected mutations are in boldface type, and numbering is according to that of HIV-1HXB2 gp41. The pocket-forming sequence in NHRand the pocket-binding domain in CHR with the M–T hook residues are underlined.

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could greatly enhance their binding and antiviral activities aswell as the genetic barriers to resistance (24, 25, 27–31).Herein, it was interesting to know the cross-resistance pro-file of SFT-induced HIV-1 mutants to the MTSFT. As shownin Table 1, the single mutations (V38A, A47I, Q52R, andN126K) conferred little resistance to MTSFT, whereas thecombined mutations (V38A/A47I, V38A/A47I/Q52R, V38A/A47I/N126K, and V38A/A47I/Q52R/N126K) resulted in theresistance with dramatically decreased -fold changes relativeto that of SFT. In particular, the single Q52R mutation couldresult in �40-fold resistance to SFT, but it had no obviouseffect on the inhibitory potency of MTSFT, verifying theimportance of the M–T hook structure in overcoming theresistance.

Afterward, we also characterized the cross-resistance ofSFT-induced mutants to the short-peptide fusion inhibitor2P23 and its lipid derivative LP-19. As illustrated in Fig. 1, 2P23is a 23-mer peptide with the M–T hook residues at its N termi-nus, and LP-19 has been further developed by conjugating aC16 fatty acid group to the C terminus of 2P23. Both inhibitorsmainly target the gp41 pocket rather than the T20-resistant siteand possess potent inhibitory activities against HIV-1, HIV-2,and simian immunodeficiency virus (SIV) (23, 32). Impres-sively, we found that all of the mutant viruses with single ormultiple mutations had no or minor resistance to 2P23 andLP-19.

Effects of the resistance mutations on the binding stability offusion inhibitors

To understand the mechanisms underlying the SFT-selectedresistance, we sought to determine the effects of the resistancemutations on the binding stability of inhibitors. To this end,the NHR-derived peptide N36 with a WT sequence (N36wt)or its mutants with single or multiple amino acid substi-tutions (N36V38A, N36A47I, N36Q52R, N36V38A/A47I, andN36V38A/A47I/Q52R) were synthesized as target surrogates, andthe interactions between SFT and target mimic peptides wereinitially measured by CD spectroscopy. As shown in Fig. 3 andTable 2, CD spectra of all of the peptide pairs displayed typicaldouble minima at 208 and 222 nm, which indicated that SFTinteracted with each of the N36 peptides to form 6-HB struc-tures. The V38A and Q52R mutations resulted in decreased�-helical contents of the 6-HBs, but the A47I mutation mightenhance the �-helicity. Defined as the midpoint of the thermalunfolding transition (Tm), the thermostability of 6-HBs demon-

strated the binding stability of SFT. It was found that V38A andQ52R severely reduced the Tm value, whereas A47I con-versely increased it.

We also examined the helical interaction and binding sta-bility of all of the other inhibitors by CD spectroscopy (Table2). Consistently, the V38A and Q52R mutations reduced the�-helical content and Tm value of the 6-HBs formed by C34,T20, and MTSFT, whereas the A47I mutation did enhancetheir �-helicity and thermostability. In sharp contrast, V38Aand Q52R had no apparent effect on the interactions of 2P23and LP-19, as shown by the �-helical contents and Tm valuesof their 6-HBs, whereas A47I still enhanced the binding sta-bility of inhibitors. As shown by N36 with combined muta-tions, the A47I mutation could counteract the negativeeffects of the V38A and Q52R mutations for the binding ofinhibitors.

Table 1Resistance profiles of HIV-1 mutants to SFT and the first-generation fusion inhibitorsThe experiment was performed in triplicate and repeated three times. Data are expressed as means � S.D. -Fold change in the IC50 was determined relative to the WT level.The t test was performed to judge the significance of the difference between the WT and mutants, and p � 0.05 values are indicated in boldface type.

HIV-1NL4-3 virus

SFT T20 C34 MT-SFT 2P23 LP-19

IC50 -Fold IC50 -Fold IC50 -Fold IC50 -Fold IC50 -Fold IC50 -Fold

nM nM nM nM nM nM

WT 1.84 � 0.27 1.00 70.74 � 0.54 1.00 1.17 � 0.01 1.00 0.93 � 0.07 1.00 0.59 � 0.11 1.00 0.09 � 0.04 1.00V38A 14.59 � 1.79 7.93 1874.68 � 252.53 26.50 14.9 � 4.07 12.79 1.78 � 0.20 1.91 0.71 � 0.21 1.20 0.10 � 0.04 1.11A47I 8.42 � 2.14 4.58 25.87 � 3.49 0.37 2.51 � 0.38 2.15 1.45 � 0.22 1.56 0.75 � 0.27 1.27 0.10 � 0.05 1.11Q52R 72.67 � 6.96 39.49 1241.32 � 242.75 17.55 40.75 � 10.45 34.98 1.62 � 0.46 1.74 2.78 � 0.42 4.71 0.08 � 0.02 0.89N126K 3.12 � 0.05 1.70 113.5 � 27.42 1.60 5.36 � 0.9 4.58 3.03 � 0.29 3.26 0.98 � 0.44 1.66 0.22 � 0.14 2.44V38A/A47I 76.56 � 13.28 41.61 962.33 � 217.73 13.60 11.21 � 2.24 9.58 4.20 � 0.37 4.52 1.10 � 0.36 1.86 0.14 � 0.06 1.56V38A/A47I/Q52R 2037.22 � 434.2 1107.19 2196.33 � 211.13 31.05 83.03 � 26.63 70.97 154.14 � 36.99 165.74 0.78 � 0.12 1.32 0.07 � 0.01 0.78V38A/A47I/N126K 149.42 � 12.36 81.21 1262.67 � 253.01 17.85 20.79 � 3.14 17.77 11.51 � 0.98 12.38 1.66 � 0.38 2.81 0.34 � 0.13 3.78V38A/A47I/Q52R/N126K 2421.46 � 827.82 1316.01 2503.34 � 227.32 35.39 142.57 � 16.63 121.85 136.27 � 27.10 146.53 1.46 � 0.53 2.47 0.15 � 0.03 1.67

Figure 3. Helical binding stability of SFT determined by CD spectroscopy.The �-helicity (A) and thermostability (B) of SFT in complexes with the NHR-derived peptide N36 or its mutants were measured with the final concentra-tion of each peptide at 10 �M in PBS. The experiments were performed twotimes, and representative data are shown.

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Effects of SFT-induced mutations on Env-mediated cell fusionand virus entry

We next focused on investigating the effects of SFT-inducedresistance mutations on the functionality of viral Env glycopro-tein by two approaches. First, the infectivity of HIV-1NL4-3pseudoviruses carrying single or combined mutations wasdetermined by a single-cycle infection assay, in which the cellentry efficiency of WT virus was normalized to 100% and therelative virus entry of other mutants was calculated accord-ingly. As shown in Fig. 4A, all of the pseudoviruses with the Envcarrying single resistance mutations (V38A, A47I, or Q52R)or combined mutations (V38A/A47I or V38A/A47I/Q52R)showed markedly decreased cell entry efficiency. Apparently,the Q52R mutation dramatically impaired the functionality ofviral Env, and the secondary N126K mutation exhibited a com-pensatory effect. Second, we measured Env-mediated cell– cellfusion by a dual split protein (DSP)-based fusion assay. It wasfound that single or combined mutations of V38A and A47I hadminor effect on the Env’s fusion activity, but Q52R resulted in adramatic reduction. Consistently, N126K mutation efficientlydisplayed a compensatory role to the V38A/A47I and V38A/A47I/Q52R mutants (Fig. 4B). The fusion capacity of diverseEnvs was also measured at different time points, and the resultssupported the observation above (Fig. 4C).

The resistance phenotypes were not associated with theexpression and processing of viral Env glycoprotein

It was critical to know whether the resistance mutationsaffected the expression and processing of the Env glycopro-teins, thereby resulting in the phenotypes of viral infectivity and

Table 2Effects of SFT-induced mutations on the �-helicity and thermostabil-ity of 6-HB structureThe �-helicity and thermostability of 6-HBs were measured by CD spectroscopy,and the experiments were repeated at least two times to verify the results. NA, notapplicable for calculation due to too low (T20) or too high (LP-19) Tm values.

Peptide complex [�]222 Helix content (%) Tm

% °CSFT–N36 �30,137 91 71SFT–N36V38A �27,794 84 64SFT–N36A47I �31,748 96 73SFT–N36Q52R �25,989 79 61SFT–N36V38A/A47I �25,419 77 66SFT–N36V38A/A47I/Q52R �22,346 68 53C34–N36 �33,087 100 64C34–N36V38A �29,791 90 54C34–N36A47I �34,560 105 73C34–N36Q52R �27,711 84 56C34–N36V38A/A47I �31,637 96 65C34–N36V38A/A47I/Q52R �24,888 75 53T20–N36 �13,889 33 52T20–N36V38A �22,250 14 NAT20–N36A47I �8,784 39 67T20–N36Q52R �10,047 15 NAT20–N36V38A/A47I �12,368 9 NAT20–N36V38A/A47I/Q52R �5,484 3 NAMTSFT–N36 �30,556 93 78MTSFT–N36V38A �28,167 85 72MTSFT–N36A47I �33,417 101 80MTSFT–N36Q52R �25,569 77 68MTSFT–N36V38A/A47I �30,181 91 76MTSFT–N36V38A/A47I/Q52R �20,611 62 652P23–N36 �24,486 78 802P23–N36V38A �23,308 76 802P23–N36A47I �24,788 79 892P23–N36Q52R �22,317 73 792P23–N36V38A/A47I �24,945 82 892P23–N36V38A/A47I/Q52R �21,294 71 86LP19–N36 �27,915 85 NALP19–N36V38A �31,068 94 NALP19–N36A47I �36,102 109 NALP19–N36Q52R �28,373 86 NALP19–N36V38A/A47I �29,237 89 NALP19–N36V38A/A47I/Q52R �24,881 75 NA

Figure 4. Effects of SFT-induced mutations on the functionality of HIV-1Env. A, the WT and mutant HIV-1NL4-3 pseudoviruses were normalized to afixed amount by p24 antigen, and their relative infectivity was determined inTZM-bl cells using a single-cycle infection assay. Forty-eight hours after infec-tion, the luciferase activity was measured and corrected for background. Theluciferase activity of WT HIV-1NL4-3 was treated as 100%, and the relative activ-ities of other mutant viruses were calculated accordingly. B, relative fusionactivity of WT and mutant HIV-1NL4-3 Envs was determined by a DSP assay. TheEnv-transfected HEK293T cells were used as effector cells, and U87-CD4-CXCR4 cells were used as target cells. Similarly, the luciferase activity of WTEnv was treated as 100%, and the relative activities of other mutant Envs werecalculated accordingly. C, fusion activity of the WT and mutant HIV-1 Envs wasdetermined at different time points by a DSP assay. For both viral entry andfusion, data were derived from the results of three independent experimentsand are expressed as mean and S.D. (error bars). t test was performed to com-pare the WT and mutants; * and **, p � 0.05 and p � 0.01, respectively.

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resistance. To this aim, we first applied the human anti-gp120antibody VRC01 and anti-gp41 antibody 10E8 in a captureELISA– based method, as both antibodies recognize the highlyconserved epitopes and possess broadly reactive virus-neutral-izing activity, and their reactivity can refer to the expressionprofile of gp160/gp120/gp41 in the culture media and lysates oftransfected cells. As shown in Fig. 5, VRC01 and 10E8 reactedequivalently with the lysates of HEK293T expressing WT andmutant Env glycoproteins that theoretically comprise gp160,gp120, and gp41. Concordantly, VRC01 detected the same levelof secreted gp120 proteins in the cell culture supernatants. Fur-ther, the expression and processing of the Env glycoproteinswere characterized by a Western blotting assay, in which thehuman anti-HIV polyclonal antibody HIV-IG or mAb HY54was used to probe the cleaved and uncleaved Env glycoproteins(Fig. 6). The results verified that the resistant mutants had noobvious changes in the protein expression and processing (Fig.S2). As expected, N126K could abolish the glycosylation site ingp41 (33). We also detected the WT and mutant Env glycopro-teins expressed on the surface of transfected cells by flowcytometry and immunofluorescence assays. As shown in Fig. 7and Fig. S3, no significant differences were observed either.Combined, the data suggested that the observed resistance phe-notypes were not determined by the expression profile of viralEnv glycoprotein.

The resistance mutations might change the conformation ofgp41 core structure

The 6-HB conformation was initially determined by theNHR-derived peptide N36 and the CHR-derived peptide C34(4), thus representing the core structure of gp41 that plays anessential role in viral fusion and entry. Here, we were intriguedto know whether SFT-selected mutations affected the function-ality of viral Env through changing the conformation of gp41core structure. We also applied two approaches that are rou-tinely used in our laboratory. First, we used a native PAGE(N-PAGE)-based method to visualize the 6-HB complexesformed between diverse N36 and C34 peptides. As shown inFig. 8, N36 and its mutants showed no band in the native gelbecause they could migrate up and off the gel due to their netpositive charges, whereas the negatively charged C34 and itsN126K mutant (C34N126K) showed specific bands. WhereasN36 or its mutants were mixed with C34 or C34N126K, thereexisted specific bands corresponding to the 6-HB complexes.Densitometric analysis suggested that all the resistancemutations had little effect on the formation of 6-HBs(Fig. S4).

Subsequently, three 6-HB conformation–specific monoclo-nal antibodies (NC-1, 17C8, and 2G8) were used as probes in anELISA-based assay (34 –36). As shown in Fig. 9, whereas theV38A mutation might not significantly affect the reactivity of6-HB with three antibodies, the A47I mutation resulted in a6-HB with largely increased reactivity, and the Q52R mutation-bearing 6-HB only displayed marginal reaction. The binding

Figure 5. Expression and processing profiles of HIV-1NL4-3 Envs deter-mined by capture ELISA. HEK293T cells were transfected by plasmidsencoding WT or mutant HIV-1NL4-3 Envs. The glyproteins (gp120/gp41) in thelysates of transfected cells (A) or culture supernatant (B) were detected bycapture ELISA, in which the sheep anti-gp120 antibody D7324 was used as acapture antibody and the human mAbs VRC01 and 10E8 were used as probes.The experiments were conducted in duplicate and repeated two times. A ttest was performed to compare the WT and mutants; * and **, p � 0.05 andp � 0.01, respectively. Error bars, S.D.

Figure 6. Expression and processing profiles of HIV-1NL4-3 Envs deter-mined by Western blotting. The viral glycoproteins in the lysates of trans-fected cells were detected with the human anti-HIV polyclonal antibodyHIV-IG (Panel 1) or the human anti-gp120 mAb HY54 (Panel 2). The reactionbands corresponding to gp160, gp120, and gp41, respectively, are marked.The gp41 subunit in the cell lysates was specifically detected by HIV-IG (Panel3). Shed gp120 subunit in the culture supernatant was detected by HY54(Panel 4). The �-actin protein in the cell lysates was detected as an internalcontrol (Panel 5). Molecular weight (MW) markers are indicated in kDa. Theexperiments were repeated at least three times, and representative data areshown.

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epitopes of three antibodies have not been finely characterized,but previous studies suggested that NC-1 might mainly targetthe gp41 NHR (37, 38), whereas the critical binding residues of17C8 and 2G8 were localized at the N-terminal portion of theNHR and the C-terminal portion of the CHR (39). Thus, thereaction patterns of diverse 6-HBs with three antibodiesimplied the increased or decreased exposure of antigenicepitopes, which might correlate with the structural changesof 6-HB core.

The resistance mutations changed the �-helicity and stabilityof NHR helix

Based on the results above, we sought to delineate the effectsof the resistance mutations on the �-helicity and thermostabil-ity of N36 peptide in the absence of inhibitors, with the inten-tion to infer the structural properties of the viral NHR helices.As measured by CD spectroscopy (Fig. 10, A and B), the WT

N36 showed an �-helical content of 79% and a Tm value of56 °C. In sharp contrast, N36 with the V38A mutation onlydisplayed 18% �-helicity, and its Tm could not be defined; N36with A47I displayed 66% �-helicity, but its Tm increased to62 °C; N36 with Q52R displayed 40% �-helicity with a Tm of46 °C. Interestingly, N36 with V38A/A47I double mutationsshowed 66% �-helicity with a Tm of 60 °C, and N36 with V38A/A47I/Q52R triple mutations had 47% �-helicity with a Tm of49 °C, which suggested that the A47I mutation could counter-act the disruptive effects by the V38A and Q52R mutations.

We recently reported the selection and characterization ofHIV-1 variants resistant to the CHR-derived fusion inhibitorsMTSC22EK and SC29EK, which also identified key resistancemutations in the inhibitor-binding NHR helices of gp41 (34 –36). To conceptualize that the resistance mutations can directlychange the conformation and stability of the gp41 NHR helices,thereby critically determining the binding affinity of inhibitors

Figure 7. Expression of WT and mutant Env glycoproteins on the surface of transfected cells. The viral Env glycoproteins expressed on the surface oftransfected HEK293T cells were detected by the human anti-gp120 mAb VRC01 with flow cytometry (A) or immunofluorescence assay (B). The experimentswere repeated three times, and representative data are shown. The red control curve was reused in the panels of A.

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and HIV-1 infectivity, we back-checked the effects ofMTSC22EK- and SC29EK-induced resistance mutations on the�-helical structure of N36 itself by CD spectroscopy. In general,the single or combined resistance mutations could markedlyreduce the �-helicity and thermal stability of N36 (Fig. 10,C–F). For example, the combination of two MTSC22EK-in-duced mutations, E49K and L57R, resulted in the �-helical con-tent and Tm value not applicable for calculation. As an excep-tion, a single N43K mutation rather increased the �-helicalcontent of N36 but it did not affect its Tm value.

The secondary N126K mutation stabilized the 6-HB structureof SFT-resistant mutants

The N126K substitution in the CHR of gp41 easily emerges inthe drug-resistance selection, and it has been considered a sec-ondary mutation that can compensate for the fusion kineticsof resistant viruses. Again, our above data indicated that theN126K mutation exhibited obvious compensatory roles to thefusion and entry capacity of the V38A/A47I and V38A/A47I/Q52R mutants. Therefore, we sought to further characterize itseffect on the binding of C34 with diverse target surrogates, fromwhich we could infer the interactions between the NHR andCHR helices of mutant viruses. As determined by CD spectros-copy, all of the 6-HBs formed between the peptides C34N126Kand N36 or its mutants exhibited relatively increased Tm values,which indicated their enhanced �-helical thermostability (Fig.11). For instance, whereas the N36V38A–C34 – based 6-HB had

a Tm of 54 °C, the N36V38A–C34N126K– based 6-HB had a Tm of59 °C, and whereas the N36Q52R–C34 – based 6-HB had a Tm of56 °C, the N36Q52R–C34N126K– based 6-HB had a Tm of 60 °C.It should be noted that the A47I mutation–mediated 6-HB

Figure 8. Visualization of 6-HB structure by native PAGE. A, the 6-HBsformed by the CHR-derived peptide C34 and NHR-derived peptide N36 or itsmutants. B, the 6-HBs formed by C34 with the N126K mutation (C34N126K) andN36 or its mutants. Each of the peptides was used at a final concentration of40 �M. The experiments were repeated three times, and representative dataare shown.

Figure 9. Effects of SFT-induced mutations on the conformation of gp41core structure. The reactivity of 6-HBs formed by C34 and N36 or its mutantswith conformation-dependent mAbs NC-1 (A), 17C8 (B), and 2G8 (C) wastested by ELISA. The peptide mixture was used to coat the plate wells at 10�g/ml, and the final concentration of a tested mAb was 5 �g/ml. Data werederived from three independent experiments and are expressed as mean andS.D. (error bars). A t test was performed to compare the WT and mutants; * and**, p � 0.05 and p � 0.01, respectively.

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thermostability could be further enhanced by the N126K muta-tion (Fig. 11C).

Structural basis of SFT-induced resistance

We previously determined the crystal structure of SFT incomplex with the target peptide N36 (Protein Data Bank acces-sion number 3VIE), which provided a structural basis for themechanism of action of SFT (26). Herein, we analyzed the resis-tance mutations based on the atomic interactions of the 6-HBstructure formed by N36 and SFT. Clearly, the residue Val38 islocated at the e position of a helical wheel model, and its hydro-phobic side chain interacts with a hydrophobic patch at theterminal portion of SFT, which is formed by the side chains ofAsn145, Glu146, and Leu149 (Fig. 12A). Because the alanine sidechain is much smaller than a valine side chain, the V38A muta-tion could significantly reduce the area of the interaction inter-face between V38A and the hydrophobic patch; hence, itaccounted for the loss of binding affinity of SFT. In the helicalwheel, the residue Ala47 stays at a g position, where its sidechain is located near a hydrophobic patch in the middle regionof SFT constituted by the side chains of Ile131 (distance 4.6 Å),Ile134 (distance 3.8 Å), and Leu135 (distance 4.4 Å) (Fig. 12B).Due to the small side chain of Ala47, there is a gap between Ala47

and the hydrophobic patch of SFT. Replacing Ala47 by isoleu-

cine increased the size of the side chain and filled the spacebetween the N36 trimer and SFT; thus, the A47I mutationserves to increase the interfacial area between the NHR trimerand SFT. Because isoleucine has a stable � branched side chainand it is deeply buried inside the hydrophobic core of 6-HB, theA47I mutation may also contribute to the stabilization of theNHR trimer itself, which is consistent with the elevated Tm

values of 6-HB or the isolated N36 bearing A47I determined byCD spectroscopy. The side chain of residue Gln52, which islocated at the e position in the CHR helix, mediates severalspecific hydrogen bonds with both SFT and the adjacentNHR simultaneously (Fig. 12C). Whereas the N�2 atom ofGln52 donates a hydrogen bond (distance, 3.0 Å; angle, 149º)to the OH group of residue Tyr132 of SFT, the O�1 atom ofGln52 accepts another hydrogen bond (distance, 2.5 Å; angle,160º) from the N�2 atom of Gln51 on the adjacent NHR helix.Additionally, the O�1 atom of Gln52 accepts the third hydro-gen bond from the C�1 atom (distance, 2.7 Å; angle, 152º) ofIle131. Therefore, Gln52 not only directly interacts with SFTbut also stabilizes NHR trimeric coiled-coil; thus, replacingGln52 with an arginine could result in the loss of bindingaffinity with SFT as well as the destabilization of the NHRtrimer itself.

Figure 10. Effects of the resistance mutations on the structure of the NHR helices. The �-helicity (A) and thermostability (B) of the NHR-derived peptideN36 and N36 carrying SFT-induced mutations, the �-helicity (C) and thermostability (D) of N36 and N36 carrying MTSC22EK-induced mutations, and the�-helicity (E) and thermostability (F) of N36 and N36 carrying SC29EK-induced mutations were determined by CD spectroscopy. The final concentration of eachpeptide in PBS was 20 �M. The experiments were performed two times, and representative data are shown.

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From the virus perspective, we analyzed the resistance muta-tions on the crystal 6-HB structure of C34/N36 (Protein DataBank accession number 1AIK), which contains the nativesequences of gp41 core. As anticipated, the V38A mutation hassimilar effects on the interaction of N36 and C34, as C34 andSFT share a hydrophobic patch formed by the side chains ofAsn145, Glu146, and Leu149. Clearly, the side chain of Val38 inter-acts with the side chain of Ile37 to form a hydrophobic layer (Fig.12D), whereas three Ile37 residues on N36 helices interact witheach other, which critically determines the conformation andstability of the inner NHR helices; thus, the V38A mutationwould dramatically destroy the helical structure of N36 itself.As compared with that of the N36 –SFT complex, the space anddistance between Ala47 on N36 helices and the hydrophobicpatch formed by Ile131 (distance 5.3 Å), Leu134 (distance 4.3 Å),and Ile135 (distance 5.2 Å) on C34 helices are much larger (Fig.12E); thus, it is conceivable that the A47I mutation wouldenhance the helical interactions of N36 and C34 more effi-ciently, consistent with the difference in the increased Tm val-ues of N36A47I–SFT (2 °C) and N36A47I–C34 (9 °C). Similar tothe structure of N36 –SFT, the side chain Gln52 can formhydrogen bonds with Gln51 on another N36 and Ile131 on C34,but it does not contact with His132 (corresponding to Tyr132 on

SFT) due to a far distance (Fig. 12F). However, the Q52R muta-tion might not only disrupt the critical hydrogen bond networkin the middle region of 6-HB but also introduce an electrostaticrepulsion with His-132.

To gain insights into the mechanism of the secondary N126Kmutation–mediated compensation for the 6-HB stability andviral infectivity, we also analyzed its potential effects on theatomic interactions of viral 6-HB as mimicked by N36 and C34peptides. As shown in Fig. 12G, Asn126 is located at the c posi-tion in the CHR helix, and its site chain faces the outside of thebinding surface of C34; thus, it is difficult to define the functionof Asn126 in the interhelical interaction of 6-HB structure; how-ever, we can see that a water molecule links Asn126 and Glu119

together via hydrogen bonds, which would stabilize the N-ter-minal PBD of C34 and thus facilitate its interactions with thedeep pocket on the NHR helices. Conceivably, the relativelylonger and positively charged side chain of lysine might disruptthe water-mediated connection but introduce salt bridges orhydrogen bonds with the negatively charged residues Glu119

and/or Glu123, which can strengthen the intra- and interhelicalinteractions. We expect a crystal structure for the N36 –C34N126K complex that can delineate the effect of the N126Kmutation in detail.

Figure 11. Compensatory effects of the secondary N126K mutation on the stability of 6-HB structure. The thermostability of 6-HB formed between C34or C34N126K and N36 (A), N36V38A (B), N36A47I (C), N36Q52R (D), N36V38A/A47I (E), or N36V38A/A47I/Q52R (F) was determined by CD spectroscopy. The final concen-tration of each peptide in PBS was 10 �M. The experiments were performed two times, and representative data are shown.

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Discussion

In this study, we dedicated our efforts to exploring thegenetic pathway and underlying mechanisms of HIV-1 resis-tance to the peptide fusion inhibitor SFT. Three primary muta-tions (V38A, A47I, and Q52R) on the NHR helix and one sec-ondary mutation (N126K) on the CHR helix of gp41 wereidentified as conferring high resistance to SFT alone or in com-bination, with a mutant virus with V38A/A47I/Q52R/N126Kpossibly dominating the resistance. The cross-resistance pro-files of SFT-induced mutants to the drug T20, the templateC34, and three newly designed fusion inhibitors (MTSFT, 2P23,and LP-19) were also characterized. Impressively, the singleand combined SFT-induced mutations did confer cross-resis-tance to T20 and C34, but they were largely tolerated by theM–T hook structure–modified SFT and short-peptide inhibi-tors mainly targeting the gp41 pocket site. In the underlyingmechanisms, the V38A and Q52R mutations could reduce thebinding stability of SFT, C34, and MTSFT, but they had noobvious effects on the binding of 2P23 and LP-19, and the A47Imutation, in sharp contrast, enhanced the binding of inhibitors.Furthermore, the results show that the primary resistancemutations could impair the functionality of viral Env to mediatecell entry/fusion and change the conformation of the gp41 coreas well as its internal NHR helices, whereas the secondary

N126K mutation did display compensatory roles in both thevirus entry and the 6-HB stability. Therefore, the present stud-ies have provided important information for understanding themechanism of HIV-1 resistance to gp41-dependent fusioninhibitors and would help in the development of new anti-HIVdrugs.

The peptide drug T20 remains the only membrane fusioninhibitor available for treatment of viral infection, which hasshown effectiveness as a salvage therapy for HIV/AIDS patientswho failed to respond to antiretroviral therapeutics that includereverse transcriptase inhibitors and protease inhibitors; how-ever, T20 behaves with relatively weak antiviral activity and alow genetic barrier to inducing drug resistance. The resistanceprofile of T20 was initially characterized by in vitro selection ofescaping HIV-1 variants, which revealed that the 36GIV38 motifin the inhibitor-binding site was a hot spot (18). Later, a numberof clinical studies demonstrated that T20-resistant mutationsappeared predominately within the amino acid Gly36–Leu45

stretch on the NHR of gp41, such as G36D/V/S, I37T, V38A/E/M, Q40H, N43D/K, and L54M mutations (13–18). Therehave been tremendous efforts to develop next-generationfusion inhibitors that can overcome the drawbacks of T20,resulting in a group of new peptides with significantly improvedpharmaceutical profiles (19 –24, 28). Meanwhile, considerableeffort was also devoted to the selection and characterization ofHIV-1 variants resistant to newly designed fusion inhibitors,which delineated their resistance evolution pathways andunderling mechanisms (34, 35, 40 – 47). Previously, Liu et al.(42) identified a panel of SFT-induced resistance mutationslargely overlapping with the T20-resistant sites, including I37T,V38A/M, Q41H/K/R, and N43K. As noticed, the concentrationof SFT was escalated to �1,000 nM after 9 –15 generations of invitro virus passage, and the most resistant HIV-1 variants withcombined mutations (e.g. I37T/V38A and I37T/N43K/N126K)displayed �100-fold increased resistance to SFT (42). In fact,our research project was originally focused on comparing thegenetic resistance barriers of the M–T hook structure–modified peptide inhibitors rather than identifying the resis-tance mutations in detail (25); however, we had been intenselycurious about the HIV-1 variants that could survive in the pres-ence of �10,000 nM SFT inhibitor. Therefore, the present studywas performed and surprisingly found a different panel of SFT-resistant mutations, including two sites (A47I and Q52R) thathad not been defined previously. In brief, three primary muta-tions (V38A, A47I, and Q52R) located at the inhibitor-bindingsite and one secondary mutation (N126K) located at the CHR ofgp41, corresponding to four HIV-1 variants, contributed to theresistance phenotype. Two mutant viruses carried a singleV38A or A47I mutation, whereas two mutant viruses evolvedwith combined mutations (V38A/A47I/N126K and V38A/A47I/Q52R/N126K). From the sequence characterization, onemight speculate a genetic resistance pathway with the followingscenario. A single V38A or A47I mutation emerged first, whichresulted in a modest resistance (5– 8-fold); subsequently, theV38A/A47I double mutations emerged, thus conferring a sig-nificantly increased resistance (�80-fold). It might require aprolonged selection, but the appearance of Q52R mutation onthe background of V38A/A47I could dramatically boost the

Figure 12. Structural basis of HIV-1 resistance to SFT. A–C, the effects ofthe resistance mutations on the binding stability of SFT were analyzed on thecrystal structure of N36 –SFT shown as a ribbon model. Three N36 peptides arecolored in gray, and three SFT peptides are colored in green. The residues Val38

(A), Ala47 (B), and Gln52 (C) on N36 (colored in magenta) and their interactingresidues on SFT are shown as stick models with labels. D–G, the effects of theresistance mutations on the interactions of viral NHR and CHR helices wereanalyzed on the crystal structure of N36 –C34 shown as a ribbon model. TheN36 peptides are colored in gray, and its residues Val38 (D), Ala47 (E), and Gln52

(F) are colored in magenta; the C34 peptides are colored in cyan, and its residueAsn126 (G) is colored in yellow. For comparison, one SFT helix and one C34helix were superimposed (E and F). The position and conformation of the inter-acting residues within 6-HB are shown in stick models with labels. Dotted linesrepresent interactions between the residues.

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resistance phenotype (�1,000-fold). Apparently, the mutantvirus with two or three combined mutations (V38A/A47I orV38A/A47I/Q52R) was accompanied by the second N126Kmutation, which also contributed to the resistance levels.Therefore, it is conceivable that the mutant virus with V38A/A47I/Q52R/N126K would finally dominate the resistance.

In this study, we found that SFT-induced primary mutations,either singly or in combination, mediated considerable cross-resistance to the drug T20 and the template peptide C34, exceptfor the A47I mutation, which could result in an increased sus-ceptibility to T20 (�3-fold). Interestingly, all of the HIV-1mutants with a single mutation (V38A, A47I, or Q52R) had noobvious resistance to the M–T hook structure–modified SFT,and all of the HIV-1 mutants with combined mutations (V38A/A47I, V38A/A47I/Q52R, V38A/A47I/N126K, and V38A/A47I/Q52R/N126K) displayed dramatically decreased resistance-fold changes (Table 1). Actually, we are still wondering why thesingle Q52R mutation could render a �40-fold resistance toSFT but did not affect the inhibitory activity of MTSFT. Moreimpressively, both the single and combined mutations did notconfer significant resistance levels to the short-peptide fusioninhibitors 2P23 and LP-19, which also contain the M–T hookstructure and mainly target the gp41 pocket site. It is worthnoting that Q52R did confer a mild resistance to 2P23, but thisphenotype could be mitigated by its combination with othermutations and by a 2P23-based lipopeptide (LP-19). Takentogether, the presented data have further verified that both theM–T hook structure and lipid conjugation are important strat-egies to design HIV-1 fusion inhibitors possessing markedlyimproved antiviral activity against WT and mutant viruses,especially for short peptides that target the gp41 pocket ratherthan the T20- and SFT-resistant sites. It will be interesting toselect and characterize HIV-1 mutants resistant to MTSFT,2P23, and LP-19, which would definitely help in our under-standing of the resistance mechanisms of diverse HIV-1 fusioninhibitors.

Previous studies have described multifaceted mechanisms ofHIV-1 resistance to peptide fusion inhibitors, including smallamino acid–mediated reduced contact, large amino acid–mediated steric obstruction, acidic amino acid–mediated elec-trostatic repulsion, basic amino acid–mediated electrostaticattraction, and disruption of hydrogen bonds and hydrophobiccontacts (48). It was also proposed that kinetically restrictedentry inhibitors have reduced sensitivity to affinity-disruptingresistance mutations (49, 50). Here, we investigated the under-lying mechanisms of SFT-induced resistance mutations fromtwo aspects: their effects on the binding stability of inhibitorsand on the functionality of viral Env glycoprotein. Clearly, theV38A and Q52R mutations reduced the binding stability ofSFT, C34, and MTSFT but not that of 2P23 and LP-19; how-ever, the A47I mutation could further stabilize the binding ofeach inhibitor. Based on the crystal structures of SFT and C34bound to a target surrogate, we can see that the residue Val38

makes critical contacts with the residues Asn145, Glu146, andLeu149 of SFT. Thus, the V38A mutation would destroy theinterhelical interactions through a mechanism of small aminoacid–mediated reduced contact; the residue Ala47 has hydro-phobic interactions with Ile131, Ile134, and Leu135 on SFT; the

A47I mutation would introduce an extended side chain thatenhances the interhelical interactions; the residue Gln52 criti-cally stabilizes the NHR helices via a hydrogen bond networkand interacts simultaneously with Tyr132 and Ile131 on SFT; andthe Q52R mutation would disrupt both the intra- and interhe-lical interactions, with a mechanism of large amino acid–mediated steric obstruction or basic amino acid–mediatedelectrostatic repulsion/attraction. From a viral perspective, theresistance mutations could severely impair the �-helicity andstability of viral NHR helices and/or 6-HB conformation, whichmight correlate with the functionality of the Env glycoproteinto mediate viral cell fusion and entry. It is conceivable from anevolutionary angle that the mutant virus would like to find abalance among the mutations, resistance, and viral fitness.

In conclusion, our studies have demonstrated the underlyingmechanism of HIV-1 resistance to SFT and verified the impor-tance of the M–T hook structure in overcoming the resistance.Finally, there are several points that should be addressed orconsidered in future studies. First, can the primary resistancemutations induced by CHR-derived fusion inhibitors univer-sally result in a structural change in the NHR region of gp41? Inother words, do the changes in the conformation and stability ofNHR essentially determine the resistance by interfering withthe binding of inhibitors and the interaction of viral NHR andCHR helices? We are in the process of examining the effects ofT20-, C34-, and SC34EK-induced resistance mutations, whichmay help to generalize a proof of concept; Second, can some ofthe resistance mutations in the NHR region also play compen-satory roles in viral infectivity? To this point, the A47I and E49Kmutations did behave like the secondary N126K mutation thatcan enhance the NHR–CHR interactions. Third, how do weexplain the fact that whereas the A47I mutation enhances thebinding of SFT, it still mediates the resistance? Now, one mayspeculate that this mutation can enhance the interaction ofviral NHR and CHR helices more efficiently, thus outcompet-ing the binding of inhibitor. When enhancing the binding ofT20 more efficiently, the A47I mutation does increase the sen-sitivity of drug (Table 1). Fourth, many mutations in the NHRand CHR of gp41 were previously reported to affect the expres-sion and processing profiles of viral Env glycoproteins, but ourstudies showed that the fusion inhibitor peptide–induced resis-tance mutations had no such effects. Thus, it will be interestingto clarify this point as a common phenomenon or a misleadingcoincidence. Fifth, it is critical to characterize whether fusioninhibitor–selected NHR mutations impact the structure andfunction of HIV-1 Rev response element, as described by pre-vious studies (51, 52). Additionally, as the fitness of the resistantviruses to SFT seems to be very poor compared with the paren-tal virus, we are wondering if these viruses can survive in vivo.

Experimental procedures

Cell lines and culture conditions

HEK293T cells were purchased from the American type cul-ture collection (ATCC) (Manassas, VA); TZM-bl indicator cellsstably expressing CD4 and CCR5 along with endogenouslyexpressed CXCR4 were obtained from John C. Kappes andXiaoyun Wu through the AIDS Reagent Program, Division of

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AIDS, NIAID, National Institutes of Health (17). Both cellswere cultured in complete growth medium that consisted ofDulbecco’s minimal essential medium supplemented with 10%fetal bovine serum, 100 units/ml penicillin-streptomycin, 2 mM

L-glutamine, 1 mM sodium pyruvate, and 1� minimum Eagle’smedium nonessential amino acids (Gibco/Invitrogen) and weremaintained at 37 °C in 5% CO2.

Synthesis of fusion inhibitor and target mimic peptides

A panel of fusion inhibitor peptides (SFT, T20, C34, MTSFT,2P23, and LP-19) and target mimic peptides (N36, N36V38A,N36A47I, N36Q52R, N36 V38A/A47I, and N36V38A/A47I/Q52R) weresynthesized with a standard solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) method as described previously (30). Allpeptides were protected by N-terminal acetylation and C-ter-minal amidation. To synthesize LP-19, the template peptide2P23 contains a lysine residue at its C terminus with a Ddeside-chain protecting group, enabling the conjugation of a C16fatty acid that requires a special deprotection step in a solutionof 2% hydrazinehydrate/N,N-dimethylformamide. The pep-tides were purified to a purity of �95% by reversed-phaseHPLC and characterized for correct amino acid composition byMS. Concentrations of the peptides were measured by UV ab-sorbance and a theoretically calculated molar extinction coef-ficient based on tryptophan and tyrosine residues.

Generation of HIV-1 Env mutants

A panel of HIV-1NL4-3 Env– based mutants was generated bysite-directed mutagenesis as described previously (53). In brief,two primers contained the desired mutation and occupied thesame starting and ending positions on opposite strands of plas-mid. DNA synthesis was performed by PCR in a 50-�l reactionvolume using 1 ng of denatured plasmid template, a 50 pM con-centration of upper and lower primers, and 5 units of the high-fidelity polymerase PrimeStar (TaKaRa, Dalian, China). PCRamplification was performed for one cycle of denaturation at98 °C for 5 min, followed by 18 cycles of 98 °C for 15 s and 68 °Cfor 15 min, with a final extension at 72 °C for 10 min. The ampli-cons were digested with DpnI at 37 °C for 1 h, and DpnI-resis-tant molecules were recovered by transforming Escherichia colistrain DH5� to antibiotic resistance. The introduced mutationswere confirmed by DNA sequencing.

Single-cycle infection assay

The backbone plasmid pSG3�env that encodes an Env-defec-tive, luciferase-expressing HIV-1 genome was obtained fromJohn C. Kappes and Xiaoyun Wu through the AIDS ReagentProgram, Division of AIDS, NIAID, National Institutes ofHealth (17). HIV-1 pseudoviruses were generated as describedpreviously (53). In brief, 293T cells were cotransfected withpSG3�env and a plasmid expressing Env glycoprotein. Superna-tants were harvested 48 h after transfection, and tissue cultureID50 values were determined in TZM-bl cells. Peptides wereprepared in graded concentrations, mixed with 100 tissue cul-ture ID50 of viruses, and then incubated for 1 h at room tem-perature. The mixture was added to TZM-bl cells (104 cells/well) containing DEAE (final concentration 15 �l/ml) andincubated 48 h at 37 °C. The luciferase activity was measured

using luciferase assay reagents and a luminescence counter(Promega, Madison, WI).

DSP-based cell fusion assay

A DSP-based cell– cell fusion assay was performed to deter-mine the fusogenic activity of viral Env as described previously(54, 55). The plasmids DSP1–7 and DSP8 –11 were kindly pro-vided by Zene Matsuda at the Institute of Medical Science ofthe University of Tokyo (Tokyo, Japan) Briefly, a total of 1.5 �104 293T cells (effector cells) were seeded on a 96-well plate,and a total of 8 � 104 U87-CXCR4 cells (target cells) wereseeded on a 24-well plate. On the following day, 293T cells weretransfected with a mixture of an Env-expressing plasmid and aDSP1–7 plasmid, and U87-CXCR4 cells were transfected with aDSP8 –11 plasmid. Twenty-four hours post-transfection, thetarget cells were resuspended in 300 �l of prewarmed culturemedium containing EnduRen live-cell substrate (Promega) at afinal concentration of 17 ng/�l and then transferred to eachwell of the effector cells with equal volume. The cell mixturewas spun down, and luciferase activity was measured by a lumi-nescence counter (Promega).

CD spectroscopy

CD spectroscopy was conducted to determine the �-helicityand thermostability of the isolated or combined peptides asdescribed previously (22). CD spectra were acquired on a Jascospectropolarimeter (model J-815) using a 1-nm bandwidth witha 1-nm step resolution from 195 to 260 nm at room tempera-ture and corrected by subtraction of a solvent blank. The �-hel-ical content was calculated from the CD signal by dividing themean residue ellipticity ([�]) at 222 nm by the value expected for100% helix formation (�33,000 degrees cm�2 dmol�1). Ther-mal denaturation was performed by monitoring the ellipticitychange at 222 nm from 20 to 98 °C at a rate of 1.2 °C/min.

Detection of viral Env glycoprotein by capture ELISA

A capture ELISA was conducted to determine the effects ofthe introduced mutations on the expression and processing ofviral Env glycoprotein, as described previously (35). Briefly, thewells of an ELISA plate were coated with a sheep anti-gp120antibody (D7324) at 10 �g/ml and blocked by 3% BSA. Celllysates or culture supernatants (50 �l) of Env-transfected cellswere added to the wells and incubated at 37 °C for 1 h. Afterextensive washes, 50 �l of human anti-gp120 mAb VRC01 oranti-gp41 mAb 10E8 was added at 10 �g/ml and incubated at37 °C for 1 h. The bound antibodies were detected by horserad-ish peroxidase– conjugated goat anti-human IgG. The reactionwas visualized by adding 3,3,5,5-tetramethylbenzidine, and theA450 was measured.

Detection of 6-HBs by conformation-specific antibodies

To detect the 6-HBs formed by N36 and C34 peptides, three6-HB conformation–specific mAbs (NC-1, 17C8, and 2G8),which react with the N36 and C34 complex but not the isolatedpeptides, were applied in an ELISA as described previously(34 –36). NC-1 was kindly provided by Shibo Jiang (Lindsley F.Kimball Research Institute of the New York Blood Center, NewYork) (56); 17C8 and 2G8 were kindly provided by Yinghua

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Chen (School of Life Sciences of Tsinghua University, Beijing,China) (39). Briefly, the isolated or mixed peptides were coatedto the ELISA wells at 10 �g/ml and blocked with 3% BSA. Afterwashing, the anti-6-HB mAb diluted at 5 �g/ml was added tothe wells and incubated at 37 °C for 1 h. After washes, the boundantibodies were detected by horseradish peroxidase– conju-gated anti-mouse IgG, the reaction was visualized by theaddition of 3,3,5,5-tetramethylbenzidine, and the A450 wasmeasured.

Western blotting

To verify the expression and processing profile of viral Envglycoprotein by Western blotting, the lysates or culture super-natants of transfected cells were centrifuged at 20,000 � g at4 °C for 15 min to remove insoluble materials. Equal amounts oftotal proteins were separated by 10% SDS-PAGE and thentransferred to a nitrocellulose membrane. After blocking with5% nonfat dry milk solution in Tris-buffered saline (TBS, pH7.4) at room temperature for 1 h, the membrane was incubatedwith the human anti-HIV polyclonal antibody HIV-IG(obtained through the AIDS Reagent Program, Division ofAIDS, NIAID, National Institutes of Health) or human anti-gp120 mAb HY54 (which was generated in our laboratory)overnight at 4 °C. The following day, the membrane waswashed three times in TBS-T and then incubated with IRDye�800CW goat anti-human IgG at room temperature for 2 h. Themembrane was then scanned using the Odyssey IR imagingsystem (LI-COR Biosciences, Lincoln, NE). Band intensitieswere analyzed using ImageJ software (National Institutes ofHealth).

Flow cytometry assay

The WT or mutant Env glycoproteins expressed on the sur-face of transfected cells were detected by flow cytometry.Briefly, HEK293T cells were transiently transfected with a spe-cific plasmid and harvested at 36 h after transfection. After twowashes with PBS, the human anti-gp120 mAb VRC01 wasadded to the cells and incubated at 4 °C for 1 h. Then cells werewashed twice and incubated with Alexa Fluor 488 anti-humanIgG (Life Technologies, Inc.) at 4 °C for 1 h. After three washes,cells were resuspended in PBS and analyzed by a FACSCalibur(BD Biosciences).

Immunofluorescence assay

An Env-encoding plasmid was transiently expressed inHEK293T cells by transfection. After 36 h, the cells were immo-bilized by 4% paraformaldehyde, and then 1% BSA was added toblock nonspecific binding of the antibodies. After washingtwice with PBS, VRC01 was added to the cells at a final concen-tration of 20 �g/ml and incubated at 4 °C overnight. After threewashes, bound antibodies were detected by Alexa Fluor 488anti-human IgG and observed under an immunofluorescencemicroscope.

N-PAGE

N-PAGE was conducted to determine the interactionsbetween N36 and C34 with native or mutant sequences asdescribed previously (22, 57). Briefly, N36 was mixed with C34

with the final concentration of each peptide at 40 �M and thenincubated at 37 °C for 30 min. The sample was added with Tris-glycine native sample buffer at a ratio of 1:1 and then loadedonto 10 � 1.0-mm Tris-glycine gels (20%) at 25 �l/well. Gelelectrophoresis was carried out with 100-V constant voltage at4 °C for 3 h. The gel was then stained with Coomassie Blue andimaged with a Bio-Rad imaging system.

Author contributions—D. Y., X. D., Z. L., X. W., Y. Z., H. W., H. C.,and S. C. investigation; D. Y., X. D., Z. L., X. W., Y. Z., H. W., H. C.,and S. C. methodology; Y. H. conceptualization; Y. H. supervision;Y. H. funding acquisition; Y. H. writing-original draft; Y. H. projectadministration.

Acknowledgments—We thank Zene Matsuda (Institute of MedicalScience of the University of Tokyo) for providing the DSP plasmids,Yinghua Chen (College of Life Science of Tsinghua University, Beijing,China) for providing the 6-HB–specific mAbs 17C8 and 2G8, andShibo Jiang (Lindsley F. Kimball Research Institute of New York BloodCenter, New York) for providing the 6-HB–specific mAb NC-1.

References1. Eckert, D. M., and Kim, P. S. (2001) Mechanisms of viral membrane fusion

and its inhibition. Annu. Rev. Biochem. 70, 777– 810 CrossRef Medline2. Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J., and Wiley, D. C.

(1997) Atomic structure of the ectodomain from HIV-1 gp41. Nature 387,426 – 430 CrossRef Medline

3. Tan, K., Liu, J., Wang, J., Shen, S., and Lu, M. (1997) Atomic structure of athermostable subdomain of HIV-1 gp41. Proc. Natl. Acad. Sci. U.S.A. 94,12303–12308 CrossRef Medline

4. Chan, D. C., Fass, D., Berger, J. M., and Kim, P. S. (1997) Core structure ofgp41 from the HIV envelope glycoprotein. Cell 89, 263–273 CrossRefMedline

5. Eckert, D. M., Malashkevich, V. N., Hong, L. H., Carr, P. A., and Kim, P. S.(1999) Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that tar-get the gp41 coiled-coil pocket. Cell 99, 103–115 CrossRef Medline

6. Chan, D. C., and Kim, P. S. (1998) HIV entry and its inhibition. Cell 93,681– 684 CrossRef Medline

7. Chan, D. C., Chutkowski, C. T., and Kim, P. S. (1998) Evidence that aprominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drugtarget. Proc. Natl. Acad. Sci. U.S.A. 95, 15613–15617 CrossRef Medline

8. He, Y. (2013) Synthesized peptide inhibitors of HIV-1 gp41-dependentmembrane fusion. Curr. Pharm. Des. 19, 1800 –1809 CrossRef Medline

9. Steffen, I., and Pohlmann, S. (2010) Peptide-based inhibitors of the HIVenvelope protein and other class I viral fusion proteins. Curr. Pharm. Des.16, 1143–1158 CrossRef Medline

10. Eggink, D., Berkhout, B., and Sanders, R. W. (2010) Inhibition of HIV-1 byfusion inhibitors. Curr. Pharm. Des. 16, 3716 –3728 CrossRef Medline

11. Lalezari, J. P., Henry, K., O’Hearn, M., Montaner, J. S., Piliero, P. J., Trot-tier, B., Walmsley, S., Cohen, C., Kuritzkes, D. R., Eron, J. J., Jr., Chung, J.,DeMasi, R., Donatacci, L., Drobnes, C., Delehanty, J., et al. (2003) Enfu-virtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection inNorth and South America. N. Engl. J. Med. 348, 2175–2185 CrossRefMedline

12. Kilby, J. M., Hopkins, S., Venetta, T. M., DiMassimo, B., Cloud, G. A., Lee,J. Y., Alldredge, L., Hunter, E., Lambert, D., Bolognesi, D., Matthews, T.,Johnson, M. R., Nowak, M. A., Shaw, G. M., and Saag, M. S. (1998) Potentsuppression of HIV-1 replication in humans by T-20, a peptide inhibitor ofgp41-mediated virus entry. Nat. Med. 4, 1302–1307 CrossRef Medline

13. Sista, 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., and Green-berg, M. L. (2004) Characterization of determinants of genotypic and phe-notypic resistance to enfuvirtide in baseline and on-treatment HIV-1 iso-lates. AIDS 18, 1787–1794 CrossRef Medline

HIV-1 resistance to sifuvirtide

12716 J. Biol. Chem. (2018) 293(33) 12703–12718

by guest on February 14, 2020http://w

ww

.jbc.org/D

ownloaded from

14. Poveda, E., Rodes, B., Labernardiere, J. L., Benito, J. M., Toro, C.,Gonzalez-Lahoz, J., Faudon, J. L., Clavel, F., Schapiro, J., and Soriano, V.(2004) Evolution of genotypic and phenotypic resistance to Enfuvirtide inHIV-infected patients experiencing prolonged virologic failure. J. Med.Virol. 74, 21–28 CrossRef Medline

15. Greenberg, M. L., and Cammack, N. (2004) Resistance to enfuvirtide,the first HIV fusion inhibitor. J. Antimicrob. Chemother. 54, 333–340CrossRef Medline

16. Baldwin, C. E., Sanders, R. W., Deng, Y., Jurriaans, S., Lange, J. M., Lu, M.,and Berkhout, B. (2004) Emergence of a drug-dependent human immu-nodeficiency virus type 1 variant during therapy with the T20 fusion in-hibitor. J. Virol. 78, 12428 –12437 CrossRef Medline

17. Wei, X., Decker, J. M., Liu, H., Zhang, Z., Arani, R. B., Kilby, J. M., Saag,M. S., Wu, X., Shaw, G. M., and Kappes, J. C. (2002) Emergence of resistanthuman immunodeficiency virus type 1 in patients receiving fusion inhib-itor (T-20) monotherapy. Antimicrob. Agents Chemother. 46, 1896 –1905CrossRef Medline

18. Rimsky, L. T., Shugars, D. C., and Matthews, T. J. (1998) Determinants ofhuman immunodeficiency virus type 1 resistance to gp41-derived inhibi-tory peptides. J. Virol. 72, 986 –993 Medline

19. He, Y., Xiao, Y., Song, H., Liang, Q., Ju, D., Chen, X., Lu, H., Jing, W., Jiang,S., and Zhang, L. (2008) Design and evaluation of sifuvirtide, a novel HIV-1fusion inhibitor. J. Biol. Chem. 283, 11126 –11134 CrossRef Medline

20. Dwyer, 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., and Delmedico,M. K. (2007) Design of helical, oligomeric HIV-1 fusion inhibitor peptideswith potent activity against enfuvirtide-resistant virus. Proc. Natl. Acad.Sci. U.S.A. 104, 12772–12777 CrossRef Medline

21. Otaka, A., Nakamura, M., Nameki, D., Kodama, E., Uchiyama, S., Naka-mura, S., Nakano, H., Tamamura, H., Kobayashi, Y., Matsuoka, M., andFujii, N. (2002) Remodeling of gp41-C34 peptide leads to highly effectiveinhibitors of the fusion of HIV-1 with target cells. Angew. Chem. Int. Ed.Engl. 41, 2937–2940 CrossRef Medline

22. He, Y., Cheng, J., Lu, H., Li, J., Hu, J., Qi, Z., Liu, Z., Jiang, S., and Dai, Q.(2008) Potent HIV fusion inhibitors against Enfuvirtide-resistant HIV-1strains. Proc. Natl. Acad. Sci. U.S.A. 105, 16332–16337 CrossRef Medline

23. Xiong, S., Borrego, P., Ding, X., Zhu, Y., Martins, A., Chong, H., Taveira,N., and He, Y. (2017) A helical short-peptide fusion inhibitor with highlypotent activity against human immunodeficiency virus type 1 (HIV-1),HIV-2, and simian immunodeficiency virus. J. Virol. 91, e01839-16Medline

24. Chong, H., Qiu, Z., Su, Y., Yang, L., and He, Y. (2015) Design of a highlypotent HIV-1 fusion inhibitor targeting the gp41 pocket. AIDS 29, 13–21CrossRef Medline

25. Chong, H., Yao, X., Qiu, Z., Sun, J., Qiao, Y., Zhang, M., Wang, M., Cui, S.,and He, Y. (2014) The M-T hook structure increases the potency of HIV-1fusion inhibitor sifuvirtide and overcomes drug resistance. J. Antimicrob.Chemother. 69, 2759 –2769 CrossRef Medline

26. Yao, X., Chong, H., Zhang, C., Waltersperger, S., Wang, M., Cui, S., andHe, Y. (2012) Broad antiviral activity and crystal structure of HIV-1 fusioninhibitor sifuvirtide. J. Biol. Chem. 287, 6788 – 6796 CrossRef Medline

27. Chong, H., Qiu, Z., Sun, J., Qiao, Y., Li, X., and He, Y. (2014) Two M-Thook residues greatly improve the antiviral activity and resistance profileof the HIV-1 fusion inhibitor SC29EK. Retrovirology 11, 40 CrossRefMedline

28. Chong, H., Yao, X., Qiu, Z., Sun, J., Zhang, M., Waltersperger, S., Wang,M., Liu, S. L., Cui, S., and He, Y. (2013) Short-peptide fusion inhibitorswith high potency against wild-type and enfuvirtide-resistant HIV-1.FASEB J. 27, 1203–1213 CrossRef Medline

29. Yao, X., Chong, H., Zhang, C., Qiu, Z., Qin, B., Han, R., Waltersperger, S.,Wang, M., He, Y., and Cui, S. (2012) Structural basis of potent and broadHIV-1 fusion inhibitor CP32M. J. Biol. Chem. 287, 26618 –26629 CrossRefMedline

30. Chong, H., Yao, X., Sun, J., Qiu, Z., Zhang, M., Waltersperger, S., Wang,M., Cui, S., and He, Y. (2012) The M-T hook structure is critical for designof HIV-1 fusion inhibitors. J. Biol. Chem. 287, 34558 –34568 CrossRefMedline

31. Chong, H., Yao, X., Qiu, Z., Qin, B., Han, R., Waltersperger, S., Wang, M.,Cui, S., and He, Y. (2012) Discovery of critical residues for viral entry andinhibition through structural insight of HIV-1 fusion inhibitor CP621-652. J. Biol. Chem. 287, 20281–20289 CrossRef Medline

32. Chong, H., Xue, J., Xiong, S., Cong, Z., Ding, X., Zhu, Y., Liu, Z., Chen, T.,Feng, Y., He, L., Guo, Y., Wei, Q., Zhou, Y., Qin, C., and He, Y. (2017) Alipopeptide HIV-1/2 fusion inhibitor with highly potent in vitro, ex vivo,and in vivo antiviral activity. J. Virol. 91, e00288-00217 Medline

33. Johnson, W. E., Sauvron, J. M., and Desrosiers, R. C. (2001) Conserved,N-linked carbohydrates of human immunodeficiency virus type 1 gp41are largely dispensable for viral replication. J. Virol. 75, 11426 –11436CrossRef Medline

34. Su, Y., Chong, H., Xiong, S., Qiao, Y., Qiu, Z., and He, Y. (2015) Geneticpathway of HIV-1 resistance to novel fusion inhibitors targeting the Gp41pocket. J. Virol. 89, 12467–12479 CrossRef Medline

35. Su, Y., Chong, H., Qiu, Z., Xiong, S., and He, Y. (2015) Mechanism ofHIV-1 resistance to short-peptide fusion inhibitors targeting the Gp41pocket. J. Virol. 89, 5801–5811 CrossRef Medline

36. Wu, X., Liu, Z., Ding, X., Yu, D., Wei, H., Qin, B., Zhu, Y., Chong, H., Cui,S., and He, Y. (2018) Mechanism of HIV-1 resistance to an electronicallyconstrained �-helical peptide membrane fusion inhibitor. J. Virol. 92,e02044-02017 Medline

37. Dimitrov, A. S., Louis, J. M., Bewley, C. A., Clore, G. M., and Blumenthal,R. (2005) Conformational changes in HIV-1 gp41 in the course of HIV-1envelope glycoprotein-mediated fusion and inactivation. Biochemistry 44,12471–12479 CrossRef Medline

38. Sackett, K., Wexler-Cohen, Y., and Shai, Y. (2006) Characterization of theHIV N-terminal fusion peptide-containing region in context of key gp41fusion conformations. J. Biol. Chem. 281, 21755–21762 CrossRef Medline

39. Li, J., Chen, X., Huang, J., Jiang, S., and Chen, Y. H. (2009) Identification ofcritical antibody-binding sites in the HIV-1 gp41 six-helix bundle core aspotential targets for HIV-1 fusion inhibitors. Immunobiology 214, 51– 60CrossRef Medline

40. Shimane, K., Kawaji, K., Miyamoto, F., Oishi, S., Watanabe, K., Sakagami,Y., Fujii, N., Shimura, K., Matsuoka, M., Kaku, M., Sarafianos, S. G., andKodama, E. N. (2013) HIV-1 resistance mechanism to an electrostaticallyconstrained peptide fusion inhibitor that is active against T-20-resistantstrains. Antimicrob. Agents Chemother. 57, 4035– 4038 CrossRef Medline

41. Wang, W., De Feo, C. J., Zhuang, M., Vassell, R., and Weiss, C. D. (2011)Selection with a peptide fusion inhibitor corresponding to the first heptadrepeat of HIV-1 gp41 identifies two genetic pathways conferring cross-resistance to peptide fusion inhibitors corresponding to the first and sec-ond heptad repeats (HR1 and HR2) of gp41. J. Virol. 85, 12929 –12938CrossRef Medline

42. Liu, Z., Shan, M., Li, L., Lu, L., Meng, S., Chen, C., He, Y., Jiang, S., andZhang, L. (2011) In vitro selection and characterization of HIV-1 variantswith increased resistance to sifuvirtide, a novel HIV-1 fusion inhibitor.J. Biol. Chem. 286, 3277–3287 CrossRef Medline

43. Eggink, D., Bontjer, I., Langedijk, J. P., Berkhout, B., and Sanders, R. W.(2011) Resistance of human immunodeficiency virus type 1 to a third-generation fusion inhibitor requires multiple mutations in gp41 and isaccompanied by a dramatic loss of gp41 function. J. Virol. 85,10785–10797 CrossRef Medline

44. Shimura, K., Nameki, D., Kajiwara, K., Watanabe, K., Sakagami, Y., Oishi,S., Fujii, N., Matsuoka, M., Sarafianos, S. G., and Kodama, E. N. (2010)Resistance profiles of novel electrostatically constrained HIV-1 fusion in-hibitors. J. Biol. Chem. 285, 39471–39480 CrossRef Medline

45. Eggink, D., Baldwin, C. E., Deng, Y., Langedijk, J. P., Lu, M., Sanders, R. W.,and Berkhout, B. (2008) Selection of T1249-resistant human immunode-ficiency virus type 1 variants. J. Virol. 82, 6678 – 6688 CrossRef Medline

46. Eggink, D., de Taeye, S. W., Bontjer, I., Klasse, P. J., Langedijk, J. P., Berk-hout, B., and Sanders, R. W. (2016) HIV-1 escape from a peptidic anchorinhibitor through stabilization of the envelope glycoprotein spike. J. Virol.90, 10587–10599 CrossRef Medline

47. De Feo, C. J., Wang, W., Hsieh, M. L., Zhuang, M., Vassell, R., and Weiss,C. D. (2014) Resistance to N-peptide fusion inhibitors correlates withthermodynamic stability of the gp41 six-helix bundle but not HIV entrykinetics. Retrovirology 11, 86 CrossRef Medline

HIV-1 resistance to sifuvirtide

J. Biol. Chem. (2018) 293(33) 12703–12718 12717

by guest on February 14, 2020http://w

ww

.jbc.org/D

ownloaded from

48. Eggink, D., Langedijk, J. P., Bonvin, A. M., Deng, Y., Lu, M., Berkhout, B.,and Sanders, R. W. (2009) Detailed mechanistic insights into HIV-1 sen-sitivity to three generations of fusion inhibitors. J. Biol. Chem. 284,26941–26950 CrossRef Medline

49. Steger, H. K., and Root, M. J. (2006) Kinetic dependence to HIV-1 entryinhibition. J. Biol. Chem. 281, 25813–25821 CrossRef Medline

50. Ahn, K. W., and Root, M. J. (2017) Complex interplay of kinetic factorsgoverns the synergistic properties of HIV-1 entry inhibitors. J. Biol. Chem.292, 16498 –16510 CrossRef Medline

51. Svicher, V., Alteri, C., D’Arrigo, R., Lagana, A., Trignetti, M., Lo Caputo, S.,Callegaro, A. P., Maggiolo, F., Mazzotta, F., Ferro, A., Dimonte, S., Aquaro,S., di Perri, G., Bonora, S., Tommasi, C., et al. (2009) Treatment with thefusion inhibitor enfuvirtide influences the appearance of mutations in thehuman immunodeficiency virus type 1 regulatory protein rev. Antimicrob.Agents Chemother. 53, 2816 –2823 CrossRef Medline

52. Nameki, D., Kodama, E., Ikeuchi, M., Mabuchi, N., Otaka, A., Tamamura,H., Ohno, M., Fujii, N., and Matsuoka, M. (2005) Mutations conferringresistance to human immunodeficiency virus type 1 fusion inhibitors arerestricted by gp41 and Rev-responsive element functions. J. Virol. 79,764 –770 CrossRef Medline

53. Chong, H., Yao, X., Zhang, C., Cai, L., Cui, S., Wang, Y., and He, Y. (2012)Biophysical property and broad anti-HIV activity of albuvirtide, a 3-maleimimidopropionic acid-modified peptide fusion inhibitor. PLoS One7, e32599 CrossRef Medline

54. Kondo, N., Miyauchi, K., Meng, F., Iwamoto, A., and Matsuda, Z. (2010)Conformational changes of the HIV-1 envelope protein during membranefusion are inhibited by the replacement of its membrane-spanning do-main. J. Biol. Chem. 285, 14681–14688 CrossRef Medline

55. Ishikawa, H., Meng, F., Kondo, N., Iwamoto, A., and Matsuda, Z. (2012)Generation of a dual-functional split-reporter protein for monitoringmembrane fusion using self-associating split GFP. Protein Eng. Des. Sel.25, 813– 820 CrossRef Medline

56. Jiang, S., Lin, K., and Lu, M. (1998) A conformation-specific monoclonalantibody reacting with fusion-active gp41 from the human immunodefi-ciency virus type 1 envelope glycoprotein. J. Virol. 72, 10213–10217Medline

57. He, Y., Cheng, J., Li, J., Qi, Z., Lu, H., Dong, M., Jiang, S., and Dai, Q. (2008)Identification of a critical motif for the human immunodeficiency virustype 1 (HIV-1) gp41 core structure: implications for designing novel anti-HIV fusion inhibitors. J. Virol. 82, 6349 – 6358 CrossRef Medline

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Huihui Chong, Sheng Cui and Yuxian HeDanwei Yu, Xiaohui Ding, Zixuan Liu, Xiyuan Wu, Yuanmei Zhu, Huanmian Wei,

membrane fusion inhibitorapproved−Molecular mechanism of HIV-1 resistance to sifuvirtide, a clinical trial

doi: 10.1074/jbc.RA118.003538 originally published online June 21, 20182018, 293:12703-12718.J. Biol. Chem. 

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