Resistance to CCR5 inhibitors caused by sequence changes in the fusion peptide of HIV-1

Cleo G. Anastassopoulou, Thomas J. Ketas, Per Johan Klasse, and John P. Moore1

Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021

Edited by Robert C. Gallo, University of Maryland, Baltimore, MD, and approved February 4, 2009 (received for review November 21, 2008) We have investigated the mechanism of resistance of a HIV type 1 VVC-resistance in a patient has also been attributed to V3 changes (HIV-1) R5 primary isolate, D1/85.16, to the small molecule CCR5 (11). The V3-route to resistance was followed when a primary inhibitor, (VVC). Unlike other resistant to this class isolate, CC1/85, was cultured with AD101, a preclinical precursor of compound, D1/85.16 lacks sequence changes in the V3 region of of VVC (6, 12). The fully resistant variant, CC101.19, was stable and the gp120 surface glycoprotein. Inspection of sequences from fit; it did not rapidly revert to sensitivity when cultured in peripheral D1/85.16 compared with those derived from the parental, inhibi- blood mononuclear cells (PBMC) without AD101 (13). CC101.19 tor-sensitive , CC1/85, revealed a cluster of 3 conservative has 4 point substitutions in V3 that are necessary and sufficient for changes in the fusion peptide (FP) of the gp41 transmembrane resistance to AD101, VVC, and similar compounds (6, 12). How- glycoprotein that tracked with the resistance phenotype. Studies ever, when CC1/85 was cultured with VVC, we identified a fully with engineered Env-chimeric and point-substituted viruses con- resistant clone with no V3 sequence changes (14). firmed that these 3 FP residues were substantially responsible for Here, we have studied the VVC-resistant D1/85.16 virus, to VVC resistance without altering coreceptor usage, as assessed in better understand the genetics of resistance and Env structure- both peripheral blood mononuclear cells and the TZM-bl line. function relationships that influence HIV-1 entry and its inhibition. VVC resistance is manifested differently in the 2 cell types, and We show that the critical sequence changes that confer resistance, there are assay-dependent complexities to the dose-response without causing coreceptor switching to CXCR4, are 3 physically curves for the engineered resistant viruses. To explain them, we proximal substitutions in the gp41 FP region. Thus, there are both created a model for resistance and generated theoretical VVC V3-dependent and V3-independent genetic pathways to the same

inhibition curves that closely mimic the experimental data for the phenotype of CCR5 inhibitor-resistance that can be followed by the MEDICAL SCIENCES resistant viruses. The basis for the model is the existence of distinct HIV-1 quasispecies of the same isolate. An additional implication forms of CCR5, with varying affinities for small molecule CCR5 is that the gp120 V3 region and the gp41 FP may become struc- inhibitors that are presumed to be present in different proportions turally or functionally associated at some stage during virus-cell on different cell types, and are used selectively by resistant HIV-1 fusion. Analysis of the behavior of resistant and sensitive clones in variants when ligated with an inhibitor. Together, the experimen- PBMC and the TZM-bl cell line led us to develop a model to explain tal results and theoretical model may help understand how HIV-1 cell type-dependent complexities in resistance to CCR5 inhibitors. uses CCR5 to enter target cells under various conditions. The model predicts the existence of at least 2 forms of CCR5 with different affinities for small molecule inhibitors that are present in vicriviroc ͉ virus entry ͉ coreceptor conformation ͉ theoretical model different relative abundances on different cell types. Results he small molecule CCR5 inhibitors (MVC) and Tvicriviroc (VVC) are now used to treat infection with HIV type VVC Resistance of D1/85.16 Maps to gp41. The D1/85.16 isolate arose 1 (HIV-1) (1). These and similar compounds target CCR5, a G under VVC selection pressure (14), and uses the inhibitor-CCR5 -coupled (GPCR) used by HIV-1 early in the complex as the basis for its resistance to VVC and related com- course of the infection (2). HIV-1 entry is mediated by the envelope pounds (5). Unlike CC101.19, D1/85.16 lacked a consistent pattern glycoprotein (Env) complex, a heterotrimer of 3 gp120 surface of sequence changes in the gp120 V3 region, and a VVC-resistant subunits noncovalently linked to 3 gp41 transmembrane subunits. clone, D1/85.16 cl.23, had a V3 sequence identical to VVC-sensitive CC1/85 clones (6, 14). To test whether resistance tracked with the The gp120 subunits contact first CD4, then CCR5; conformational ϭ rearrangements within the trimer drive insertion of the gp41 fusion gp120 or gp41, we swapped these subunits from D1/85.16 cl.23 ( R) with the corresponding components of the parental clone peptide (FP) region into the host cell membrane, leading to fusion ϭ and the initiation of infection (1). MVC or VVC binding within a CC1/85 cl.6 ( S). Therefore, the R/S chimeric virus contains gp120 cavity in the CCR5 transmembrane helices stabilizes the extracel- from R and gp41 from S, and conversely for the reverse chimera, S/R [for virus nomenclature, Table S1]. Both chimeric viruses were lular domains in a conformation that is not efficiently recognized by 4 5 wild-type gp120; they inhibit entry via a noncompetitive or allo- replication-competent in PBMC with titers of 10 to 10 steric mechanism (1). TCID50/mL on day 7 postinfection (p24, 3–5 ng/mL). VVC resis- The clinical use of small molecule CCR5 inhibitors could, tance in these cells tracked with gp41. Thus, the R/S chimera theoretically, drive the emergence of more pathogenic HIV-1 X4 behaved identically to the sensitive isolate and clone S, the S/R variants that use CXCR4 for entry (1). Although expansion of chimera to the resistant isolate and clone R (Fig. 1). The complex preexisting X4 or dual-tropic (R5X4) variants during has

been reported (3), the principal escape mechanism is the evolution Author contributions: C.G.A., P.J.K., and J.P.M. designed research; C.G.A. and T.J.K. per- of mutations that permit HIV-1 to continue to use CCR5 despite formed research; P.J.K. contributed new reagents/analytic tools; C.G.A., P.J.K., and J.P.M. the presence of the inhibitor; fully resistant viruses can use both the analyzed data; and C.G.A., P.J.K., and J.P.M. wrote the paper. inhibitor-CCR5 complex and free CCR5 for entry (4, 5). The The authors declare no conflict of interest. genetic determinants of resistance in vitro have been mapped, This article is a PNAS Direct Submission. almost invariably, to the gp120 V3 region (4, 6–9). This localization Data deposition: The env sequences of the D1/85.16-derived viruses reported in this paper is consistent with the variable nature of V3 and its function as a have been deposited in the GenBank database (accession nos. FJ713453–FJ713458). component of the CCR5-binding site (1, 10). VVC-resistant viruses 1To whom correspondence should be addressed. E-mail: [email protected]. generated in vitro contained various sequence changes in gp120 This article contains supporting information online at www.pnas.org/cgi/content/full/ including, but not limited to, V3 (7), and the emergence of 0811713106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811713106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 30, 2021 CC1/85 cl.6 = S R/S gp41 start, position 512 of HXB2 gp160 I S/R D1/85.16 cl.23 = R Parental CC1/85 cl.16 AVGIGAMFLGFLGAAGSTMGAAS CC1/85 cl.11 ...... V...... CC1/85 cl.10 ...... 100 CC1/85 cl.9 ...... V...... A CC1/85 cl.8 ...... CC1/85 cl.7 ...... 75 CC1/85 cl.6 ...... D1/85.16 cl.23 ....V.VI...... VVC- D1/85.16 ....V.VI...... 50 Resistant D1/85.16R7 ....V.VI...... D1/85.16R8 ....V.VI...... D1/85.16R10 ....V.VI...... 25 D1/85.16R19 ....V.VI...... CC101.19 cl.3 ...... V...... A AD101- CC101.19 cl.4 ...... V...... A Resistant 0 CC101.19 cl.7 ...... V...... A CC101.19 cl.15 ...... V...... A CC101.19 cl.18 ...... V...... A -25 CC101.19 cl.20 ...... V...... A -2 -1 0 1 2 3 4

Vicriviroc (log nM) Fig. 2. Alignment of N-terminal gp41 sequences from VVC-sensitive and VVC-resistant viruses. The first 23 amino acid residues from the gp41 N Fig. 1. VVC resistance maps to gp41. Clonal chimeric viruses R/S and S/R terminus are shown for 7 clones from the CC1/85 parental virus, 6 VVC- (Table S1) bearing gp120 and gp41 subunits derived either from the parental, resistant isolates or clones based on D1/85.16, and 6 clones from the AD101- VVC-sensitive clone S, or the VVC-resistant clone R were tested for VVC resistant CC101.19 isolate. The sequences are recorded relative to that of sensitivity in a multicycle PBMC-based replication assay measuring p24 pro- CC1/85 cl.16 (top line), with dashes indicating amino acid sequence identity. duction 7 days postinfection. The data shown are mean values derived from 7 Amino acid numbering is based on HXB2 Env, with the first residue of gp41 at to 15 independent experiments Ϯ SEM. position 512. The 3 conservative substitutions of hydrophobic residues, G516V, M518V, and F519I, in the FP region of resistant viruses are highlighted in bold. Among the 7 parental clones, S (GenBank accession no. AY357338) is the most structure to the VVC-inhibition curve for S/R is analyzed further closely related to R; these 2 clones, which were used for subsequent genetic below. Similar results for both chimeric viruses were also obtained studies, are boxed. The env sequences of the D1/85.16-derived viruses have when AD101 was used. been deposited in GenBank (accession nos. FJ713453–FJ713458).

Sequence Changes in the FP Region of D1/85.16. We sequenced the env genes from isolate D1/85.16 and its clone R, as well as from 4 and R/S ϩ 3FP engineered clones were tested for VVC sensitivity reversion culture isolates (D1/85.16R7, D1/85.16R8, D1/85.16R10, by using PBMC, they were all resistant to high VVC concentrations and D1/85.16R19; Fig. S1). These sequences were compared with (Fig. 3). As previously, the S and R/S viruses were VVC-sensitive, clones derived from the CC1/85 parental virus and the AD101- whereas R was fully resistant. The S/R reverse chimera and the S resistant CC101.19 isolate (6). The only consistent pattern of changes involved the FP region of gp41. Thus, all 6 D1/85.16 isolates or clones contained 3 changes that were rare or absent from the CC1/85 cl.6 = S S+3FP parental CC1/85 clones: G516V, M518V, and F519I (Fig. 2). Of R/S+3FP these, 516V and 519I were absent from all of the CC1/85 or D1/85.16 cl.23 = R CC101.19 sequences, whereas 518V was present in 2/7 CC1/85 clones and in all 6 CC101.19 clones. This pattern suggests 518V is 100 a naturally occurring polymorphism that becomes enriched under AD101 or VVC selection pressure. All 3 changes involve the conservative substitution of hydrophobic amino acids, consistent 75 with their location in the FP, and each introduces a C-␤ branched amino acid (Val or Ile). No virus containing all 3 altered amino 50 acids is present in the Los Alamos HIV Sequence Database (www..lanl.gov/, as per 10.28.2008), implying that their occur- 25 rence in D1/85.16 did not arise by chance. All of the CC101.19 clones also contained an Ala substitution at position 534, but 6/7 parental CC1/85 clones and all of the D1/85.16-derived se- 0 quences contained Ser, suggesting this position is irrelevant to VVC resistance. -25 -2 -1 0 1 2 3 4 Site-Directed Mutagenesis of Specific Residues in the FP Region. To Vicriviroc (log nM) test whether the G516V, M518V, and F519I changes confer VVC resistance, site-directed mutagenesis was performed on the sensi- Fig. 3. The 3 gp41 FP amino acid substitutions confer resistance to VVC. tive parental clone S. This clone was chosen because it was the most Site-directed mutagenesis was performed to introduce the 3 changes (G516V, closely related to the resistant clone R (14). The same 3 substitu- M518V, and F519I) into the VVC-sensitive parental clone S and the R/S chimera ϩ ϩ tions were also introduced into the sensitive R/S chimera to (Table S1). The engineered mutant viruses, S 3FP and R/S 3FP, were then investigate their actions in a different gp120 context. The engi- tested for VVC sensitivity in a multicycle PBMC-based replication assay mea- ϩ ϩ suring p24 production 7 days postinfection. For comparison, S and the VVC- neered clones S 3FP and R/S 3FP were replication-competent resistant clone R were also tested. In the same experiments, the R/S and S/R in PBMC; their titers were several-fold lower than the other test chimeras behaved comparably with S and R, respectively, but the curves are 3 4 viruses’ ranging from 10 to 10 TCID50/mL on day 7 postinfection not shown, for clarity. The depicted results are the average of 6–10 indepen- (p24, Ϸ3 ng/mL). When the reverse chimera S/R and the S ϩ 3FP dent experiments, with the error bars indicating the SEM.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811713106 Anastassopoulou et al. Downloaded by guest on September 30, 2021 ϩ ϩ 3FP and R/S 3FP mutants were also cross-resistant to AD101. CC1/85 cl.6 = S R/S Thus, the 3 FP changes are sufficient to confer VVC (and AD101) D1/85.16 cl.23 = R S/R resistance on a sensitive virus, and without causing CXCR4 usage S+3FP R/S+3FP (Fig. S2). However, the same 3 FP changes do not have this effect 100 when introduced into a different, VVC-sensitive virus, JR-FL, implying that they act only in a defined Env context (Fig. S3). Studies on the roles played by the individual changes and their 75 combinations in conferring resistance on CC1/85 are now in progress. 50 However, there were intriguing aspects to the VVC dose- response curves for the resistant viruses. Thus, the replication of S/R, S ϩ 3FP and R/S ϩ 3FP was partially inhibited (40–60%) by 25 low VVC concentrations (Ϸ0.3–2 nM), but enhanced by higher ␮ concentrations (up to 5 M; see Figs. 1 and 3). This pattern of 0 partial, biphasic inhibition at low VVC concentrations was also observed, albeit less so, with R (Figs. 1 and 3). The replication- enhancing effect of higher VVC concentrations appeared to be -25 more pronounced for the R/S ϩ 3FP virus than for S ϩ 3FP (Fig. -2 -1 0 1 2 3 4 3). Therefore, the gp120 component derived from R, which is Vicriviroc (log nM) present in the former but not the latter virus, may contribute to the resistance phenotype, particularly at high inhibitor concentrations. Fig. 4. VVC resistance is manifested differently in a cell line-based assay. The In contrast, the enhancing effects of higher VVC concentrations same infectious, clonal viruses studied in PBMC were tested in TZM-bl cells ϩ with a luciferase reporter gene endpoint. The VVC inhibition curves (dashed were similar for the reverse chimera S/R and the S 3FP mutant black line for the resistant virus R) asymptote to a plateau that is high, but (Figs. 1 and 3); therefore, this phenotypic effect of the gp41 subunit consistently lower (upper limit of 95% C.I. Ͻ100%) for the resistant than the from R may be attributable to the 3 specific FP changes. A possible sensitive viruses. The data shown are the average of 3 independent experi- biological explanation for the biphasic inhibition curve is modeled ments. Resistance-related parameters derived from these experiments are below. summarized in Table 1.

Cell Type Dependence of VVC-Resistance. Resistance to small- MEDICAL SCIENCES molecule CCR5 inhibitors varies with the cell type (4, 5, 7). When has the paramount role in resistance, principally due to the 3 specific viruses that are fully resistant to a compound such as VVC and FP changes. The gp120 context can have an additional influence, MVC in PBMC are tested in cell lines engineered to express CCR5 particularly at high inhibitor concentrations. Thus, the MPI values (e.g., U87.CD4.CCR5 or TZM-bl), they tend to be inhibited but are almost identical for the S/R and S ϩ 3FP viruses, and lower for only up to a maximum value [the maximum percent inhibition R/S ϩ 3FP (Table 1). The complexities of CCR5 usage that may (MPI)]. The extent of inhibition is then unaffected by a further underlie these observations are discussed below. increase in inhibitor concentration (‘‘the plateau effect’’). The MPI reflects the relative ability of the virus to use the inhibitor-bound Modeling the Cell Type-Dependency of Resistance. The dose- and inhibitor-free forms of CCR5 for entry; the higher the MPI response curves for the resistant clones depicted in Figs. 1, 3, and value, the less efficiently a resistant virus recognizes the inhibitor- 4 are clearly both complex and cell type-dependent. To try to CCR5 complex (4, 5). Therefore, we tested the engineered, clonal understand the underlying biology, we developed a model (Fig. S5). infectious viruses for their sensitivity to VVC in TZM-bl cells, to We postulate that 2 different subpopulations of CCR5 receptors further characterize their resistance profiles (Fig. 4). coexist on the cell surface in proportions that vary between cell Each virus was replication-competent in TZM-bl cells, with types, and that the infectivity of a viral variant is a linear function relative luminescence units (RLU) varying within a 10-fold range of the amount of a CCR5 form(s) that can support entry of that (Table 1). The parental clone S and the sensitive chimera R/S variant. One CCR5 subpopulation (designated CCR5-A) is as- behaved similarly; each was fully inhibited by VVC (MPI Ͼ99% in sumed to bind VVC, and related inhibitors, with a low affinity, both cases), with half maximum (IC50) values of 20 and 7.8 nM, whereas the second form (CCR5-B) binds them with a higher respectively (Fig. 4, Table 1). The R and S/R viruses had MPI values affinity. Wild-type HIV-1 can have a preference for one of the free of 93 and 94% with reduced IC50 values of 2.4 and 2.0 nM; although forms of CCR5, but cannot use either inhibitor-bound form for these MPI values are high, the upper limits of their 95% C.I. were entry. A resistant variant can use inhibitor-CCR5-A complexes consistently Ͻ100% (Table 1). The MPI values for the engineered while losing the capacity to enter via the free form of CCR5-A, but resistant viruses S ϩ 3FP and R/S ϩ 3FP were also consistently retaining or increasing its ability to use the free form of CCR5-B. Ͼ Ͻ100% (95 and 90%, respectively), but their IC50 values were This version of the model is the simplest; in reality, there can be 2 reduced even further, to 0.70 and 0.66 nM (Fig. 4, Table 1). Similar subpopulations of CCR5. The mathematical formulation of the results for the S, R, and S ϩ 3FP viruses were also obtained by using AD101. Therefore, the engineered mutant viruses, which are strongly Table 1. Indicators of VVC-resistance in TZM-bl cells Ϸ resistant in PBMC (Fig. 3), are 30-fold more sensitive (as judged Virus MPI, % (95% C.I.) IC50, nM RLU by IC50 values) than the parental clone to low VVC concentrations ϭ ϫ 5 when tested in TZM-bl cells (Fig. 4, Table 1). However, these CC1/85 cl.6 S 100 (94–110)* 20 1.4 10 ϭ ϫ 5 viruses do display the noncompetitive resistance indicator that is D1/85.16 cl.23 R 93 (89–96) 2.4 1.6 10 ϫ 4 characteristic of assays based on engineered cell lines, i.e., MPI R/S 100 (93–110)* 7.8 6.7 10 ϫ 4 significantly Ͻ100% (4, 5, 7). Experiments using U87.CD4.CCR5 S/R 94 (90–98) 2.0 4.1 10 ϩ ϫ 4 cells and pseudotyped viruses bearing Env from clones S S 3FP 95 (93–97) 0.70 4.2 10 ϩ ϫ 4 and R yielded similar findings, including the replication of a small R/S 3FP 90 (87–93) 0.66 1.9 10 viral fraction in the presence of high VVC concentrations and a Data are mean values derived from 3 independent experiments. *, P Ͻ 0.01 reduced IC50 for R compared with S (Fig. S4). Also, the principal for R vs. S and for R vs. R/S MPI values; P Ͼ 0.05 between the MPI for R and each finding from both the cell line- and PBMC-based assays is that gp41 other clone.

Anastassopoulou et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 30, 2021 when fitting the function to the PBMC data. However, to avoid A PBMC creating excessively wide C.I. when modeling the TZM-bl cell data, IC50 values were estimated for S and S ϩ 3FP from the sigmoid dose-response plots derived experimentally by using these cells. The resulting IC50 ratio of 37 was then used as a constant affinity ratio in a 2-parameter function (SI Materials and Methods). The resulting functions were then fitted to the experimental data (Fig. 5). As noted above (Fig. 3), the experimental VVC dose-response curves against the resistant S ϩ 3FP mutant in PBMC have complex shapes. Inhibition is maximal at intermediate VVC concentrations, but a plateau arises at higher concentrations that varies from partial inhibition to enhanced infectivity (negative inhibition) in experi- ments on PBMC from different donor pools (Fig. 5 A and B). B Similar data profiles, as well as intermediate forms, arise with PBMC PBMC from other donor pools. We have yet to determine whether the various curve shapes are donor-specific, and how they relate to variation in CCR5-expression levels; studies on cells from individ- ual donors, rather than the pooled cells from 2 or 3 donors that we routinely use, will be necessary. The difference between the plateaus of inhibition at the highest VVC concentrations for the sensitive and resistant viruses is much smaller in the TZM-bl cell assay than with PBMC (Fig. 5C). However, as with the PBMC-based data, the experimental datasets from TZM-bl cells can be closely mimicked by the nonlinear- regression-generated curves based on the model (Fig. 5 and Fig. C S6). Thus, in both the experimental data and the theoretical curves, inhibition of the VVC-resistant virus is incomplete at the plateau, TZM-bl but the IC50 is lower than for the wild-type virus; these 2 apparently conflicting observations are otherwise hard to reconcile with each other. Fitting the function derived from the model to the experimental PBMC data yields values for 3 parameters: the affinities of VVC for CCR5-A and CCR5-B and w, which reflects the relative amount of the form of CCR5 that is infection-permissive when ligated by VVC; also, w can be interpreted as expressing a variable capacity of a test virus to use these VVC-CCR5 complexes (SI Materials and Methods). For the PBMC-derived data, w is close to 0 for the wild-type S virus (0–0.0040; Table S2); for the resistant S ϩ 3FP Fig. 5. Fitting of model function to empirical data from PBMC and TZM-bl virus, it varies above and below 1 between experiments on cells from cell assays. The relative inhibition of virus infectivity is expressed in percentage different donor pools; it is Ͻ1 (0.34 Ϯ 0.029) for the data showing on the y axis as a function of the VVC concentration in nM on the x axis. The Ϯ black circles represent data for wild-type virus (S); data for the VVC-resistant partial inhibition at the plateau, it approaches 1 (1.0 0.037) when S ϩ 3FP mutant are shown as gray squares and triangles (error bars, SEM in there was no inhibition at the highest VVC concentrations (Fig. 5A, each case). The theoretical curves derived from the model are shown as Table S2), but it exceeds 1 (1.8 Ϯ 0.059) when high concentrations continuous lines in the shades of the dataset each is fitted to. (A) Inhibition of of VVC enhance infection (Fig. 5B, Table S2). In each of the 3 cases, the S ϩ 3FP (gray) and S (black) viruses in PBMC from 2 different donor pools. the estimated dissociation constant of VVC for CCR5-A was The resistant virus is partially or negligibly inhibited at the highest VVC distinct from that for CCR5-B (9.6–50 and 0.30–1.6 nM, respec- concentrations. Datasets from other donor pools fall between these profiles tively, with ratios of 7.5–170; Table S2). The modeled Kd values for when plotted similarly. (B) An example of enhancement of S ϩ 3FP virus S on PBMC from 3 donor pools were in an intermediate range of infection (gray) at the highest VVC concentration is contrasted with inhibition 1.3–6.4 nM. of S virus (black) on PBMC from different donor pools. Different relative Modeling the data for S from the TZM-bl cell assay yielded a amounts of the CCR5-A and CCR5-B modulate the different levels of inhibition ϭ seen at the highest VVC concentrations in the different experiments. (C) value of w close to 0 (w 0.0095), similar to the one derived from Inhibition of TZM-bl cell infection by S ϩ 3FP (gray) and S (black). The model PBMC with the same virus (w ϭ 0.0040) (Table S2). The w value fits the data well when the VVC affinities of CCR5-A and CCR5-B are assumed for infection of TZM-bl cells by the resistant S ϩ 3FP virus was to differ by Ͼ37-fold, a value that was predetermined by measuring the IC50 7-fold higher (Ϸ0.06) than for S on those cells, but it was lower than values for the respective viruses on TZM-bl cells. The small residual infectivity for S ϩ 3FP on PBMC (0.34–1.8). A dissociation constant for VVC of S ϩ 3FP at the highest VVC concentrations results from an excess of CCR5-B binding to CCR5-A on TZM-bl cells could not be precisely esti- (high-affinity for VVC) over CCR5-A (lower affinity for VVC). This excess would mated, but it must exceed 25 nM; for CCR5-B on the same cells, it Ϸ ϩ be 20-fold if the efficiency of S 3FP viral entry were the same for free could be estimated to be 0.67 Ϯ 0.045 nM. Therefore, the corre- CCR5-B as for VVC-CCR5-A complexes. sponding ratio of Ͼ37 may fall within the range derived from the PBMC experiments (7.5–170). model is presented in SI Materials and Methods, and the different Discussion scenarios are depicted schematically in Fig. S5. The principles of the Resistance to small molecule CCR5 inhibitors, exemplified by modeling results are shown in Fig. S6. MVC and VVC, is now well documented in vitro, and has arisen in We used nonlinear regression to fit the general function of the vivo (4, 6–9, 11). Cross-resistance within the class is usually ob- model (15) to experimental PBMC and TZM-bl data (Fig. 5). In served (5, 6, 14), but sometimes not (4). Contrary to initial this analysis, we made no assumptions about the relative propor- assumptions, the principal resistance pathway does not involve tions of the 2 CCR5 forms, or their absolute affinities for VVC, switching to CXCR4; the virus instead acquires the ability to

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811713106 Anastassopoulou et al. Downloaded by guest on September 30, 2021 recognize the inhibitor-CCR5 complex while retaining use of the between Env and the inhibitor-CCR5 complex by changing the free coreceptor (4, 5). Entry/replication of some resistant viruses normal requirements for both V3 and the bridging sheet to contact can increase when an inhibitor is present, because they recognize different regions of CCR5 (ECL2 and the Nt). In principle, the FP the inhibitor-CCR5 complex more efficiently than free CCR5, substitutions may act analogously, via allosteric effects on Env and/or because more inhibitor-CCR5 complexes are available on conformations that functionally mimic the consequences of the V3 the cell (5, 11). The resistant viruses replicate efficiently, and their changes. An alternative hypothesis is that the FP substitutions lower phenotype is stable, at least in PBMC cultures, exemplified by the the threshold for fusion triggering, such that bridging-sheet inter- lack of reversion of both the CC101.19 (13) and the D1/85.16 isolate actions with the CCR5 Nt now suffice to drive the Env refolding (Fig. S1) after prolonged culture in the absence of AD101 and that mediates fusion. Although structural information on the native VVC, respectively. Therefore, the altered Env configuration en- Env trimer would clarify intersubunit effects, there is now consid- abling D1/85.16 to use the VVC-CCR5 complex must also recog- erable information about how gp41 sequence changes affect gp120 nize free CCR5 approximately as efficiently as Env from the structure and function. The first study of how Env responds to a parental CC1/85 isolate. The same applies to the AD101-resistant selection pressure, a serum from a HIV-1-infected individual, isolate CC101.19 and clones (5, 6). involved a point substitution in gp41 (A582T) that created a How the Env complex adjusts to use the inhibitor-CCR5 complex IIIB-variant resistant to neutralizing (NAbs) that spe- is under investigation. The generally accepted model of gp120- cifically target CD4-binding site-associated gp120 (19, 20). CCR5 interactions involves the crown of the V3 region contacting Other influences of sequence changes in the gp41 ectodomain and the second extracellular loop (ECL2), whereas the bridging sheet cytoplasmic tail on neutralization by gp120 ligands are now known and more conserved residues in the V3 stem bind the tyrosine- (21–23). The VVC- and AD101-resistant D1/85.16 and CC101.19 sulfated amino-terminal domain [N terminus (Nt)] (1, 10, 16). The isolates are somewhat more NAb sensitive than their parental virus, available evidence suggests that resistant viruses become less de- CC1/85, further suggesting that the FP and V3 changes affect the pendent on, or even independent of, the V3-ECL2 interaction (8, overall geometry of the Env complex. The greatest difference in 17). The genetic pathway typically involves accumulating multiple sensitivity between D1/85.16 and CC1/85, Ϸ58-fold, was to NAb sequence changes in V3 that, presumably, impede its binding to 2G12 against a conformational comprising gp120 N-linked ECL2 (4, 6–9, 11). However, we described a VVC-resistant clone glycans (24). How this difference arises is yet another unknown that with no V3 sequence changes (14); its phenotype was comparable will probably require Env trimer structural information. Alterations with the AD101-resistant (and VVC-cross resistant) viruses derived in gp41, including the cytoplasmic tail, can lead to CD4 indepen- from the same parental isolate under similar experimental condi- dence and reduced CCR5 dependence (25), and secondary resis- tions (6, 12). Thus, there is a second genetic pathway to the same tance substitutions affecting small molecule inhibitors of gp120- MEDICAL SCIENCES phenotype. CD4 binding were also mapped to gp41 (26). Here, we show that this alternative pathway not only does not The manifestation of resistance to CCR5 inhibitors is assay- involve V3, but that the critical changes do not even lie within dependent; the relative efficiency with which the resistant virus uses gp120; instead, they are located within the FP of gp41. This finding the inhibitor-bound and inhibitor-free forms of CCR5, the viral was unexpected. The 3 FP changes are collectively compatible with inoculum size, and the CCR5 surface density are all relevant fusion; the resistant viruses are replication-competent, as is the variables (4, 5, 7, 27). In PBMC, resistance usually appears as a corresponding, inhibitor-sensitive JR-FL mutant. However, no rightwards shift in the inhibitor dose-response curve (IC50 in- naturally occurring viruses in the HIV Sequence Database contain crease), whereas in CCR5-expressing cell lines such as TZM-bl all 3 changes, although some subtype C strains have 2 there is a plateau effect, and resistance is defined by MPI values (G516VϩM518V or M518VϩF519I). Of note is that one of the FP Ͻ100% (4, 5, 7). In general, the engineered FP mutants behaved changes, G516V, was recently shown to confer CXCR4 usage on a similarly to the naturally resistant virus, but with some informative clonal R5 virus (18). Perhaps the coreceptor switching of that virus exceptions. Thus, replication of the S/R, S ϩ 3FP, and R/S ϩ 3FP and the altered CCR5 interactions of the VVC-resistant CC1/85 viruses in PBMC was partially inhibited (40–60%) by low VVC variants involve similar mechanisms. However, the 3 FP changes concentrations (Ϸ0.3–2 nM), but enhanced by higher concentra- combined did not enable CC1/85 variants to use CXCR4 in PBMC, tions (up to 5 ␮M), and there was considerable variation in the and CXCR4 usage was not responsible for VVC resistance in extent of resistance among donor pools (Figs. 1, 3, and 5 A and B). TZM-bl cells (Fig. S2). We are now investigating whether any of the Replication of the engineered viruses in TZM-bl cells was hyper- 3 FP changes individually (or in pairs) affect VVC sensitivity, fusion sensitive to low VVC concentrations, compared with the parental efficiency, and coreceptor usage. However, the effect of the FP and naturally resistant clones, but there was a small yet consistent changes is context-dependent. Thus, a JR-FL variant engineered to persistent fraction of viruses replicating in the presence of very high have the same FP sequence as resistant D1/85.16-derived variants VVC concentrations (Fig. 4). The manifestation of resistance of remained VVC-sensitive (Fig. S3); likewise, the V3 changes that some CXCR4-using viruses to the small molecule inhibitor confer resistance on CC1/85 have no such effect on JR-FL. AMD3100 has also been shown to involve plateau heights that vary Although the gp120 subunit is not responsible for CCR5 inhibitor with the cell type and among PBMC from different donors (28). resistance, gp120 sequence changes may influence the phenotype of The explanation may be similar to what we propose here for CCR5; D1/85.16 and some mutants derived from it. We are assessing thus, the derivation of analogous models could be informative. whether compensatory changes elsewhere in Env influence the Our model of CCR5-inhibitor resistance explains the complex stability and fitness of the FP-engineered viruses; they may subtly curve shapes arising for the VVC-resistant clones in PBMC and alter how CCR5 is recognized, particularly at high inhibitor con- TZM-bl cells. Its key feature is the existence of 2 CCR5 forms, centrations (compare S ϩ 3FP and R/S ϩ 3FP in Figs. 3 and 4; see CCR5-A and CCR5-B, which bind VVC and related inhibitors with Table 1). low and high affinity (in reality, there may be Ͼ2 forms; see SI How FP changes alter the gp120-CCR5 interaction remains to be Materials and Methods and Fig. S5). The model also assumes that understood, but it seems reasonable to assume they act at a wild-type and resistant HIV-1 variants differentially use free and distance; there are no grounds to believe that the FP contacts the inhibitor-bound conformations of these CCR5 forms. By varying coreceptor before or during fusion. When unligated by an inhibitor, the proportions of CCR5-A and CCR5-B and their affinities for CCR5 provides a stimulus in a chain of conformational changes in VVC, we can derive infection-inhibition curves that mimic data Env that culminate in membrane fusion. This stimulus is abrogated generated in both PBMC and TZM-bl cells. The similarities be- for wild-type Env, but not the resistant variant if an inhibitor is tween the experimental results and the model curves support the bound. The V3 sequence changes may facilitate an interaction central hypothesis that free and VVC-ligated forms of different

Anastassopoulou et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 30, 2021 CCR5 subpopulations, respectively, can mediate entry of the re- the binding site for the small molecule inhibitors in the transmem- sistant S ϩ 3FP variant, whereas the wild-type S virus can use both brane helices. Overall, we hope that our experimental results, and free CCR5 forms to various degrees. Thus, the model explains the the model they have helped generate, will drive new lines of distinct inhibition profiles of the 2 viruses on the 2 cell types. research into how HIV-1 uses its coreceptors under different How reasonable are the model and its underlying assumptions? conditions, and into the general biology of GPCRs. -reactivity profiles show that multiple CCR5 conforma- Materials and Methods tions are present in proportions that vary between cell types (16). These different conformations may arise because CCR5 can be Viruses and Inhibitors. The study viruses are listed in Table S1 (5, 6, 14). Infectious stocks were prepared by transient transfection of 293T cells with pNL4-3/env coupled intracellularly to G proteins, which affects the shape of the proviral plasmids by using Lipofectamine 2000 (Invitrogen), and titrated before ligand-binding sites in the extracellular loops and Nt (29). Also, the testing. Full-length env genes were sequenced as described (12, 14), and aligned CCR5 Nt is tyrosine-sulfated at 3 positions posttranslation, 2 of with MacVector 10.0.2. VVC (SCH-D, SCH-417690), and AD101 (SCH-350581) (12) which (Tyr-10 and Tyr-14) are required for recognition by wild-type were provided by Julie Strizki, AMD3100 by Gary Bridger. HIV-1 (30). Perhaps overexpressing CCR5 in engineered cell lines like TZM-bl and U87.CD4.CCR5 saturates the tyrosine sulfotrans- Construction of Chimeric NL4-3/env Proviruses. The env genes with interchanged ferases, creating partially sulfated CCR5 proteins that are antigeni- gp120/gp41 subunits from viruses S and R were constructed by using the unique MfeI sites, sequenced, then subcloned into pNL4-3 to produce chimeric infectious cally and functionally distinct. The Tyr-sulfation requirements of viruses (6). Site-directed mutagenesis was performed with QuikChange II (Strat- the various resistant isolates remain to be investigated. Overall, the agene), using pBluescript KS(ϩ) plasmids containing EcoRI/XhoI fragments that postulates that different forms of CCR5 exist in different propor- were then subcloned into pNL4-3. tions on different cell types and are recognized with different efficiencies by wild-type and resistant HIV-1 variants seem plau- Infection-Inhibition Assays. Virus sensitivity to VVC or AD101 was assessed by sible. Quite what forms correspond to our theoretical CCR5-A and using PBMC from 2 or 3 random blood donors, as described (6, 14, 27). TZM-bl cells ϫ 4 CCR5-B remains to be investigated experimentally; based on the were seeded at 1 10 per well in 96-well plates and allowed to adhere overnight; inhibitors were added for 1h before virus infection. After culture for curve shapes in Figs. 4 and 5, we argue that the CCR5-B, which has 48h, the supernatant was removed, cells lysed, and luciferase expression quan- a high affinity for VVC and related inhibitors, will be overexpressed tified as RLU by using the Bright-Glo Luciferase Assay System (Promega). Back- relative to CCR5-A in engineered cells like TZM-bl and, by ground values from mock-infected cells, which were also cultured with each VVC extrapolation, U87.CD4.CCR5. Also, because CCR5-B may be or AD101 dose to control for cell viability, were subtracted. A 4-parameter used less efficiently by wild-type HIV-1 in those cells, it could sigmoidal function was fitted to the inhibition data by nonlinear-regression (Prism, Graphpad). In testing theoretical models, we fit different functions as perhaps be a variant that is under-sulfated on the Nt tyrosines. The described (SI Materials and Methods). HIV-1 replication (p24 production) in relative usage of the CCR5 Nt and ECL2 does appear to alter U87.CD4.CCR5 and U87.CD4.CXCR4 cells was measured as described (6, 12). between wild-type and resistant HIV-1 variants, the latter becom- For modeling CCR5 inhibitor resistance, see SI Materials and Methods. ing more dependent on the Nt (8). However, why under-sulfation of the Nt would increase the affinity of CCR5 for small molecule ACKNOWLEDGMENTS. We thank Antonia Thomas, Nicole Labutong, and Sam- inhibitors, as this interpretation of our model implies, remains to be son Jacob for technical support; Min Lu, Reem Berro, Rogier Sanders, and Antu Dey for helpful discussions; and past contributions by Andre Marozsan, Pavel understood. Perhaps intermolecular interactions involving CCR5 Pugach, Julie Strizki, and Shawn Kuhmann to work on VVC-resistant viruses. This and/or other cellular proteins involve the Nt and indirectly modify work was supported by National Institutes of Health Grant R01 AI41420.

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