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Sensitivity of viruses with the wild-type HIV-1AD8 Env or mutant Envs to antibody neutralization. (A) Recombinant single-round luciferase-expressing viruses with the wild-type HIV-1AD8 Env or mutant Envs were incubated with Cf2Th-CD4/CCR5 cells for 2 days. For comparison, the infectivity of recombinant luciferase-expressing virus containing the HIV-1JR-FL J9 mutant Env is shown. The infectivity is expressed as the ratio of relative luciferase units (RLU) of infected cells normalized to the reverse transcriptase activity (cpm). (B) The recombinant, luciferase-expressing viruses with the indicated HIV-1AD8 Env variants (or the HIV-1JR-FL J9 mutant Env) were incubated with antibody prior to addition of the virus-antibody mixture to Cf2Th-CD4/CCR5 cells. Viral infection is measured as a percentage of the luciferase activity observed in the absence of antibody. Shown are means and standard deviations from duplicate samples in a typical experiment.
Although some primary human immunodeficiency viruses can utilize low levels of CCR5 to enter cells (Sterjovski et al., 2010), there are no reports of coreceptor-independent HIV-1 in the literature. In this work, by extensive passaging of HIV-1NL4.3(JR-FL), a virus that can efficiently use modest levels of CCR5 for entry into host cells, we obtained virus variants that could detectably replicate in cells with less than ~1300 CCR5 molecules on the surface. Additional passaging of the adapted virus failed to generate viruses capable of replicating on cells with lower or no CCR5 expression. This outcome contrasts with the multiple successes in generating CD4-independent HIV-1 variants (Kolchinsky et al., 1999; Edwards et al., 2001; Dumonceaux et al., 1998; Zhang et al., 2002). The stringent requirement for CCR5, CXCR4 or related chemokine receptors is consistent with the notion that these coreceptors likely served as the primary receptor for primordial lentiviruses (Willett et al., 1998; Poeschla and Looney, 1998; Dealwis et al., 1998). In this scenario, viruses evolved to bind CD4 as a means of forming and exposing the coreceptor-binding site. Therefore, in State 1, the highly conserved coreceptor-binding site on gp120 is unavailable to host antibodies. When Env binding to target cell CD4 exposes the coreceptor-binding site, steric factors limit the access of host antibodies to this element (Labrijn et al., 2003). Thus, the use of two receptors provides significant advantages for lentiviruses that establish persistent infections in the host.
The mechanistic contributions of CCR5 binding to the HIV-1 entry process are incompletely understood. Inferences about the structural influence of CCR5 on Env have centered on the physical interactions of CCR5 with gp120. Crystal structures of gp120 cores bound to sCD4 and 17b Fab revealed a highly conserved gp120 surface element composed of the bridging sheet and V3 base and tip that is thought to interact with the N-terminus and second extracellular loop of CCR5 (Kwong et al., 1998; Rizzuto et al., 1998; Wyatt et al., 1998). The interaction of this gp120 element with CCR5 serves to bring the CD4-bound Env trimer close to the host cell membrane. In addition to this attachment function, CCR5 binding serves to orient the Env spike. Mixing studies of HIV-1 Env mutants have suggested that at least two gp120 subunits in the Env trimer must engage CCR5 to allow virus entry (Yang et al., 2006; Salzwedel et al., 2009). Analogous to the events that occur after CD4 binding, CCR5 binding has been proposed to trigger additional conformational changes in Env that drive the formation of the gp41 six-helix bundle. How gp120-CCR5 binding leads to conformational changes in gp41 is still a matter of speculation. Presumably, CCR5-induced triggering of Env function involves a release of restraints that allows the gp41 to refold into a highly stable six-helix bundle.
Insights into the interaction of HIV-1 Env with CCR5 have been obtained by generating HIV-1 variants that escape from inhibitors of CCR5 binding. In these cases, the virus does not switch tropism, but rather adapts to use the inhibitor-bound CCR5 as a coreceptor. Adaptation of HIV-1D1/85.16 to replicate in the presence of maraviroc involved changes in the gp120 V3 region that influence CCR5 binding, as well as changes in the gp41 fusion peptide (G516V, M518V and F519I) that enhance viral membrane fusion with the target cell membrane (Anastassopoulou et al., 2012). HIV-1JR-CSF and HIV-111-121 adapted readily to escape the inhibitory effects of the 2D7 antibody against the second extracellular loop of CCR5, with the resistant viruses exhibiting a change (D167N) in the gp120 V2 region (Aarons et al., 2001). These studies indicate that HIV-1 can adapt to an inhibitor-bound CCR5 by making changes within as well as outside the V3 region.
HIV-1 has also been adapted to replicate in cells that express low levels of CCR5. HIV-1YU2 was adapted to replicate in SupT1 cells expressing a low level of CCR5 from a transfected gene (Garg et al., 2016). The adapted virus continued to use CCR5 as a coreceptor, and exhibited changes in V3 (N302Y), which made a large contribution to the phenotype, and in V2 (E172K), which added to the N302Y phenotype. The N302Y and N302Y/E172K viruses were ~5-fold more resistant to maraviroc than the parental HIV-1YU2. HIV-1KP-5 adapted to replicate in PM1 cells expressing low CCR5 levels without a switch in coreceptors (Yoshimura et al., 2014). Changes in V3 (D321E) as well as outside of V3 (D141N in V1 and I463T in V5) were associated with the adaptation. The adapted viruses were ~10-fold more resistant to maraviroc than the parental HIV-1KP-5, and exhibited increased sensitivity to a CD4-induced antibody and a slight increase in sensitivity to b12, a CD4-binding site antibody. The adapted virus was as sensitive to neutralization by a V3-directed antibody as the parental virus. These two studies suggest that one adaptive approach to low CCR5 levels is to increase CCR5-binding affinity through V3 changes, with auxiliary changes in the gp120 V1/V2 region. The gp120 V1/V2 region has been suggested to mask the coreceptor-binding site in primary Envs in State 1 (Wyatt et al., 1993; Kolchinsky et al., 2001b; Binley et al., 1998). Changes in the V1/V2 region can lead to increased Env transitions to State 2 (Herschhorn et al., 2016).
Our study generated HIV-1JR-FL variants that replicated in Cf2Th-CD4/CCR5 cells with ~1300 CCR5 molecules per cell. As seen in the Garg et al. and Yoshimura et al. studies, the adapted virus did not switch coreceptors. In contrast to the outcomes of these earlier studies, the adapted HIV-1JR-FL exhibited no V3 changes and did not exhibit altered sensitivity to maraviroc; we found no evidence for an increase in the affinity of the adapted virus gp120 for CCR5. In fact, Env changes in gp41 (R564H or E662K) were essential for the adapted virus’ ability to replicate on cells with low CCR5 levels. The adapted viruses exhibited an increased sensitivity to neutralization by F105 (a CD4-binding site antibody), 17b (a CD4-induced antibody) and 19b (a V3 antibody); thus, the adapted viruses are more prone to expose normally cryptic epitopes near the receptor-binding regions. Compared with the parental HIV-1JR-FL, the adapted viruses were slightly more sensitive to neutralization by sCD4 and small-molecule CD4-mimetic compounds; consistent with this observation, the activation of infection of CD4-negative CCR5-positive cells by sCD4 and CD4-mimetic compounds was slightly more efficient for the adapted viruses than for wild-type HIV-1JR-FL. The adapted viruses did not appreciably differ from the parental virus in T20 sensitivity.
Mechanistic insights into the HIV-1JR-FL adaptation to low-CCR5 utilization were obtained by assessing the contribution of Env changes, both individually and in combination, to this phenotype. The molecularly cloned J3 virus with three Env changes (S115N, R564H and E662K) replicated in low-CCR5 cells, demonstrating the sufficiency of these three changes to account for this phenotype. Two combinations, S115N + E662K and R564H + E662K, also allowed HIV-1JR-FL to replicate in cells with low CCR5 expression. The E662K change alone was insufficient to allow virus replication in low-CCR5 cells, indicating the importance of the S115N and R564H changes to the phenotype. Of interest, the S115N + E662K and R564H + E662K Envs differed in a number of ways, suggesting that distinct, parallel pathways of replication in low-CCR5 cells can be utilized by HIV-1JR-FL. In the first pathway, exemplified by the S115N + E662K virus, the activation barriers separating State 1 from downstream Env conformations are apparently lowered. These Envs are more prone to opening receptor-binding gp120 Env regions, as evidenced by the increased susceptibility to sCD4, CD4-mimetic compounds and antibodies. An expected consequence of these Env changes is an increase in the amount of the CD4-bound Env intermediate (State 3) present in the virus-target cell synapse. Thus, an increased concentration of coreceptor-binding-competent Envs is available to utilize low CCR5 levels efficiently. In the second pathway, exemplified by the R564H + E662K virus, the Env does not exhibit phenotypes associated with a more “open” conformation. Rather than increasing the overall amount of State 3 Env potentially available for CCR5 binding or increasing Env-CCR5 binding affinity, these gp41 changes apparently affect the responsiveness of Env to CCR5 binding.
What might account for the different strategies used by HIV-1 to adapt to low CCR5 levels in our study and in previous studies, where Envs with increased affinity for CCR5 were selected (Garg et al., 2016; Yoshimura et al., 2014)? One possibility is the nature of the starting virus. The HIV-1YU2 used by Garg and colleagues is more readily triggered by CD4 and CD4-mimetic compounds than HIV-1JR-FL (Madani et al., 2017), so the latter virus’ infectivity in low-CCR5 cells could benefit more from an increase in CD4 responsiveness. Another explanation relates to the cells used for virus adaptation. The Cf2Th-CD4/CCR5 cells used in our experiments are adherent and thus allow significant cell-cell transmission to occur as a result of cellular contact. Both Garg et al. and Yoshimura et al. used lymphocyte target cells grown in suspension, where cell-free virus transmission may be the predominant mode of infection. Optimizing Env binding to scarce CCR5 may be more important in increasing the efficiency of encounters between cell-free virions and target cells. In adherent cells, the budding virions have a greater opportunity to interact with target cells regardless of CCR5 levels; the rate-limiting factor in this case may be the efficiency with which each Env encounter with CCR5 actually triggers functional membrane fusion events leading to infection.
The gp120 and gp41 changes responsible for HIV-1JR-FL adaptation to low-CCR5 cells potentially involve dynamic regions of Env involved in receptor-induced conformational changes. Structural explanations for the effects of these changes are complicated by the possibility that the different observed Env changes may operate at distinct stages of the HIV-1 entry process. Given the considerable gaps in our structural information on the different stages of HIV-1 entry, our interpretation of the Env determinants of low-CCR5 utilization must be considered tentative. With that caveat, we examined the location of these determinants in available HIV-1 Env structures. The S115N change contributed to the increased sensitivity of the adapted viruses to inhibition by antibodies directed against the receptor-binding sites of gp120. Ser 115 is located at the carboxy-terminal end of the α1 helix of gp120 (Kwong et al., 1998); based on current Env structures, any consequences of the S115N change on neutralization sensitivity would be mediated by indirect mechanisms. Although Ser 164 is predicted to make interprotomeric gp120 contacts in structures of the HIV-1BG505 sgp140 SOSIP.664 Env trimer (Julien et al., 2013; Lyumkis et al, 2013; Pancera et al., 2014), we found that the S164N change is not essential for the ability of the adapted HIV-1JR-FL to use low levels of CCR5. Another study failed to observe the viral phenotypes predicted by the sgp140 SOSIP.664 Env trimer structure for changes in this interprotomer region (Madani et al., 2016), adding to uncertainty about the relationship of this structure to functional Env conformations (Alsahafi et al., 2015; Kesavardhana and Varadarajan, 2014; Pacheco et al., 2017). The gp41 residue Arg 564 faces the amino-terminal end of the gp120 α1 helix in the structure of the HIV-1JR-FL Env trimer bound to the PGT151 neutralizing antibody (Lee et al., 2016). Therefore, alteration of Arg 564 could potentially influence gp120-gp41 interaction and the triggering of Env conformational changes by CCR5 binding. Because the membrane-proximal region of gp41 is either missing or disordered in current Env structures (Julien et al., 2013; Lyumkis et al., 2013; Pancera et al., 2014), the consequences of the E662K change on HIV-1 Env structure are not interpretable. A full understanding of the mechanistic basis for the low-CCR5 replication phenotype awaits additional structural information on the functional Env conformations.
Natural variation in the ability of HIV-1 to use different levels of target cell CCR5 has been suggested to influence viral tropism and sensitivity to inhibitors (Etemad et al., 2009; Gorry et al., 2002; Karlsson et al., 2004; Koning et al., 2003; Pfaff et al., 2010; Repits et al, 2005; Taylor et al., 2008). Understanding the mechanisms whereby HIV-1 modulates CCR5 requirements will assist intervention efforts.
Adaptation of HIV-1NL4.3(JR-FL) to replicate on CD4-positive cells expressing low levels of CCR5 has revealed residues in gp120 and gp41 responsible for this phenotype. The Env changes do not apparently increase gp120 affinity for CCR5, but promote two parallel pathways of adaptation: 1) increased propensity of Env to assume a more open coreceptor binding-competent conformation; and 2) increased Env responsiveness to CCR5 binding.
Materials and Methods
Generation of cell lines
293T and Cf2Th cells were obtained from the American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium with 10% tetracycline-free fetal bovine serum (DMEM-10, Clontech). Cf2Th cell lines stably expressing human CD4 and CCR5 (Cf2Th-CD4-R5) were grown in DMEM-10 in the presence of 400 μg/mL G418 and 200 μg/mL hygromycin (Invitrogen).
Cf2Th cell lines with regulatable CCR5 expression were generated in the following manner: Cf2Th cells were transfected with Tet-On® Advanced Vector (Clontech), passaged in DMEM-10 and 0.6 ug/mL puromycin, and single cells selected by limiting dilution in 96-well plates. Single-cell clones were expanded and tested for expression of the tTA protein after treatment with 0–1 ug/mL doxycyline by Western blotting of whole cell lysates with a monoclonal antibody against TetR (Clontech). tTA-expressing cells were transfected with pGL422-tetO-CCR5, passaged in the presence of 0.6 μg/mL G418, and selected for single cells. Cells were expanded and tested for doxycycline-regulated CCR5 expression via flow cytometry as follows: after incubation with R-Phycoerythrin-conjugated mouse anti-human CCR5 (PE-anti-CD195, BD Biosciences), ~1 × 106 cells were analyzed with a BD FACSCanto II flow cytometer (BD Biosciences). Clones with the least background and greatest dynamic range of induced CCR5 expression were then transfected with the pcDNA-CD4 plasmid expressing human CD4, passaged in the presence of 0.025 μg/mL zeocin, and cloned. Cells were tested for doxycycline regulation of CCR5 expression and stable expression of CD4 using flow cytometry with PE-anti-CD195 and fluorescein isothiocyanate-conjugated mouse anti-human CD4, clone RPA-T4 (FITC-anti-CD4, BD Biosciences), respectively.
Two clones that express high levels of CD4 and low or high ranges of cell-surface CCR5 expression upon doxycycline treatment, herein called R5-Low and R5-High, were selected for HIV-1 adaptation to low levels of CCR5 (Fig. 1A). Levels of CCR5 were measured by QuantiBritePE (Fig. 1B, BD Biosciences) and vary within a physiological range (Fig. 1C).
Generation of replication-competent HIV-1 and adaptation of virus to low levels of CCR5 in R5-Low cells
Replication-competent HIV-1 was generated by transfecting 2 million 293T cells with 20 μg of a plasmid containing the pNL4.3 provirus with the HIV-1JR-FL env (between the SalI and BamHI sites) using Effectene transfection reagent (Qiagen). The supernatant was harvested after two days and frozen in aliquots. Reverse transcriptase (RT) activity in the supernatant was measured using 3H-labelled nucleotide triphosphates for initial viral production assays and 32P-labelled nucleotide triphosphates for all viral adaptation assays, by a previously described method (Rho et al., 1981). To produce the virus in the Cf2Th cell line, 100,000 Cf2-T4R5 cells were infected with 3000 cpm (3H) of HIV-1NL4.3(JR-FL) virus made in 293T cells as described above. Cells were passaged every 3–4 days by lifting them from the plate using 5 mM EDTA in PBS and diluting them 1:5. Supernatants were collected at each passage to measure RT. Additional studies of virus replication in Cf2Th-CD4/CCR5 cells used 6600 cpm (32P) of virus from the time point with peak RT in the presence of 2 μg/mL polybrene.
For all subsequent rounds of adaptation, 25,500 cpm (32P) RT units of supernatant, collected at the time point with peak RT activity, was used to infect R5-High cells and then R5-Low cells at decreasing expression levels of CCR5 (i.e., lower concentrations of doxycycline). Serial passage of the virus was continued until three independent assays with triplicate samples containing cells expressing the lowest amount of CCR5 yielded no detectable RT activity.
Quantification of p24 levels in cell supernatants was performed using a p24 ELISA kit (Advanced BioScience Laboratories).
Sequencing env from genomic DNA of infected cells
Proviruses were sequenced from the genomic DNA of infected cells. Genomic DNA was isolated from cells frozen at peak RT using the GIAamp DNA blood minikit (Qiagen) followed by genome amplification using a published protocol (Gall et al., 2012). In summary, the HIV-1 genome was PCR-amplified by primers SK145 and OFM19 using the PrimeSTAR GXL DNA polymerase (Clontech). The viral env was then amplified from the PCR reaction using the Pan-HIV-1_4 forward and reverse primers. PCR fragments were purified using the QIAquick PCR Purification kit (Qiagen) and sequenced by the Sanger method.
Env cloning and mutagenesis
Changes in the HIV-1JR-FL env that arose during passage of the virus were introduced into the pNL4.3(JR-FL) proviral DNA or the Env expressor plasmid pSVIIIenv(JR-FL) by site-directed mutagenesis using the PfuUltra II Hotstart PCR Master Mix (Agilent). Mutagenesis was confirmed by automated DNA sequencing.
Gp120-CCR5 binding assay
293T cells were transfected with pSVIIIenv (JR-FL) plasmids using Qiagen Effectene and supernatants were harvested after 2 days. Approximately 0.5 ml of supernatant was incubated with 30 ng/mL sCD4 with or without 1uM Maraviroc for 30 minutes at 37 degrees. Two T-175 flasks of confluent Cf2Th-CCR5 cells were lifted using 5 mM EDTA, spun down and resuspended in 20 mL DMEM-10. One ml of the Cf2Th-CCR5 cell suspension was incubated with the gp120-containing supernatants for 1 hour at 37°C. Cells were washed twice with DMEM-10 and lysed in 500 μL 10 mM Tris pH 7.5, 0.5 M NaCl, 5% Igepal. Cell lysates were spun at 14,000 rpm for 30 minutes at 4°C and the supernatants were incubated on a rotating platform overnight with 4 μL serum from HIV-1-infected individuals and 80 μL 1:1 resuspended Protein A-Sepharose beads. Beads were washed twice with 10 mM Tris pH 7.5, 0.5 M NaCl, 5% Igepal and then washed once with 10 mM Tris pH 7.5, 1.5 mM NaCl. Proteins were eluted by boiling beads in 30 uL 4x NuPAGE LDS Sample Buffer plus ß-mercaptoethanol at 100°C. Samples were run on SDS-polyacrylamide gels, transferred to Hybond ECL nitrocellulose membranes (GE Healthcare Life Sciences) using a Trans-blot semi-dry transfer cell (Bio-rad), and blotted with 1:200 anti-gp120 antibody-HRP (Abcam). Blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and imaged on the GelDoc XR+ System (Bio-rad).
Viral replication assay
To test the replication capacity of NL4.3 viruses expressing wild-type HIV-1JR-FL Env or the adapted virus Envs, we introduced mutations into the HIV-1NL4.3(JR-FL) proviral DNA and transfected them into target cells. Site-directed mutagenesis was performed using the QuikChange II XL Site-Directed Mutagenesis protocol (Agilent) and the PfuUltra Hotstart DNA Polymerase (Agilent). Approximately 4×104 Cf2Th-CD4/CCR5 or R5-Low cells preincubated with 50 or 100 ng/mL doxycycline were transfected with 100 μg proviral DNA and passaged for 3 weeks. Supernatants of each cell sample were collected regularly throughout the culture period and evaluated for RT activity.
Generation and purification of single-round recombinant viruses expressing luciferase
Single-round recombinant HIV-1 expressing firefly luciferase was generated by transfecting 293T cells with pSVIIIenv(JR-FL) constructs, pLuciferase, and the pCMVΔP1ΔenvpA HIV-1 Gag-Pol packaging construct at a ratio of 1:2:1 micrograms of DNA using Effectene transfection reagent. The virus-containing supernatant was harvested after 3 days and used in all assays without freezing.
To test viral infectivity, virus-containing supernatants were incubated with target cells in 96-well plates for three days in a 37°C CO2 incubator. The activation of HIV-1 infection of CD4-negative Cf2Th-CCR5 cells by sCD4 or CD4-mimetic compounds was assessed as described (Madani et al., 2016). Cells were lysed with 30 μL passive lysis buffer (Promega) and luciferase activity was measured with a Mithras LB 940 luminometer (Berthold Technologies).
To test levels of Env expression on virions, viral particles were pelleted by ultracentrifugation through a 20-percent sucrose cushion at 4°C for 2 hours in a Beckman SW55 rotor at 30,000 rpm. Pellets were lysed in 4x NuPAGE LDS Sample Buffer plus ß-mercaptoethanol and frozen. Samples were run on SDS-polyacrylamide gels, transferred to Hybond ECL nitrocellulose membranes (GE Healthcare Life Sciences) using a Trans-blot semi-dry transfer cell (Bio-rad), and blotted with a 1:200 dilution of an anti-HIV-1 gp120 antibody-HRP. Blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and imaged on the GelDoc XR+ System (Bio-rad). Membranes were stripped with Western Blot Stripping Buffer (Thermo Fisher) and then blotted with a 1:2000 dilution of anti-HIV-1 p55 + p24 + p17 antibody (Abcam) and secondary HRP-Protein A/G, redeveloped and imaged.
Cell fusion assays
To test the capacity of wild-type HIV-1JR-FL Env and the adapted virus Envs to initiate cell fusion (syncytium formation), we used an alpha complementation assay (Holland 2004). In this assay, COS-1 cells were plated in a 96-well plate and transfected the next day with 0.1 μg/well pSVIIIenv(JR-FL), 0.01 μg/well pTat, and 1.1 μg/well of a plasmid containing the N-terminal fragment of beta-galactosidase, using the Effectene protocol. Cf2Th-CD4/CCR5 cells were plated in a T-75 flask and transfected with 10 μg of a plasmid containing the C-terminal fragment of β-galactosidase using the Effectene protocol. After 2 days of incubation at 37°C, Cf2Th-CD4/CCR5 cells were lifted with 5 mM EDTA in PBS and diluted to 2×105 cells per mL DMEM-10. To each well of COS-1 cells preincubated with increasing concentrations of Maraviroc was added 100 μL Cf2Th-CD4/CCR5 cells. Cells were incubated at 37°C for 8 hours, washed in PBS, and lysed with 20 μL Galactostar Lysis Buffer (ThermoFisher). Plates were frozen and thawed three times at −20°C to improve cell lysis. Plates were then warmed to room temperature and incubated with 100 μL 1:100 Galactostar substrate in Reaction Buffer Diluent. β-galactosidase enzymatic activity was measured in the luminometer, reading 1 second per well.
Cell-to-cell HIV-1 transmission assay
293T cells producing single-round luciferase recombinant virus were lifted with 5 mM EDTA in PBS and added to cultured Cf2Th-CD4/CCR5 cells. Cocultures were passaged every 3–4 days. At passaging, the remaining cells were diluted in DMEM-10 on a 96-well plate and incubated overnight. At various time points, cells were lysed with 30 μL passive lysis buffer (Promega) and luciferase activity was measured with a Mithras LB 940 luminometer (Berthold Technologies).
Single-round luciferase-expressing recombinant viruses were incubated with antibodies for 1 hour at room temperature and then incubated with Cf2Th-CD4/CCR5 cells in a 96-well plate for 2 hours at 37°C. The virus and antibody mixtures were incubated with the cells in DMEM-10 for 3 days in a 37°C CO2 incubator. Cells were lysed with 30 μL passive lysis buffer (Promega) and luciferase activity was measured with a Mithras LB 940 luminometer (Berthold Technologies).
Data representation and statistical analysis
Microsoft Excel and Prism 6.0 (GraphPad Software, L1 Jolla CA) was used to graph and analyze numerical data. Statistical tests as reported in the figure legends were performed using Prism software.
- HIV-1 can adapt to CD4-positive cells expressing progressively lower levels of CCR5.
- The adapted virus’ Env changes are located outside of the gp120 CCR5-binding region.
- The gp120 of the adapted virus did not increase its affinity for CCR5.
- The Env changes enhance HIV-1 replication at all levels of CCR5 expression.
- The adapted virus Envs exhibit increased propensity to change conformation.
We thank Elizabeth Carpelan for manuscript preparation. We thank Vlad Novitsky for providing the protocol for proviral sequencing from genomic DNA. This study was supported by the National Institutes of Health (AI24982, GM56550 and AI100645) and the late William F. McCarty-Cooper. N.E. was supported by a predoctoral fellowship from the National Institutes of Health (AI112404).
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