Macrophage-tropic HIV-1 variants from brain demonstrate alterations in the way gp120 engages both CD4 and CCR5

Hamid Salimi; Michael Roche; Nicholas Webb; Lachlan R Gray; Kelechi Chikere; Jasminka Sterjovski; Anne Ellett; Steve L Wesselingh; Paul A Ramsland; Benhur Lee; Melissa J Churchill; Paul R Gorry

doi:10.1189/jlb.0612308

. 2013 Jan;93(1):113–126. doi: 10.1189/jlb.0612308

Macrophage-tropic HIV-1 variants from brain demonstrate alterations in the way gp120 engages both CD4 and CCR5

Hamid Salimi ^*,^†, Michael Roche ^*, Nicholas Webb ^‡, Lachlan R Gray ^*,^§, Kelechi Chikere ^‡, Jasminka Sterjovski ^*, Anne Ellett ^*, Steve L Wesselingh ^‖, Paul A Ramsland ^¶,^**,^††, Benhur Lee ^‡, Melissa J Churchill ^*,^†,^‡‡, Paul R Gorry ^*,^‡‡,^§§,¹

PMCID: PMC3525831 PMID: 23077246

Along with an enhanced interaction with CD4, highly M-tropic HIV-1 Envs have an altered mechanism of engagement with CCR5.

Keywords: Env, Affinofile, CNS, signature, phenotype

Abstract

BR-derived HIV-1 strains have an exceptional ability to enter macrophages via mechanisms involving their gp120 Env that remain incompletely understood. Here, we used cell-based affinity-profiling methods and mathematical modeling to generate quantitative VERSA metrics that simultaneously measure Env-CD4 and Env-CCR5 interactions. These metrics were analyzed to distinguish the phenotypes of M-tropic and non-M-tropic CCR5-using HIV-1 variants derived from autopsy BRs and LNs, respectively. We show that highly M-tropic Env variants derived from brain can be defined by two distinct and simultaneously occurring phenotypes. First, BR-derived Envs demonstrated an enhanced ability to interact with CD4 compared with LN-derived Envs, permitting entry into cells expressing scant levels of CD4. Second, BR-derived Envs displayed an altered mechanism of engagement between CD4-bound gp120 and CCR5 occurring in tandem. With the use of epitope mapping, mutagenesis, and structural studies, we show that this altered mechanism is characterized by increased exposure of CD4-induced epitopes in gp120 and by a more critical interaction between BR-derived Envs and the CCR5 N-terminus, which was associated with the predicted presence of additional atomic contacts formed at the gp120-CCR5 N-terminus interface. Our results suggest that BR-derived HIV-1 variants with highly efficient macrophage entry adopt conformations in gp120 that simultaneously alter the way in which the Env interacts with CD4 and CCR5.

Introduction

HIV-1 establishes infection in the CNS, causes HIV-1-associated neurological disorders in infected subjects, and presents a barrier to effectively treating HIV-1 infection [1, 2]. Microglia and perivascular macrophages are target cells for HIV-1 replication in the brain [1, 3]. They are less susceptible to the cytopathic effects of HIV-1 than activated CD4⁺ T cells [4, 5], so they may continue to shed virus for the duration of their normal lifespan. As most antiretroviral therapies have poor CNS penetration [6], the brain is a significant reservoir for viral persistence within macrophage-lineage cells.

The genetic evolution of HIV-1 within the CNS is distinct from that in lymphoid tissues and other organs [7–20]. Specific sequences within the viral Env, particularly the gp120 V3 region, have been associated with CNS infection [12, 13, 19, 21–26], although no conserved genetic signature sequence has been identified. The genetic compartmentalization of viral variants in the CNS, which has been shown recently to occur very early in HIV-1 infection [16], suggests that adaptive changes may occur in response to unique constraints of the CNS microenvironment, such as different target cell populations and immune-selection pressures.

The HIV-1 Env is organized into trimers on virions and consists of the gp120 surface and gp41 transmembrane subunits. HIV-1 entry into cells is initiated by a high-affinity interaction between gp120 and CD4 [27], which induces a conformational change in gp120 that exposes the binding site for a chemokine coreceptor, either CCR5 or CXCR4 [28, 29]. Current models of gp120 binding to coreceptor suggest that the gp120 V3-loop tip interacts principally with the coreceptor ECL2 region, whereas the V3-loop stem and the gp120 bridging sheet, which is formed among the C1, C2, and C4 domains of gp120 after CD4 binding, interact with the coreceptor N-terminus [30–33]. The V3-loop of gp120 is the primary determinant of coreceptor specificity [34, 35]. Whereas the coreceptor N-terminus and ECL2 region appear to be important for gp120-coreceptor binding, the ECL1 and ECL3 regions may also influence coreceptor function of CCR5 and CXCR4 [36–38]. The interaction of CD4-bound gp120 with coreceptor induces additional conformational changes in gp120, which leads to a structural rearrangement in gp41 that enables fusion and virus entry.

The tropism of HIV-1 for particular target cell populations in different tissue compartments is influenced by the coreceptor used by HIV-1 Env for virus entry [3, 39, 40]. Macrophage-tropic HIV-1 primarily uses CCR5 (R5) as a coreceptor [41–43], whereas T cell line tropic viruses use CXCR4 (X4) [44]. Dual-tropic viruses can use both coreceptors (R5X4) [45, 46]. Thus, the coreceptor specificity of primary HIV-1 isolates is frequently, but incorrectly, used to define cellular tropism. For example, R5 viruses are often collectively grouped as M-tropic viral strains [40]. However, several studies have demonstrated the presence of non-M-tropic R5 viruses, which were replication-competent in primary CD4⁺ T cells but could not productively infect MDMs [10, 47–51]. Thus, there is a notable distinction between HIV-1 tropism and coreceptor use [40, 52, 53]; whereas most M-tropic viruses use CCR5 for HIV-1 entry, not all R5 viruses are M-tropic [40, 53, 54].

In fact, recent studies have shown that M-tropism is relatively rare among R5 HIV-1 variants isolated from blood and lymphoid tissues but that most R5 viruses isolated from the CNS are highly M-tropic [10, 15, 49–51, 55–58]. However, M-tropic R5 variants can be detected in blood of some subjects with advanced infection [47, 48, 59]. The CNS, therefore, selects for viral variants that have characteristics favorable for efficient entry into macrophage-lineage cells or facilitates their evolution via a unique microenvironment that may involve reduced immune surveillance and low anti-HIV-1-neutralizing antibody concentrations.

The mechanisms underlying the efficient macrophage entry of BR-derived HIV-1 variants are incompletely understood. Several studies have shown that the gp120 Env glycoproteins of CNS-derived viruses have conformational alterations within the CD4-binding domain that increase the efficiency of the interaction between gp120 and CD4, thus permitting these variants to scavenge relatively low levels of CD4 expressed on the surface of macrophages and microglia [15, 49, 50, 55–58, 60–64]. Other studies have shown that certain BR-derived variants have Env glycoproteins that exhibit increased affinity for CCR5 [11]. Moreover, our recent studies have shown that efficient CCR5-mediated HIV-1 entry of certain blood-derived variants into macrophages involves an altered mechanism of Env-CCR5 engagement, characterized by reduced dependence of Env on the CCR5 N-terminus and increased dependence on the CCR5 ECL2 region [65, 66].

In the present study, we used an innovative and highly quantitative HIV-1 receptor affinity-profiling system and mathematical modeling combined with epitope mapping studies, mutagenesis, and structural modeling to more completely understand the virus–cell interactions underlying efficient macrophage entry by BR-derived HIV-1 Env variants. We show that highly M-tropic HIV-1 variants derived from the brain have alterations in the way their Env glycoproteins engage CD4 and CCR5, characterized by a highly efficient interaction between gp120 and CD4 occurring in tandem with a modified mechanism of engagement between CD4-bound gp120 and CCR5. Our findings more completely define the macrophage-tropic phenotype of HIV-1 variants derived from the brain.

MATERIALS AND METHODS

Cells

293T cells, JC53 cells [67], and NP2-CD4 cells [68] were cultured in DMEM supplemented with 10% (vol/vol) FCS and 100 μg penicillin and streptomycin/ml. The dually inducible 293-Affinofile cell line [69], in which expression of CD4 and CCR5 can be induced and regulated by the addition of minocycline or ponA, respectively, was maintained in DMEM supplemented with 10% (vol/vol) FCS, 100 μg penicillin and streptomycin/ml, 50 μg blasticidin/ml, and 200 μg G418/ml. PBMCs were purified from blood of healthy HIV-1-negative donors by density gradient centrifugation, stimulated with 10 μg PHA (Sigma, St. Louis, MO, USA)/ml for 3 days, and cultured in RPMI-1640 medium, supplemented with 10% (vol/vol) FCS, 100 μg penicillin and streptomycin/ml, and 20 U IL-2 (Roche, Basel, Switzerland)/ml. Monocytes were purified from PBMC by plastic adherence and allowed to differentiate into MDM by culturing for 5 days in IMEM, supplemented with 10% (vol/vol) pooled AB+ human sera, 100 μg penicillin and streptomycin/ml, and 12.5 ng M-CSF/ml.

HIV-1 Env plasmids

This study characterized six Envs cloned from HIV-1 viruses isolated from autopsy brain of three subjects who died from AIDS (subjects Macs2, Macs3, and UK1), as well as six Envs cloned from HIV-1 viruses isolated from autopsy LN of two of the same subjects (subjects Macs2 and Macs3; LN-derived Envs from UK1 were not available). The subjects, including their clinical and neuropathological details, have been described previously [10, 11]. The Env clones used were Macs2-BR-2, Macs2-BR-8, Macs3-BR-1, Macs3-BR-8, UK1-BR-2, UK1-BR-10, Macs2-LN-2, Macs2-LN-3, Macs2-LN-5, Macs2-LN-6, Macs3-LN-8, and Macs3-LN-9. JR-CSF and YU2 Envs were included as controls. All of the Envs are cloned into the pSVIII-Env mammalian expression vector [70] and have been described previously [25, 26].

Production and quantitation of Env-pseudotyped luciferase reporter viruses

Env-pseudotyped, luciferase reporter viruses were produced by transfection of 293T cells with pCMVΔP1ΔenvpA, pHIV-1Luc, and pSVIII-Env plasmids using Lipofectamine 2000 (Invitrogen, Life Technologies, Grand Island, NY, USA) at a ratio of 1:3:1, as described previously [71–73]. Supernatants were harvested 48 h later, filtered through 0.45 μm filters, and stored at −80°C. The TCID₅₀ of virus stocks was determined by titration in JC53 cells [67], as described previously [25, 74, 75].

Single-round HIV-1 entry assays

For single-round entry assays using JC53 cells, 293-Affinofile cell populations, or NP2-CD4 cells expressing WT or mutant CCR5 coreceptors, 2 × 10⁴ cells cultured in 96-well plates, were inoculated with 200 TCID₅₀ of Env-pseudotyped luciferase reporter virus (equating to a MOI of 0.01) in a volume of 100 μl for 12 h at 37°C. The cells were washed twice with culture medium to remove residual inoculum and incubated for a further 60 h at 37°C. For single-round entry assays using MDM, cell monolayers, which were ∼90% confluent in 48-well tissue-culture plates, were inoculated with 1500 TCID₅₀ of Env-pseudotyped luciferase reporter virus in a volume of 300 μl for 12 h at 37°C. The MDMs were then washed twice with culture medium to remove residual inoculum and incubated for a further 96 h at 37°C. For single-round entry assays in PBMC, 2 × 10⁵ cells were inoculated with 2000 TCID₅₀ of Env-pseudotyped luciferase reporter virus in a volume of 200 μl for 12 h at 37°C. The cells were then washed twice with culture medium to remove residual inoculum and incubated a further 72 h at 37°C. In all cell types, the level of HIV-1 entry was measured by luciferase activity in cell lysates (Promega, Madison, WI, USA), according to the manufacturer's protocol. Luminescence was measured using a FLUOStar Optima microplate reader (BMG Labtech GmbH, Germany). Negative controls included mock-infected cells that were incubated with culture medium instead of virus, and cells inoculated with luciferase reporter virus pseudotyped with the nonfunctional ΔKS Env [76].

Affinofile cell assays and quantitative vector analysis

Infection of 293-Affinofile cells with Env-pseudotyped luciferase reporter viruses was performed as described previously [69]. Briefly, 48 populations of cells expressing different combinations of CD4 and CCR5 levels were generated by inducing the cells with twofold serial dilutions of minocycline (0.156–5.0 ng/ml, resulting in six induction levels of CD4 increasing linearly from 750 to 95,000 CD4 molecules/cell) and ponA (0.0156–2.0 μM, resulting in eight induction levels of CCR5 increasing linearly from 3500 to 110,000 CCR5 molecules/cell). CD4 and CCR5 concentrations were determined by quantitative flow cytometry (quantitative FACS), as described previously [69, 77]. The induced cell populations were then inoculated with equivalent amounts of Env-pseudotyped reporter virus and analyzed for levels of HIV-1 entry as described above. The relative level of virus entry achieved by each Env tested was expressed as a percentage of that achieved in 293-Affinofile cells expressing the highest concentrations of CD4 and CCR5. The robustness of the 293-Affinofile assay for measuring alterations in CD4 and CCR5 dependence was validated using a CD4-independent virus (SIV 316), which was highly sensitivity to alterations in the expression levels of CCR5 but not CD4, and with a CXCR4 using virus (IIIB), which was highly sensitivity to alterations in the expression levels of CD4 but not CCR5 (data not shown). After normalization of the virus entry data, the relative dependence of Env-pseudotyped reporter viruses on CD4 and CCR5 expression levels was mathematically modeled using the VERSA computational platform (http://versa.biomath.ucla.edu), as described previously [69, 78]. With the use of this model, viral infectivity is quantified using a single vector. The vector magnitude and mean induction reflect the efficiency of virus entry, and the vector angle represents the relative dependence on CD4 or CCR5. The mathematical derivation of these metrics has been described in detail previously [69] and is summarized in Supplemental Fig. 1. In theoretical extremes, viruses that have the greatest possible sensitivity to alterations in CD4 expression but are unaffected by alterations in CCR5 expression have a vector angle of 0°, and conversely, viruses that have the greatest possible sensitivity to alterations in CCR5 expression but are unaffected by alterations in CD4 expression have a vector angle of 90°.

gp120-binding assays

293T cells were transfected with Env expression plasmids using Lipofectamine 2000 (Invitrogen, Life Technologies), according to the manufacturer's protocol, and stained for surface gp120 expression using pooled polyclonal HIV+ sera BB10, as described previously [66, 72]. Approximately 2 × 10⁵ 293T cells transfected with each Env expression plasmid were used in binding reactions. In binding reactions with the Env mAb 17b [79–82], cells were preincubated in FACS buffer [PBS containing 10% (vol/vol) FCS and 0.05% (wt/vol) sodium azide], with or without 20 μg sCD4/ml (Progenics Pharmaceuticals, Tarrytown, NY, USA) for 1 h at room temperature. Cells were then washed twice with 200 μl FACS buffer and resuspended in 50 μl FACS buffer containing 10 μg 17b [79–84]/ml. This concentration was empirically determined to be within the linear range of binding [66]. Following incubation for 1 h at room temperature, cells were washed twice with 200 μl FACS buffer and resuspended in 50 μl FACS buffer containing a 1:200 dilution of FITC-conjugated anti-human IgG F(Ab)₂ fragment (Millipore, Billerica, MA, USA). Cells were incubated for 1 h at room temperature, prior to being washed twice with 200 μl FACS buffer and resuspended in 150 μl PBS containing 4% (wt/vol) PFA and analyzed by flow cytometry as described previously [85].

HIV-1 neutralization/inhibition assays

The ability of the Env mAb b12 or sCD4 to neutralize/inhibit the infectivity of Env-pseudotyped luciferase reporter viruses was assayed using JC53 cells. Two hundred TCID₅₀ of each Env-pseudotyped luciferase reporter virus (equating to a MOI of 0.01) were incubated with tenfold-increasing concentrations of b12 (0.0005–50 μg/ml) or sCD4 (Progenics Pharmaceuticals; 0.001–100 μg/ml) for 45 min at 37°C. The virus-inhibitor mixtures were then used to inoculate JC53 cells for 12 h at 37°C. Cells were rinsed twice with culture medium to remove residual virus inoculum and incubated a further 60 h at 37°C. Virus infectivity was then measured by assaying luciferase activity in cell lysates (Promega), according to the manufacturer's protocol. Negative controls included mock-infected cells that were incubated with culture medium instead of virus. After subtracting background luciferase activity, the amount of luciferase activity in the presence of antibody or inhibitor was expressed as a percentage of the amount produced in control cultures containing no antibody or inhibitor. The percent inhibition was calculated by subtracting this number from 100. Data were fitted with a nonlinear function, and IC₅₀ values were calculated by least squares regression analysis of inhibition curves, as described previously [11, 48, 72, 86].

gp120 structural modeling

3D protein structures of BR- and LN-derived gp120 sequences were prepared using the Discovery Studio suite, version 3.0 (Accelrys, San Diego, CA, USA), as we have described previously [65, 66, 75, 87]. The crystal structure of YU2 gp120 bound to CD4, and mAb 17b [88] was used as a template to generate homology models of Macs2-LN-2 and Macs2-BR-2 gp120 sequences. The interface between gp120 and CD4 or 17b was mapped to atoms predicted to be within 4 Å of the ligand. PNGS were identified by the amino acid sequon “NXS or T”, where X does not equal a proline residue. The crystal structure of CD4-bound YU2 gp120 containing the V3-variable loop and docked with the NMR structure of an N-terminal peptide of CCR5 (residues 2–15; kindly provided by Peter D. Kwong [89]) was used as a template to generate homology models of BR-derived (Macs2-BR-2, UK1-BR-10) and LN-derived (Macs2-LN-2) gp120 sequences. Harmonic restraints were applied prior to optimization using the steepest descent protocol, which incorporates iterative cycles of conjugate-gradient energy minimization against a probability density function that includes spatial restraints derived from the template and residue-specific properties [90].

In silico glycosylation was performed using the GlyProt web-based server (http://www.glycosciences.de/modeling/glyprot/php/main.php) [91]. Homology models of Macs2-LN-2 and Macs2-BR-2 gp120 proteins generated using the 17b-bound gp120 crystal structure template were submitted for analysis. Briefly, potential glycosylation sites were identified in the amino acid sequence of the 3D structures, and spatially accessible sites were identified. The core regions of basic glycoforms were then added to the predicted glycosylation sites in orientations using the preferred values for the χ₁, χ₂, Φn, and Ψn dihedral angles observed in 3000 experimentally determined 3D structures of N-glycan chains from glycoproteins.

RESULTS

Highly M-tropic Env variants derived from the CNS

In this study, we characterized the HIV-1 entry mechanisms of CCR5-using (R5) Env variants derived from BR and LN. These Envs and the HIV-1 isolates and subjects from whom they were derived have been described in detail previously [10, 11, 25, 26]. We first determined the ability of the Envs to enter MDMs when pseudotyped onto luciferase HIV-1 reporter viruses (Fig. 1). As controls, we used luciferase reporter viruses pseudotyped with JR-CSF or YU2 Envs, which have poorly M-tropic and highly M-tropic phenotypes, respectively [66], and virus pseudotyped with the nonfunctional ΔKS Env [76] to determine background levels of luciferase activity. Our results show that all of the BR-derived Envs tested are highly M-tropic, exhibiting entry levels in MDM similar to those achieved by YU2, and that all of the LN-derived Envs tested are poorly M-tropic with entry levels in MDM lower than that achieved by JR-CSF (Fig. 1A). In contrast, the BR- and LN-derived Envs showed similar levels of HIV-1 entry into PBMC (Fig. 1B) and the JC53 cell line that expresses high levels of CD4 and CCR5 (Fig. 1C).

Figure 1. — Luciferase reporter viruses pseudotyped with BR- or LN-derived Envs were produced and titrated as described in Materials and Methods, and equivalent infectious units were used to infect cultures of MDM (A), PBMC (B), or JC53 cells (C). Controls included luciferase reporter viruses pseudotyped with JR-CSF or YU2 Envs or with the nonfunctional ΔKS Env [76]. The results shown are means of triplicate wells and are representative of four independent experiments. For experiments in MDM and PBMC, the independent experiments were conducted in cells obtained from different donors. Error bars represent sd.

Highly M-tropic Envs can efficiently scavenge low levels of cell-surface CD4

To better understand the virus–cell interactions underlying efficient MDM entry, we next used the 293-Affinofile affinity-profiling system [69] to quantify the CD4- and CCR5-usage efficiencies of the BR- and LN-derived Envs. In this system, CD4 and CCR5 expression is controlled by separate inducible promoters, permitting independent variation of CD4 and CCR5 expression over a physiological concentration range [69]. When 48 differentially induced cell populations are subjected to single-round entry assays with Env-pseudotyped luciferase reporter viruses, and data sets are analyzed quantitatively by mathematical modeling using the VERSA computational platform [69], three vector metrics are generated that capture the essential phenotypic characteristics of the Env. This is graphically illustrated in Supplemental Fig. 1. The vector angles measure the degree of CD4 and CCR5 dependence, and the vector magnitude measures the efficiency of virus entry characterized by the overall responsiveness to changes in CD4 and CCR5 levels; in other words, it measures the overall steepness or gradient of the surface plot. Lastly, the vector mean induction measures the efficiency of virus entry characterized by the level of infection averaged across the entire matrix of distinct CD4 and CCR5 expression levels.

These quantitative metrics can be used to dissect simultaneously occurring gp120-CD4/CCR5 interactions. For example, a concordant increase in vector angle and mean induction signifies that the increased infection response to CCR5 levels is a result of a comparative increase in the efficiency of CCR5 usage, whereas a “discordant” increase in vector angle accompanied by a decrease in the mean induction indicates that the increase in CCR5 dependence is a result of a decrease in CCR5 usage efficiency, as would occur when an Env exhibits efficient infection only at higher levels of CCR5 [69]. Thus, the combined analysis of vector angle with the vector magnitude or mean induction permits the VERSA metrics to determine how efficiently a particular Env can interact with CD4 and CCR5. The following analysis of our BR- and LN-derived Envs will exemplify the physiological meaning of these vector metrics.

Figure 2A shows the inducible levels of CD4 and CCR5 expression achieved by minocycline or ponA, respectively. The 293-Affinofile profiles of control JR-CSF and YU2 Envs show that the highly M-tropic YU2 Env can enter cells expressing scant levels of CD4, where it achieves 20–25% of maximal entry when low or high levels of CCR5 are present, whereas the poorly M-tropic JR-CSF Env is dependent on much higher levels of CD4 to enter cells (Fig. 2B). The individual 293-Affinofile plots of the BR- and LN-derived Envs are shown in Supplemental Fig. 2, and the corresponding surface plots are shown in Supplemental Fig. 3. These data are compiled together in Fig. 2C. Together, these results show that all of the BR-derived Envs efficiently use very low levels of CD4 to enter cells, similar to YU2, whereas all the LN-derived Envs are dependent on higher levels of CD4 to enter cells, similar to JR-CSF. The entry of the BR-derived Envs and YU2 Env into 293-Affinofile cells could be inhibited completely by the CCR5 antagonist MVC (Supplemental Fig. 4), confirming the specificity of the entry levels by these Envs at low CD4 concentrations.

These results are reflected quantitatively by the VERSA metrics (Fig. 3). For example, the BR-derived Envs had significantly higher vector angles and lower vector magnitudes compared with the LN-derived Envs (P<0.001; Fig. 3A and B). These data indicate that the BR-derived Envs are less dependent on CD4 (angle; ∼24° vs. ∼10° for BR- and LN-derived Envs, respectively), and as a consequence of their higher baseline infection at low CD4 levels (see Fig. 2C), their overall responsiveness to increasing CD4/CCR5 levels is also comparatively muted (magnitude; ∼55 vs. ∼80 for BR- and LN-derived Envs, respectively). The BR-derived Envs also had significantly higher vector mean inductions than the LN-derived Envs (P<0.001; Fig. 3C), also principally a result of their increased ability to enter 293-Affinofile cells expressing lower levels of CD4 (see Fig. 2C). Interestingly, when the alternative VERSA metrics were plotted on a 3D axis, the primary Envs studied formed two distinct tissue-specific clusters (Supplemental Fig. 5). For these particular Envs, this occurred in the absence of intersubject genetic compartmentalization [26], suggesting that VERSA metrics could be applied in future studies to reveal novel Env phenotypic signatures. The altered responsiveness to increasing CD4 levels by the BR- and LN-derived Envs in the presence of low or high levels of CCR5 is illustrated further when the 293-Affinofile data are analyzed with five-parameter logistics (Fig. 3D and E). In this case, it is apparent that the increased vector magnitude is a reflection of the steeper slopes exhibited by the LN-derived Envs. Together, the 293-Affinofile data and quantitative VERSA metrics confirm that highly M-tropic Envs have the ability to scavenge low levels of CD4 on the cell surface to mediate HIV-1 entry, whereas non-M-tropic Envs do not.

Figure 3. — The VERSA metrics were calculated from the 293-Affinofile data as described in Materials and Methods. A graphical representation of the VERSA metrics comparing vector angles and vector magnitudes is shown in A. The shaded wedges represent the sem of the vector angles, and the boxes represent the sem of the vector magnitudes. Statistical analysis was conducted using an unpaired t-test to compare the differences in vector angle (B) and vector mean induction (C) between the BR- and LN-derived Envs. P values < 0.05 were considered statistically significant. The response of the BR- and LN-derived Envs to increasing CD4 levels when CCR5 levels were low (D) or high (E) was determined by five-parameter logistical analysis of the 293-Affinofile data, using Prism, version 5.0 (GraphPad Software, San Diego, CA, USA).

Highly M-tropic Envs have greater exposure of the CD4bs

To further understand the Env determinants contributing to highly efficient MDM entry, we next conducted virus inhibition studies in JC53 cells with sCD4 and the Env mAb b12 [92–94] whose epitope in gp120 overlaps the CD4bs.

The individual inhibition curves of Env-pseudotyped luciferase reporter viruses by sCD4 are shown in Fig. 4A and B, and the IC₅₀ for the BR- and LN-derived Envs are plotted in Fig. 4C. These results show that the BR-derived Envs are significantly more sensitive to inhibition by sCD4 than the LN-derived Envs. The results also show that the BR-derived Envs exhibit a largely homogeneous response to inhibition by sCD4, whereas the LN-derived Envs show a high degree of heterogeneity in their sensitivity to inhibition by sCD4. We also observed a direct relationship between the sensitivity of virus inhibition by sCD4 and the ability of the Env to enter 293-Affinofile cells, expressing low levels of CD4 together with low (Fig. 4D), medium (Fig. 4E), or high levels of CCR5 (Fig. 4F).

Figure 4. — Virus inhibition assays were conducted in JC53 cells, as described in Materials and Methods (A and B), and the sCD4 IC₅₀ values for viruses pseudotyped with BR- or LN-derived Envs were calculated from the virus inhibition curves using Prism, version 5.0 (GraphPad Software; C). Statistical comparisons were made with a nonparametric Mann-Whitney U-test. The ability of the Envs to enter 293-Affinofiles expressing low levels of CD4 and low (D), medium (E), or high (F) levels of CCR5 was plotted against the sCD4 IC₅₀ values using Prism, version 5.0 (GraphPad Software). The data shown are means of triplicates and are representative of two independent experiments. The Spearman correlation coefficients (r) and P values are shown. P values < 0.05 were considered statistically significant.

The individual neutralization curves of Env-pseudotyped luciferase reporter viruses by b12 are shown in Supplemental Fig. 6A and B, and the IC₅₀ values are shown in Supplemental Fig. 6C. Interestingly, the BR- and LN-derived Envs from subject Macs3 were resistant to neutralization by b12 (Supplemental Fig. 6A–C), which is consistent with the results of previous studies of other Envs cloned from this subject [61]. When these Envs were excluded from analysis, the remaining BR-derived Envs showed significantly greater sensitivity to neutralization by b12 compared with the remaining LN-derived Envs (Supplemental Fig. 6D). Together, the results of the sCD4 and b12 inhibition studies suggest that the ability of highly M-tropic Envs to scavenge low levels of cell-surface CD4 is principally due to the Envs existing in a conformation that has increased exposure of the CD4bs in gp120.

Evidence that highly M-tropic Envs have altered presentation of the CD4-induced CCR5-binding domain in gp120

The preceding studies illustrate the use of the 293-Affinofile affinity-profiling system and VERSA metrics for demonstrating enhanced Env-CD4 interactions by highly M-tropic Envs derived from brain, thus unifying the results of recent studies [15, 49, 50, 55–58, 60–64] in a highly quantitative fashion. However, our recent studies also show that certain blood-derived M-tropic Envs have increased exposure of CD4-induced epitopes in gp120 that is associated with an altered interaction with CCR5 [65, 66] and may not be immediately evident from the 293-Affinofile data. We therefore next compared the ability of the BR- and LN-derived Envs to bind to the 17b mAb, whose epitope overlaps the CD4-induced CCR5-binding site in gp120. After incubation with sCD4, collectively, the BR-derived Envs showed significantly greater binding to 17b compared with the LN-derived Envs (Fig. 5A), although at the individual level, it is clear that two of the BR-derived Envs (Macs3-BR-1 and -8) have 17b-binding profiles that are similar to those of the LN-derived Envs. In the absence of sCD4, there was equivalent, low-level binding to 17b by the BR- and LN-derived Envs (data not shown). Similar levels of gp120 were expressed on the surface of the cells used in the binding assays, as shown by equivalent levels of staining with polyclonal HIV+ sera (Fig. 5B). These data indicate that the highly M-tropic BR-derived Envs from subjects Macs2 and UK1, but not those from Macs3, have increased exposure of the CD4-induced 17b epitope.

Figure 5. — Binding assays between Env expressed on 293T cells and the mAb 17b (A) or polyclonal HIV+ sera (B) were conducted as described in Materials and Methods. The data shown are means of triplicates and are representative of three independent experiments. Statistical comparisons were made with a nonparametric Mann-Whitney U-test, and P values < 0.05 were considered statistically significant. The gp120 V4 amino acid sequence of Macs2-LN-2, Macs2-BR-2, and UK1-BR-10 Envs and location of PNGS are shown in C. Homology models of Macs2-LN-2 and Macs2-BR-2 gp120 proteins were generated using the 17b-bound gp120 crystal structure template, as described in Materials and Methods (D). The molecular surface of gp120 is shown in gray, and the CD4bs and the 17b epitope are shown in pink and aqua, respectively. The N-glycan core of PNGS at positions N386 (Macs2-LN-2) and N397 (Macs2-BR-2) is shown as orange space-filling sphere representations.

To investigate the potential mechanisms by which these BR-derived Envs have greater presentation of the 17b epitope, we first compared the gp120 sequences of representative BR (Macs2-BR-2 and UK1-BR-10)- and LN (Macs2-LN-2)-derived Envs that had differences in 17b-binding profiles. Sequence analysis identified a conserved shift in the pattern of PNGS within the gp120 V4-loop region that segregated the BR- from LN-derived Envs (Fig. 5C). The Macs2-LN Env clones have a PNGS at Asn386, whereas the Macs2-BR and UK1-BR Env clones have Asp at this position, eliminating the PNGS, but contain a small insertion of 3–4 aa in V4 that reintroduces the PNGS at Asn397. To determine whether this shift in N-linked glycosylation may potentially influence 17b binding, we next produced 3D homology models of Macs2-LN-2 and Macs2-BR-2 gp120 proteins in their 17b-bound conformations using the 17b-bound gp120 crystal structure as template [88] and modeled the glycosylation at positions Asn386 or Asn397 (Fig. 5D). For Macs2-LN-2, the glycan is located proximal to the 17b epitope and therefore, has the potential to obscure part of the CCR5-binding domain. In contrast, for Macs2-BR-2, repositioning the glycan to aa 397 places it distal to the 17b epitope, thus facilitating greater exposure of the CCR5-binding domain. Supplemental Fig. 7 shows that glycosylation at Asn386 can also potentially occlude the 17b-binding site on gp120 and modulate 17b binding in the context of other glycosylated residues. Similar results were obtained from models of 17b-bound gp120 of UK1-BR-10 (data not shown). Although further mutagenesis studies are required to determine the role of V4-loop glycan alterations in exposing or occluding the 17b epitope, these analyses provide evidence that the majority of the highly M-tropic Envs exists in a conformation that has greater presentation of CD4-induced epitopes, which may alter the interaction between CD4-bound gp120 and CCR5.

Highly M-tropic Envs derived from brain display an altered mechanism of engagement with CCR5

To determine whether the BR-derived Envs have an altered interaction with CCR5, we next elucidated the mechanism of gp120-CCR5 engagement by the BR- and LN-derived Envs. Single-round entry assays were conducted in NP2-CD4 cells expressing WT CCR5 or CCR5 containing various mutations in the N-terminal domain or ECL regions, using luciferase reporter viruses pseudotyped with each of the BR- and LN-derived Envs (Fig. 6). The levels of virus entry in cells expressing CCR5 mutants were expressed as percentages of that attained in cells expressing equivalent levels of WT CCR5, which was verified by flow cytometry. The results show that the BR-derived Envs have significantly increased dependence on Tyr10, Asp11, Tyr14, Tyr15, Gly18, and Lys22 within the CCR5 N-terminus compared with the LN-derived Envs. In particular, most of the BR-derived Envs are critically dependent on the sulfated Tyr10, Tyr14, and Tyr15 residues in the CCR5 N-terminus, whereas most of the LN-derived Envs are not. The results also show that the BR-derived Envs have increased dependence on His88 and His181 in the CCR5 ECL1 and ECL2 regions, respectively. No differences in dependence on Val5 in the CCR5 N-terminus, Tyr89 in the CCR5 ECL1 region, or Tyr184 and Gln188 in the CCR5 ECL2 region were observed between the BR- and LN-derived Envs (data not shown). Together, these results illustrate an increased reliance of the BR-derived Envs on the CCR5 N-terminus and novel interactions with charged elements of the CCR5 ECL1 and ECL2 regions.

Figure 6. — Luciferase reporter viruses were used to infect NP2-CD4 cells expressing equivalent levels of WT CCR5 or CCR5 with alternative mutations in the CCR5 N-terminus (Y10A, D11A, Y14F, Y15A, E18A, K22A), ECL1 (H88A), or ECL2 regions (H181A), as described in Materials and Methods. Entry levels were expressed as a percentage of those attained in cells expressing WT CCR5. The results shown are means of triplicates and are representative of three independent experiments. Statistical comparisons were made with a nonparametric Mann-Whitney U-test. *P < 0.05; **P < 0.01. P values < 0.05 were considered statistically significant.

Potential structural basis for the altered engagement between BR-derived Envs and the CCR5 N-terminus

Sequence analysis identified 7 aa polymorphisms in gp120, located within or immediately adjacent to coreceptor-binding sites that segregated Macs2-LN from Macs2-BR and UK1-BR Envs. These polymorphisms include Arg/Gln328 and Gly/Arg335 within or near the gp120 V3-loop; Asn/Asp386 within the gp120 V4-loop; and Arg/Lys121, Arg/Lys432, Leu/Gln442, and Val/Ile443 within or near the gp120 bridging sheet (Fig. 7A). To identify the potential molecular interactions associated with the increased reliance of the BR-derived Envs on the CCR5 N-terminus and the potential influence of these amino acid polymorphisms on such interactions, we next produced full-length 3D homology models of UK1-BR-10, Macs2-BR-2, and Macs2-LN-2 gp120 proteins based on the crystal structure of CD4-bound YU2 gp120 [89]. This structure is particularly useful, as it is docked to a sulfated CCR5 N-terminal peptide (CCR5_2–15) and thus, permits the investigation of the molecular interactions at the gp120-CCR5 N-terminus interface. Analysis of the potential atomic contacts at the gp120-CCR5 N-terminus interface shows that Gly441 and Thr303 of all three gp120 structures are predicted to interact directly with sulfated Tyr14 of the CCR5 N-terminus (Fig. 7B). However, Macs2-BR-2 and UK1-BR-10 gp120 structures can potentially form several additional contacts with the CCR5 N-terminus. Specifically, Macs2-BR-2 has additional predicted atomic contacts between Ile326 and sulfated Tyr10, Arg327, and Asn13 and Asn300 and sulfated Tys14 of gp120 and the CCR5 N-terminus, respectively. UK1-BR-10 has additional predicted atomic contacts between Arg327 and sulfated Tyr10, Arg327, and Asn13 and Asn302 and sulfated Tyr14 of gp120 and the CCR5 N-terminus, respectively. These results suggest that the BR-derived Envs may rely on more atomic contacts with the CCR5 N-terminus than the LN-derived Envs to enter cells. Further mutagenesis studies are required to determine precisely whether this is the case. However, our modeling studies provide a potential structural basis for why the entry of BR-derived Envs into cells is more readily abrogated by CCR5 N-terminus mutations.

Figure 7. — Amino acid polymorphisms associated with BR-derived Envs were identified within or near CCR5-binding sites in gp120, as indicated in the multiple sequence alignment (A). The locations of common alterations occurring in Macs2-BR-2 and UK1-BR-10 Envs are highlighted. 3D homology models of gp120 of UK1-BR-10, Macs2-BR-2, and Macs2-LN-2 Envs, in their CD4-bound conformation and docked to a CCR5_2–15 N-terminal peptide, were produced as described in Materials and Methods (B). The α-carbon atoms of amino acids, occurring at positions 121, 328, 335, 386, 432, 442, and 443, are shown as blue space-filled models. gp120 is shown as gray ribbon, CD4 as purple ribbon, and the CCR5 N-terminal peptide (aa 7–15) as yellow ribbon. The CD4bs, as defined by gp120 amino acids within 4 Å of the CD4 molecule [66, 87], is colored orange. In the enlarged boxes, the amino acids in gp120 making hydrogen-bond contacts with the CCR5 N-terminus peptide are shown as magenta stick models. The CCR5 N-terminus hydrogen-bond partners are shown as yellow stick models, with sulfate groups on Tyr10 and Tyr14 (Tys10 and Tys14, respectively) colored orange. Hydrogen bonds are shown as black dotted lines. Residues are numbered according to the HXB2 gp120 sequence.

DISCUSSION

Although it is well-established that M-tropic HIV-1 variants are more likely to persist in the CNS than in peripheral tissues and that several studies have illustrated an enhanced interaction between gp120 of M-tropic viruses and CD4 (reviewed in ref. [54]), we still do not have a firm understanding of the broader virus–cell interactions that contribute to M-tropism of BR-derived HIV-1 variants. Here, we took advantage of the 293-Affinofile affinity-profiling system [69] to model, in a quantitative fashion, the tandem interactions between BR-derived Envs and both CD4 and CCR5, which are associated with efficient macrophage entry. In addition to demonstrating a dramatically reduced dependence of BR-derived Envs on CD4 levels, our results identify an alteration in the way BR-derived Envs interact with CCR5. In the majority of cases, this alteration was characterized by increased exposure of the CD4-induced CCR5-binding domain in gp120, which was associated with repositioning of the glycan shield within the V4 region, and by increased dependence on the CCR5 N-terminus and on charged elements within the CCR5 ECLs, which was associated with the predicted formation of additional atomic contacts at the gp120-CCR5 N-terminus interface. When viewed in context with the 293-Affinofile profiles and VERSA metrics, these results suggest that the highly M-tropic BR-derived Envs have an altered but not necessarily more efficient interaction with CCR5 that occurs in tandem with an enhanced interaction with CD4.

In addition to providing a more detailed understanding of how M-tropic Envs interact with cellular receptors, our results further illustrate the distinction between coreceptor use of HIV-1 and cellular tropism. An important conclusion that we draw is that CCR5 use of HIV-1 per se is insufficient to confer M-tropism. This interpretation is consistent with our original classification made more than 10 years ago—that a significant proportion of CCR5-using HIV-1 strains can be termed non-M-tropic R5 viruses based on their ability to enter and replicate in PBMC but not in MDM and primary cultures of human fetal microglia [10]. In fact, there is now evidence to suggest that most of the circulating R5 HIV-1 strains and those residing in lymphoid tissues are T cell-tropic rather than M-tropic [10, 15, 49–51, 55]. In addition, a subset of highly M-tropic HIV-1 strains has been shown to enter macrophages efficiently via CXCR4 [10, 25, 65]. These results underscore that the determinants of M-tropism by HIV-1 strains are significantly more complex than the coreceptor specificity of their Env glycoproteins (reviewed in ref. [40]).

Whereas there is convincing evidence that reduced CD4 dependence permits the ability of BR-derived Envs to scavenge low levels of CD4 expressed on the surface of macrophages and microglia [15, 49, 50, 55–58, 60–64], it is presently unclear how an altered interaction between gp120 and the CCR5 N-terminus may also contribute to the entry of BR-derived Envs into these cells. It is possible that BR-derived Envs have undergone adaptations to reflect the differences in CCR5 conformation [28, 95] and/or post-translational modifications, such as sulfation [32] or O-linked glycosylation [32, 96, 97], which exist among macrophages, resting T cells, and activated T cells.

The pattern of increased reliance on the CCR5 N-terminus and on charged elements within the CCR5 ECLs exhibited by the BR-derived Envs is remarkably similar to the pattern of altered CCR5 engagement that we and others have shown to be the major mechanism by which HIV-1 can develop allosteric resistance to CCR5 antagonists, including MVC [75, 78, 98–101]. This raised the possibility that the BR-derived Envs may exhibit baseline resistance to MVC. However, we showed that all of the BR- and LN-derived Envs are completely inhibited by MVC (Supplemental Fig. 8). As resistance to CCR5 antagonists is characterized by plateaus of virus inhibition below 100% rather than by shifts in IC₅₀ values [102], none of the Envs tested exhibited resistance to MVC. In fact, the majority of the BR-derived Envs could be inhibited completely by ∼20-fold lower concentrations of MVC than the majority of the LN-derived Envs (Supplemental Fig. 8). From these results, we can conclude that the BR-derived Envs remain highly sensitive to inhibition by MVC, despite having a mechanism of CCR5 engagement that could be considered predictive of CCR5 antagonist resistance. These findings underscore the strain-dependent nature of Env phenotypes and suggest that CCR5 antagonists could effectively treat CNS infection, should they be developed with suitable pharmacological properties that allow sufficient CNS penetrance and bioavailability.

The increased dependence of the highly M-tropic BR-derived Envs on the CCR5 N-terminus was an intriguing, unexpected result. We recently characterized the association between the efficiency of macrophage entry by certain blood-derived Envs and the ability of the Envs to tolerate mutations in the CCR5 N-terminus and showed that efficient CCR5-mediated macrophage entry was associated with reduced, rather than increased, dependence on elements within the CCR5 N-terminus, which included Asp11, Tyr14, Tyr15, and Glu18 [65, 66]. In these studies, we also saw correlations between the efficiency of macrophage entry and the ability of the Env to enter 293-Affinofile cells expressing low levels of CCR5 and also with the MVC IC₅₀, suggesting that unlike the BR-derived Envs, the M-tropic blood-derived Envs studied had a more efficient interaction with CCR5. Thus, there appears to be dichotomous mechanisms of altered Env-CCR5 engagement by M-tropic Envs, depending on whether they were derived from brain or from blood—the former involving increased dependence on the CCR5 N-terminus that does not increase the efficiency of the Env-CCR5 interaction and the latter involving reduced dependence on the CCR5 N-terminus that increases the efficiency of the Env-CCR5 interaction. Thus, the R5 M-tropic phenotype or “color”, as termed recently by Swanstrom and colleagues [53], may indeed be comprised of at least two distinct M-tropic subphenotypes.

Although all the BR-derived Envs studied had reduced CD4 dependence that was associated with increased sensitivity to inhibition by sCD4, two of the BR-derived Envs—Macs3-BR-1 and Macs3-BR-8—were resistant to neutralization by b12, as were the Envs derived from LNs of the same subject (see Supplemental Fig. 6). The determinants of b12 resistance by these Envs are unclear, but it is noteworthy that other Envs, which were amplified and cloned directly from tissues of this subject, were also shown to be resistant to b12 [61]. The BR-derived Envs from Macs3 also showed reduced exposure of the CD4-induced 17b epitope than the other BR-derived Envs, and they also had less pronounced dependence on the CCR5 N-terminus than the other BR-derived Envs. These results indicate that some highly M-tropic BR-derived Envs can efficiently engage CD4 through a mechanism that does not involve greater exposure of the b12 epitope. The results also suggest that a subset of BR-derived Envs may not necessarily exist in conformations that have greater presentation of the 17b epitope nor exhibit an altered interaction with CCR5. Further studies are required to determine how frequently this occurs among highly M-tropic BR-derived Envs.

In summary, the results of our study provide new mechanistic insights into the virus–cell interactions involved in macrophage tropism of HIV-1 variants derived from brain, using quantitative affinity-profiling techniques and mathematical modeling of biological data that account for the simultaneous alterations in the way the Env glycoproteins of M-tropic viruses interact with both CD4 and CCR5.

Supplementary Material

Supplemental Data

supp_93_1_113__index.html^{(882B, html)}

ACKNOWLEDGMENTS

This study was supported, in part, by grants from the NHMRC to P.R.G., M.J.C., and S.L.W. (#603708 and #1006534) and by a grant from U.S. National Institutes of Health/NIAID to B.L. (R21 AI092218). H.S. is supported by a postgraduate research scholarship from the Iranian Ministry of Health and Medical Education. P.A.R. is the Sir Zelman Cowen Senior Research Fellow (Sir Zelman Cowen Fellowship Fund, Burnet Institute). P.R.G. is the recipient of an Australian NHMRC Level 2 Biomedical Career Development Award. L.R.G. is the recipient of an Australian NHMRC Early Career Research Fellowship. The authors gratefully acknowledge the contribution to this work of the Victorian Operational Infrastructure Support Program received by the Burnet Institute. We thank J. Sodroski for providing JR-CSF, YU2, ΔKS Env, pCMVΔP1ΔenvpA, and pHIV-1Luc plasmids. We thank D. Kabat for providing JC53 cells, H. Hoshino for permission to use NP2-CD4 cells, and D. Mosier and R. Nedellec for supplying the NP2-CD4 cells. We also thank J. Sodroski and R. Doms for providing CCR5 mutants, D. Gabuzda for providing primary HIV-1 isolates, and D. Burton for providing the b12 mAb. The following reagent was obtained through the U.S. National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID: HIV-1 gp120 mAb (17b) from Dr. James E. Robinson.

The online version of this paper, found at www.jleukbio.org, includes supplemental information.

3D: three-dimensional
BR: brain
CD4bs: CD4-binding site
ECL: extracellular loop
Env: HIV-1 envelope glycoprotein
M-tropic: macrophage tropic
MVC: maraviroc
NHMRC: Australian National Health and Medical Research Council
NIAID: National Institute of Allergy and Infectious Diseases
PNGS: potential N-linked glycosylation site
ponA: ponasterone A
sCD4: soluble CD4
TCID₅₀: 50% tissue culture-infective dose
VERSA: viral entry receptor sensitivity analysis

AUTHORSHIP

H.S., M.R., L.R.G., J.S., and A.E. performed experiments. N.W., K.C., S.L.W., P.A.R., B.L., M.J.C., and P.R.G. analyzed data. P.A.R., B.L., M.J.C., and P.R.G. designed experiments. P.R.G. and B.L. wrote the manuscript. All authors edited the manuscript.

DISCLOSURES

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PERMALINK

Macrophage-tropic HIV-1 variants from brain demonstrate alterations in the way gp120 engages both CD4 and CCR5

Hamid Salimi

Michael Roche

Nicholas Webb

Lachlan R Gray

Kelechi Chikere

Jasminka Sterjovski

Anne Ellett

Steve L Wesselingh

Paul A Ramsland

Benhur Lee

Melissa J Churchill

Paul R Gorry

Abstract

Introduction

MATERIALS AND METHODS

Cells

HIV-1 Env plasmids

Production and quantitation of Env-pseudotyped luciferase reporter viruses

Single-round HIV-1 entry assays

Affinofile cell assays and quantitative vector analysis

gp120-binding assays

HIV-1 neutralization/inhibition assays

gp120 structural modeling

RESULTS

Highly M-tropic Env variants derived from the CNS

Figure 1. BR-derived Envs are highly M-tropic.

Highly M-tropic Envs can efficiently scavenge low levels of cell-surface CD4

Figure 2. BR-derived Envs have reduced CD4 dependence.

Figure 3. VERSA metrics distinguish BR- and LN-derived Envs.

Highly M-tropic Envs have greater exposure of the CD4bs

Figure 4. BR-derived Envs have greater sensitivity to inhibition by sCD4.

Evidence that highly M-tropic Envs have altered presentation of the CD4-induced CCR5-binding domain in gp120

Figure 5. The majority of the BR-derived Envs has greater presentation of the CD4-induced 17b epitope.

Highly M-tropic Envs derived from brain display an altered mechanism of engagement with CCR5

Figure 6. BR-derived Envs have reduced dependence on the CCR5 N-terminus and on charged elements of CCR5.

Potential structural basis for the altered engagement between BR-derived Envs and the CCR5 N-terminus

Figure 7. BR-derived Envs have structural alterations at the gp120-CCR5 N-terminus interface.

DISCUSSION

Supplementary Material

ACKNOWLEDGMENTS

AUTHORSHIP

DISCLOSURES

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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