An allosteric rheostat in HIV-1 gp120 reduces CCR5 stoichiometry required for membrane fusion and overcomes diverse entry limitations

Abstract

Binding of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120 to the CCR5 co-receptor reduces constraints on the metastable transmembrane subunit gp41, thereby enabling gp41 refolding, fusion of viral and cellular membranes, and infection. We previously isolated adapted HIV-1(JRCSF) variants that more efficiently use mutant CCR5s, including CCR5(Delta18) lacking the important tyrosine sulfate-containing amino terminus. Effects of mutant CCR5 concentrations on HIV-1 infectivities were highly cooperative, implying that several may be required. However, because wild-type CCR5 efficiently mediates infections at trace concentrations that were difficult to measure accurately, analyses of its cooperativity were not feasible. New HIV-1(JRCSF) variants efficiently use CCR5(HHMH), a chimera containing murine extracellular loop 2. The adapted virus induces large syncytia in cells containing either wild-type or mutant CCR5s and has multiple gp120 mutations that occurred independently in CCR5(Delta18)-adapted virus. Accordingly, these variants interchangeably use CCR5(HHMH) or CCR5(Delta18). Additional analyses strongly support a novel energetic model for allosteric proteins, implying that the adaptive mutations reduce quaternary constraints holding gp41, thus lowering the activation energy barrier for membrane fusion without affecting bonds to specific CCR5 sites. In accordance with this mechanism, highly adapted HIV-1s require only one associated CCR5(HHMH), whereas poorly adapted viruses require several. However, because they are allosteric ensembles, complexes with additional co-receptors fuse more rapidly and efficiently than minimal ones. Similarly, wild-type HIV-1(JRCSF) is highly adapted to wild-type CCR5 and minimally requires one. The adaptive mutations cause resistances to diverse entry inhibitors and cluster appropriately in the gp120 trimer interface overlying gp41. We conclude that membrane fusion complexes are allosteric machines with an ensemble of compositions, and that HIV-1 adapts to entry limitations by gp120 mutations that reduce its allosteric hold on gp41. These results provide an important foundation for understanding the mechanisms that control membrane fusion and HIV-1's facile adaptability.

Fig 1

Properties of CCR5(HHMH)-adapted viruses. (A) Infectivities of CCR5(HHMH)-adapted HIV-1_JR-CSF. Viruses adapted to grow in HeLa-CD4 clones expressing medium or low amounts of CCR5(HHMH) were titered in these same cells and compared to wild-type HIV-1. Relative infectivities were obtained by dividing the titer obtained in CCR5(HHMH) cells by those obtained in JC.53 cells (N=5). Error bars are S.E.M. (B) Syncytia formation by unadapted and adapted viruses. The average numbers of nuclei per focus of infection were counted in cells with wild-type CCR5, HHMH-med, or HHMH-low. The data were obtained by examining 50–200 foci from a representative experiment. Error bars are S.E.M. N.D., (not done) due to the low titers on these cells. (C) Contributions of adaptive gp120 mutations to CCR5(HHMH) use. Viruses pseudotyped with single adaptive mutations or combinations thereof were used to infect HeLa-CD4 cells containing low, medium, or high amounts of CCR5(HHMH). Relative infectivity values (i_rel) were measured. The data represent the average of four independent experiments performed in duplicate. Error bars are S.E.M. Inset: Schematic showing locations of CCR5(HHMH)-adaptive mutations in JRCSF gp120. Variable regions are shown in gray (V1–V5), while branched structures denote glycosylation sites. Virus initially adapted in cells expressing medium amounts of CCR5(HHMH) had adaptive mutations in V3 (F313L), V4 (N403S) and C4 (A428T) (red). After further adaptation in cells a with low amount of CCR5(HHMH), the C4 A428T mutation disappeared and the V3 mutation S298N accumulated (blue).

Fig 2

Structural modeling of the trimeric gp120/CD4 complex showing the adaptive mutations as space-filling objects. The structure of HIV-1 JR-FL gp120 core protein containing the third variable region (V3) was used to generate the trimeric model in accordance with previous evidence. Frame A, side view of the gp120 trimer (green shaded) with associated CD4s (in grey) and indicating positions of the adaptive mutations near the top and gp41 at bottom. Frame B shows a top view in low magnification, with adaptive mutations clustered at the gp120 interfaces overlying the central channel above gp41. Frame C shows the top view at a higher magnification. This clustering of adaptive mutations supports the hypothesis that they collaboratively control allosteric changes in quaternary structure that prevent gp41 refolding.

Fig 3

Facile use of CCR5 domains by adapted HIV-1. (A) HIV-1 infections mediated by wild-type, mutant and murine CCR5s. Wild-type HIV-1_JRCSF and the variant adapted to a low amount of CCR5(HHMH) were compared for abilities to infect 293T cells transiently expressing CD4 and murine CCR5 (mu CCR5), CCR5(Δ18), CCR5(G163R) or the double mutant CCR5(Δ18, G163R). The data are means of two independent experiments performed in triplicate. Error bars are S.E.M. (B) CCR5(Δ18) and CCR5(HHMH) use by HIV-gpt viruses containing adaptive gp120 mutations. Pseudotyped viruses were used to infect HeLa-CD4 cells expressing CCR5(Δ18) (2.7 × 10⁴ molecules/cell), CCR5(HHMH)-low, or CCR5(HHMH)-high. Adaptive gp120 mutations in the virus pseudotypes were as follows: CCR5(HHMH)-Ad: S298N/F313L/N403S; CCR5(Δ18)-Ad minus N300Y: S298N/I307M/F313L/T315P/N403S; CCR5(Δ18)-Ad:S298N/N300Y/I307M/F313L/T315P/N403S. The data are from 2 independent experiments performed in duplicate. Error bars are S.E.M. (C) Infections mediated by CCR5(Δ18) plus sulfated N-terminal CCR5 peptide. HeLa-CD4 cells expressing 2.7 × 10⁴ CCR5(Δ18) molecules/cell were infected in the presence of varying concentrations of CCR5 peptide (0, 25, 100, and 200 μM), and infectivities (i_rel) were measured relative to JC.53 cells. The replication competent CCR5(Δ18)-adapted, CCR5(HHMH)-adapted, and wild-type JRCSF (blue, green, and red curves, respectively) isolates were tested. The graph shows a representative experiment performed in duplicate. Error bars are the range.

Fig 4

Relationship of CCR5(HHMH) concentrations and viral infectivities. (A) Infectivities of wild-type and CCR5(HHMH)-adapted viruses in a HeLa-CD4 panel expressing distinct amounts of CCR5(HHMH). The infectivities of HIV-gpt viruses pseudotyped with wild-type envelopes or envelopes with the adaptive mutations designated in the figure were tested. Relative infectivities compared to JC.53 cells were plotted versus CCR5(HHMH) concentrations. The data represents the average of three independent experiments performed in triplicate. Error bars are S.E.M. (B) Normalization of the infectivity data in panel A to i_{rel max} values. The infectivity data for each virus was normalized to its maximum value, and the curves were drawn using Kaleidagraph (version 3.6) employing the least squares method. The infectivity data for wild-type virus with low titers on CCR5(HHMH) cells were easily measured using concentrated virus samples (average colony numbers on the highest expressing CCR5(HHMH) cell line ranged from 30 to 70 per well at a 1/5 virus dilution).

Fig 5

Role of allostery in HIV-1 infections. (A, left panel) Activation Energy Model. The activation energy model (Eq 4) was fit to the infectivity data (Fig 4) for the virus with S298N/F313L/N403S/A428T that is highly adapted to CCR5(HHMH). (A, right panel) Monod-Wyman-Changeux Model. The latter model also fit to the same infectivity data. Both models closely fit the data and gave similar estimates for the contributions to infectivity of the viral complexes with different CCR5(HHMH) stoichiometries, of 1 (N=1, R₁), 2 (N=2, R₂), or 3 (N=3, R₃). (B) Infection triggered by a tyrosine sulfated N-terminal CCR5 peptide. The N-terminal CCR5 peptide induced infections of HeLa-CD4 cells expressing low (2.7 × 10⁴) or high (6.6 × 10⁴) amounts of CCR5(Δ18) by the virus highly adapted to use CCR5(HHMH)-low. (B,left panel) Fit of infectivity data (i_rel values) using the activation energy allosteric model equation 4. The i_{rel max} values for both cell clones were 0.5. (B, middle and right panels) Deconvolution analysis of the peptide triggering data for cells with high (middle panel) and low (right panel) CCR5(Δ18), showing how complexes with 1, 2, or 3 peptides contributed to infection. Parameters obtained by fitting the data using equation 4 are given in Table 1.

Fig 6

TAK-779 and T-20 sensitivities of HIV-gpt viruses pseudotyped with envelopes containing wild-type gp120 or gp120 highly adapted to use CCR5(HHMH). (A) TAK-779 inhibition curves. Left: TAK-779 dose response assays performed using HeLa-CD4 cells expressing a high amount of CCR5(HHMH) (1.4 × 10⁵ molecules/cell). Right: TAK-779 dose response assays using HeLa-CD4 cells expressing large amounts of wild-type CCR5 (1.9 × 10⁵/cell). (B) T-20 inhibition curves. Left: T-20 dose response assays using HeLa-CD4 cells expressing high amounts of CCR5(HHMH). Right: T-20 dose response assays using HeLa-CD4 cells expressing high (1.9 × 10⁵ molecules/cell) or low (6.0 × 10³ molecules/cell) amounts of wild-type CCR5. Data are averages of 3 independent experiments performed in duplicate. Error bars are S.E.M.