Adaptive mutations in the V3 loop of gp120 enhance fusogenicity of human immunodeficiency virus type 1 and enable use of a CCR5 coreceptor that lacks the amino-terminal sulfated region - PubMed (original) (raw)
Adaptive mutations in the V3 loop of gp120 enhance fusogenicity of human immunodeficiency virus type 1 and enable use of a CCR5 coreceptor that lacks the amino-terminal sulfated region
E J Platt et al. J Virol. 2001 Dec.
Abstract
To identify sites in gp120 that interact with the CCR5 coreceptor and to analyze the mechanisms of infection, we selected variants of the CCR5-dependent JRCSF molecular clone of human immunodeficiency virus type 1 (HIV-1) that adapted to replicate in HeLa-CD4 cells that express the mutant coreceptor CCR5(Y14N) or CCR5(G163R), which were previously shown to bind purified gp120-CD4 complexes only weakly. Correspondingly, these mutant CCR5s mediate infections of wild-type virus only at relatively high cell surface concentrations, demonstrating a concentration-dependent assembly requirement for infection. The plots of viral infectivity versus concentration of coreceptors had sigmoidal shapes, implying involvement of multiple coreceptors, with an estimated stoichiometry of four to six CCR5s in the active complexes. All of the adapted viruses had mutations in the V3 loops of their gp120s. The titers of recombinant HIV-1 virions with these V3 mutations were determined in previously described panels of HeLa-CD4 cell clones that express discrete amounts of CCR5(Y14N) or CCR5(G163R). The V3 loop mutations did not alter viral utilization of wild-type CCR5, but they specifically enhanced utilization of the mutant CCR5s by two distinct mechanisms. Several mutant envelope glycoproteins were highly fusogenic in syncytium assays, and these all increased the efficiency of infection of the CCR5(Y14N) or CCR5(G163R) clonal panels without enhancing virus adsorption onto the cells or viral affinity for the coreceptor. In contrast, V3 loop mutation N300Y was selected during virus replication in cells that contained only a trace of CCR5(Y14N) and this mutation increased the apparent affinity of the virus for this coreceptor, as indicated by a shift in the sigmoid-shaped infectivity curve toward lower concentrations. Surprisingly, N300Y increased viral affinity for the second extracellular loop of CCR5(Y14N) rather than for the mutated amino terminus. Indeed, the resulting virus was able to use a mutant CCR5 that lacks 16 amino acids at its amino terminus, a region previously considered essential for CCR5 coreceptor function. Our results demonstrate that the role of CCR5 in infection involves at least two steps that can be strongly and differentially altered by mutations in either CCR5 or the V3 loop of gp120: a concentration-dependent binding step that assembles a critical multivalent virus-coreceptor complex and a postassembly step that likely involves a structural rearrangement of the complex. The postassembly step can severely limit HIV-1 infections and is not an automatic consequence of virus-coreceptor binding, as was previously assumed. These results have important implications for our understanding of the mechanism of HIV-1 infection and the factors that may select for fusogenic gp120 variants during AIDS progression.
Figures
FIG. 1
Schematic diagram of adapted HIV-1 JRCSF virus production by passage in cells expressing mutant coreceptors. High-titer HIV-1 JRCSF was propagated on HeLa-CD4 cells expressing either CCR5(Y14N) or CCR5(G163R), and the envelope genes of emergent variants were cloned and sequenced as described in Materials and Methods.
FIG. 2
Immunoprecipitation-Western blot analysis of wild-type and mutant JRCSF envelope proteins. Cell extracts were harvested from transfected COS-7 cells and subjected to immunoprecipitation-Western blot analysis as described previously (49). Mock-transfected cell extract and well-characterized SVIII_env_ were included as negative and positive controls, respectively. Mutant envelope nomenclature is defined in Table 2.
FIG. 3
HIV-1 infections mediated by mutant CCR5 coreceptors. (A and B) Infection of the CCR5(Y14N) clonal panel by pseudotyped HIV-gpt virions bearing the wild-type envelope or the mutant JRCSF envelopes described in Table 2. Infections were performed and quantitated as described in Materials and Methods. Relative infectivities were determined by dividing the titer measured on a given cell clone by the titer determined on a HeLa-CD4 clone expressing high levels of wild-type CCR5 (clone JC.53). (C and D) Infection of the CCR5(G163R) clonal panel by the same pseudotyped viruses as in panels A and B. Data points represent means of four independent assays, and error bars represent the standard error of the mean. The CCR5(Y14N) and CCR5(G163R) clonal panels were described previously (32).
FIG. 4
Mathematical analyses of infectivity data generated on the CCR5(Y14N) panel. The data in Fig. 3A and B were analyzed in accordance with the mathematical model derived by Kuhmann et al. (32). The _i_rel at the highest concentration of mutant CCR5 that was assayed was used as the _E_rel value, and all of the other _i_rel values that were defined were plotted as log [_i_rel/(_E_rel − _i_rel)] versus log [CCR5]. Only data points where CCR5 is at subsaturating concentrations can be plotted in this analysis, because when _i_rel approaches _E_rel, the difference becomes inaccurate. Therefore, the number of data points is not the same for each pseudotyped virus but represents all of the significant information.
FIG. 5
Syncytium induction by wild-type and adapted JRCSF envelopes on cells expressing CCR5(Y14N) and CCR5(G163R). 293T cells that had been transfected with JRCSF envelope expression vectors (described in Materials and Methods and Table 2) were cocultured for 6 h with HeLa-CD4 cells expressing either the wild-type or a mutant CCR5 coreceptor and then fixed and stained, and the syncytia in each well were counted. Percent syncytia compared to JC.53 was calculated for each envelope construct by dividing the number of syncytia obtained on cells expressing mutant coreceptors by the number of syncytia generated on JC.53 cells expressing wild-type CCR5 and multiplying by 100. These values were, in turn, normalized to the fusogenicity of wild-type JRCSF obtained on JC.53 cells (see values below). YB8 cells express 1.7 × 105 CCR5(Y14N) molecules per cell. JYN.4 cells express 6.0 × 104 CCR5(Y14N) molecules per cell. JGR.H4 cells express 1.9 × 104 CCR5(G163R) molecules per cell. JC.53 cells express 1.9 × 105 CCR5 molecules per cell. A representative assay, performed in triplicate, is shown, and the error bars represent standard deviations. The numbers of syncytia caused by the different envelope glycoproteins with JC.53 cells were similar but not identical. Thus, in a representative experiment, the normalized fusogenicities on the JC.53 cells were as follows: wild-type JRCSF, 1; S298N, 1.4; F313I, 1.2; F313L, 1.6; N300Y, 0.7; SNFL, 1.7; SNNY, 3.1; NYFL, 2.1; SNNYFL, 1.8. The expression levels of the envelope constructs were approximately equal, as measured by Western blot analysis (data not shown).
FIG. 6
Infections of HeLa-CD4 cells expressing CCR5 coreceptors with amino-terminal deletions. Populations of HeLa-CD4 cells expressing CCR5 with 16 (R5d16) or 18 (R5d18) amino-terminal residues removed, wild-type CCR5, or Y14N(CCR5) were generated by transduction with retroviral vectors (see Materials and Methods for details). Transduced cells were infected with pseudotyped viruses bearing the wild-type envelope or one of the mutant envelopes described in Table 2. Cells were placed in selective medium 48 h after infection. Drug-resistant colonies were stained and counted. The percentages of HeLa-CD4 cells expressing wild-type or mutant CCR5, determined by fluorescence-activated cell sorter analysis, were as follows: wild-type CCR5, 7.5%; CCR5(Y14N), 9.2%; R5d16, 8.2%; R5d18, 5.1%. Relative titers for each envelope construct in cells expressing mutant coreceptors were calculated by normalizing to the wild-type coreceptor activity and expression level with the following formula: (titer on mutant CCR5/titer on wild-type CCR5) × (wild-type CCR5 transduction efficiency/mutant CCR5 transduction efficiency). The average of three independent assays is shown, and error bars represent the standard error of the mean.
FIG. 7
sCD4 inactivation of pseudotyped HIV-gpt. HIV-gpt pseudotyped with the wild-type or mutant JRCSF envelope was subjected to treatment with various concentrations (5, 25, and 50 μg/ml) of sCD4 for 30 min prior to infection as previously described (49). Relative infectivities are plotted versus sCD4 concentrations with values in the absence of sCD4 equal to 1. All of the mutant envelopes described in Table 2 were tested, and the data have been divided into two panels (A and B) for ease of interpretation. SVIII_env_ was included as a positive control for sCD4 sensitivity.
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