Mutations in the V3 stem versus the V3 crown and C4 region have different effects on the binding and fusion steps of human immunodeficiency virus type 1 gp120 interaction with the CCR5 coreceptor

Abstract

The current model for HIV-1 envelope-coreceptor interaction depicts the V3 stem and bridging sheet binding to the CCR5 N-terminus while the V3 crown interacts with the second extracellular loop, which is the coreceptor domain that appears to be relatively more important for fusion and infection. Our prediction based on this model is that mutations in the V3 crown might consequently have more effects on cell-cell fusion and virus entry than mutations introduced in the V3 stem and C4 region. We performed alanine-scanning of the V3 loop and selected C4 residues in the JRFL envelope and tested the capacity of the resulting mutants for CCR5 binding, cell-cell fusion, and virus infection. Our cross comparison analysis revealed that residues in C4 and in both the V3 stem and crown were important for CCR5 binding of gp120 subunits. Contrary to our prediction, mutations in the V3 crown had less effect on membrane fusion than mutations in the V3 stem. The V3 stem thus appears to be the most important region for CCR5 utilization since it affected both coreceptor binding and subsequent fusion and viral entry. Our data raises the possibility that some residues in the V3 crown and in C4 may play distinct roles in the binding and fusion steps of envelope-coreceptor interaction.

Fig. 1

(A) Schematic drawing of the proviral clones used in this study. The open rectangle represents sequences from the subtype B molecular clone HXB2RU3. The hatched rectangle in ConB represents the consensus V3 sequence of HIV-1 subtype B NSI viruses in the HXB2RU3 backbone. The filled rectangle in HXB2-JRFL represents the SalI-BamHI env fragment (positions 5789 to 8451) derived from the R5 subtype B strain JRFL. The EspI (8840) site used in the cloning procedures is also indicated. (B) V3 sequences of the proviral clones, based on the JRFL numbering. Stem (positions 297–305 and 321–329) and crown (positions 306–320) regions are indicated. Naturally occurring alanines in the V3 of HXB2-JRFL that were mutated to glycine are indicated in bold-face. (C) Coreceptor utilization by the proviral clones as measured by p24 levels in virus-infected cultures at day 7. The results represent the means and standard deviations (SD) of data from three independent experiments.

Fig. 1

(A) Schematic drawing of the proviral clones used in this study. The open rectangle represents sequences from the subtype B molecular clone HXB2RU3. The hatched rectangle in ConB represents the consensus V3 sequence of HIV-1 subtype B NSI viruses in the HXB2RU3 backbone. The filled rectangle in HXB2-JRFL represents the SalI-BamHI env fragment (positions 5789 to 8451) derived from the R5 subtype B strain JRFL. The EspI (8840) site used in the cloning procedures is also indicated. (B) V3 sequences of the proviral clones, based on the JRFL numbering. Stem (positions 297–305 and 321–329) and crown (positions 306–320) regions are indicated. Naturally occurring alanines in the V3 of HXB2-JRFL that were mutated to glycine are indicated in bold-face. (C) Coreceptor utilization by the proviral clones as measured by p24 levels in virus-infected cultures at day 7. The results represent the means and standard deviations (SD) of data from three independent experiments.

Fig. 1

(A) Schematic drawing of the proviral clones used in this study. The open rectangle represents sequences from the subtype B molecular clone HXB2RU3. The hatched rectangle in ConB represents the consensus V3 sequence of HIV-1 subtype B NSI viruses in the HXB2RU3 backbone. The filled rectangle in HXB2-JRFL represents the SalI-BamHI env fragment (positions 5789 to 8451) derived from the R5 subtype B strain JRFL. The EspI (8840) site used in the cloning procedures is also indicated. (B) V3 sequences of the proviral clones, based on the JRFL numbering. Stem (positions 297–305 and 321–329) and crown (positions 306–320) regions are indicated. Naturally occurring alanines in the V3 of HXB2-JRFL that were mutated to glycine are indicated in bold-face. (C) Coreceptor utilization by the proviral clones as measured by p24 levels in virus-infected cultures at day 7. The results represent the means and standard deviations (SD) of data from three independent experiments.

Fig. 2

Effect of V3 and C4 mutations on CCR5-dependent cell-cell fusion as measured by a reporter gene activation assay. β-Gal activity is expressed as a percentage of that seen with the wild-type (WT) clone, HXB2-JRFL. White bars indicate values (mean plus one standard deviation) less than 50%. Gray bars indicate mean values that are 50%. Data shown represent the means and standard deviations of data from at least three independent experiments.

Fig. 3

(A) Effect on gp120-sCD4 binding, as measured by ELISA, for V3 and C4 mutations that were found to reduce cell-cell fusion by approximately 2-fold or more. The gray bar indicates a mean value less than 50% of WT. Data shown represent the means and SD of data from three independent experiments. (B) A representative western blot of envelope proteins in viral lysates for V3 and C4 mutants that decreased CCR5-dependent cell fusion by approximately 2-fold or more. Pooled sera from individuals infected with HIV-1 subtype B was used to detect the expression of envelope proteins. The positions of the gp120 and p24 bands are labeled.

Fig. 3

(A) Effect on gp120-sCD4 binding, as measured by ELISA, for V3 and C4 mutations that were found to reduce cell-cell fusion by approximately 2-fold or more. The gray bar indicates a mean value less than 50% of WT. Data shown represent the means and SD of data from three independent experiments. (B) A representative western blot of envelope proteins in viral lysates for V3 and C4 mutants that decreased CCR5-dependent cell fusion by approximately 2-fold or more. Pooled sera from individuals infected with HIV-1 subtype B was used to detect the expression of envelope proteins. The positions of the gp120 and p24 bands are labeled.

Fig. 4

Effect of V3 and C4 mutations on CCR5 binding of soluble gp120-CD4 complexes, as measured by cell-based ELISA. Values are expressed as a percentage of WT. White bars indicate values (mean plus one standard deviation) less than 50%. Gray bars indicate mean values that are approximately 50%. Data shown represent the means and standard deviations of data from at least three independent experiments, with each experiment carried out in duplicate.

Fig. 5

Effect of V3 and C4 mutations on virus infection, as measured by p24 levels in virus-infected cultures at day 7. White bars indicate values (mean plus one standard deviation) less than 50%. Gray bars indicate mean values that are approximately 50%. Data shown represent the means and standard deviations of data from at least two independent experiments, with each experiment carried out in duplicate.