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. 2004 Jan;78(1):524-30.
doi: 10.1128/jvi.78.1.524-530.2004.

Intrapatient alterations in the human immunodeficiency virus type 1 gp120 V1V2 and V3 regions differentially modulate coreceptor usage, virus inhibition by CC/CXC chemokines, soluble CD4, and the b12 and 2G12 monoclonal antibodies

Alexey A Nabatov  1 , Georgios Pollakis, Thomas Linnemann, Aletta Kliphius, Moustapha I M Chalaby, William A Paxton
Affiliations

Intrapatient alterations in the human immunodeficiency virus type 1 gp120 V1V2 and V3 regions differentially modulate coreceptor usage, virus inhibition by CC/CXC chemokines, soluble CD4, and the b12 and 2G12 monoclonal antibodies

Alexey A Nabatov et al. J Virol. 2004 Jan.

Abstract

We studied human immunodeficiency virus type 1 (HIV-1) chimeric viruses altering in their gp120 V1V2 and V3 envelope regions to better map which genetic alterations are associated with specific virus phenotypes associated with HIV-1 disease progression. The V1V2 and V3 regions studied were based on viruses isolated from an individual with progressing HIV-1 disease. Higher V3 charges were linked with CXCR4 usage, but only when considered within a specific V1V2 and V3 N-linked glycosylation context. When the virus gained R5X4 dual tropism, irrespective of its V3 charge, it became highly resistant to inhibition by RANTES and highly sensitive to inhibition by SDF-1alpha. R5 viruses with higher positive V3 charges were more sensitive to inhibition by RANTES, while R5X4 dualtropic viruses with higher positive V3 charges were more resistant to inhibition by SDF-1alpha. Loss of the V3 N-linked glycosylation event rendered the virus more resistant to inhibition by SDF-1alpha. The same alterations in the V1V2 and V3 regions influenced the extent to which the viruses were neutralized with soluble CD4, as well as monoclonal antibodies b12 and 2G12, but not monoclonal antibody 2F5. These results further identify a complex set of alterations within the V1V2 and V3 regions of HIV-1 that can be selected in the host via alterations of coreceptor usage, CC/CXC chemokine inhibition, CD4 binding, and antibody neutralization.

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Figures

FIG. 1.
FIG. 1.
Viruses used in this study and their coreceptor usage profiles. (A) Schematic representation of the chimeric molecularly cloned viruses generated from patient ACH168 and used in this study (11, 12, 38). (B) Coreceptor usage patterns of the viruses on U87.CD4 cells expressing CCR3, CCR5, or CXCR4. CCR3 is represented by grey bars, CCR5 is represented by white bars, and CXCR4 is represented by black bars. Cells of the U87.CD4+ line (3.0 × 104) expressing CCR3, CCR5, or CXCR4 were infected with the viruses from each panel (0.1 to 1 μg of p24/ml). Cells were infected for 18 h before being washed twice with phosphate-buffered saline and fed with fresh medium. On day 10 of culture, the p24 levels in the culture supernatants were determined by using a standard enzyme-linked immunosorbent assay. Data are expressed as mean p24 values of triplicate wells, and the intersample standard deviation did not exceed 10%.
FIG. 2.
FIG. 2.
CC/CXC chemokine inhibitions. (A) Bulk inhibition of the X (R5) panel of viruses with a high concentration of RANTES (200 nM) and of the X.10 (R5X4) panel of viruses with a high concentration of RANTES (200 nM) or SDF-1α (2 μg/ml). The experiments were performed with CD4+-enriched lymphocytes isolated from a CCR5+/+ individual. The white bars represent percent inhibition by RANTES, and the black bars represent percent inhibition by SDF-1α. (B) Inhibition of the X and X.10 panels of viruses carrying the different V3 charges (symbols: diamonds, +3 V3 charge; squares, +4 V3 charge; triangles, +5 V3 charge; circles, +6 V3 charge) by RANTES and SDF-1α, respectively. (C) Inhibition of the +5 V3 viruses carrying the different glycosylation pattern (symbols: diamonds, X.10; squares, X.10ΔgV1; triangles, X.10ΔgV3). CD4+-enriched lymphocytes isolated from a CCR5+/+ individual were used in the RANTES inhibition assays, while CD4+-enriched lymphocytes isolate from a CCR5−/− individual were used in the SDF-1α inhibition experiments. The respective CD4+ lymphocytes (2.0 × 105) were preincubated with serial dilutions of the chemokine for 1 h, and then the viruses were added. The day chosen for calculating inhibiting response was based on the day the p24 value of the positive control well peaked (not longer than 14 days), and percent inhibition was calculated by determining the reduction in p24 production in the presence of the agent compared to that for the cultures with virus only. For each inhibition experiment, a positive control, virus incubated with cells in the absence of the agent, and a negative control, virus in the absence of cells, was included. The negative control p24 concentration was subtracted from all of the test results. Each experiment was repeated at least twice with CD4+-enriched lymphocytes from different donors. Data are expressed as mean p24 values of triplicate wells, and the intersample SD did not exceed 10%.
FIG. 3.
FIG. 3.
sCD4 neutralization of viruses altering in V3 charge and pattern of gp120 N-linked glycosylation (symbols: closed circles, X; open circles, X.10; open squares, X.10ΔgV1; closed squares, X.10ΔgV3). The assays were carried out by using a scheme similar to that described for the chemokine inhibition assay (Fig. 2) but with preincubation of the viruses with sCD4, followed by addition of CD4+-enriched lymphocytes.
FIG. 4.
FIG. 4.
Virus neutralization by MAbs 2F5, b12, and 2G12. The +5 V3 array of viruses (symbols: closed circles, X; open circles, X.10; open squares, X.10ΔgV1; closed squares, X.10ΔgV3) were tested for neutralization by MAbs 2F5 (A), b12 (B), and 2G12 (C). Other MAbs were tested (15e, 17b, and 48d), but none was shown to possess neutralizing activity (data not shown). The assays were carried out by using the same scheme as described for the sCD4-mediated neutralization assay.
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