CCR5, GPR15, and CXCR6 are major coreceptors of human immunodeficiency virus type 2 variants isolated from individuals with and without plasma viremia

Abstract

Human immunodeficiency virus type 2 (HIV-2) is generally considered capable of using a broad range of coreceptors. Since HIV-2 variants from individuals with nonprogressive infection were not studied previously, the possibility that broad coreceptor usage is a property of variants associated with progressive infection could not be excluded. To test this, we determined the coreceptor usage of 43 HIV-2 variants isolated from six long-term-infected individuals with undetectable plasma viremia. Using GHOST indicator cells, we showed for the first time that the only coreceptors efficiently used by low-pathogenic HIV-2 variants are CCR5, GPR15 (BOB), and CXCR6 (BONZO). Surprisingly, control HIV-2 variants (n = 45) isolated from seven viremic individuals also mainly used these three coreceptors, whereas use of CCR1, CCR2b, or CCR3 was rare. Nearly a quarter of all HIV-2 variants tested could infect the parental GHOST cells, which could be partially explained by CXCR4 usage. Use of CXCR4 was observed only for HIV-2 variants from viremic individuals. Thirty-eight variants from aviremic and viremic HIV-2-infected individuals were additionally tested in U87 cells. All except one were capable of infecting the parental U87 cells, often with high efficiency. When virus production in parental cells was regarded as background in the coreceptor-transduced cell lines, the results in U87 cells were largely in agreement with the findings in GHOST cells. HIV-2 isolates from aviremic individuals commonly use as coreceptors CCR5, GPR15, and CXCR6, as well as an unidentified receptor expressed by U87 cells. Broad coreceptor usage, therefore, does not appear to be associated with pathogenicity of HIV-2.

FIG. 1.

Evaluation of GHOST cells. (A) GFP-expression was analyzed by flow cytometry. Three days postinfection, the cells were harvested and live cells were gated based on their forward and side scatter. Instrument settings were adjusted to reduce the mean background signals (caused by constitutive expression of GFP) to <10¹, and the negative cutoff was set to 0.15% using mock-infected cells. The values obtained were used to calculate the RTCN. Shown are two examples of GHOST-CCR5 cells infected with two different HIV-2 variants. (B) To compare GFP expression (RTCN) with p26 production, GHOST-CCR5 (solid circles), GHOST-CXCR6 (darkly shaded circles), GHOST-CCR2b (lightly shaded circles), and GHOST-CCR3 (open circles) cells were infected with 11 HIV-2 variants, each virus-cell combination in two parallel cultures. Supernatants were harvested and stored on days 0 and 3 (culture A) and days 5 and 7 (culture B) and analyzed for p26 content using a SIV core antigen enzyme-linked immunosorbent assay (Beckman-Coulter Netherlands BV, Mijdrecht, The Netherlands). Culture A was terminated on day 3, when the cells were harvested for analysis of GFP expression. The RTCN and p26 concentrations are shown for four representative viruses (i, RH2-1 A8; ii, RH2-14 1D1; iii, RH2-3 4C5; iv, RH2-4 4B2). (C) The GHOST assay is highly reproducible. Shown are the results of infection with a control X4 HIV-1 variant (ACH208) that was included in multiple independent experiments.

FIG. 2.

Correlation between RTCN and percentages of CXCR4⁺ cells. Eight GHOST cell lines were exposed to an HIV-1 X4-restricted variant, and the RTCN, reflecting virus production, was determined. Each symbol represents a cell line transduced with the indicated chemokine receptor. Parental cells are not transduced with a chemokine receptor. The values depicted are the mean RTCN from nine independent experiments. The vertical bars represent the standard errors.

FIG. 3.

T22 sensitivities of HIV-2 variants with tropism for parental GHOST cells. Fourteen HIV-2 variants capable of infecting parental GHOST cells were tested for their sensitivity to T22 (Bachem, Bubendorf, Switzerland). A R3X4 HIV-1 variant was included as a control. The cells were preincubated in 250 μl of medium containing 6 or 0 μM T22 for 2 h, after which 350 μl of virus inoculum was added, resulting in 2.5 or 0 μM T22 and 1 ng of RT ml⁻¹. After infection, the virus was removed, and selection medium containing 3 μM T22 was added. Each bar represents a cell line. The background bars (gray) represent values obtained in the absence of T22. The overlaid bars represent values obtained in the presence of T22 and are therefore not visible when inhibition was complete.

FIG. 4.

HIV-2 coreceptors and phenotypes determined using GHOST cells. (A) The frequency with which chemokine receptors are used as HIV-2 coreceptors is indicated by the percentages of HIV-2 variants capable of using the different chemokine receptors, which are specified on the x axis. Use of a potential unidentified coreceptor expressed by GHOST cells is indicated as GH-CoR. Depicted are the percentages of coreceptor-using variants among all successfully characterized HIV-2 variants (total) and of variants from aviremic and viremic individuals separately. (B) Using GHOST cells in combination with T22, phenotypes were determined; occurrences of the different phenotypes are depicted as a proportion of total HIV-2 variants and of variants from aviremic and viremic individuals separately. The abbreviations X4, R5, G15, and X6 represent the different chemokine receptors used by the variant phenotypes.

FIG. 5.

Infection in parental versus coreceptor-transduced U87 cells. Thirty-seven HIV-2 variants were tested on all U87 cell lines (Table 6), and these variants were grouped based on the level of virus production in the parental cells. The resulting four groups are specified on the x axis, with the number of HIV-2 variants in each group above the bars. Shown are the group median levels of RT production in the coreceptor-transduced U87 cells, with each bar representing a cell line as indicated.

FIG. 6.

Infection of parental U87 cells. The susceptibilities to HIV-2 infection of three batches of parental cells with different origins and culture histories were compared. Shown is the virus production by 13 HIV-2 variants, with each symbol representing a variant. The horizontal bars represent median levels of virus production in each cell batch. The four control HIV-2 variants are indicated by black symbols. The relative susceptibilities of the batches of parental U87 cells were analyzed by assigning ranks based on the level of RT production for each virus individually and comparing the average ranks using the Kendal W test. The dashed line indicates the lower cutoff value of the assay (10 pg/ml).