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. 2000 Oct;106(8):1039-52.
doi: 10.1172/JCI7953.

Differential coreceptor expression allows for independent evolution of non-syncytium-inducing and syncytium-inducing HIV-1

R P van Rij  1 , H Blaak, J A Visser, M Brouwer, R Rientsma, S Broersen, A M de Roda Husman, H Schuitemaker
Affiliations

Differential coreceptor expression allows for independent evolution of non-syncytium-inducing and syncytium-inducing HIV-1

R P van Rij et al. J Clin Invest. 2000 Oct.

Erratum in

  • J Clin Invest 2000 Dec;106(12):1569

Abstract

We demonstrated previously that CD45RA(+) CD4(+) T cells are infected primarily by syncytium-inducing (SI) HIV-1 variants, whereas CD45RO(+) CD4(+) T cells harbor both non-SI (NSI) and SI HIV-1 variants. Here, we studied evolution of tropism for CD45RA(+) and CD45RO(+) CD4(+) cells, coreceptor usage, and molecular phylogeny of coexisting NSI and SI HIV-1 clones that were isolated from four patients in the period spanning SI conversion. NSI variants were CCR5-restricted and could be isolated throughout infection from CD45RO(+) CD4(+) cells. SI variants seemed to evolve in CD45RO(+) CD4(+) cells, but, in time, SI HIV-1 infection of CD45RA(+) CD4(+) cells equaled infection of CD45RO(+) CD4(+) cells. In parallel with this shift, SI HIV-1 variants first used both coreceptors CCR5 and CXCR4, but eventually lost the ability to use CCR5. Phylogenetically, NSI and SI HIV-1 populations diverged over time. We observed a differential expression of HIV-1 coreceptors within CD45RA(+) and CD45RO(+) cells, which allowed us to isolate virus from purified CCR5(+) CXCR4(-) and CCR5(-) CXCR4(+) CD4(+) cells. The CCR5(+) subset was exclusively infected by CCR5-dependent HIV-1 clones, whereas SI clones were preferentially isolated from the CXCR4(+) subset. The differential expression of HIV-1 coreceptors provides distinct cellular niches for NSI and SI HIV-1, contributing to their coexistence and independent evolutionary pathways.

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Figures

Figure 1
Figure 1
The clinical course of HIV-1 infection by CD4+ T-cell number (filled circles) and serum RNA load (open triangles). Filled triangles at the x axis indicate time points selected for isolation and analysis of virus. Open triangles at the x axis indicate time points of virus isolation after cell sort based on HIV-1–coreceptor expression. SI conversion occurred 16, 62, 14, and 75 months after seroconversion in patient ACH039, ACH171, ACH208, and ACH490, respectively.
Figure 2
Figure 2
Tropism for CD45RA+ and CD45RO+ CD4+ T cells during NSI to SI HIV-1 conversion. Patient PBMCs were FACS sorted in CD45RA+ CD45RO– CD4+ and CD45RA– CD45RO+ CD4+ T cells, and clonal virus isolation was performed on these T-cell subsets. (a) The frequency of infected cells with either NSI or SI HIV-1 variants (NSI or SI load) in total (top), CD45RO+ (middle), and CD45RA+ (bottom) CD4+ T cells relative to SI conversion in patient ACH171 (left panels) and 490 (right panels). Filled symbols indicate SI clones, and open symbols indicate NSI clones. In the top panel, serum RNA load is indicated by triangles. (b) Ratio of NSI load in CD45RO+ to NSI load in CD45RA+ CD4+ T cells (left) and ratio of SI load in CD45RA+ to SI load in CD45RO+ CD4+ T cells (right) of patient ACH171 (filled circles) and ACH490 (open circles). (c) Prevalence of SI phenotype among HIV-1 clones isolated from CD45RA+ (circles) or CD45RO+ (squares) CD4+ T-cell subset in ACH171 and ACH490.
Figure 3
Figure 3
Longitudinal analysis of coreceptor usage of NSI and SI HIV-1 clones. Based on the ability to infect MT2 cells, viral clones were subdivided in NSI (left panels) and in SI (right panels) phenotypes. Coreceptor usage was tested by the ability to productively infect U87 cells transfected with CD4 and one of the HIV-1 coreceptors CCR3, CCR5, or CXCR4 after cell-free infection of these cells. Bars indicate in vitro specificity for HIV-1 coreceptors: CCR5 (R5), open bars; CCR5 and CXCR4 (R5X4), gray bars; CCR3, CCR5, and CXCR4 (R3R5X4), light gray bars; CCR3 and CXCR4 (R3X4), dark gray bars; and CXCR4 (X4), filled bars.
Figure 4
Figure 4
Envelope gp120 V3 sequence analyses of NSI and SI HIV-1. (a) Sequence alignment of the deduced amino acid sequences of the gp120 V3 loop. Sequences were aligned with the consensus sequence of the first time point from each patient. SI capacity in MT2 cells, coreceptor usage in U87 cells, the ability to infect CCR5 Δ32/Δ32 PBMCs, and the absolute number of positively charged and negatively charged amino acids in the V3 loop are indicated. Although more sequence data are available, the V3 loop is depicted for clarity only. (b) Number of positively charged amino acids (pos.) and net positive charge (net.) of the gp120 V3 loop of biological clones by coreceptor usage. Horizontal bars reflect median values. R3R5X4 clones are combined with R5X4 clones and R3X4 clones with X4-restricted HIV-1 clones. For patient ACH490, clones with a discordant phenotype (MT2 tropic, but unable to infect CCR5 Δ32/Δ32 PBMCs [R5MT2+]) are also shown.
Figure 5
Figure 5
Molecular phylogeny of NSI and SI HIV-1 based on gp120 V3-spanning region. (a) Neighbor joining phylogenetic trees of gp120 V3 regions. Bootstrap values, based on 100 bootstrap analyses, indicate in how many replicate trees the depicted clustering is observed. Coreceptor usage of the clones is indicated by the color of the symbols: R5, open symbols; R3R5X4 or R5X4, light-gray symbols; R3X4, dark-gray symbols; and X4-restricted clones, filled symbols. Asterisks indicate HIV-1 clones that were MT2 tropic but were unable to infect U87-CXCR4 cells or CCR5 Δ32/Δ32 PBMCs. (b) Pairwise hamming distances between R5 clones to R5 clones (R5), between R5 clones to R5X4 clones (R5X4), and between R5 clones to X4 clones (X4). Uncorrected hamming distances between each pair of clones were displayed in a box plot, depicting tenth, 25th, 50th, 75th, and 90th percentiles. Circles depict fifth and 95th percentiles.
Figure 6
Figure 6
SI HIV-1 clones isolated from CD45RA+ and CD45RO+ CD4+ T cells are phenotypically and evolutionarily indistinguishable. (a) Coreceptor usage of SI clones isolated from CD45RA+ and CD45RO+ CD4+ T cells from ACH171 and ACH490. Virus clones were pooled from the time points in which SI clones were obtained from both subsets (10.5, 19.5, and 27 months for ACH171 and 4.4, 19.2, and 25.1 months for ACH490). Bar descriptions are as in Figure 3. (b) Pairwise hamming distances were calculated between clones obtained from CD45RA+ CD4+ T cells (RA vs. RA), between clones obtained from CD45RO+ CD4+ T cells (RO vs. RO), and between clones isolated from CD45RA+ to clones from CD45RO+ CD4+ T cells (RA vs. RO). Uncorrected hamming distances between each pair of sequences were calculated and are depicted in a box plot as described in Figure 5.
Figure 7
Figure 7
Expression of CCR5 and CXCR4 on CD45RO– and CD45RO+ CD4+ T cells. Cryopreserved PBMCs from HIV-1–infected patients and a healthy donor were incubated with mAb’s against CD4, CD45RO, CCR5, and CXCR4. The CD45RO– (top) and CD45RO+ CD4+ T cells (bottom) were gated. Within these subsets, CCR5 and CXCR4 expression was determined. Numbers in the figures depict percentage of gated cells in the corresponding quadrants. A representative healthy donor from eight unrelated donors is shown.
Figure 8
Figure 8
CD4+ T-cell subsets with distinct HIV-1–coreceptor expression provide distinct niches for NSI and SI HIV-1. Cryopreserved PBMCs were sorted based on expression of CD45RO and HIV-1 coreceptors CCR5 and CXCR4, and virus was isolated by cocultivation with PHA-PBMCs under limiting dilution conditions. In addition to SI-infected patients ACH039, ACH171, ACH208, and ACH490, one patient (ACH001) who was infected solely with NSI HIV-1 variants, was studied. (a) Frequency of CD45RO– CXCR4+ CCR5–; CD45RO+ CXCR4+ CCR5–; and CD45RO+ CXCR4– CCR5+ CD4+ T cells productively infected with HIV-1. NT, not tested. (b and c) Phenotype of virus clones from CD45RO– CXCR4+ (left panel), CD45RO+ CXCR4+ (middle), and CD45RO+ CCR5+ (right) cells as defined by the ability to infect MT2 cells (b) or CCR5Δ32/Δ32 PBMCs (c). Filled bars reflect the percentage of clones able to infect the indicator cells, whereas gray bars reflect clones unable to infect these cells. (d) The difference in the proportion of clones with SI phenotype (left panel) and clones able to infect CCR5 Δ32/Δ32 PBMCs (right panel) isolated from CD45RO+ CXCR4+ and CD45RO+ CCR5+ cells were compared pairwise by Wilcoxon signed rank test (P = 0.07, in both panels).
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