CXCR4-using viruses, but not R5 viruses, exhibit differences in their sensitivity to chemokines in the course of HIV-1 infection (Related to Fig 1).
A and B Inhibition by CXCL12 of early (viruses # 1 and 16) and late (viruses # 6 and 28) Envs from PBMCs of two patients of the ACS (P#39 (A) and P#208 (B)) (Related to Fig 1A). Data points (means ± SEM of triplicate determinations) are expressed as percent infection of CD4TL relative to control infection measured in the absence of CXCL12 (100%) and were fitted to a sigmoidal dose-response model with a variable slope. IC50s of CXCL12 were derived from inhibition curves using GraphPad Prism 6. Representative experiments out of at least three independent experiments carried out on CD4TL from distinct healthy donors are shown. C and D Percent infection of PHA/IL-2-activated PBMCs from healthy donors with 20 ng p24 of viruses pseudotyped with Envs isolated at the stage of PHI or the early or late R5 Envs shown in Fig 1B, in the presence of 20 nM CCL3 (C) or 10 nM CCL5 (D). Each data point represents the mean infection measured for a given virus (n = 3 determinations), expressed relative to infection in the absence of chemokine (100%). One (C) and two (D) out of three independent experiments are shown. Error bars represent the SD to the means. ns, not significant; **P < .01 in the Mann-Whitney test. E Coreceptor usage of recombinant virus populations pseudotyped with the Envs depicted in Fig 1C, isolated at the time of diagnosis, at the chronic or late stage of infection. Results (means ± SEM of triplicate determinations) represent infectivity of viruses measured 48 h post-inoculation of R5X4JT cells (with 20 ng of p24), in the presence or absence (black bars, 100%) of 10 μM maraviroc (MVC, light green bars), 10 μM AMD3100 (AMD, orange bars) or a mixture of both antagonists at 10 μM each (pink bars). A representative experiment out of three is shown. Group-1 and group-2 refer to late Envs that are not, or are, significantly more resistant to inhibition by CXCL12, as compared to chronic Envs and NL4-3. F Correlation analysis (Spearman two-tailed test) between the IC50s of CXCL12 for inhibiting the plasma-derived Envs shown in Fig 1C and the CD4TL count in the blood of patients. Error bars are means ± SEM of 2 to 3 independent determinations. ns, not significant. G Coreceptor usage of recombinant viruses pseudotyped with the Envs depicted in Fig 1D, isolated at the stage of PHI. Results (means ± SEM of triplicate determinations) represent infectivity of viruses measured 48 h post-inoculation of activated CD4TL (with 20 ng of p24), in the presence or absence (black bars, 100%) of 10 μM maraviroc (MVC, light green bars), 10 μM AMD3100 (AMD, orange bars) or a mixture of both antagonists at 10 μM each (pink bars). Luciferase activity in the lysates of infected cells, expressed as relative light units (RLU) was used to quantify virus infectivity. One representative experiment out of three is shown. H Inhibition by CXCL12 of recombinant viruses pseudotyped with the indicated Envs isolated at the time of PHI. Data points (means ± SEM of triplicate determinations) are expressed as percent infection of activated CD4TL relative to control infection measured in the absence of CXCL12 (100%) and were fitted to a sigmoidal dose-response model with a variable slope. IC50s were calculated using Prism 6. A representative experiment out of three independent experiments carried out on CD4TL from distinct healthy donors is shown. I and J Inhibition by CXCL12 of infection of CD4TL with viruses pseudotyped with chimeric Envs containing gp120 from the early (#1 and #16) or late (#6 and #28) viruses of P#39 (A) and P#208 (B), combined with NL4-3 gp41. Equal amounts of viruses were used (50 ng of Gag p24). Inhibition curves were fitted according to a sigmoidal dose-response model with a variable slope. Data points (means ± SEM of triplicate determinations) are expressed as percent infection of CD4TL relative to control infection measured in the absence of CXCL12 (100%). Representative experiments out of three independent experiments carried out on CD4TL from distinct donors are shown. K Fusion of chimeric viruses containing late gp120s (red bars) with CD4TL is more resistant to CXCL12 inhibition, compared with viruses with early gp120s (green bars). βLactamase (BLaM)-Vpr-containing viruses (500 ng of Gag p24) were incubated with CD4TL loaded with the fluorescent BLaM substrate CCF2. Fusion was quantified by flow cytometry by counting the number of cells with cleaved CCF2. Results are expressed as percent fusion in the presence of 30 or 300 nM CXCL12 relative to control fusion measured in the absence of the chemokine (100%). Shown are the means ± SEM of two independent experiments carried out with the viruses containing gp120s #1 (early), #6 (late), #16 (early) and #28 (late). **P < .01, Mann-Whitney test.
(PPTX)
