Article Figures & Data
Figures
- FIG. 1.
Coreceptor usage of HIV-1 group M and group O isolates. The ability of the expanded virus stocks to utilize the major coreceptors CXCR4 and/or CCR5 for infection was examined on TZM-bl reporter cells in the presence or absence of the indicated coreceptor antagonists (see Materials and Methods for details). For each isolate, luciferase counts determined for lysates from untreated, infected cells were set to 100%. Shown are the arithmetic means ± SDs (n = 4).
- FIG. 2.
Replication of HIV-1 group M and group O viruses in single-donor PBMCs, a PBMC donor pool, and a culture of human tonsil tissue ex vivo. PBMCs from a single donor (A), a pool of PBMCs from four different donors (B), and an HLAC from a routine therapeutic tonsillectomy (C) were infected in triplicates with identical amounts of prototypic HIV-1 M strains (X4: NL4-3; R5: 49.5), HIV-1 group O primary isolates (X4: 2901; R5: 1639, 2549, 6778, 8913, 9435, 13127, and 13740), or HIV-1 group M primary isolates (X4: 2005, 2044, 2044, and ELI; R5: IN22, RU570, RW9, and D117) (each with 5 ng of p24 per well). Viral replication was monitored by assessing the concentration of p24 in the culture medium between successive medium changes on days 1, 4, 7, and 11 p.i. and is depicted for six selected viruses (left panels). Given are the arithmetic means ± SDs from one of three to four different donors analyzed for each primary cell system. The corresponding cumulative p24 production over the culture period was determined for each virus as the AUC of the mean replication kinetic (left panels), in principle as reported previously (6, 26), and is shown in the panels on the right.
- FIG. 3.
Depletion of CD4 T cells in HLACs from tonsil by HIV-1 group M and O viruses. CD4 T-cell depletion in infected HLACs (HLAC donor 4) was assessed on day 11 p.i. by measuring the relative percentage of CD4 T cells and CD8 T cells by flow cytometry. (A) FACS dot plots of dispersed HLACs costained for CD3-phycoerythrin and CD8-allophycocyanin. Viable lymphocytes were identified by forward scatter (FSC) and side scatter (SSC) characteristics (gate R1) and then classified as CD4 T cells (CD3+ CD8−) or CD8 T cells (CD3+ CD8+). The relative percentage of these T-cell subsets of all lymphocytes is indicated next to the FACS gates. CD3− CD8− cells to large extent represent B cells and NK cells (data not shown). (B) The CD4/CD8 ratios of HLACs infected with the different HIV-1 strains and primary isolates were calculated and plotted as percent CD4 T-cell depletion relative to the ratios observed in mock-infected cultures. Shown are the arithmetic means ± SDs from one of four different donors analyzed for each virus, performed in triplicate.
- FIG. 4.
HIV-1 group O isolates 2549 and 13740 do not replicate on CCR5-negative A3.01 T cells. (A) Human A3.01 T cells (CD4+ CXCR4+ CCR5−) or (B) a blasted PBMC donor pool were challenged with either the HIV-1 group O isolates 2901, 2549, or 13740 or with the group M reference viruses NL4-3 (X4) or 49.5 (R5) (5 ng of p24 per well). After overnight infection, washed cells were cultivated for 11 to 12 more days, and replication was monitored at the indicated time points by quantification of the p24 concentration in culture supernatants. Shown are the arithmetic means ± SDs (n = 3).
- FIG. 5.
Cross-donor and HIV-1 group-stratified analyses of HIV-1 replication. The analysis of HIV-1 replication kinetics based on p24 concentrations in culture supernatants and corresponding AUC determinations were performed on primary cells from multiple donors as described in the legend to Fig. 2. Box plots were used as a descriptive statistical representation of the AUC values obtained for each virus (A) in a combined analysis of results for single-donor PBMCs (n = 3) and multidonor pools of PBMCs (n = 3) and in a combined analysis of results for HLACs established from four different donors (C). A stratification of results in panels A and C was performed according to the viruses' HIV-1 group classification and is represented as box plots for PBMCs (B) and HLACs (D). Box plots depict the median (horizontal line) and upper and lower quartiles. Whiskers define the border for the 1.5× interquartile distance. Circles represent outliers. Values given within panels B and D represent the relative differences between all group M and group O viruses analyzed: M > O, 7.9-fold (PBMCs; P = 7.8 × 10−8) (B) and 5.2-fold (HLACs; P = 2.9 × 10−5) (D).
- FIG. 6.
Cross-donor, HIV-1 group-, and coreceptor-stratified analyses of HIV-1-mediated CD4 T-cell depletion. CD4 T-cell depletion in HIV-infected tonsil HLACs from four patients was quantified as described in the legend to Fig. 3. Combined depletion analysis of results for individual R5 viruses (n = 14) (A) or X4 viruses (n = 5) (B) from prototypic group M strains, group O primary isolates, or group M primary isolates are represented as box plots. (C) Stratification of results in panels A and B according to the viruses' coreceptor usage (C) or HIV-1 group classification (D). ***, P < 0.0001; n.s., not significant.
- FIG. 7.
R5 primary isolates from HIV-1 groups O and M differ strikingly in their relationship of CD4 T-cell depletion and viral replication in ex vivo cultures from human tonsil. Mean values for the cumulative p24 production (AUC) (Fig. 5C) and for the cumulative CD4 T-cell depletion (Fig. 6A and B) of individual viruses in HIV-infected tonsil HLACs from four patients were plotted against each other and are shown for R5 viruses (A) or X4 viruses (B). Each elliptical symbol depicts one isolate and is numbered according to increasing values for cumulative CD4 T-cell depletion (group O) or increasing values for cumulative p24 production (group M). Lines within the elliptical symbols reflect the SD of values calculated for both parameters.
Tables
- TABLE 1.
Classification and characterization of virus stocks of HIV-1 group M and group O isolates used in this study
Virus isolate Group Subtype Coreceptor usagea p24 CA level (ng/ml)b TCID50 (IU/ml)c Infectivity (IU/ng of p24 CA)d NL4-3 M B X4 35,144 68,080 1.94 2005 M B X4 3,827 1,540 0.40 2044 M B X4 1,743 1,778 1.02 ELI M D X4 4,570 178 0.04 JR-FL M B R5 4,315 41,248 9.56 Ba-L M B R5 9,400 316,228 33.64 IN22 M C R5 1,448 2,610 1.80 RU570 M G R5 442 562 1.27 RW9 M A R5 440 1,000 2.27 D117 M B R5 2,870 1,778 0.62 2901 O X4 55 1,540 28.00 1639 O R5 56 562 10.04 2549 O R5 117 562 4.80 6778 O R5 405 178 0.44 8913 O R5 58 178 3.07 13127 O R5 193 3,162 16.38 13740 O R5 124 562 4.53 9435 O R5 500 ND ND ↵ a Coreceptor usage was determined as described in Materials and Methods, and representative data are shown in Fig. 1.
↵ b CA, capsid protein.
↵ c The infectious titer was determined on a pool of phytohemagglutinin/IL-2-activated PBMCs from six donors by end-point limiting dilution 5 days after inoculation as reported previously (32). ND, not determined; IU, infectious units.
↵ d The arithmetic mean ± standard error of the mean of relative infectivity (IU/ng of p24 CA) was 5.26 ± 3.27 for group M isolates and 9.6 ± 3.65 for group O isolates. ND, not determined; IU, infectious units.
