Fractionation of blood from HIV-1-infected patients for obtaining plasma and RBC_native in the presence or absence of EDTA.

<p>Whole blood collected from a patient in the presence (A,B) or absence (C,D) of EDTA, and separated into four plasma and erythrocyte fractions, A, B, C, and D, as shown. Fraction D was treated with 5 mM EDTA, resulting in Fraction E. All of the samples were used immediately for co-culture with PBMC to detect capability for causing HIV-1 infection of the PBMC. Aliquots were stored at −20°C for viral RNA measurements. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081002#s4" target="_blank">Materials and Methods</a> for further details.</p

HIV-1 cell-cell infection of PBMC is dependent on PLT.

<p><b>A</b>. RBC, PLT-RBC, and PLT were separated by sorting with FACS as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081002#pone-0081002-g003" target="_blank">Fig. 3B</a>. After incubation with HIV-1_Bal, followed by washing 3 times to remove unbound HIV-1, the cells were co-incubated with PBMC to determine HIV-1 infection. **Cell-cell infection with PLT-bound HIV was higher than with RBC-bound HIV (p = 0.0332) (one-way Anova with Tukey's multiple comparisons test); ***cell-cell infection with PLT-RBC-bound HIV was higher than with RBC-bound HIV (p = 0.0124) (one-way ANOVA with Tukey's multiple comparisons test). <b>B</b>. Mixtures of normal RBC_native enriched with PLT-RBC were prepared by pre-incubation of 0.3 ml containing 10⁹ normal RBC_native with the indicated numbers of PLT in 0.3 ml RPMI dilutions of platelet-rich plasma, and then washed 3 times. The cells were then pre-incubated with HIV-1_Bal, washed 3 times, and examined for HIV-1 infection of PBMC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081002#s4" target="_blank">Materials and Methods</a>. Normal RBC_native not pre-incubated with PRP served as a control. *Cell-cell infection was increased as a function of the number of platelets added to RBC_native (p<0.0001, one way Anova).</p

Enrichment of PLT-RBC as a subpopulation of normal (uninfected) cells by adding platelets to a preparation of RBC_native from a normal volunteer.

<p>A. Normal RBC_native obtained from citrated blood from a normal (uninfected) volunteer were incubated with platelet-rich plasma, washed, and analyzed by flow cytometry. B. RBC, PLT-RBC, and PLT were separated by sorting the populations shown in frame A. C–E. Light microscopy of Giemsa-stained mixtures of normal RBC_native enriched with PLT-RBC. Magnification: 400X and 1000X, respectively. Arrows in frame C indicate platelets bound to RBC. F. Fluorescent microscopy of PLT-RBC visualized at 630X magnification. Left panel, bright field; 2^nd panel, visualization of both FITC and PE signals; 3^rd panel, visualization of FITC; right panel, visualization of PE signal. Arrows point to platelets.</p

Cell-cell infection of PBMC by RBC_native from HIV-positive patients.

<p><b>A</b>. EDTA-free RBC_native from 11 chronically-infected patients were obtained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081002#pone-0081002-g001" target="_blank">Fig. 1D</a> and co-incubated with PBMC to examine infection of the PBMC. 25 µl of EDTA-free RBC_native were diluted in 75 µl of IL-2 medium and added to 50 µl of 1.5×10⁵ PHA-stimulated PBMC/well in IL-2 medium. <b>B</b>. 100 µl of EDTA-free plasmas from the patients, obtained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081002#pone-0081002-g001" target="_blank">Fig. 1C</a> and supplemented with 20 U/ml of recombinant IL-2, did not infect co-incubated PBMC. <b>C</b>. The RBC_native shown in frame A were treated with 5 mM EDTA followed by washing 3 times in IL-2 medium before infecting PBMCs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081002#pone-0081002-g001" target="_blank">Fig. 1E</a>) showed no HIV-1 infection of co-incubated PBMC. The infection exhibited by the group of EDTA-free RBC_native (<b>A</b>) was significantly higher using a paired t-test, than the infection exhibited both by the group of EDTA-free plasma (<b>B</b>) and by the group of EDTA-stripped RBC_native (<b>C</b>) at 8 days post-infection (p = 0.0404) and 10 days post-infection (p = 0.038).</p

Binding of HIV-1 to RBC_native as well as cell-cell infection of PBMC is inhibited by anti-DC-SIGN monoclonal antibody.

<p>A. RBC_native was pre-incubated with HIV-1_Bal in the absence or presence of anti-DC-SIGN mAb in a final 50 µg/ml concentration, washed 3 times, and bound p24 was measured by ELISA. The binding was inhibited by pre-incubation of the cells with the monoclonal antibody (p<0.01, t-test), but not inhibited by pre-incubation of the virus with the monoclonal antibody. B. Cell-cell infection of PBMC was also inhibited in a dose-dependent manner by anti-DC-SIGN mAb (p<0.02 and p<0.01 respectively, paired t-test).</p

HIV-1 RNA in plasma and RBC_native from chronically infected HIV-1 patients.

a,b,c,d<p>Fractions were obtained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081002#pone-0081002-g001" target="_blank">Fig. 1A, 1B, 1C, and 1D</a>, respectively.</p

PLT-RBC is a subpopulation of cells in whole blood and in preparations of RBC_native from HIV-1-infected patients.

<p>A–B. Flow cytometry was performed with EDTA-anticoagulated whole blood obtained from an HIV-positive individual. A. Representative dot plot shows scatter properties of the RBC, PLT-RBC, and PLT. CD41a+/CD235a+ cells (red) represent PLT-RBC; CD41a+/CD235a- cells (green) represent free PLT; CD41a-/CD235a+ cells (blue) represent RBC; and CD41a-/CD235a- particles (gray) represent small vesicles or debris. B. Quantification of cells shown in frame A that express CD41a and/or CD235a. C–D. Flow cytometry was performed with an EDTA-free RBC_native preparation (see fraction D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081002#pone-0081002-g001" target="_blank">Figure 1</a>) obtained from an HIV-positive individual. RBC_native were analyzed as in frames A–B.</p

HIV-1 binds to erythrocytes, erythrocytic ghosts, and leukocytes.

<p>(A) After incubation of 151,286 pg of HIV isolate 89BZ_167 with the indicated preparation containing 2.5×10⁹ erythrocytes, the mean p24 bound (± SD of triplicate measurements) was determined. The erythrocytes were then hemolyzed and the p24 bound to the ghosts and the leukocytes was separately measured. (B) After incubation of 105,900 pg of HIV-1 isolate 89BZ_167 with the indicated erythrocyte preparation (3.5×10⁹ erythrocytes), or with purified ghosts previously depleted of leukocytes, from each donor, the mean bound p24 (± SD of triplicate measurements) was determined.</p

Binding of a HIV-1 isolate to erythrocytes.

<p>(A) Increasing amounts of HIV-1 isolate 90US_873 (as quantified by p24) were incubated with 5×10⁷ erythrocytes (donor Q6), and binding of p24 to the cells was determined. (B) Dose-dependent binding of the HIV-1 isolate (8,486 pg p24) with increasing numbers of erythrocytes. The experiment shown is representative of 3 separate experiments. In each experiment HIV-1 was bound to erythrocytes in triplicate, washed, and the triplicates were pooled for p24 determination.</p

Release of erythrocyte-bound HIV-1 by treatment with EDTA.

<p>(A) HIV-1 isolate 89BZ_167 (75,643 pg) was incubated with 5×10⁸ erythrocytes (donor S4) in RPMI, washed three times, and then treated with either EDTA or RPMI (no EDTA). The bound HIV-1 was completely removed by treatment with EDTA. The dashed line shows the limit of detection of the p24 assay. (B) After incubation of HIV-1 with erythrocytes from 3 different donors, following washing, the cells were incubated with different amounts of EDTA (or no EDTA), and washed either in the absence or presence (control) of Ca²⁺ and Mg²⁺.</p