Human erythrocytes selectively bind and enrich infectious HIV-1 virions

Abstract

Although CD4(+) cells represent the major target for HIV infection in blood, claims of complement-independent binding of HIV-1 to erythrocytes and the possible role of Duffy blood group antigen, have generated controversy. To examine the question of binding to erythrocytes, HIV-1 was incubated in vitro with erythrocytes from 30 healthy leukapheresis donors, and binding was determined by p24 analysis and adsorption of HIV-1 with reduction of infectivity for CD4(+) target cells. All of the cells, regardless of blood group type, bound HIV-1 p24. A typical preparation of erythrocytes bound <2.4% of the added p24, but erythrocytes selectively removed essentially all of the viral infectivity as determined by decreased infection of CD4(+) target cells; however, cell-associated HIV-1 was approximately 100-fold more efficient, via trans infection, than unadsorbed virus for infection of CD4(+) cells. All of the bound HIV-1 p24 was released by treatment of the cells with EDTA, and binding was optimized by adding Ca(2+) and Mg(2+) during the washing of erythrocytes containing bound HIV-1. Although the small number of contaminating leukocytes in the erythrocyte preparation also bound HIV-1 p24, there was no significant binding to CD4, and it thus appears that the binding occurred on leukocytes at non-CD4 sites. Furthermore, binding occurred to erythrocyte ghosts from which contaminating leukocytes had been previously removed. The results demonstrate that erythrocytes incubated in vitro with HIV-1 differentially adsorb all of the infectious HIV-1 virions (as opposed to non-infectious or degraded virions) in the absence of complement and independent of blood group, and binding is dependent on divalent cations. By analogy with HIV-1 bound to DC-SIGN on dendritic cells, erythrocyte-bound HIV-1 might comprise an important surface reservoir for trans infection of permissive cells.

Figure 1. Binding of a HIV-1 isolate to erythrocytes.

(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.

Figure 2. Release of erythrocyte-bound HIV-1 by treatment with EDTA.

(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⁺.

Figure 3. HIV-1 binds to erythrocytes, erythrocytic ghosts, and leukocytes.

(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.

Figure 4. HIV-1 bound to the erythrocyte cell preparation is not internalized by contaminating leukocytes at 37°C.

(A) Erythrocytes (donor R9) were first incubated with HIV-1 (isolate 89BZ_167) at 4°C for 2 hr and binding was determined. The cells were then incubated further at either 4°C or 37°C, and at the indicated times 10 ml of 5 mM EDTA (Na salt) were added and p24 associated with the cells was measured. The experiment shown was performed twice with similar results. (B) In a further experiment, containing a positive control, erythrocytes (donor T4) and PBMC were first incubated with HIV-1 (isolate 89BZ_167) either at 4°C or 37°C for 2 hr and binding was determined. The cells were then incubated for 4 hours at either 4°C or 37°C then treated with 5 mM EDTA (Na salt). Cells were washed and p24 associated with the cells was measured.

Figure 5. Erythrocyte preparations selectively adsorb most of the infectious HIV-1 virions.

HIV-1 isolate 89BZ_167 (105,900 pg) was adsorbed with erythrocytes the indicated numbers of times by adding 3.5×10⁹ erythrocytes from the indicated donors. After removing the erythrocytes by centrifugation, non-adsorbed virus in the supernatant was assayed for the ability to infect CD4(+) TZM-bl cells in trans. The relative degree of infection, determined by the mean p24 (± SD of triplicate measurements), was compared with infection by the original free virus (shown as 100%).

Figure 6. Erythrocyte-adsorbed virus is more infectious, based on p24, than free virus.

To compare the infectivity of cell-bound virus with free virus-1, HIV isolate 90US_873 was bound to erythrocytes (sample no. Q6). Increasing numbers of erythrocytes containing bound virus were then added to a PBMC culture, and virus production was determined on day 4 post-infection. The amount of virus production was then compared with that obtained by incubating the original free virus with PBMC. A similar result was obtained in a second experiment with a different donor (no. T1) and HIV-1 isolate 89BZ_167.