Human immunodeficiency virus type 1 replication in dendritic cell-T-cell cocultures is increased upon incorporation of host LFA-1 due to higher levels of virus production in immature dendritic cells

Abstract

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here, we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1), we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together, our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore, the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures.

FIG. 1.

HIV-1 production in DC-T-cell cocultures. IM-MDDCs (A) and M-MDDCs (B) (1 × 10⁵ cells) were pulsed for 60 min with similar concentrations of isogenic JR-CSF either lacking or bearing host-derived LFA-1 (2 ng of p24^Gag). After three washes with PBS, the cell-virus mixture was divided into three aliquots and cocultured with autologous CD4⁺ T cells at a DC-to-T-cell ratio of 1:3. Virus production was determined by measuring p24^Gag levels in the culture supernatants at the indicated time points (D.P.I., day postinfection). The data shown are the means ± the standard deviations (SDs) of the results for triplicate samples and are representative of three independent experiments. *, P < 0.05; **, P < 0.01. A 2- to 10-fold increase in virus production at 4 days following initiation of the coculture was observed with LFA-1-bearing virions for the three donors tested.

FIG. 2.

Virus replication in activated CD4⁺ T cells. CD4⁺ T cells (3 × 10⁵ cells) were exposed for 2 h to similar concentrations of isogenic NL4-3balenv, JR-CSF, or NL4-3 either lacking or bearing host-derived ICAM-1 or LFA-1 (15 ng of p24^Gag). Virus production was determined by measuring p24^Gag levels in the culture supernatants at day 3 postinfection. The data shown are the means ± the SDs of the results for triplicate samples and are representative of four independent experiments. ***, P < 0.001.

FIG. 3.

Virus uptake by IM-MDDCs. IM-MDDCs (2 × 10⁵ cells) were pulsed for 60 min with identical concentrations of isogenic JR-CSF virions either lacking or bearing host-derived LFA-1 (20 ng of p24^Gag). After three washes with PBS, the cells were subjected to a hypotonic lysis buffer or analyzed by flow cytometry. The uptake of virions by IM-MDDCs was estimated by measuring the p24^Gag content in cell lysates (A) or quantifying the percentage of IM-MDDCs positive for both cell surface DC-SIGN and intracellular p24 (B). The data shown are the means ± the SDs of the results for triplicate samples, and these results are representative of at least five independent experiments for panel A and two for panel B.

FIG. 4.

Early and late transfer of HIV-1 particles by IM-MDDCs. (A) Schematic representation showing early- and late-transfer processes of HIV-1 by DCs. iDC, immature DCs. (B) IM-MDDCs (1 × 10⁵ cells) were either left untreated (empty bars) or treated with the antiviral agent AZT (10 μM; filled bars) for 10 min. The cells were then pulsed for 60 min with similar concentrations of isogenic NL4-3balenv either lacking or bearing host-encoded LFA-1 (10 ng p24^Gag). After three washes with PBS, the cell-virus mixture was divided into three aliquots and cocultured with autologous CD4⁺ T cells at a DC-to-T-cell ratio of 1:3. Virus production was determined by measuring p24^Gag levels in the culture supernatants at day 3 following initiation of the coculture. (C) The late transfer phase was assayed as follows. First, IM-MDDCs (5 × 10⁵ cells) were pulsed with similar concentrations of isogenic NL4-3balenv either lacking or bearing host-encoded LFA-1 (50 ng p24^Gag). Next, 5 days later, IM-MDDCs were cocultured with autologous CD4⁺ T cells at a DC-to-T-cell ratio of 1:3. Virus production was determined by measuring p24^Gag levels in the culture supernatants at day 6 following initiation of the coculture. The data shown are the means ± the SDs of the results for triplicate samples and are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

FIG. 5.

Virus replication in IM-MDDCs. IM-MDDCs (1 × 10⁶ cells) were pulsed for 60 min with similar concentrations of isogenic NL4-3balenv either lacking or bearing host-derived LFA-1 (100 ng of p24^Gag). After three washes with PBS, the cells were maintained in complete culture medium supplemented with GM-CSF and IL-4. (A) Virus production was assessed by measuring p24^Gag levels in the culture supernatants at the indicated time points. D.P.I., day postinfection. (B) The amounts of integrated viral DNA were quantified at day 12 after virus exposure with real-time PCR assays, as described in Materials and Methods. The data shown represent the means ± the SDs of the results for duplicate samples and are representative of at least four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) The percentage of IM-MDDCs positive for both cell surface DC-SIGN and intracellular p24 was quantified by flow cytometry at 9 days postinfection.

FIG. 6.

Effects of PKA and PKC inhibitors on virus replication in IM-MDDCs. IM-MDDCs (1 × 10⁶ cells) were either left untreated or treated with the PKA inhibitor H89 (30 μM) or the PKC inhibitor Ro-318220 (1 μM) for 10 min. Next, IM-MDDCs were pulsed for 60 min with similar concentrations of isogenic NL4-3balenv either lacking (A) or bearing (B) host-derived LFA-1 (100 ng of p24^Gag). After three washes with PBS, the cells were cultured in complete culture medium supplemented with GM-CSF and IL-4. Virus production was determined by measuring p24^Gag levels in the culture supernatants at the indicated time points. The amount of integrated viral DNA was quantified at day 12 after virus exposure with real-time PCR assays, as described in Materials and Methods (C). The data shown are the means ± the SDs of the results for triplicate samples and are representative of at least four independent experiments. **, P < 0.01; DPI, day postinfection.

FIG. 7.

Specificity of the LFA-1-mediated effect on virus production in DC-T-cell cocultures. NL4-3balenv (10 ng of p24^Gag) either lacking or bearing host-derived LFA-1 was produced in 293T cells. Virus stocks were first incubated for 15 min at 4°C with the F(ab′)₂ fragments of an anti-CD11a antibody (0.5 μg). The virus-antibody mixture was diluted in four volumes of RPMI medium before being incubated with IM-MDDCs (1 × 10⁵ cells) for 60 min. After three washes with PBS, IM-MDDCs were cocultured with autologous CD4⁺ T cells at a DC-to-T-cell ratio of 1:3. Virus production was evaluated at day 2 by measuring p24^Gag levels. The data shown are the means ± the SDs of the results for triplicate samples and are representative of at least two independent experiments. **, P < 0.01.

FIG. 8.

Biological significance of the LFA-1-mediated effect. NL4-3balenv (10 ng of p24^Gag for panel A and 100 ng of p24^Gag for panel B) produced in PBMCs was first incubated for 15 min at 4°C with the F(ab′)₂ fragments from the listed antibodies (0.5 μg each in panel A and 5 μg each in panel B). The virus-antibody mixture was diluted in four volumes of RPMI medium before being incubated with IM-MDDCs (1 × 10⁵ cells in panel A and 1 × 10⁶ cells in panel B) for 60 min. After three washes with PBS, IM-MDDCs were either divided into three aliquots and cocultured with autologous CD4⁺ T cells at a DC-to-T-cell ratio of 1:3 (A) or cultured in complete culture medium supplemented with GM-CSF and IL-4 (B). Virus production was evaluated at either day 2 (A) or day 8 (B) by measuring p24^Gag levels. The data shown are the means ± the SDs of the results for triplicate samples and are representative of at least two independent experiments. *, P < 0.05; **, P < 0.01.