We next examined the inhibition of trans
infection. Briefly, CD14+
cells were isolated as described previously (11
). The CD14−
cells were cryopreserved, while the CD14+
cells were differentiated into mature DC (11
). DC were incubated overnight at 37°C with 10,000 tissue culture infective doses/ml of virus, washed five times, resuspended in PBMC culture medium (36
), seeded at 2 × 105
cells/well in 96-well plates, and incubated with CD4-IgG2 or T-20 for 1 h at 37°C. CD14−
cells were thawed, stimulated as described previously (36
), and incubated with PRO 140 or RANTES. CD14−
cells (1.4 × 105
cells/well) were then added to DC, and HIV-1 replication was determined as described above.
Each inhibitor effectively blocked trans infection. In no case did the ICs for a given inhibitor vary by more than threefold between direct and trans infection of PBMC (Table and Fig. ). Thus, during trans infection, DC-associated virus is sensitive to inhibition throughout the fusion cascade.
Thus DC-mediated trans
infection is inhibited by CD4-IgG2 as well as HIV-1-neutralizing monoclonal antibodies (13
). In contrast, follicular DC-mediated trans
infection reportedly is unaffected by neutralizing antibody (15
). The divergent results could reflect differences both in how DC versus follicular DC process immune-complexed virus and in assay methodology.
Similarly, we observed that T-20 efficiently blocks DC-mediated trans
infection. Our findings stand in apparent contrast to those reported for trans
infection mediated by intestinal epithelial cells, where T-20 was inactive (22
). However, the different outcomes may again reflect methodology differences. In the earlier study, T-20 was removed from the epithelial cells prior to the addition of CD4+
target cells (22
), whereas T-20 was present in our DC-T-cell cocultures.
In the present study, DC were added in excess of T cells such that most of the T cells could be infected in trans during the first round of infection. To explore the potential importance of secondary rounds of infection, inhibitors were washed away prior to the combination of DC and T cells. CD4-IgG2 was fully active in the washout setting (data not shown), indicating that the agent blocks trans infection. These findings support the notion that the inhibitors block trans infection.
Our findings shed light on the mechanism of HIV-1 infection in trans
. Notably, during this process, virus is sensitive to each of the known classes of entry inhibitors. This result is surprising for two reasons. Firstly, lentiviruses are internalized in prominent vacuoles in mature DC (11
) and internalization may be critical for infection (20
). Secondly, trans
infection may occur at polarized interfaces between the cells (19
) and internalized virus moves to this immunologic synapse within minutes of cell contact (D. McDonald, D. Unatmaz, V. KewalRamani, and T. Hope, Keystone Symp. HIV Pathog., abstr. 121, 2002). Nonetheless, our findings indicate that virus remains vulnerable to inhibition by membrane-impermeable drugs.
In summary, we report that the main classes of HIV-1 entry inhibitors block the direct and trans infection of multiple primary cell types. Although minor cell-type-specific differences in activity were observed, CD4-IgG2, PRO 140, and T-20 were effective in each setting. Our findings also offer new insights into the nature of the DC/T-cell synapse during trans infection.