Our studies demonstrate that the maturation of MDDCs both decreases the magnitude and kinetically slows the rate of HIV virion fusion. Specifically, laboratory-adapted 81A virions and virions pseudotyped with various primary envelopes fused with lower efficiency to mature than immature MDDCs, and the fusion kinetics of 81A and NL4-3 were slower in mature MDDCs than in immature MDDCs.
These findings confirm and extend the results of Granelli-Piperno et al., who showed by quantification of the early products of reverse transcription that maturation of MDDCs decreased the entry of the R5-tropic Ba-L (19
). However, our results contrast with the increased entry of Ba-L and the X4-tropic LAI observed in mature DCs derived in vitro from CD34+
hematopoietic progenitor cells (9
) and with the changes in Ba-L and LAI entry in MDDCs (4
). The origin of the viruses used in our studies cannot easily account for these differences, since 81A contains the V1 to V3 loop of Ba-L (40
) and LAI and NL4-3 encode identical envelopes (1
). These contradictory results could reflect the nature of the DCs and differences in procedures for inducing DC maturation and measuring viral fusion. In our experiments, immature MDDC resembled primary Langerhans cells and dermal DCs in their sensitivity to 81A and NL4-3 fusion, indicating that MDDCs function as an acceptable surrogate for DCs present in tissues in vivo. Furthermore, treatment of MDDCs with poly(I:C) and TNF-α induced complete phenotypic maturation, avoiding the generation of a heterogenous population which could potentially mask the effect of maturation on HIV fusion. Finally, our measurement of viral fusion is not affected by the abundance of virion endocytosis that characterizes DCs, since the HIV virion-based fusion assay, but not the use of early products of reverse transcription as an “entry marker”, measures viral fusion independently of viral endocytosis (11
). Therefore, our results strongly suggest that in vivo fusion of HIV to mature DCs is also impaired.
HIV-1 binding to alternative receptors like C-type lectin receptors influences HIV-1 replication in DCs (26
). We found that mannan, one of the ligands of the C-type lectin receptor, significantly decreased fusion of R5-tropic virions to immature MDDC; the effect was not as pronounced for the X4-tropic NL4-3 virions. Mannan had no effect on HIV-1 fusion to mature MDDCs, consistent with the lower amount of C-type lectin receptors on mature DCs (14
). Thus, C-type lectin receptors appear to mainly play a role in the efficient fusion of R5-tropic HIV-1 to immature MDDCs. Previous studies based on immunohistochemical and confocal microscopy had indicated that DC-SIGN, one of the C-type lectin receptors, colocalizes with CD4 and CCR5 on alveolar macrophages (26
), suggesting that C-type lectin receptors could focus HIV-1 virions in a location that favors successful engagement of CD4 and CCR5 and not CXCR4. This location could be the lipid rafts, since DC-SIGN, CD4, and CCR5 are preferentially enriched in lipid rafts compared to CXCR4 (7
In addition to the changes in the overall level of HIV fusion, laboratory-adapted strains of HIV also fused more rapidly to immature MDDCs than to mature MDDCs. The rapid fusion kinetics displayed by R5-tropic 81A virions relates in part to the high density of CCR5 receptors at the plasma membrane, since slower kinetics were observed when available CCR5 receptors were reduced by the addition of graded doses of the CCR5 antagonist TAK-779. Maturation of MDDCs, which is intrinsically associated with a decline in CCR5 expression (13
), also led to lower levels and slower kinetics of R5-tropic 81A fusion. Interestingly, although DC maturation induced by poly(I:C) and TNF-α did not decrease cell surface expression of CXCR4 or CD4, the kinetics of fusion of the X4-tropic NL4-3 to mature MDDCs was also delayed in these cells. Morphological changes induced by DC maturation could be responsible for this finding. Specifically, the development of cellular dendrites in mature MDDCs (10
) could separate microclusters of CD4, usually present at the tip of the dendrite, from the coreceptors, which usually localize near the base of the dendrite (34
). Consequently, gp120/gp41 complexes bound to CD4 might require more time to effectively engage the coreceptors and trigger the fusion reaction.
Surprisingly and in sharp contrast to the laboratory-adapted viruses, primary envelopes with CXCR4 tropism mediated fusion to immature MDDCs with efficiencies similar to those of R5-tropic primary envelopes. These results contrasted with the ~20- to 70-fold difference in fusion observed in immature MDDCs with the R5-tropic 81A and X4-tropic NL4-3 laboratory viruses. Despite lower levels of fusion, the four X4-tropic primary envelopes and the seven R5-tropic envelopes mediated comparable levels of fusion in immature MDDCs obtained from multiple donors. It is very unlikely that the pseudotyping procedure differentially altered the properties of R5- and X4-tropic envelopes. Therefore, these surprising results suggest that primary viruses with tropism for either CCR5 or CXCR4 may fuse similarly in vivo to immature DCs, such as Langerhans cells. Previous studies of HIV replication in Langerhans cells relied on the use of laboratory-adapted strains of HIV and showed high replication of R5-HIV and not X4-HIV, suggesting that immature DCs might play a role in the preferential transmission of R5-HIV (20
). However, another study noted that immature MDDCs exposed to HIV-1 isolates with mixed tropism did not exclusively replicate the R5-tropic isolates (43
). Although our results with laboratory-adapted strains of HIV support a role of immature DCs in the preferential transmission of R5-HIV, our more physiologically relevant analysis of fusion with primary envelopes does not support this model.
In summary, we have shown that the maturation of dendritic cells is associated with a marked decline and slowing of HIV fusion. Since DC maturation also alters HIV transcription (4
), the fusion defect could be the first of several blocks encountered by viruses in these cells that culminate in reduced viral replication in mature DCs. This defect in fusion, which leaves HIV virions intact, could potentially facilitate handling of the virus by endocytosis with later transfer of these intact virions to interacting CD4 T cells.