In this study, we have demonstrated that DC-SIGN, a C-type lectin on DCs that mediates HIV-1 uptake and transfer to T cells, interacts with LSP1, an actin-binding cytoskeletal protein. HIV-1 uptake is dependent on the cytoplasmic domain of the DC-SIGN molecule (). By using immunoprecipitation for biochemical purification and identification by mass spectrometry, we showed that LSP1 interacts specifically with this cytoplasmic region. Our results also show that LSP1 interacts with other C-type lectins, L-SIGN and Langerin, which are present on the human DC. Because these C-type lectins have been shown to mediate HIV-1 transfer independently of DC-SIGN (39
), LSP1 may be involved with these C-type lectins' interaction with HIV-1. Langerin, unlike DC-SIGN, contains a proline-rich region that is likely responsible for binding and rapid internalization of pathogens. CD2 and CD40L both contain this proline-rich region, possibly providing the region necessary for interaction with LSP1. Down-regulation of LSP1 siRNAs in human DCs or murine lsp1−/−
BMDCs showed a dramatic increase in the amount of virus transferred from the DC to the susceptible T cell, as LSP1 was shown to facilitate proteasomal degradation of HIV-1.
A leukocytic protein, LSP1 (also known as WP34, pp52, and leufactin) is a 52-kD F-actin binding phosphoprotein expressed in all human leukocytes and leukocytic cell lines (40
). The basic C-terminal domain contains amino acid sequences homologous to two known F-actin binding proteins, caldesmon and the villin headpiece (36
). Although LSP1 is an F-actin binding protein, it is also a very important regulator of microfilamentous cytoskeleton dynamics (34
). After HIV-1 uptake in the DCs, it is internalized into a specialized viral endosome, which is distinct of early and late endosomal vesicles (43
), where a fraction of virus remains undigested and polarizes to the infectious synapse between the targeted T cells (24
). HIV-1 virus that does not polarize is subjected to lysosomal processing and MHC II antigen presentation, or it is degraded by the proteasome (30
). Because LSP1 interacts specifically with full-length DC-SIGN and not a truncated cytoplasmic domain mutant, this finding suggests that it is involved with trafficking HIV through the DC. LSP1 has proven important in polarizing the actin cytoskeleton and aiding in motility of the cell. Using proteasome inhibitors and confocal microscopy, we show that LSP1 helps to shuttle the HIV-1 virus into the proteasome, promoting its degradation, a process independent of its interaction with DC-SIGN. In the absence of LSP1, HIV-1 degradation decreases and more virus is able to recycle to the surface, promoting transfer to T cells. We do not know why the proteasomes in murine DCs were not susceptible to bafilomycin as they are in human DCs. Because they are isolated differently from the human cells and are grown in cytokines, it is possibly a difference in the patterns of gene expression in these cells, although we cannot exclude a species effect. Proteasomal inhibitors can affect ubiquitin levels in the cells, which could explain the decrease in HIV-1 degradation; however, in the absence of LSP1, there was less colocalization of HIV-1 to the proteasome and no significant difference in HIV-1 degradation in the lsp1−/−
BMDCs compared with wt
cells treated with proteasomal inhibitors, suggesting that this effect was independent of ubiquitin effects. This experiment confirmed the role of LSP1 in this degradative process.
Our study reveals new insights into HIV-1 trafficking through DCs leading to the enhancement of T cell infection by DC-SIGN–internalized virus. The role of DC-SIGN in trans-infection is not completely understood, in part because other C-type lectins may be involved in the process in some DC populations, and blocking of DC-SIGN with mAbs does not always completely inhibit HIV-1 transfer. Although one previous study contradicted the report of Kwon et al. (27
) that point mutants in the tyrosine and dileucine motifs of the cytoplasmic domain of DC-SIGN do not affect gp120 binding, it is important to recognize that the latter study analyzed internalization with gp120 protein rather than virus (44
), and the significance of this assay for virus internalization and transfer is questionable. Nonetheless, consistent with the present work, a mutant with a deletion of the cytoplasmic domain in that study showed the same loss of function as seen here examined by transmission of the lentiviral vector.
The discovery that LSP1, an actin-binding molecule, interacts with DC-SIGN has implications for understanding the trans-enhancement of T cell infection by DCs, possibly leading to ways of blocking transfer. Sequestering actin and the cytoskeleton may lead to decreased transfer of HIV-1, but not without possible serious effects on DC viability. Antigen internalized by DCs has been shown to lead to classical MHC II processing, peptide loading, and surface presentation (45
). We find that increased transfer of HIV-1 to T cells in the absence of LSP1 is due to decreased HIV-1 degradation in the proteasome; however, insights into the effect of LSP1 on peptide processing and antigen presentation have yet to be investigated. Collectively, these data suggest a role for LSP1 trafficking of HIV-1 to the proteasome for viral degradation. Continued elucidation of HIV-1 trafficking in DCs provides us with a greater understanding of how C-type lectins, such as DC-SIGN, mediate viral uptake and transfer to susceptible target cells.