The cell type dependency of DC-SIGN-mediated HIV-1 transmission provides a useful model system to investigate the mechanisms underlying DC-mediated viral transfer (61
). We previously discovered that monocytic THP-1 cells do not support DC-SIGN-mediated HIV-1 transmission, although it was widely assumed in the literature that such cells recapitulated DC transmission of HIV-1 (61
). Here, we identify an underlying mechanism for the monocytic cell restriction of DC-SIGN-mediated HIV-1 transmission and present a model by which HIV-1 might exploit primary cells with similar characteristics to potentiate viral spread to CD4+
In this report, we show that CD4 expression levels regulate DC-SIGN and MDDC transmission of HIV-1. Monocytic cell type restriction of DC-SIGN-mediated HIV-1 transmission correlates with CD4 expression, and this impairment can be blocked with CD4 antibodies that prevent interactions with HIV-1 Env. This restriction can be re-created by the expression of CD4 in Raji/DC-SIGN cells, which are otherwise permissive to DC-SIGN-mediated HIV-1 transmission. In the presence of an appropriate HIV-1 coreceptor, cells engineered to coexpress CD4 and DC-SIGN are preferentially infected. By contrast, CD4/DC-SIGN double-positive cells lacking a viral coreceptor efficiently internalize and trap HIV-1. Using primary cells expressing CD4 and DC-SIGN, we observed that HIV-1 infection changes the transmission properties of these cells. Specifically, infection by Nef-positive HIV-1 efficiently promotes DC-mediated HIV-1 transmission, which correlates with CD4 downregulation in these cells. Collectively, these data suggest that modulations of CD4 levels may play an important regulatory role in DC-mediated HIV-1 transmission.
DC-mediated HIV-1 transmission to CD4+
T cells involves at least two pathways (60
), namely, HIV-1 trans
infection without viral infection in DCs, and long-term viral transfer depended on the HIV-1 replication in infected DCs (6
). It has been reported that certain DC subsets and macrophages in vivo express DC-SIGN, CD4, and HIV-1 coreceptors (24
), and both cell types were proposed to facilitate mucosal transmission of HIV-1 (47
). Thus, it is conceivable that these types of cells are more susceptible to HIV-1 infection due to the coexpression of CD4, DC-SIGN, and viral coreceptors, which may augment viral dissemination by DCs and macrophages using the cis
infection pathway. Upon HIV-1 infection, the Nef protein may convert these cells into more potent HIV-1 transmitters through a significant downregulation of CD4. CD4 and DC-SIGN molecules have dileucine-based internalization motifs in the cytoplasmic domains (5
), and the dileucine motifs of CD4 and DC-SIGN are critical for endocytosis function and regulation by Nef (51
). Although MDDCs have provided a convenient tool to study DC-HIV interactions, further examinations of bona fide DC subsets ex vivo for viral infection and transmission would be beneficial for understanding the contribution of DCs to AIDS pathogenesis.
Internalization of HIV-1 and distinct viral trafficking have been suggested to be important for DC-mediated HIV-1 trans
); however, it is unclear how internalized virions remain infectious, recycle back to the cell surface, and are transferred to the T cells. By contrast, our viral binding and electron microscopy results suggest that HIV-1 internalization in Raji/DC-SIGN cells is not required for efficient HIV-1 transmission. Consistent with our observations, Burleigh and colleagues recently reported that DC-SIGN-mediated HIV-1 internalization is dispensable for both trans
infection of T cells and the retention of viral infectivity (6
). We found that the coexpression of CD4 and DC-SIGN in Raji cells significantly promoted viral internalization and altered viral trafficking to late endosomal compartments (also known as multivesicular bodies [MVBs]). Although endocytosis-mediated HIV-1 entry can lead to productive viral infection (15
), only X4 HIV-1, but not R5 HIV-1, replicated in CD4-expressing Raji cells. These results suggest that the fusion-mediated entry of X4 HIV-1 leads to productive infection in these cells; however, endocytosed R5 HIV-1 is trapped or inactivated in acidified endosomes and is eventually degraded in lysosomes. Indeed, compared with Raji/DC-SIGN cells, we have observed significant colocalization of the HIV-1 p24 protein with lysosome-associated membrane protein 1 in Raji/DC-SIGN/CD4 cells (data not shown), suggesting enhanced HIV-1 degradation within lysosomes in CD4-DC-SIGN-expressing cells.
Interestingly, it was shown that HIV-1 captured by immature MDDCs is also internalized to MVBs, and infectious HIV-1 is constitutively released into the extracellular milieu in association with the exosomes to initiate viral transfer to T cells (58
). Similarly, HIV-1 particles internalized by mature MDDCs are also sequestered into MVBs (17
). In contrast to the Raji/DC-SIGN/CD4 cells, it remains unclear how internalized HIV-1 circumvents the intracellular degradation machinery in DCs and recycles back to the DC surface to be transferred to the T cells. Further investigations of HIV-1 trafficking in DCs in comparison to Raji/DC-SIGN/CD4 cells may help us to understand this unique feature of DC-mediated viral transmission.
The blocking of exposed CD4 on DCs with specific mAbs promoted HIV-1 trans
infection, most likely by impairing virus internalization and intracellular degradation. Similar results were confirmed using DCs generated from different donors and using different CD4 mAbs in independent experiments (data not shown). However, the enhancement of HIV-1 transmission by CD4 mAb blocking appears to be limited, with 2.4-fold and 4-fold increases in MDDCs and various cell lines, respectively. It is conceivable that other regions of CD4 outside the gp120 binding domain contribute to the inhibition of DC-SIGN-mediated HIV-1 transmission. In addition, during the 2-day coculture with T-cell targets, the newly synthesized CD4 proteins on the donor cell surfaces diminished the blockade effect of CD4 mAb. In DCs, CD4 mAb alone may not completely block HIV-1 internalization due to the coexpression of DC-SIGN or other C-type lectins. These results also suggest that CD4 expression is not the sole cellular determinant that controls the efficiency of DC-SIGN-mediated HIV-1 trans
infection. For example, we previously reported that CD4-negative human erythroleukemic K562 cells do not efficiently transmit HIV-1 despite high levels of exogenous DC-SIGN expression (62
). Presumably, another unidentified cellular factor(s) can also influence DC-SIGN-mediated HIV-1 transmission, given its cell type dependence in CD4-negative cells (61
Nef-expressing HIV-1-infected DCs promoted viral transmission to cocultured T cells. Indeed, Nef modulation of DC-SIGN and CD4 expression was observed despite the limited levels of DC-SIGN upregulation. These results provide an additional context to consider previous observations of Nef facilitating DC-mediated HIV-1 spread to T cells (37
). It has been reported that HIV-1 transmission efficiency can be enhanced by the maturation of DCs (32
). To examine the possibility that Nef-promoted HIV-1 transmission results from Nef-induced DC maturation, maturation markers (such as HLA-DR, CD83, and CD86) of WT and Δnef
HIV-1-infected DCs were compared, but no Nef-induced upregulation of the maturation markers was observed (unpublished data). It has also been reported that Nef-expressing immature MDDCs stimulate T-cell activation but without upregulating DC maturation markers (33
). We confined our analyses to a transformed CD4+
T cell that is constitutively activated. With resting, primary CD4+
T cells, it is likely that Nef may also facilitate DC-mediated HIV-1 transmission by promoting DC-T-cell interactions or by enhancing T-cell clustering (51
In summary, we find that CD4 coexpression with DC-SIGN enhances HIV-1 internalization and retention but strongly impairs HIV-1 transmission to T cells. The blocking of CD4 on the DC surfaces promotes HIV-1 trans infection. Significantly, Nef facilitates DC-mediated HIV-1 transmission, which correlates with Nef-induced downregulation of CD4. These data provide a novel insight into cellular characteristics that influence DC-mediated HIV-1 dissemination and highlight Nef's role as a multifunctional pathogenic factor capable of regulating these processes.