Elucidating the mechanisms underlying DC-mediated HIV infection and viral transmission will enhance our understanding of HIV interactions with host cells (52
). Here we provide evidence that cis
- and trans
-infections of HIV-1 mediated by DCs are dissociable pathways. HIV-1 cis
-infection is restricted in mDC-LPS and impaired in mDC-TNF-α; however, these cells efficiently promoted HIV-1 trans
-infection of CD4+
T cells, suggesting that, in vivo, the various DC subsets facilitate HIV-1 dissemination to different extents via cis
- and trans
-infections. It has been proposed that DCs transfer HIV-1 to CD4+
T cells in two phases (46
). In the first phase (1 dpi), DC-captured HIV-1 is spread to CD4+
T cells via the trans
-infection pathway. The second phase (2 to 3 dpi) occurs after de novo HIV-1 production by cis
-infected DCs (46
). However, our findings of dissociable cis
- and trans
-infections mediated by DCs suggest that these two processes are not necessarily sequential or interdependent.
Our results highlight the potential significance of DC-mediated HIV-1 transmission in vivo. In submucosal tissues, HIV-infected iDCs may act as a viral reservoir to further the spread of infection. HIV-1 coinfection with other sexually transmitted pathogens may induce DC maturation through inflammatory responses, such as upregulated cytokine production (43
). Given their proximity to CD4+
T cells in lymphoid tissues, mDCs may potently stimulate HIV-1 transmission to these activated CD4+
T cells. A recent study indicated that significantly increased plasma LPS levels in HIV-infected humans correlate with systemic immune activation and AIDS progression (6
). It is conceivable that increased LPS in HIV-infected individuals may induce DC maturation, thereby stimulating HIV-1 dissemination in vivo. Similarly, more recent reports indicated that LPS-activated CD34+
cell-derived Langerhans cells mediate efficient trans
-infection of HIV-1 (10
). Despite the convenience of using monocyte-derived DCs, these cells may not fully mimic the physiological functions of the DC subsets that are involved in HIV infection in vivo. Thus, further studies are required to confirm these in vitro observations, using myeloid DCs, plasmacytoid DCs, Langerhans cells, and autologous CD4+
T cells from HIV-infected individuals.
DCs in vivo would likely be exposed to HIV-1 before or simultaneously to a maturation stimulus. It has been reported that HIV-infected blood myeloid DCs and monocyte-derived DCs fail to mature in culture (24
). Moreover, DCs from individuals with acute HIV infection have reduced expression of the costimulatory molecules CD80 and CD86, and this might influence DC-induced T-cell responses (31
). These results suggest that productive HIV infection of DCs undermines the direct induction of T-cell-mediated immunity. In contrast, other studies have indicated that monocyte-derived DCs from HIV-infected individuals can efficiently induce cytotoxic T cells to respond to various antigens (15
). In addition, no functional defects in cytokine production were observed following stimulation of HIV-infected myeloid DCs and plasmacytoid DCs (45
). In our study, to better compare HIV-1 infections among various types of DCs, viral infection was performed after generating mDCs from iDCs with different stimuli.
We found that distinct mechanisms contributed to postentry blocks of HIV-VSV-G and replication-competent HIV-1 in mDC-LPS. Thus, it is critical to consider the potential differences in using HIV-VSV-G instead of authentic HIV-1 to study postentry infection. Although endocytosis of both types of viruses was increased in mDC-LPS compared with that in other types of DCs, different viral trafficking pathways might affect the various steps in the viral life cycle. HIV-1-VSV-G enters cells through a low-pH-dependent endocytic pathway (1
). Relative to those in other types of DCs, increased RT and efficient integration were detected in HIV-Luc/VSV-G-infected mDC-LPS, indicating that endocytosed HIV-VSV-G can complete uncoating, RT, and integration in mDC-LPS, while impaired gene expression in mDC-LPS could eventually block viral expression of the luciferase reporter in the infected cells. In contrast, in HIV-1NLAD8
-infected mDC-LPS, restricted RT appeared to be an important component of the postentry block, and impaired gene expression in these cells might also play a role. For LPS-induced mDCs, it has been shown that HIV-1 is internalized into nonconventional, nonlysosomal, acidic compartments (20
), which may restrict the RT process of endocytosed HIV-1NLAD8
In addition to the restriction of RT, it has been proposed that there is a postentry block of HIV-1 in mDCs at the transcriptional level (4
). Using nucleofection of HIV-1 proviral DNA in DCs to overcome any restrictions in the early steps of the HIV-1 life cycle, we found that impaired gene expression in mDC-LPS contributed to the potent block of HIV-1 replication in these cells. Moreover, we observed that LPS-treated CD4+
T cells showed 1.2- to 1.4-fold increases in the efficiency of a single-cycle HIV-1 infection (not shown), suggesting that LPS does not generally induce restriction of HIV-1 infection in permissive cells. Consistent with this observation, an early study demonstrated that LPS is a potent stimulator of HIV-1 expression in monocytes and macrophages (39
). The impaired HIV-1 gene expression in mDC-LPS might be due to inefficient transcription and/or translation. A comprehensive gene expression analysis showed that 225 transcripts among a total of 31,837 tag sequences were statistically different between iDCs and mDC-LPS (26
). Moreover, treatment of iDCs with LPS activates several signal transduction pathways, including the NF-κB pathway (2
). Therefore, the impaired gene expression in mDC-LPS is unlikely to be specific for HIV-1.
Although 2-LTR circles have been used as a marker for nuclear import of HIV-1 DNA, it is not always technically correct to interpret the lack of 2-LTR circles as an indicator of blocked nuclear import of viral DNA (57
). Our data showed that 2-LTR circles were undetectable in HIV-VSV-G-infected mDC-LPS, while 1-LTR circles and integrated proviral DNA were detected in these cells. This might be due to the low abundance and degradation of 2-LTR circles in mDC-LPS. It has been reported that in HeLa-CD4 cells infected with HIV-1 at 2 dpi, most viral DNA is fully processed into integrated provirus (≈55%) and 1-LTR circles (≈35%), while only trace amounts of 2-LTR circles (<5%) can be detected (58
). We estimated the abundance of LTR circles and integrants in various types of DCs infected with HIV-VSV-G. Interestingly, only 0.1 to 1% of the late RT products were converted into 2-LTR circles, while 0.3 to 22% and 4 to 35% of the late RT products were converted into 1-LTR circles and integrated proviral DNAs, respectively. These data suggest that the ratio of HIV-1 DNA to integrants might be cell type dependent.
The inefficient block of HIV-1 uptake into DCs by the fusion inhibitor T-20 suggests that HIV-1 entry into DCs occurs primarily through endocytosis rather than fusion. Nobile et al. reported that DC-SIGN, a C-type lectin molecule highly expressed on monocyte-derived DCs, facilitates HIV-1 capture and intracellular accumulation but inhibits viral fusion (36
). This may help to explain the low levels of HIV-1 fusion and replication in DCs. We recently reported that mDC-LPS are more efficient than iDCs at transmitting HIV-1 to various types of target cells, independent of C-type lectins, including DC-SIGN (48
). In addition, we observed that HIV-1 uptake by mDC-LPS was slightly increased (around 30%) by T-20 treatment for 2 to 12 h (Fig. and ), suggesting that there is a balance between HIV-1 fusion and endocytosis. The blockade of fusion-mediated HIV-1 entry by T-20 might decrease the degradation of Gag p24 in the cytosol, thereby indirectly increasing the amount of internalized HIV-1 in vesicles of mDC-LPS. A similar compensatory link between HIV-1 fusion and endocytosis has been reported for CD4+
primary T cells (42
). In that study, blockade of CXCR4-tropic HIV virion fusion with AMD3100, a CXCR4-specific entry inhibitor, increased virion entry into CD4+
T cells via the endocytic pathway.
Although endocytosis-mediated HIV-1 entry can lead to productive viral infection in certain cell types, including human primary macrophages (13
), our results suggest that efficient RT and productive HIV-1 infection in DCs require fusion-mediated viral entry. A recent report indicated that HIV-1 enters Langerhans cells primarily by multiple-receptor-mediated endocytosis and that virions persist intact within the cytoplasm for several days without productive replication (27
). The majority of endocytosed HIV-1 confined in intracellular compartments in DCs will eventually be degraded. In fact, rapid intracellular HIV-1 degradation has been reported for both iDCs and mDCs (35
CD40L-induced mDCs appeared to be more susceptible to HIV-VSV-G infection than were other types of DCs. This was likely due to more efficient nuclear import and integration of viral DNA in mDC-CD40L, as more 1-LTR and 2-LTR circles and proviruses were detected in the infected cells. In contrast, HIV-1NLAD8
-infected mDC-CD40L and iDCs showed similar levels of viral replication, although viral uptake in mDC-CD40L was slightly lower than that in iDCs (Fig. ). These data suggest that the fusion of HIV-1NLAD8
is less efficient in mDC-CD40L. It has been shown that productive HIV-1 infection of DCs is triggered by CD40L treatment (16
); however, other studies have reported that HIV-1 infection is decreased in CD40L-induced mDCs (4
). These discrepant results might be due to different DCs and recombinant CD40L used in these studies.
Cellular restriction of HIV-1 infection in DCs may reflect innate antiviral immunity of the antigen-presenting cells. It has been reported that the antiretroviral factor APOBEC3G/3F (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G and 3F) mediates postentry resistance to HIV-1 infection in monocyte-derived iDCs (38
). In addition, LPS-induced mDCs have reduced susceptibility to HIV-1 infection, which correlates with increased APOBEC3G levels (38
). A recent report suggests that langerin-mediated HIV-1 internalization results in viral degradation, thus inhibiting HIV-1 replication in Langerhans cells (12
). A better understanding of intrinsic antiretroviral immunity in DCs will provide new insights into more effective intervention strategies against HIV-1 infection and dissemination mediated by DCs.