Previous studies indicated that IFNα treatment partially inhibits post-entry HIV-1 replication and cell-to-cell transmission in CD4+ T cells and macrophages [18–20], suggesting type I IFN-mediated anti-HIV responses. Compared with iDCs, mDC-LPS do not support productive HIV-1 replication, but mediate highly efficient cell-to-cell transmission [2, 6, 9, 11]. To better understand the innate immune response of DCs to HIV-1 infection, we investigated the effect of IFNα on HIV-1 replication and cell-to-cell transmission using primary human DCs. We found that DC-mediated HIV-1 transmission and viral replication were impaired in mDC-IFNα. IFNα induced DC maturation but did not affect overall expression levels of HIV-1 receptors and the attachment factor DC-SIGN, suggesting that mDC-IFNα can mediate HIV-1 binding and entry. Compared with mDC-LPS, mDC-IFNα-mediated transmission of HIV-1 to CD4+ T cells was significantly lower. Our data suggest that IFNα treatment of DCs induces an antiviral response to block HIV-1 replication and cell-to-cell transmission.
IFNα is a major inducer of tetherin expression [27, 61], and other pathogenic stimuli have been suggested to stimulate tetherin expression as part of the innate immune response . We thus evaluated any link between tetherin and the inhibition of HIV-1 replication and cell-to-cell transmission in DCs by assessing the levels of tetherin expression in iDCs and mature DCs. We observed that iDCs were extremely low or negative for tetherin expression, while mDC-LPS showed high levels of tetherin expression at the cell membrane. By contrast, mDC-IFNα were negative, or expressed very low levels of surface tetherin despite high levels of whole cell tetherin expression, indicating that IFNα-induced tetherin is mainly confined to the intracellular compartment in mDC-IFNα. Given the different expression levels and apparent sub-cellular localization patterns of tetherin within DCs, we therefore investigated any link between the high levels of tetherin induced in mature DCs and the HIV-1 replication and cell-to-cell transmission phenotypes observed in these cells.
Tetherin localization and co-localization with HIV-1 is vital to its restriction function, as it must tether the newly formed HIV-1 virions to the cell membrane [37, 49, 54, 55, 61]. The localization of HIV-1 in mDC-IFNα may contribute to restricted HIV-1 transmission to CD4+ T cells. HIV-1 and CD81 strongly co-localized in mDC-LPS, with clear evidence of concentration of CD81 at the site of HIV-1 binding, as expected [12, 57, 58]. Co-localization of HIV-1 and CD81 was also observed in mDC-IFNα, but there was no evidence of a concentration of CD81 at the sites of HIV-1 binding, suggesting that the localization of HIV-1 is distinct from that observed in mDC-LPS. The lack of co-localization between HIV-1 and LAMP-1 in mDC-LPS and mDC-IFNα indicates that HIV-1 did not traffic to the lysosome for degradation in either cell type at 2 h post-infection. In both mDC-LPS and mDC-IFNα, co-localization of tetherin and HIV-GFP-Vpr was observed, which raises questions about whether tetherin affects incoming HIV-1 captured by mature DCs.
We sought to investigate the direct effect of tetherin on the replication and DC-mediated transmission of HIV-1 by silencing tetherin in mDC-LPS and mDC-IFNα. Recent studies of the tetherin function in HIV-1 cell-to-cell spread have focused on viral transmission from infected CD4+ T cells to uninfected cells [48–50]. However, in DC-mediated HIV-1 transmission, DC-captured virus is concentrated at, or near, the cell surface and can be transmitted to CD4+ T cells without productive replication in DCs [2, 6, 9–12, 62]. Furthermore, the major role of tetherin is to prevent the release of fully formed HIV-1 virions from the cell surface [27, 37, 55], rather than affecting incoming virions. In a single-cycle HIV-1 transmission assay, tetherin knockdown in mDC-LPS and mDC-IFNα resulted in a modest increase of viral transmission to CD4+ T cells, suggesting that high levels of induced tetherin in mature DCs may partially impair DC-mediated transmission of incoming HIV-1 to CD4+ T cells. It is possible that tetherin siRNA may have potential off-target effect, which should be considered in transient tetherin downregulation in primary DCs. Recent studies indicated that tetherin knockdown in CD4+ T cells reduces the formation of the virological synapse and HIV-1 cell-to-cell transmission [48, 49]. Whether tetherin knockdown affects the formation of the virological synapse between DCs and CD4+ T cells remains to be established.
A recent study indicated that tetherin expressed on target Sup-T1 cells can promote HIV-1 cell-cell transmission . In this study, we focused on the role of tetherin in donor DCs and used Hut/CCR5 T cells as targets in DC-mediated HIV-1 transmission assays. Hut/CCR5 cells express high levels of endogenous tetherin (data not shown), while primary human CD4+ T cells express variable levels of tetherin [49, 63]. Using Hut/CCR5 cells avoided donor variations of tetherin expression in primary CD4+ T cells. However, it remains to be investigated whether tetherin expressed in CD4+ T cell targets affects DC-mediated HIV-1 transfer.
Tetherin inhibits HIV-1 release from cells, and its function is antagonized by Vpu [27, 37]. We investigated HIV-1 replication and release in mature DCs using tetherin knockdown and a Vpu-defective mutant. The effect of tetherin knockdown on HIV-1 release from mature DCs appears to be dependent upon the maturation stimulus used and on the expression of Vpu by the virus. Our data demonstrate that tetherin expression alone is not responsible for restriction of WT HIV-1 replication in mature DCs. Indeed, previous work has identified other mechanisms responsible for post-entry restriction of HIV-1 replication in mDC-LPS . The restriction of HIV-1 replication in mDC-IFNα may be due to multiple restriction factors. For example, APOBEC3G can block HIV-1 infection in DCs, and its expression is upregulated by IFNα and LPS [24, 64].
In iDCs, which do not express high levels of endogenous tetherin, there was a significant increase in tetherin expression in response to the infection with WT and ΔVpu HIV-1. This is consistent with earlier studies that endogenous tetherin in macrophages can be upregulated by HIV-1 infection . When mDC-LPS and mDC-IFNα were infected with WT or ΔVpu HIV-1, tetherin expression was maintained longer than that in mock-infected controls, which is presumably due to stabilization of tetherin or replenishment by tetherin induction. The maintenance of tetherin expression within mature DCs does not appear to be affected by Vpu expression.
Upregulation of surface tetherin in macrophages by HIV-1 infection appears to be induced by Nef , and HIV-1 replication in a human CD4+ cell line causes tetherin induction after an initial down-modulation of tetherin . Thus, we investigated the role of both HIV-1 replication and Nef protein in the transient induction of tetherin in iDCs. AZT treatment and deletion of Nef blocked the HIV-1-mediated tetherin upregulation in iDCs. These data suggest a role of newly synthesized Nef in the transient upregulation of tetherin in iDCs. HIV-1 Nef can cause induction of pro-inflammatory cytokines from human DCs and macrophages [65, 66], so it is possible that these cytokines act in an autocrine manner to induce transient tetherin expression in DCs as part of an innate immune response to HIV-1 infection. Moreover, an increase in cellular content of tetherin may reflect its stabilization or a slow turn-over upon HIV-1 infection and expression of Nef. The mechanisms by which Nef induces tetherin expression in DCs remain to be elucidated.
Of note, a recent study indicated that HIV-1 infection of MDDCs undermines the IFN induction pathway via interferon regulatory factor 1 (IRF1) and blocks type I IFN production, although HIV-1 infection in DCs induces a subset of ISGs . In agreement with this report, we were not able to detect the release of IFNα or IFNβ in the supernatants from HIV-1-infected iDCs at 1 to 5 dpi despite significant increases of IFIT-1 mRNA expression.
It is unclear as to why HIV-1 has not evolved a mechanism to block Nef-dependent induction of tetherin in DCs in addition to expressing Vpu as an antagonist of tetherin. Given the apparent transient nature of the Nef-induced tetherin expression in DCs, it is possible that as the tetherin level naturally diminishes over time, it does not affect HIV-1 release at time points of significance. HIV-1-induced tetherin expression also has the potential to ensure that HIV-1 remains in close association with the cell. In the case of DCs, this may allow HIV-1 to stay in close association with the cells during trafficking to the lymph node and subsequent transmission to CD4+ T cells at late time points, as is suggested to occur in vivo [2, 5].
In summary, we have investigated the role of IFNα and tetherin in DC-mediated HIV-1 infection and transmission. Our data suggest that tetherin is not a major restriction factor for WT HIV-1 replication in DCs or DC-mediated cell-to-cell transmission of HIV-1 to CD4+ T cells. Interestingly, we found that HIV-1 infection of iDCs induces Nef-dependent tetherin expression, suggesting an intrinsic antiviral mechanism in DCs triggered by productive HIV-1 infection and the pathogenic factor Nef. Further studies of this mechanism in DCs will provide a better understanding of the innate immune response against HIV-1 infection.