This is the first study that has analyzed the function of DC-SIGN from a natural host species of SIV. Previous studies have only reported interactions between DC-SIGN from human and rhesus macaques expressed on THP-1 or 293T cells with HIV-luc pseudotyped with Env from SIVagm or SIVsm (38
). Importantly, interactions of SIV with human molecules or vice versa of HIV with simian molecules do not necessarily reflect interactions of the virus with the homologous host-specific molecule. It was shown, for instance, that SIVmac, although able to use human STRL33/Bonzo, does not efficiently use the macaque molecule for entry (39
). Similarly, HIV-1 uses AGM CCR5 only inefficiently due to the G163R difference between the human and AGM protein, whereas SIVagm is adapted to efficiently use the wild-type AGM CCR5 (26
). It is thus important to analyze the interactions of receptors and viruses derived from the same species. Here we describe the efficient interaction between DC-SIGN derived from AGMs (C. sabaeus
) and its host-specific SIV (SIVagm.sab). In order to be close to physiological conditions, we used a viral isolate and not a molecular clone. Furthermore, we performed infections with low viral doses (MOIs of 10−3
). We showed on human cell lines that DC-SIGNagm can act as a viral attachment factor like its human and macaque counterparts despite structural differences among these molecules. Consequently, this suggests that the lower viral load in LNs during nonpathogenic SIVagm infection (10
) compared to pathogenic HIV/SIVmac infections (4
) cannot be explained by structural differences in the AGM molecule. We cannot totally exclude variations either among SIVagm strains or among dc-signagm
alleles that might be detected by analyzing more donors. However, even if such variations exist, they could not explain the consistently nonpathogenic outcome of SIVagm infection.
In view of the above results, additional factors must be involved in the low viral load in LNs during SIVagm infections. Efficiency of transmission to T cells in vitro depends on the levels of DC-SIGN and ICAM-1 expression on DC (38
). Alternative splicing of dc-sign
mRNA might represent a mechanism that could regulate the expression of the dc-sign
gene. Our study reveals a nonexhaustive repertoire of DC-SIGN in LNs of AGMs. We have not detected dc-sign
mRNA encoding potentially soluble isoforms of DC-SIGNagm, whereas transcripts coding for these isoforms have been detected by using only a single RT-PCR step in human cells, such as PBMC, DC derived from CD34+
peripheral blood hematopoietic progenitors, and resting CD14+
). Indeed, the expression pattern of dc-sign
transcripts may depend on the origin, as well as the maturation or activation state, of the cells (35
). It remains unknown whether transcripts coding for potentially soluble isoforms also exist in human LN or in other nonhuman primates such as macaques. Although soluble isoforms do not seem to be able to mediate enhancement of infection (27
), they may regulate expression of the membrane-associated forms of DC-SIGN (35
). However, we demonstrate here that AGM MDDC show high surface-expressing levels of DC-SIGN protein. This contrasts with one study which reported very low levels of DC-SIGN on rhesus macaque MDDC (61
). The expression levels of DC-SIGN may vary according to the primate species and/or the donor (25
). Numbers of DC-SIGN molecules on AGM MDDC did indeed vary according to the AGM donor, as shown for human MDDC (1
). However, the AGM myeloid DC (MDDC) from all studied AGM donors expressed at least 100,000 DC-SIGN molecules and often more than 200,000 molecules per cell, a level sufficient to promote viral transmission to T cells in vitro.
In vivo, DC-SIGN+
cells could be easily detected within the medulla sinuses and the paracortex of AGM LNs. We cannot exclude the possibility that the detection of positive cells in the medulla sinuses could also be due to the related L-SIGN2 (3
). However, the distribution of DC-SIGN+
cells in the LNs of AGMs was very similar to that described in macaques (8
). Rhesus macaque antigen-presenting cells in the medulla and cortex/paracortex in organized lymphoid tissues (LNs and spleen) have been shown to express DC-SIGN, although the number of positive cells has not been quantified (8
). The nature of the AGM DC-SIGN+
cells is unknown, but the cells exhibited a morphology characteristic of DC. It has been suggested in similar studies in humans and rhesus macaque tissues that the DC-SIGN+
cells likely correspond to interdigitating DC and sinusoidal macrophages (14
). The distribution of DC-SIGN+
cells in the medulla and paracortex was similar to that of cells expressing a marker of the monocyte/macrophage lineage (CD68). However, only CD68+
cells were detected in germinal center, showing that not all CD68+
cells express DC-SIGN. Furthermore, it has been shown that in macaque spleens and LNs all DC-SIGN+
cells exhibiting a dendritic morphology express CD68 (45
). Altogether, our data show a similar morphology and distribution of DC-SIGN+
cells compared to these cells in humans and macaques. We also observed that mRNA coding for functional DC-SIGN is abundantly expressed in AGM LNs. This suggests that DC-SIGN expression on DC in AGM LN is not lower than in humans and macaques.
We cannot totally exclude a distinct regulation of DC-SIGN expression in response to SIVagm infection. The level of human DC-SIGN at the cell surface is indeed influenced by HIV-1 proteins. A dramatic increase of lymphocyte adhesion to HIV-1-infected DC in vitro due to an upregulation of DC-SIGN mediated by Nef has been reported (50
). This phenomenon, however, is not consistently observed for all donors (31
). It is not excluded that Nef encoded by SIVagm interacts differentially with host-specific cellular proteins compared to HIV/SIVmac Nef in human and macaque cells, which could result in lower levels of DC-SIGN expression and/or lower T-cell activation profiles in SIVagm-infected AGMs. Lower T-cell activation states are indeed observed in nonpathogenic SIV infections in their natural hosts (7
). However, there are no signs thus far of SIVagm replication within AGM MDDC with at least up to 10−3
/cell and after p27 values up to 2 weeks. This might also be dependent on the SIVagm strains used. Further studies are needed to determine the susceptibility of DC to SIV in nonpathogenic SIV infection models.
Finally, the expression level might depend on the cytokine environment in vivo after SIV infection. Several studies have reported that IL-4 induces DC-SIGN expression on human peripheral blood monocytes in vitro (35
). This IL-4-dependent expression is negatively regulated by alpha interferon (IFN-α), transforming growth factor β, and IFN-γ (41
). Recently, it has been shown in another model of nonpathogenic SIVinfection, SIVsm infection in the sooty mangabeys, that the chronic phase of infection is characterized by higher levels of IL-4 and lower levels of IFN-γ production (47
). Thus, there are as yet no data that could support any downregulation of DC-SIGN expression after SIV infection in the natural host.
We show here that MDDC from AGMs not only express high levels of DC-SIGN but also efficiently transmit SIVagm to T cells. This has been confirmed here for MDDC from 10 distinct AGM donors. Furthermore, as shown by inhibition assays with anti-DC-SIGN MAb, viral transmission by AGM MDDC is DC-SIGN dependent. This has been confirmed with MDDC from six distinct AGM donors. This result supports the findings of an earlier study of Chinese macaque species, M. fascicularis
, in which viral transmission is also largely DC-SIGN dependent (25
). However, it contrasts with a study of M. mulatta
MDDC that indicated no discernible role of DC-SIGN in viral transmission (61
). These controversial results might be explained by variations of DC-SIGN expression according to individuals, especially if only a few animals were studied, and/or according to monkey species. Interestingly, we observed that the viral transmission by AGM MDDC was significantly blocked by anti-DC-SIGN MAbs only when a low MOI (10−5
) was used. It has been reported that the affinity of HIV-1 gp120 for DC-SIGNhu is higher than for CD4hu (9
), and some data suggested that it is also higher than for the mannose receptor (56
). Thus, the use of high viral infectious doses could probably allow attachment to other molecules as well. This might also explain why in previous studies anti-DC-SIGN MAb or mannan could not consistently block viral entry or Env/DC-SIGN interaction in MDDC. Indeed, in these previous studies, very high doses of HIV AD8 (MOI of 45) (57
) or a high amount of gp120 (twofold more than the predetermined concentration for cellular saturation) (56
) were used.
DC seem to be a “hiding place” for HIV-1 (58
). However, the mechanism of viral transfer via DC-SIGN has yet to be fully elucidated. The function of DC-SIGN in the transmission of HIV-1 depends on its cellular context (54
). DC-SIGN expressed by DC or the monocytic cell line THP-1 but not 293 and HOS cells internalizes and retains HIV-1Bal
in a highly infectious state for more than 5 days (13
). Immature human MDDC promote HIV-1Bal
transfer by a mechanism that is DC-SIGN dependent (40 to 100% according to the donors tested) at least up to 2 days after viral exposure (54
). Recently, others suggest most endocytosed virus is already degraded after 24 h (55
). We demonstrated here that sufficient amounts of infectious SIVagm particles remain intact in AGM MDDC up to 3 days, even when low doses of virus are used. This efficiency might be due to the fact that we used a virus isolate and MDDC to assess viral transfer to highly permissive T cells.
We analyzed here for the first time the virological functions of DC-SIGN from an animal species resistant to AIDS. The present study revealed similar activities for DC-SIGNagm regarding trans infection compared to previous reports on human and macaque DC-SIGN. We demonstrated that DC-SIGN+ cells are present in AGM LNs and that AGM MDDC express levels of DC-SIGN similar to those expressed by human MDDC. We showed that AGM MDDC are able to efficiently transmit SIVagm to T cells. Finally, we showed that SIVagm transmission is largely DC-SIGN dependent at low MOIs. Altogether, our data indicate that the virus-binding properties of DC-SIGN are not directly associated with viral load levels in LNs. In conclusion, additional factors, such as the frequency in LNs of major target cells (activated T CD4+ lymphocytes), are more likely determinating factors for viral replication levels in LNs.