We show here that after uptake by DCs, HIV virions can be directly transferred to lymphocytes but only during a short period of time (i.e., a few hours). There is no long-term storage of captured virions by DCs or by DC-SIGN-expressing cell lines. After a few days, virus progeny is transmitted to lymphocytes.
We have examined the cellular and virological mechanisms underlying these phenomena. We focused a large part of our study on the behavior of X4 strains, which were widely believed not being able to replicate in iDCs (14
). For both X4 and R5 isolates, the maturation state of DCs apparently regulates viral replication. It has been reported that mature DCs (mDCs) display a decreased capacity for the production of HIV, which may be due to a postentry block (4
) and/or to variations in CXCR4 and CCR5 expression (14
). Whatever the maturation state of these cells, X4 and R5 isolates readily enter DCs, perform RT, and are transmitted to lymphocytes (3
). It was thus puzzling that productive infection was not detected with X4 strains in iDCs. We provide here three lines of evidence that X4 strains replicate, albeit at covert levels, in iDCs. First, small amounts of viral production were measured in supernatants of cells exposed to the laboratory-adapted NL4.3 strain, as well as two isolates carrying primary envelope glycoproteins. Flow cytometry analysis confirmed that DCs were productively infected. Viral production at the peak was about 10-fold lower with NL4.3 than in the isogenic R5 strain NLAD8, which differs only in the envelope gene. Second, by using a single-cycle X4 virus expressing the luciferase reporter gene, we detected low levels of luciferase expression in these cells. The viral cycle was apparently slow, since the signal was significantly detectable at 96 h and not at 48 h p.i. Third, quantitative PCR analysis demonstrated that NL4.3 performed RT in iDCs. Both early and late viral DNA products were synthesized at 20 to 40-fold-lower levels than with NLAD8. Therefore, X4 HIV strains replicate covertly in monocyte-derived iDCs, and this is the consequence of an inefficient early event of the viral cycle occurring at the entry or postentry step.
In iDCs, as well as in DC-SIGN-expressing cell lines, a large portion of incoming virions is internalized in intracellular vesicles (9
). We show here that in DCs, proviral DNA synthesis is blocked by T-20, a viral fusion inhibitor, and does not occur with a mutant HIV carrying nonfusogenic viral envelope glycoproteins. Furthermore, by using HeLa cells expressing either DC-SIGN, CD4, or both molecules, we demonstrate that DC-SIGN by itself does not allow RT but significantly enhances viral DNA synthesis in cells expressing appropriate viral receptors (CD4 and CXCR4). Altogether, these results indicate that efficient proviral DNA synthesis requires access of incoming virions to the cytosol. After capture by DC-SIGN and internalization in a vesicular compartment, the so-called natural endogenous RT process (61
) does not occur and is therefore not involved in the trans
enhancement of viral infectivity from DCs to lymphocytes.
In what form is viral infectivity is transmitted from DCs and what is the role of DC-SIGN in this process? By using single-cycle HIV-Luc virions, we show that incoming particles are transferred from iDCs, as well as from Raji DC-SIGN and C1RA2 DC-SIGN cells, to target cells immediately after viral exposure. Parental Raji and C1RA2 cells did not transfer infectivity, confirming the role of the lectin in this process. However, no increase of luciferase signal was detected if target cells were added 48 h after viral exposure, indicating that DC-SIGN-expressing cells do not protect virus inoculum in the long term. Interestingly, in Raji DC-SIGN cells, a significant luciferase activity was detected even in the absence of targets, a situation reminiscent of primary iDCs. Low-level proviral DNA synthesis was detected in Raji DC-SIGN cells (with a 60-fold-lower efficiency than HeLa CD4+
cells), confirming the occurrence of surreptitious HIV replication in these cells. Interestingly, replicative HIV and not single-cycle virus was transmitted from Raji DC-SIGN cells 48 h after virus exposure. Altogether, these results indicate that in the long term, only progeny virus is transmitted from Raji DC-SIGN cells or from iDCs to lymphocytes. Turville et al. recently reported that DCs transfer R5 HIV to CD4+
lymphocytes in two distinct phases. By using replicative R5 HIV, they showed that transfer of infectious virus shortly after uptake does not require de novo synthesis, whereas the second phase of transfer is inhibited by AZT and is dependent on productive infection of iDCs (57
). Our experiments confirm and extend these observations. We demonstrate here that the transfer of X4 strains follows the same rules, with an immediate phase of transmission of incoming virions and a second phase of delivery of neosynthesized virus. Moreover, we point out that the role of DC-SIGN is mainly at the phase of virus uptake. Most of the incoming virions are rapidly degraded (within hours), whereas only a small fraction reaches the contact zone when DCs interact with T cells. DC-SIGN does not protect incoming virions over the long term, at least in iDCs and the cell lines studied here. The situation may be different in mDCs, which efficiently transmit HIV in the absence of detectable productive infection (21
). Upon DC maturation, DC-SIGN is down-regulated, the endocytic capacity of the cell is decreased, and HIV virions accumulate in vacuoles which differ in size and intracellular localization from iDCs (18
). The half-lives of incoming virions are also short in mDCs, but the rate of viral decay is slightly slower and less extensive than in iDCs (57
). It is thus conceivable that mDCs retain the ability to transmit captured virions for longer periods of time. On the other hand, it will be worth reexamining whether productive infection occurs at particularly low levels in these cells.
Our results provide a simple explanation for the cell-type-dependent effect of DC-SIGN on long-term retention of viral infectivity (54
). We show here that Raji DC-SIGN cells share with iDCs the capacity to replicate HIV at low levels and thereby to transfer infection to virus inoculum a few days after the initial exposure. In other cell lines, such as HeLa DC-SIGN, 293 DC-SIGN, or C1RA2 DC-SIGN, the absence of productive infection precludes any retention of viral infectivity.
DC-SIGN also promotes the so-called trans
enhancement of HIV infection, through a poorly characterized mechanism. Small amounts of virus, insufficient to allow the direct infection of T cells, become infectious after transiting by DCs or DC-SIGN-expressing cells (19
). DCs enhance infection through the formation of an infectious synapse, which brings virus and receptors closer together at the contact zone (41
). Formation of this synapse is important for virions in transit, but also for transfer of newly synthesized virus particles (29
). On the other hand, it has been proposed that DC-SIGN-mediated internalization of incoming HIV is required for trans
). In this last report, trans
enhancement was blocked by concanamycin A, an inhibitor of vesicular acidification (36
). In contrast, we show here that neither concanamycin A nor bafilomycin A1 inhibited transfer of replicative virus from iDCs to lymphocytes or transmission of single-cycle virus from Raji DC-SIGN to HeLa-CD4 cells. Of note, we used experimental conditions (low doses, pulse incubation, and extensive washing) to minimize the toxicity of these compounds. These drugs are known to affect cell viability, and various side effects have been reported that may bias the interpretation of experiments aimed at raising acidic pH (7
). We conclude that virus transfer from iDCs or from DC-SIGN-expressing cell lines does not require the low-pH environment encountered in vesicular compartments. DC-SIGN-mediated enhancement has been previously observed with single-cycle virus and thus involves transfer of incoming virions (19
). However, our results indicate that in donor cells where virus replicates at low levels, such as iDCs or Raji DC-SIGN cells, a large part of the trans
enhancement process is due to the dissemination of freshly produced virus.
R5 strains are preferentially transmitted among humans (39
). This restriction process is likely multifactorial and has been suggested to take place at the stage of DC infection (49
). We show here that X4 strains replicate in iDCs, albeit at much lower levels than R5 viruses, and are then efficiently transmitted to lymphocytes. Low levels of productive X4 HIV replication have also been observed in Langerhans cell-like DCs (33
), as well as in more complex models of HIV dissemination, such as ex vivo culture explants of cervical tissue (25
). This should be taken into account when designing strategies aimed at blocking HIV-1 uptake by DCs or other cells within genital mucosa (5