Here, we provide a global view of the diverse modes of HIV cell-to-cell transfer. Quantitative analysis indicated that single synapses, polysynapses, and thin membrane protrusions mediate viral spread in lymphocytes. Close interactions between infected and target cells, leading to a polarization of HIV Gag proteins at the contact zone, were frequently observed (25% and 15 to 20% of infected cells engaged in single and polysynapses, respectively). Filopodial bridges (46
) and nanotube-like structures (50
) transporting HIV-1 material were less frequent (<10% of infected cells), at least in our experimental setting, but may promote viral transfer to targets that are not in the immediate vicinity of infected cells. We show that HIV (in part through Env expression) manipulates cell-cell interactions and enhances these three types of contacts.
We identified a mechanism by which HIV-1 spreads in cultures from one infected cell to multiple (two to six) recipients, through polysynapses. These rosette-like structures, displaying multifocal Gag accumulation, were observed in infected lymphocytes and DCs. That Env-expressing cells can enter in contact with several cells was not unexpected. Images illustrating these multiple contacts appear sometimes in the literature (46
), but this phenomenon has not been the subject of a detailed investigation. The classical VS, initially described for HTLV-1 (20
), corresponds to a single donor engaging a single recipient, with a polarization of the MTOC and viral budding toward the site of contact. Obviously, this model cannot apply to HIV polysynapses, which require a more flexible mode of formation. Rather, because GM1, as well as CD63, Thy-1, and CD59 frequently colocalized with HIV-1 Gag at the sites of contacts, it is likely that the multifocal capping of assembled or budding virions required for polysynapse formation is promoted by tetraspanins and plasma membrane mobility via lipid rafts. Released HIV-1 particles incorporate various cellular molecules, including GM1, Thy-1, and CD59 (38
), implicating rafts as platforms of HIV budding (30
). Our results extend these observations and highlight a major role for rafts in polysynapse formation. The involvement of lipid rafts was further supported by our observation that cholesterol depletion of infected cells with β-methyl-cyclodextrin strongly inhibited both cell contacts and Gag polarization in polysynapses (not shown). Actin and microtubules are also likely involved in transporting Gag proteins toward the plasma membrane or in movement of surface viral proteins or particles toward polysynapses, because nocodazole, cytochalasin D, and latrunculin B inhibited polysynapse formation. It is well documented that retroviruses have evolved means to hijack actin as well as microtubule skeletal networks to traffic (8
). Whether the cytoskeleton provides concomitant routes of HIV transport to various regions of the surface or oscillates toward the different target cells will require further investigation. However, our combined use of IF, correlative microscopy, and time-lapse imaging strongly suggests that the multiple viral transfer zones of polysynapses can be formed simultaneously, rather than one after another.
In the immune synapse formed between one cytotoxic T lymphocyte (CTL) and its target, the MTOC is drawn vectorially to the contact site by a microtubule sliding mechanism and then oscillates laterally (29
). Thus, highly dynamic movements of the cytoskeleton likely promote formation of virological polysynapses. Assembly of individual virions at the plasma membrane is a rapid phenomenon (5 min) (27
). A fast viral assembly may be instrumental for polysynapse formation.
It is noteworthy that we report here a supramolecular structure of Gag proteins which accumulate in a ring-shaped zone at the junctions. This structure was observed in VS as well as in polysynapses and resembles the pSMAC of adhesion molecules in the immune synapse (6
). We report here that LFA-1, an integrin previously known to facilitate HIV replication, also enhances cell-to-cell viral transfer and formation of single synapses and polysynapses. Whether other adhesion molecules, like ICAM-1 (52
), and whether the recently described HIV-1 Env-α4β7 integrin interaction (2
) stabilize Gag rings and polysynapses remain to be determined. It will be also worth examining the role of viral accessory proteins (32
) as well as that of the MVB-associated HIV budding machinery (35
) in formation of Gag rings and polysynapses. Interestingly, a central clustering of Env was documented, in a situation where this viral protein is situated in planar bilayers that contact uninfected T cells (52
). Furthermore, quantitative 3D videomicroscopy revealed the formation of micrometer-sized “buttons” of Gag at the junction zones of infected Jurkat cells and primary CD4 T cells (18
). The links that may exist between the topology of Env proteins and of Gag rings and/or buttons will deserve further investigation.
Viruses exploit the normal physiology of cells to spread. The identification of virological polysynapses suggests that immune cells may simultaneously exchange signals and information with multiple adjacent cells. Examples of such functional multiple contacts are scarce. For instance, CD4 T cells can integrate signals from multiple antigen-presenting cells simultaneously (4
), whereas cytotoxic T lymphocytes polarize lytic granules toward different cells and may kill more than one target at a time (19
). Polysynapses likely represent an underestimated mode of communication in immune cells, and it will be of interest to investigate the role of signaling events (48
) in the establishment of virological polysynapses.
In lymphoid and mucosal tissues, a high cell density and a low speed of lymphocytes (10 μm/min) allow multiple encounters between cells. That clusters of infected cells are detected in such tissues suggests that virological polysynapses are biologically relevant. Polysynapses could play a critical role in the exponential explosive propagation that characterizes the acute phase of infection (14
). It is also tempting to speculate that polysynapses, by enhancing locally the MOI, provide a means for HIV and other viruses to escape innate, intrinsic, and adaptative immune responses (32