shown that direct contact between HIV-1-infected PBMCs and the apical epithelial surface played a critical role in HIV-1 binding and endocytosis in epithelial cells (Phillips et al., 1994
; Bomsel, 1997
). The present work presents evidence for morphological, structural, and functional similarities between such HIV-1-infected cell-epithelial cell adhesion and the neuronal and immune synapses that have evolved for efficient communication between pre- and postsynaptic cell surfaces. Indeed, the site of contact between HIV-infected cells and the mucosal epithelial surface, when visualized at the ultrastructural level, closely resembles neuronal and immune synapses and requires the synapse-specific scaffolding molecule agrin. Furthermore, the process of information transfer from the HIV-1-infected cells to the epithelial cells shares most of the important steps involved in the formation and function of both neuronal and immunological synapses. We propose, therefore, a new efficient mechanism for mucosal entry and efficient transcytosis of HIV-1 through its epithelial target cell via the formation of a “virally mediated synapse” between HIV-infected and epithelial cells.
In the virally mediated synapse described here, it is the infectious process that confers to the HIV-1-infected cell the capacity to act as epithelial cell partner. Several factors could confer to the HIV+ cell the ability to establish a synapse: first, the virally encoded envelope glycoproteins gp120/gp41; second, an increased expression of cell surface adhesion molecules on HIV+ cell as compared with uninfected cells (Shattock et al., 1996b
); and third, an actin cytoskeleton-mediated process through adhesion-induced polarized secretion of HIV-1 from activated mononuclear cells onto epithelia.
The present data, therefore, not only have described the synapse formed between an HIV-1-infected cell and an epithelial cell, one of the principal primary target cells of HIV upon sexual and vertical transmission, but also have allowed us to unravel the machinery on which the virally mediated synapse is based.
Three epithelial molecules, namely, agrin as HIV-1 attachment receptor, beta-1 integrin/RGD containing factors and GalCer, the previously described endocytic receptor for HIV-1 in epithelial cells (Bomsel, 1997
; Alfsen et al., 2001
), are required for virally mediated synapse formation, stabilization, and initiation of efficient HIV-1 endocytosis/transcytosis.
The presence of the scaffolding HSPG agrin in the synaptic cleft is a hallmark of immunological and neuronal synapses (Bezakova and Ruegg, 2003
). We now show that agrin is expressed with a polar distribution on the apical epithelial surface and around the microvilli on a polarized monolayer of HT29 or HEC-1 cells. Agrin is also present on primary epithelial cells purified from human colon (). Agrin participates in the virological synapse and its signaling, because anti-agrin antibodies inhibit HIV-1 transcytosis (). As in the immunological synapse (Batista et al., 2001
; Bromley et al., 2001
), agrin would act in an autocrine manner in the virally mediated synapse. However, in contrast to the immunological synapse, but similar to the neuronal synapse, only glycosylated agrin is active at the virological synapse between PBMCs and epithelial cells.
In addition to a scaffolding role within the synapses (Dustin and Colman, 2002
), agrin appears to function as an HIV-1 attachment receptor. Indeed, colocalization of gp41 and agrin is detected in a punctuated pattern at the interface of the HIV+ cell and the apical epithelial cell surface (), most likely representing the synaptic cleft. In addition, newly budded HIV-1 particles are detected to colocalize with agrin on microvillosities at the ultrastructural level () but cell-free HIV-1 also bind epithelial cells at 4°C in an agrin-dependent manner ().
The gp41 region interacting with agrin appears restricted to the conserved region P1, as demonstrated by biochemical and biophysical studies. P1 contains epitopes critical for HIV neutralization by one of the rare IgG antibodies (2F5) able to neutralize HIV primary isolates in vivo (Baba et al., 2000
) and by the other gp41 specific neutralizing IgG, 4E10 (Zwick et al., 2001
) as well as by mucosal IgA from seropositive individuals (Bomsel et al., 1998
; Alfsen et al., 2001
) and from highly exposed but persistently IgG seronegative subjects resistant to AIDS (Devito et al., 2000
). The present data demonstrate a direct interaction of P1 with HSPG and specifically with agrin, before P1 binding to its epithelial endocytic receptor GalCer (Figures and ). Such an interaction between agrin and P1 provides a novel explanation for the exceptional antiviral properties of 2F5-like antibodies. Actually, the negative charge of agrin HS would modify the orientation of the P1 lectin site orientation, in turn strengthening P1 interaction with GalCer. Accordingly, only glycosylated agrin bound to P1 and strengthened P1 binding to GalCer. As in CNS neurons (Hilgenberg et al., 1999
), there was no distinction between B/z+ and B/z- agrin isoforms. Moreover, the agrin core protein also appeared critical in this activity as neither syndecan-1 or similar HS bound to another protein core, nor other IgA glycans were effective ().
The long chains of polysaccharides in HSPG provide much versatility in biological information storage and transfer which justifies the new concept of “sugar code”(Gabius et al., 2002
). How the organization of these long polysaccharide chains on a specific protein core adds to the specificity of the molecule, remains to be investigated.
Binding of HIV-1 to its agrin attachment receptor is not sufficient to induce endocytosis/transcytosis. Hence, the attachment efficiency of cell-free HIV-1 to the epithelial membrane is several times higher than the efficiency of transcytosis (compare Figures and ). An additional signal provided by the virological synapse framework, possibly initiated by RGD-containing proteins, is required.
Cell-cell adhesion leading to recognition between and locking of the two cell surfaces is due, at least in part, to integrin-disintegrin interactions between epithelial cells and HIV-1-infected PBMCs. Indeed, function-blocking beta-1 integrin antibodies or a monomeric RGD disintegrinlike peptide, applied at the epithelial cell apical surface, inhibit HIV-1 transcytosis induced upon contact of HIV-1-infected PBMCs with epithelial cells (). Reciprocally, because beta-1 integrin ligand is RGD dependent, a multibranched RGD (octamer) peptide is able to promote efficient cell-free HIV-1 transcytosis (). Functional integrins are alpha/beta heterodimers and the beta-1 subunit can only associate with a limited set of alpha subunits, namely alpha V, 8 and 5 (Hartner et al., 1999
; Skrzypczak et al., 2001
; Proulx et al., 2003
) and bind to RGD-containing ligands. These three alpha subunits are expressed on epithelial cells including endometrial cells and therefore could participate in the virally mediated synapse. Integrins are not constitutively active. The interaction of the disintegrin with the alpha/beta interface induces a conformational change in the integrin dimer and propagation of signaling. Many function-blocking and -activating antibodies bind the same part of the interface, also suggesting a propagated conformational change in this region related to integrin activity (Hynes, 2002
). Here, disintegrin on HIV-1-infected cells PBMCs, by interactions with epithelial cell integrin, could activate the integrin conformational change.
Within the neuromuscular junction synapse, integrins not only promote cell-cell contact but also interact with agrin to trigger acetylcholine (ACh) receptor clustering on the postsynaptic muscle cell in an RGD-independent manner; agrin here appears as an aggregating factor (Martin and Sanes, 1997
). In the current model, because cell-free HIV-1 does not efficiently transcytose despite the presence of agrin on the epithelial surface, an RGD-independent interaction of agrin with integrin does not appear sufficient to initiate HIV-1 transcytosis.
However agrin and integrin are both required for the virally mediated synapse formation and signaling, probably not in a direct synergistic way. Rather, the interaction of integrin with the RGD-containing molecules on the infected cell membrane or delivered in the synaptic cleft as soluble factors by the HIV+ cell (Rusnati and Presta, 2002) could initiate the signaling pathway.
Regarding the results reported for cell-free virus transcytosis (Hocini et al., 2001
) the inoculum needed is 100-1000 fold higher in p24 U than that required for transcytosis of virus generated when HIV-1-infected PBMC are in contact with epithelial cells. These data suggest that transcytosis in such cell-free conditions occurs nonspecifically by fluid phase transcytosis (Bomsel et al., 1989
), and therefore no mechanistic comparison can be made with the present data.
Taken together, the present data suggest that direct contact of HIV-1-infected PBMC with epithelial cells, leading to the virally mediated synapse formation, induces a segregation and concentration of molecules such as adhesion molecules (Shattock et al., 1996b
), with the recruitment of raft microdomains in both cell membranes (Nguyen and Hildreth, 2000
; Alfsen et al., 2001
), in the synaptic cleft as defined for the prototypic synapse (Dustin and Colman, 2002
). Additionally, HIV-1, through gp41, attaches to an epithelial HSPG, as do several other viruses with epithelial tropism (reviewed in Rabenstein, 2002
; Bomsel and Alfsen, 2003
), here further identified as agrin. Such interaction modifies the gp41-lectin site (P1) orientation toward GalCer, the HIV-1-endocytic receptor in epithelial cells, and strengthens the P1/GalCer interaction. In turn this would favor and stabilize the recruitment of GalCer to apical raft microdomains (Alfsen and Bomsel, 2002
) together with the additional interaction of GalCer with HIV-1 envelope gp120 (Harouse et al., 1991
). Therefore agrin appears not only as a HIV-1 attachment receptor but also as an aggregating factor that mediates GalCer recruitment into raft microdomains via an interaction with gp41. The modifications induced in both cell membranes, together with HIV-1 binding, allow signal transduction from the infected PBMC to the epithelial cell to promote efficient HIV-1 transcytosis.
Hence, our present results indicate that viruses can induce and opportunistically utilize the specific molecular synapse scaffold and molecular machinery for entry into and transcytosis through their initial target cells at mucosal site, the epithelial cells.