We have shown previously that the interaction of uninfected target CD4 T cells with HIV-1 envelope gp120 and ICAM-1 on laterally mobile planar bilayers results in transient formation of VS, which share some common morphological features with the better defined IS. The present study provides the first molecular details about the initial assembly of VS, the local signaling triggered and the cytoskeleton organization underlying the VS. Similar to the TCR MCs found at the early stages of IS, the VS is initiated with gp120 MCs that converge centripetally to form the cSMAC. As in the IS, the formation and centripetal transport of the gp120 MCs are actin dependent, as they are sensitive to inhibitors of actin polymerization. Unlike the TCR MCs (71
), once formed, gp120 MCs remain actin dependent, although similar to the IS cSMAC (71
), the VS cSMAC is actin independent. In addition to being a domain where virus antigens and cellular receptors are highly concentrated, one notable feature of the VS cSMAC is the cortical actin rearrangement that creates an F-actin-depleted zone immediately beneath the cSMAC (Fig. ), which can be important for postentry events. Previous studies in which F-actin was visualized at the VS in cell-cell systems lacked the resolution needed to evaluate F-actin clearance from a 2- to 3-μm-size cSMAC region (41
). Our data showing actin-depleted cSMACs are consistent with the idea recently presented by Liu et al. (51
) suggesting that the interaction of CD4 T cells with gp120 triggers cytoskeleton reorganization where actin first polymerizes to form a cortical F-actin zone and then depolymerizes so an F-actin-depleted zone is formed underneath the gp120-rich region. This model provides an explanation for the seemingly contradictory roles of F-actin in HIV infection: cortical actin polymerization is essential for the initial assembly of the VS and for the capping process of virus receptors (3
) that facilitates viral transfer and binding to the target CD4 T cells, but a zone depleted of cortical F-actin is then needed to enable the virus core to move from the plasma membrane to the nucleus. Nevertheless, the postentry steps in HIV-1 infection affected by cortical F-actin and its inhibitory mechanisms are still unknown. It is plausible that HIV-1 evolves to exploit the molecular segregation and signaling as induced physiologically in the IS for creating an actin-depleted zone in the VS cSMAC and possibly relieving postentry blocks.
Our model would suggest that HIV Gag should accumulate to induce viral budding opposite the F-actin-depleted cSMAC in the target cell. However, the exact localization of virus budding and entry in the context of the VS substructures (i.e., cSMAC versus pSMAC) remains unclear and should be further investigated. In one study, Gag was reported to accumulate in a ring-shaped structure (61
) that resembles a pSMAC, while in another study, it was shown to be in micrometer-sized buttons (36
) that resemble the cSMAC. It is also not clear whether Gag and Env colocalize at the same substructures of VS, though they have been suggested to colocalize at the plasma membranes of virus-infected cells (34
) and at the VS interface (41
). Furthermore, viral transfer across the VS has been suggested to occur via endocytosis (8
), and cell-free virions have also been found to enter cells via fusion with endosomal membranes in a dynamin-dependent mechanism (55
). Nevertheless, the IS cSMAC is a site of bidirectional membrane traffic (50
) despite being relatively F-actin depleted (43
). Therefore, if virions are to be endocytosed at the cSMAC prior to fusion, it is likely that there is a sufficient F-actin level in the VS cSMAC to mediate this process while also allowing movement of the virus core to the nucleus.
The molecular basis for the formation of the F-actin-depleted zone underneath the VS cSMAC is not fully understood, though activation of Lck is clearly required for its formation (Fig. ). A recent study by Yoder et al. (74
) demonstrates that gp120 interaction with CXCR4 activates cofilin, an actin-depolymerizing factor, which facilitates HIV-1 postentry replication events in resting T cells, which otherwise are refractory to HIV-1 infection. Cofilin is an important component for F-actin dynamics at the periphery of the IS (64
) and may also play a role in F-actin depletion in the center of the IS (43
). In the IS, full TCR signaling is terminated in the cSMAC, but it is possible that some partial signaling remains to form the F-actin-depleted central zone (11
). Whether cofilin plays a role in creating the F actin-depleted zone in the VS is not known, and the signaling pathway connecting Lck activity and F-actin depolymerization remains to be determined. Nevertheless, our data provide clear evidence for active local membrane-proximal TCR signaling at the VS. Lck is associated with gp120 MCs and is activated within minutes after CD4 T cells contact gp120 on the bilayer.
Evidence for downstream activation of the TCR signaling pathway at the VS is presented by the activation and recruitment of CD3ζ, ZAP70, LAT, SLP76, Itk, and PLCγ. Jolly et al. have shown before that CD3
was partially recruited to the interface in 15% of cells forming VSs and thus concluded that the TCR is not involved (41
). However, it is well established that TIRFM is much more sensitive for detection of TCR than cell-cell imaging methods (10
). Indeed, our experiments showed relatively weak recruitment of CD3
and αβTCR to the VS interface compared to CD3ζ. Either preassociation of CD4 and TCR (44
) or induced association following CD4 clustering (54
) could account for the observed recruitment of TCR components following CD4 engagement by gp120. Moreover, evidence for TCR-CKR physical association upon chemokine engagement (45
) further strengthens the possibility that once gp120 binds to CD4 and CKR, the TCR-CD3 ITAMs may be recruited to the VS and become available for phosphorylation by Lck.
Nevertheless, full T-cell activation as classically triggered via TCR is not induced at the VS, as indicated by the absence of PKCθ recruitment, intracellular Ca2+
influx, and CD69 upregulation. In contrast, soluble gp120 and cell-free virions have been shown to induce Ca2+
influx in T cells in a CKR-dependent manner (53
). The organization of gp160 trimers on virions could activate signals through a specific cross-linking geometry that might not be generated by monomeric gp120 in the supported planar bilayers. The mechanism by which HIV-1 virions would induce signals that are not induced by gp120 in the planar bilayers is not clear. The organization of molecules in the VS may inhibit signals that are induced by engagement of CD4 and CKR, or the soluble gp120 in the earlier studies may contain some aggregates. It should be noted that in our planar bilayer system, the target cell engages only gp120 and ICAM-1, while HIV-1 virions can acquire host cell surface molecules like CD28, CD152, CD80, and CD86 (30
) that can interact with their ligands on the target cells and modulate T-cell activation together with the viral Env. In addition, gp120 can engage α4β7 (1
) and other CKRs on T cells and other types of cells (26
), all of which may contribute directly or indirectly to Ca2+
influx detected in the T cells. Another possible reason is the use of activated CD4 T cells in our experiments compared to nonactivated CD4 T cells in the other studies. In the quiescent G0
-phase cells, robust activation signals are needed to release postentry blocks and allow integration (67
), whereas in the G1
-phase resting cells, a subthreshold stimulus is sufficient to support infection, albeit at a reduced level compared to activated CD4 T cells (69
Akt phosphorylation is also induced upon VS formation, but only at one of the two phosphorylation sites associated with Akt activation. PI3K has been shown to regulate HIV-1 replication following viral entry in CD4 T cells and to trigger Akt activation in response to soluble gp120 and cell-free virions (27
). The PI3K/Akt pathway is known to be crucial for cell survival (12
) and has also been implicated to play a key role in prolonging survival of target cells and replication of many viruses other than HIV-1 (39
). However, it is yet to be determined whether the prosurvival PI3K/Akt pathway is triggered by the VS in a manner that promotes target cell survival and early HIV-1 replication events. Considering that gp120 interaction with the CKR has been shown to induce apoptosis in CD4 T cells (6
), induction of a prosurvival pathway by the VS may serve as a counter mechanism to protect the target cells from gp120-mediated apoptosis.
In conclusion, the VS is generated by the convergence of gp120 MCs into a cSMAC. TCR signaling is initiated in MC and is sustained in the cSMAC. The signaling does not lead to canonical T-cell activation and proliferation but marks an F-actin-depleted zone in the VS center and thus may play a critical role in removing the cytoskeletal barriers for viral entry and infection.