The clustering and organization of proteins and lipids in the plasma membrane is vital for membrane-based processes. A variety of interactions, most notably lipid-lipid, protein-lipid, and protein-protein interactions, have been described to contribute to the lateral organization of membrane domains (for a review, see reference 84
). The HIV-1 Gag protein has recently been shown to reorganize preexisting membrane microdomains and to alter the dynamics of such domains (33
). Because the regulation of Env fusion is critical for efficient viral spread, we investigated how the presence of Gag affected the nanoscale organization and dynamics of Env in the plasma membrane. Our data revealed that Gag, at viral assembly sites, traps Env in a CT-dependent manner and that such trapping correlates with the inhibition of Env-induced fusion of producer and target cell membranes.
It is well established that Env forms trimers, with each subunit consisting of a heterodimeric complex formed by a surface subunit (gp120) and a transmembrane subunit (gp41). Using STORM, we found that WT Env trimers form small clusters at the plasma membrane and that upon Gag assembly, these Env clusters become significantly larger, suggesting that the assembly process draws multiple small Env clusters to these sites. Although we cannot determine the stoichiometry of Env molecules in these clusters, previous work (high-pressure liquid chromatography [85
] and electron tomography studies [86
]) suggested that each nascent virion incorporates 7 to 21 Env trimers. Therefore, the small clusters we observed outside Gag assembly sites likely consisted of fewer than 7 trimers and perhaps as few as one. Note that, to some extent, the clustering of individual (or small groups of) Env trimers resembles the Gag-induced reorganization of tetraspanins and raft lipid-containing nanodomains mentioned above. It will be interesting to determine the sequence of recruitment of these and other host cell assemblages (for example, see reference 34
), including the ones containing phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2
], which has recently been shown to form discrete but very small domains (71
Another superresolution study (53
), which was in progress in parallel to the investigations presented here, also documents that Env trimers cluster at Gag sites. In addition, both studies document that, unlike WT Env, ΔCT Env formed equal-size clusters throughout the plasma membrane that were not dependent on the presence of Gag. Thus, Gag apparently does not affect ΔCT Env clustering. Regarding the situation in viral particles, it was recently hypothesized that such differential accumulation of Env trimers is due to differences in Env mobility and, more specifically, that the immature Gag lattice inhibits Env from moving freely in the viral membrane. It was speculated that Env can form a single focus (consisting of multiple Env trimers) only upon particle maturation (i.e., Gag processing) because Env trimers then become mobile and can polarize toward the CD4 clusters on the target cell (18
). However, directly measuring whether altered Env mobility is indeed responsible for altered accumulation of the viral fusogen cannot yet be done in viral particles as, to the best of our knowledge, no appropriate biophysical methods have been implemented to do that. Methods to analyze the mobility of integral membrane proteins in cellular membranes, however, are available, and we therefore set out, using FRAP, to measure the mobility of WT and ΔCT Env at Gag sites in the plasma membrane of producer cells. Our data demonstrate that the immature Gag lattice completely traps Env. Upon deletion of its CT, Env can move freely even at sites of Gag accumulation (i.e., at assembly/release sites), strongly suggesting that immature Gag traps Env via an interaction (direct or indirect) with the CT. The restoration of Env mobility (by deletion of the CT) was accompanied by a significant increase in Env-triggered cell-cell fusion, comparable to how CT deletion in particle-associated Env leads to a pronounced fusion increase for immature virions (14
). Our data thus strongly support the idea that the regulation of Env's mobility is linked with Env's ability to fuse membranes. Exactly how increased Env mobility (following CT deletion) ultimately results in increased fusion, however, remains to be analyzed. At a minimum, lateral Env mobility allows for the recruitment of multiple Env trimers to the site of interaction with CD4 (18
), i.e., it increases the likelihood that Env binds the receptor (and thus increases the likelihood that fusion is triggered). Additionally, the Gag maturation status and/or the presence of Env's CT likely also directly regulates the fusion process, either through modulating how Env and Gag interact with components of the cytoskeleton by regulating membrane stiffness (87
), or perhaps, through changes that occur in the extracellular domain of Env. Studies have shown, for example, that Gag maturation and/or mutations in the CT, through a so-called “inside-out” mechanism, lead to changes in epitope exposure of gp120 (e.g., see references 88
) and result in altered fusogenicity of Env.
Interestingly, while ΔCT Env, unlike WT Env, was still mobile at viral assembly sites, its mobility at those sites was reduced compared to its mobility elsewhere in the membrane. Also, the restriction of ΔCT Env mobility at assembly sites was dependent on Gag being able to multimerize. This finding is consistent with the hypothesis, recently put forward by Lucas et al., that the local accumulation of retroviral Gag creates a membrane environment that restricts Env glycoprotein mobility, perhaps facilitating Env incorporation into nascent particles (32
). In that report, the authors measured how murine leukemia virus (MLV) and HIV-1 Gag competed for the acquisition of limited amounts of MLV Env. They documented that MLV Gag outcompeted HIV-1 Gag but, also, that the differences in incorporation decreased drastically if MLV Env was expressed without its cytoplasmic tail. Lucas et al. concluded that there are at least two mechanisms that allow for Env recruitment, a specific mechanism (which depends on Env's cytoplasmic domain) and a generic, tail-independent mechanism. Our FRAP data provide further support for the existence of such a generic mechanism, as they document that ΔCT Env mobility is restricted at sites of assembly. Together with the previously mentioned reports (33
), our data document that Gag multimerization, besides being obviously critical for particle morphogenesis, also adjusts the immediate membrane milieu, e.g., for the capturing of Env.
In summary, we have used superresolution microscopy in combination with live-cell analyses to define the clustering and dynamics of Env in the plasma membrane. Our results reveal nanoscale differences in Env clustering that correlate with Env's fusion activity. Regulation of this activity is critical: a substantial amount of Env at the plasma membrane does not get incorporated into particles (53
; also data not shown). While Env situated adjacent to budding sites has been hypothesized to promote the formation and maintenance of the virological synapse (VS) (53
), if unhindered by Gag, host cell proteins, and/or restrictions imposed by the topology of membrane areas where Env is situated, Env would likely trigger the fusion of producer and target cells, thus leading to the formation of syncytia. Some syncytium formation may be tolerable or (under certain circumstances) even beneficial for virus spread, as evidenced by in vitro
analyses, as well as a recent study of the migration of infected cells in lymph nodes of humanized mice (13
). The latter study, however, also documented that only a small subset of infected lymphocytes fused to form syncytia, suggesting that the virus evolved mechanisms that allow it to prevent excessive formation of these multinucleated entities.