|Home | About | Journals | Submit | Contact Us | Français|
In vitro propagation studies have established that human immunodeficiency virus type 1 (HIV-1) is most efficiently transmitted at the virological synapse that forms between producer and target cells. Despite the presence of the viral envelope glycoprotein (Env) and CD4 and chemokine receptors at the respective surfaces, producer and target cells usually do not fuse with each other but disengage after the viral particles have been delivered, consistent with the idea that syncytia, at least in vitro, are not required for HIV-1 spread. Here, we tested whether tetraspanins, which are well known regulators of cellular membrane fusion processes that are enriched at HIV-1 exit sites, regulate syncytium formation. We found that overexpression of tetraspanins in producer cells leads to reduced syncytium formation, while downregulation has the opposite effect. Further, we document that repression of Env-induced cell-cell fusion by tetraspanins depends on the presence of viral Gag, and we demonstrate that fusion repression requires the recruitment of Env by Gag to tetraspanin-enriched microdomains (TEMs). However, sensitivity to fusion repression by tetraspanins varied for different viral strains, despite comparable recruitment of their Envs to TEMs. Overall, these data establish tetraspanins as negative regulators of HIV-1-induced cell-cell fusion, and they start delineating the requirements for this regulation.
The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is incorporated into released virus particles and enables the virus to attach to and fuse with target cells in order to initiate the infectious cycle. Before Env mediates the fusion of viral and cellular membranes, i.e., while it is still incorporated in the plasma membrane of the infected cell, it drives the adhesion between virus producer cell and target cells, which gives rise to the formation of the so-called virological synapse (VS) (21, 24, 35, 36). The VS shares certain characteristics with the immunological synapse, including an accumulation of specific cellular membrane proteins and lipids (see, e.g., reference 5), and it provides efficient and secure transfer of virus particles from infected to uninfected cells (8). Importantly, the two adhering cells, like the pre- and postsynaptic cells that form an immunological synapse, typically do not fuse during such cell-to-cell transfer events. At first glance this seems surprising, as HIV-1 Env, unlike many other viral envelope proteins, can induce membrane fusion at physiological pH. Also, adhesion of producer and target cell, which can be initiated when the uropod of the infected cell contacts the uninfected cell (8), followed by reorganization of the cytoskeleton (25) and formation of full-fledged synapses, can extend over minutes (see, e.g., reference 20). This process should allow enough time to trigger cell-cell fusion. However, it is now well established that newly synthesized Env is efficiently internalized upon its arrival at the host cell plasma membrane, unless it is recruited into budding structures by viral Gag (see, e.g., reference 11; also discussed in references 3 and 6). Further, and likely also contributing to the prevention of producer-target cell fusion, immature Gag at the host cell plasma membrane represses Env-driven fusion, and this repression is lost only once Gag is processed in released virions (9, 22, 23, 31, 50). Finally, because syncytia are clearly not required for the transmission of virus from cell to cell in vitro and are possibly detrimental to virus spread in vivo, we hypothesize that HIV-1 cooperates with cellular membrane proteins to prevent cell-cell fusion.
Members of a group of cellular proteins known as tetraspanins play an important role as regulators of cellular fusion processes, including myotube formation and fertilization (28, 30, 44; reviewed in, e.g., reference 17). As membrane organizers, these proteins homo- and heteromultimerize and associate with other cellular proteins to form variably sized but discrete microdomains, the so-called tetraspanin-enriched microdomains (TEMs) (29) (also called TERMs  or TEAs ). Knowledge of the molecular mechanisms through which tetraspanins regulate the fusion of cellular membranes is still lacking, though the available evidence strongly suggests (i) that these proteins are not themselves fusogens but rather that they coordinate the fusion activity of other cellular proteins and (ii) that they can act both as positive and negative regulators of cellular fusion processes. For instance, several in vivo studies unequivocally showed that CD9 expression in oocytes is essential for sperm-egg fusion (27, 28, 30), but CD9 and CD81 ablation in monocytes enhances the formation of multinucleated phagocytes that are involved in immune defense against certain microbes (45). Interestingly, the same two tetraspanins are also known to regulate virus-induced fusion processes. CD9 is involved in regulating cell-cell fusion driven by canine distemper virus, as the anti-CD9 antibody K41 inhibits syncytium formation by this virus (42), and CD81 is a necessary cofactor for infection of cells by hepatitis C virus (see, e.g., references 2 and 52). Finally, tetraspanins on uninfected (target) cells inhibit HIV-1-induced cell-cell fusion (14). This fusion regulation is likely due to interactions of CD9 and CD81 with CD4 and coreceptors at the surface of target cells, though the tetraspanin CD63 has also been implicated in the trafficking of CXCR4 to the plasma membrane (51).
Because tetraspanins in HIV-1-producing cells are enriched at budding sites (4, 10, 13, 15, 33, 46, 49) and at the VS (26), we hypothesized that they regulate Env-driven fusion at the VS. Here, we document that tetraspanins in HIV-1-producing cells can indeed restrict syncytium formation. We also define some of the requirements for this fusion inhibition, thus laying the necessary groundwork for future mechanistic analyses. In addition, the characterization of cell-cell fusion regulation parameters in this study will allow the fusion-inhibitory activities to be distinguished from other regulatory functions exerted by tetraspanins, such as the modulation of virion infectivity and the regulation of cell-to-cell transmission of HIV-1.
The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl cells from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc.; Jurkat clone E6-1 cells from Arthur Weiss; reverse transcriptase inhibitors zidovudine and efavirenz; and the antibodies monoclonal antibody to HIV-1 p24 (AG3.0) from Jonathan Allan, HIV-IG from NABI and NHLBI, HIV-1 p24 hybridoma (183-H12-5C) from Bruce Chesebro, and HIV-1 gp120 monoclonal antibody (immunoglobulin G1 [IgG1] b12) from Dennis Burton and Carlos Barbas.
HeLa, 293T and TZM-bl cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. Jurkat T cells were maintained in RPMI supplemented with 10% fetal bovine serum.
The following proviral plasmids were used: pNL4-3, Fyn-ΔMA, EnvΔCT, (a gift of Eric Freed, NCI), NL4-3-iGFP WT, EnvΔCT, and ΔMA (from Benjamin Chen, NYU ). The C-terminally green fluorescent protein (GFP)-tagged Env was created by cloning the HxB2 Env from the pSRα Env expressor plasmid into pEGFP-N1 vector (Clontech). pJRFL and pJRCSF, as well as tetraspanin and empty vector expression plasmids (in pCMV-Sport6 vector), were provided by Y. Koyanagi (Kyoto University, Japan) (41). The FG12 short hairpin RNA (shRNA) delivery vector system was developed in David Baltimore's laboratory (38).
HeLa cells (105) were plated in 24-well plates and then transfected with GFP-tagged pNL4-3 or GFP-tagged Env (and cotransfected with tetraspanin plasmids where appropriate). Twenty-four hours later, 106 Jurkat cells were labeled with Cell Tracker Blue CMAC dye (Molecular Probes) and cocultured with Env-expressing HeLa cells for 4 h. Excess target cells were washed off, and the remaining cells were fixed in 4% paraformaldehyde (PFA) and analyzed by fluorescence microscopy.
Cell Tracker Blue stains both the cytoplasm and the nucleus. Upon fusion, the cytoplasm of stained target cells merges with that of the producer cell, yet because relative to the producer cell the Jurkat (target cell) cytoplasm is small, it contributes little fluorescence after fusion. The target cell nucleus however, from which the dye leaks slowly, can be readily viewed in the blue channel (and appears as a black hole in the green channel).
Analyses of such images allow fusion activity to be precisely quantified because the quantification of fusion activity goes beyond merely counting syncytia. In cases where a single HeLa cell fused with multiple Jurkat cells, the total number of blue Jurkat nuclei within a syncytium was counted to represent multiple fusion events. Also, because HIV-1-positive cells can be distinguished from nonexpressing cells, the relative fusion activity can be calculated by dividing the number of fusion events by the number of Env- or HIV-1-positive cells. For our quantitative analyses, we randomly selected multiple fields (n ≥ 20).
HeLa cells (105) were plated in 24-well plates and then either transfected with pNL4-3 or cotransfected with Env and Tat plasmids with or without a Gag expressor (and cotransfected with tetraspanin plasmids where appropriate). Twenty-four hours later, 106 TZM-bl cells were cocultured with Env-expressing HeLa cells for 3 h. Excess target cells were washed off, and the remaining cells were incubated for additional 14 h. Cells were lysed, and β-galactosidase activity was measured colorimetrically (EL800; Bio-Tek) using All-in-One mammalian β-galactosidase reagent (Pierce). In parallel, p24 production was measured in lysates by a homemade enzyme-linked immunosorbent assay, as described previously (48). β-Galactosidase activity was divided by p24 production to yield relative fusion activity and was further normalized to the mock condition (normalized fusion). Env surface expression was quantified by flow cytometry as shown in Fig. Fig.2C.2C. Equal levels of surface Env expression for different conditions were achieved by adjusting levels of transfected DNA and/or by adding empty vector DNA as a stuffer in cotransfection protocols.
To knock down tetraspanins, small interfering RNA (siRNA) oligonucleotide transfection was done using siPORT NeoFX reagent (Ambion) according to the manufacturer's instructions. Briefly, RNA oligonucleotides were diluted to a final concentration of 10 nM and mixed with siPort NeoFX in 24-well plates, and 105 HeLa cells per ml were then added. Cells were transfected with Env or provirus vectors at 48 h after siRNA transfection. Twenty-four hours later, target cells were added, and fusion assays were carried out as described above. Knockdown efficiency was measured by Western blotting and flow cytometry. Oligonucleotide sequences used were from reference 13 for CD9 and Ambion's predesigned CD63 oligonucleotide (no. 10412) and silencer negative control (AM4635).
The lentiviral shRNA system used has been previously described (38), and most procedures were carried out essentially as described. Briefly, stock lentiviruses were produced by transfecting 293T cells with the FG12 shRNA plasmids, vesicular stomatitis virus G, and packaging plasmid pDeltaR8.1. Supernatants were concentrated by centrifugation at 50,000 × g for 2 h and then stored at −80°C. The titer was determined by infecting Jurkat cells with serial dilutions of virus and then determining the percentage of GFP-positive cells by flow cytometry. Infections were done in the presence of 10 μg/ml DEAE-dextran to enhance transduction efficiency. Knockdown efficiency was confirmed by Western blotting and flow cytometry. shRNA sequences were based on previously described siRNA sequences for CD9 and CD81 (14) and Ambion's predesigned CD63 (no. 10412).
Lentiviral vector-mediated overexpression of tetraspanins was done as described previously (51), with similar modifications as described above.
For fusion experiments, 5 × 105 Jurkat cells were coinfected with NL4-3 virus and the appropriate lentiviruses and cultured in 24-well plates for 3 days. Target Jurkat cells (106) were labeled with Cell Tracker Blue and added to the infected cells. Sixteen hours later, cells were resuspended, allowed to adhere to Cell-Tak (BD Biosciences)-coated coverslips for 2 h, and then fixed with 4% PFA and stained for Gag. Syncytium counting was carried out as described above.
In brief, HeLa or Jurkat cells were suspended in phosphate-buffered saline, incubated for 30 min with appropriate antibodies (IgG1 b12 for Env labeling) at 4°C, fixed with 4% PFA, and stained with secondary antibody. Flow cytometry was performed on an Epics XL/XL-MCL flow cytometry system with a 488-nm argon laser.
Imaging and colocalization analyses were performed essentially as described previously (33). Briefly, cells were surface stained with anti-Env (IgG1 b12) and anti-CD63 (H5C6) antibodies on ice and then fixed with 4% PFA and labeled with Alexa Fluor 488- and 594-conjugated secondary antibodies (Invitrogen). Cells were then imaged on a DeltaVision deconvolution microscope, and the colocalization analysis was carried out using Volocity software (v3.7). Twenty cells per condition were included, and the bottom z sections were used for the analysis.
Student's t test was used to determine statistical significance. P values of <0.05 were considered significant.
Numerous previous studies have shown that tetraspanins are enriched at HIV-1 budding sites, recruited to VSs, and incorporated into budding virions. Because of their well-known role in regulating membrane fusion processes, we set out to test whether tetraspanins in HIV-1-producing cells regulate syncytium formation.
To measure cell-cell fusion activity, we implemented two different assays. In the first assay, HeLa cells expressing either GFP-tagged provirus or Env, or infected Jurkat cells, served as producers. These cells were cocultured with fluorescently labeled target Jurkat cells, and syncytia could be readily detected and counted by fluorescence microscopy (Fig. (Fig.1A,1A, left panel). In the second assay, used in parallel, HeLa cells expressing either provirus or Env and Tat were used as producer cells, and indicator cells (TZM-bl), which contain the β-galactosidase gene under the control of the HIV-1 long terminal repeat, were used as target cells (Fig. (Fig.1A,1A, right panel). We confirmed that in the short time frame used in this second assay (16 h), all the reporter gene expression was due to producer-target cell fusion and not productive infection; i.e., it was not due to activation upon integration of virus into the genome of target/reporter cells, as the addition of the reverse transcriptase inhibitor efavirenz did not lower the signal (data not shown). Tetraspanins CD9, CD63, and CD81 were either overexpressed or knocked down in HIV-1-producing HeLa cells, and cell-cell fusion activity was quantified using the assays described above. Surface Env levels in HIV-1-producing cells were adjusted to be equal, as measured by flow cytometry (data not shown), by optimizing the transfection protocol with appropriate changes in the amount of empty vector DNA administered. As shown in Fig. Fig.1B,1B, higher levels of tetraspanins in HIV-1 producer cells resulted in significant reductions of cell-cell fusion activity, whereas silencing of CD9, CD63, and CD81 yielded the opposite effect, though these latter effects were less pronounced, likely because other (endogenously expressed) members of the tetraspanin family can partially compensate for the tetraspanin(s) that is downregulated (Fig. 1B and C). Also, the inhibitory functions of tetraspanins were recapitulated when Jurkat T cells were used as producer cells (Fig. (Fig.1C).1C). Finally, fusion repression was dose dependent (Fig. (Fig.1D1D).
HIV-1 Env drives virus-induced cell-cell fusion, and this protein can also induce the fusion of membranes if expressed independently of other viral components. However, we previously documented that Env expressed alone poorly colocalizes with tetraspanins and that coexpression of Gag greatly enhances this colocalization (33). Thus, we hypothesized that Env-driven fusion repression by tetraspanins would be enhanced by coexpression of Gag. As shown in Fig. 2A and B, when Env was expressed alone, neither overexpression nor knockdown of CD9 and/or CD63 resulted in a significant change of fusion activity. However, when Env was expressed together with Gag, fusion repression by tetraspanins was restored. This was not due to altered Env surface levels that resulted from Gag coexpression, as we adjusted the experimental conditions such that fusion activities were measured at comparable Env levels (Fig. (Fig.2C;2C; see Materials and Methods).
Because Gag recruits Env into virions via the interaction between the Env cytoplasmic tail (CT) and the matrix (MA) domain of Gag (see reference 32 and references therein), we surmised that this interaction would also be required for the recruitment of Env into TEMs and the subsequent fusion repression by tetraspanins. To test this hypothesis, we utilized mutant viruses carrying MA or CT deletions, (ΔMA and ΔCT, respectively). Because these mutants display different Env expression at the plasma membrane, the input of proviral DNA was adjusted to achieve equal surface Env levels (see Materials and Methods). We observed the expected significant decrease in colocalization between Env and Gag (not shown), which was paralleled by a similar reduction of Env-tetraspanin colocalization (Fig. (Fig.3B).3B). The fusion activities of these mutants were no longer repressed by tetraspanin overexpression (Fig. (Fig.3A).3A). Interestingly, not only did CD9 lose its fusion repression for the ΔCT mutant virus, but overexpression of this tetraspanin even led to increased syncytium formation.
As tetraspanins can inhibit HIV-1 NL4-3 Env-mediated membrane fusion if they colocalize with Env, one should expect that they also reduce the infectivity of virions when incorporated into particles. This is indeed the case, as reported previously by the Koyanagi group (41) and as confirmed in our laboratory (D. N. Krementsov et al., unpublished data). However, Sato et al. (41) also reported that virus-cell fusion mediated by Env of a different HIV-1 strain, JRCSF, was repressed by tetraspanin overexpression in producer cells, while a similar R5 strain, JRFL, was insensitive to such repression. We thus set out to test if such specificity also applied to cell-cell fusion.
Interestingly, while our initial experiments indicated that JRFL Env-driven cell-cell fusion was not inhibited by tetraspanins, upon increasing the levels of tetraspanin expression, we found that JRFL Env-mediated cell-cell fusion could also be repressed. Hence, JRFL Env-mediated cell-cell fusion was merely less sensitive to tetraspanin inhibition than NL4-3 Env-mediated cell-cell fusion (Fig. (Fig.4A).4A). JRCSF exhibited an intermediate phenotype.
To address the question of why repression of JRFL Env-induced fusion required the expression of higher tetraspanin levels, we tested whether the different Envs have different intrinsic fusion activities. The data presented in Fig. Fig.4B4B reveal that this is indeed the case: cells expressing equal levels of the different Envs display different fusogenicities.
Because colocalization of Env with tetraspanins is a prerequisite for fusion repression, we also tested whether diminished Env-tetraspanin colocalization contributed to the reduced susceptibility of JRFL Env. As documented in Fig. Fig.4C,4C, quantitative imaging analysis revealed that this was not the case: all three viral Envs (NL4-3, JRCSF, and JRFL), when expressed together with Gag, displayed comparable levels of colocalization with the tetraspanin CD63.
Gordon-Alonso et al. (14) previously reported that overexpression of CD9 and CD81 in target cells repressed HIV-1-induced membrane fusion. To test whether the fusion repression functions of tetraspanins situated in producer and target cells, respectively, could complement or negate each other, we overexpressed CD9, CD63, and CD81 in target cells, producer cells, or both. Overexpression of these tetraspanins in target cells repressed cell-cell fusion, as previously reported (14), and overexpression of these tetraspanins in both producer and target cells showed a cumulative effect (Fig. (Fig.5),5), suggesting that these fusion-regulatory proteins operate independently of each other at the presynapse and the postsynapse.
In this study, we demonstrated that tetraspanins in virus-producing cells prevent the formation of syncytia, which are multinucleated cells that result from fusion of virus producer cells with one or more target cells. We also documented that this fusion prevention depends on the presence of Gag and the recruitment of Env into TEMs. Further, intrinsically more fusogenic Envs require higher levels of tetraspanin expression for the repression of their fusion activity.
Accumulation of tetraspanins at HIV-1 budding sites is well documented, but their function as budding cofactors remains unclear (7, 16, 40, 41; Krementsov et al., unpublished data). Because of their well-established role in membrane fusion regulation, here we hypothesized that these proteins regulate HIV-1-induced fusion. In agreement with this hypothesis, we found that overexpression or ablation of individual members of the tetraspanin family in producer cells resulted in fusion repression or enhancement, respectively. This held true when either HeLa cells or more physiologically relevant Jurkat T cells served as virus producers (see Fig. Fig.1).1). We also document that this fusion regulation by tetraspanins is dose dependent, a finding that has bearing on the strain-specific sensitivity differences shown in Fig. Fig.44 (discussed below).
One member of the tetraspanin family, CD82, has previously been shown to inhibit cell-cell fusion mediated by the envelope glycoprotein of another retrovirus (human T-cell leukemia virus type 1) (37). In that study, fusion mediated by HIV-1 Env (expressed independently from other HIV components) was also tested, but it turned out to be insensitive to the inhibitory action of that tetraspanin. We know now that CD82 can indeed repress HIV-1 Env-mediated membrane fusion (data not shown), but this repression, like the fusion inhibition by CD9 and CD63 (Fig. (Fig.2),2), requires the presence of Gag. This dependence on Gag coexpression is likely due to the fact that Env colocalizes (and thus can either directly or indirectly associate with tetraspanins) only if Gag recruits it to TEMs (33). Since immature Gag also represses the fusogenicity of Env (9, 22, 23, 31, 50), how can we distinguish between fusion repression by uncleaved Gag and fusion regulation by tetraspanins? Strongly arguing that the two mechanisms are indeed different is the finding that tetraspanin overexpression also leads to repression of fusion mediated by virion-associated Env, i.e., in the context of processed Gag (as discussed below).
The findings reported here in Fig. Fig.11 and and22 are in line with a recent report by Sato et al. (41), as well as our own findings, which show that tetraspanins in virions inhibit virus-cell fusion, thus suggesting that these tetraspanins repress Env-mediated fusion in virions and in producer cells via a similar mechanism. However, we also note important differences. Sato et al. show that virus-cell fusion of ΔCT Env mutant NL4-3 can be inhibited by CD63 overexpression. In contrast, and as shown in Fig. Fig.3A,3A, cell-cell fusion driven by ΔCT Env is insensitive to CD63. This discrepancy is most likely due to the fact that though ΔCT Env is “nonspecifically” incorporated into particles, once it is acquired by the virions, it is situated in close proximity to tetraspanins and thus can be repressed by them. In contrast, the majority of ΔCT Env at the cell surface is not in proximity to tetraspanins (as documented in Fig. Fig.3B)3B) and hence is not repressed. We conclude that the Gag-Env interaction is not required if tetraspanins and Env are already proximal (as in the case of ΔCT Env in virions). Why overexpression of CD9 not only fails to repress but even enhances ΔCT Env-driven fusion is unclear at this point, although it is evident that such enhancement does not require colocalization of CD9 and Env. It seems plausible that overexpression of CD9 diverts or sequesters some other cellular factor which would otherwise prevent fusion mediated by tail-less Env. The fact that only CD9 and not CD63 enhances ΔCT Env-driven fusion can serve as starting point for a genetic analysis aiming at the identification of such a hypothetical cellular factor.
Another apparent discrepancy between previously published data (41) and our own data is displayed in Fig. Fig.4.4. There, we show that cell-cell fusion mediated by Env of the R5 virus JRFL is inhibited by overexpression of the tetraspanin CD63. We also found that JRFL infectivity could be partially inhibited by expressing larger amounts of CD63 or CD9 (data not shown), suggesting that this virus is simply less sensitive to tetraspanin-mediated fusion inhibition than NL4-3. Interestingly however, and consistent with the results of Sato et al. (41), both JRFL and NL4-3 were less sensitive to tetraspanin overexpression when the viruses were produced in 293T cells (in fact, JRFL was not inhibited at all at intermediate tetraspanin concentrations) (data not shown). Presumably, higher levels of virus production (relative to tetraspanins) in 293T cells together with apparently higher intrinsic fusion activity of JRFL Env (Fig. (Fig.4B)4B) explain why the repression of virus-cell fusion was not observed previously.
Finally, we asked whether the fusion-preventive functions of tetraspanins in producer cells (described in this report) were independent of their entry-inhibiting activities in target cells (14, 18, 47, 51). The data presented in Fig. Fig.55 suggest that this is the case, as neither synergism nor mutual negative interference was observed in these overexpression experiments. These results are also in agreement with the fact that so far tetraspanins have be found to functionally interact with each other only in cis, i.e., when situated within the same lipid bilayer, and not in trans. Nevertheless, to formally exclude the latter possibility, we will still need to test whether simultaneous ablation of tetraspanins in producer and target cell shows they same, nonsynergistic outcome.
Did HIV-1 evolve to utilize tetraspanins in producer cells to prevent syncytium formation? To date, it remains unclear whether syncytia are beneficial for virus replication. Some reports answered that question positively by pointing to the facts that, in vitro, these multinucleated entities can shed virions at a high rate (see, e.g., reference 43), that they can be formed at high levels in cocultures of infected and uninfected lymphocytes, and that they produce infectious virus for a short time prior to their death (39). However, consistent with the idea that HIV-1 ultimately does not benefit from syncytium formation, syncytia are typically detected (if at all) only in late-stage AIDS, during which they are thought to contribute to the cytopathology of HIV-1. Also in agreement with the idea that syncytia are dispensable for virus spread is the finding that LFA-1-negative peripheral blood mononuclear cells do not form syncytia yet support HIV-1 replication (34). Nevertheless, and unexpectedly at first sight, ablation of tetraspanins, which leads to increased syncytium formation as shown in this report, does not impede virus transmission in vitro (Krementsov et al., unpublished data). We reason that this is due to the fact that in our transmission assay, as in other currently used in vitro replication assays, producer and target cells are far less densely packed than in lymphoid tissue. Indeed, also compared to the two fusion assays used in this study, syncytia are rarely formed in the transmission assay. Consequently, even a considerable enhancement of syncytium formation, due to tetraspanin ablation, would not be expected to reduce the number of successfully infected target cells to a significant degree. Still, we hold that fusion of producer and target cells, in vitro as well as in vivo, is not likely to be beneficial for HIV-1, and this report now unequivocally shows that one of the functions of tetraspanins at viral exit sites is to prevent syncytium formation. Further, it characterizes some of the requirements for this regulation, thus providing the groundwork for understanding its mechanism.
We thank Yoshio Koyanagi, Kei Sato, Wes Sundquist, Hyo-Young Chung, Clarisse Berlioz-Torrent, Eric Freed, Benjamin Chen, and Marie Lambelé for gifts of plasmids and/or helpful discussions and advice and the NIH AIDS Reagent program for providing many high-quality research reagents. We also thank the reviewers of an NIH grant proposal for their thoughtful critiques, which helped to clarify some of our working hypotheses.
This work was supported by UVM COM Bridge Support and NIH grants RO1 AI47727, R56 AI047727, and R01 AI080302, as well as a UVM VGN graduate fellowship and training grant T32 AI055402 to D.N.K.
Published ahead of print on 20 May 2009.