Interaction between Simian Virus 40 Major Capsid Protein VP1 and Cell Surface Ganglioside GM1 Triggers Vacuole Formation

ABSTRACT Simian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40.

the most recently discovered human tumor virus, Merkel cell polyomavirus (11). The icosahedral SV40 capsid consists of 360 molecules of the major capsid protein VP1, which assemble into 72 pentamers that form the outer capsid shell (12). Two closely related minor capsid proteins, VP2 and VP3, are localized to the interior of the capsid.
Dramatic cytoplasmic vacuolization is the most distinguishing feature of lytic SV40 infection of African green monkey kidney cells and certain other permissive monkey cells. SV40 is the only known polyomavirus with significant vacuolizing activity. However, 56 years after the discovery of SV40, the mechanism of SV40induced vacuolization is not known. Vacuolization normally occurs late during infection, coincident with the release of progeny virus. In addition, transient, early cellular vacuolization (ECV) occurs in primary African green monkey kidney cells infected at a high input multiplicity by infectious or UV-inactivated SV40, suggesting the capacity to induce early vacuolization resides in a structural component of the SV40 virion and does not require replication of SV40 DNA (13). The relationship between ECV and classic vacuolization at late times is not known. Neither the viral nor the cellular components required for early or late vacuolization have been identified.
The cell surface ganglioside GM1 is the receptor for SV40, while other polyomaviruses utilize different gangliosides for virus binding and entry (14)(15)(16)(17). The carbohydrate moiety of these gangliosides binds directly to binding pockets generated by pentamerization of VP1 (18). The pentameric nontoxic B subunit of cholera toxin (CTXB) also binds to GM1 (19). Helenius and coworkers showed that binding of SV40 capsids or isolated pentameric VP1 to cell surface GM1 induced dramatic membrane curvature that led to the formation of tight-fitting, virus-filled invaginations and tubules of the plasma membrane of monkey CV-1 cells (20). This process appears to provide a route of virus internalization during cellular uptake of SV40.
Amino acid substitutions in VP1 can alter the receptor specificity of polyomaviruses (16,21). Previously, by introducing mutations in the GM1 binding site on SV40 VP1, we and others have isolated viable mutant viruses that no longer use GM1 for infection but appear to use alternative ganglioside receptors (21,22). These mutations also inhibit the ability of SV40 to induce vacuolization in permissive monkey cells without impairing virus replication, raising the possibility that the VP1-GM1 interaction plays a role in mediating vacuolization. Here, we show that SV40induced vacuolization is triggered by the massive amount of released progeny viruses during the late stage of permissive infection and the resulting binding of higher-order VP1 pentameric structures to GM1 at the cell surface.

SV40 mutants defective for GM1 binding fail to induce vacuole formation in CV-1 cells.
Ganglioside GM1 is the cell surface receptor that mediates entry of wild-type SV40 during permissive infection (14,15). Previously, we identified several viable SV40 mutants that were defective for GM1 binding because of amino acid substitutions in the GM1 binding site of VP1 (21). Interestingly, these mutants replicate well (apparently by utilizing a different ganglioside to support infection) but fail to induce vacuole formation in permissive CV-1 monkey cells. An SV40 mutant partially defective for GM1 binding was also partially defective for vacuole induction, and Murata and colleagues reported a different mutation in the GM1 binding site of VP1 that appeared to affect GM1 binding and also prevented vacuolization (21,23). Here, we focused on the VP1 mutant that has the lowest GM1 binding ability. This mutant contains 3 amino acid substitutions, alanine 70 to leucine, phenylalanine 75 to leucine, and histidine 129 to glutamine, that appear to occlude the GM1 binding site (designated here the LLQ mutant) (21). As shown in Fig. 1A, many CV-1 cells infected at a multiplicity of infection (MOI) of 10 with wild-type SV40 but not the LLQ mutant displayed dramatic vacuolization 72 h postinfection. It is important to note that the wild-type and mutant viruses entered cells and replicated to similar extents with the same kinetics, as shown by large T-antigen expression levels at different times postinfection (see Fig. S1A in the supplemental material). In addition, a second GM1 binding-defective VP1 mutant with a similar vacuolization-defective phenotype accumulated abundant virus particles in the nucleus of infected cells, confirming that these mutants do not display an entry or replication defect (21) (see Fig. S1B). These results suggest that the ability of SV40 to induce vacuole formation is related to GM1 binding.
To confirm the role of GM1 in triggering vacuolization, we performed small interfering RNA (siRNA) transfection to knock down GM3 synthase, an enzyme required for the synthesis of GM3, the precursor of GM1. Cell surface GM1 levels were not affected at 24 h post-siRNA transfection but were depleted approximately 4-fold by 72 h posttransfection, as assessed by flow cytometry for CTXB binding (Fig. 1B). Wild-type SV40 was inoculated at an MOI of 10 at 24 h post-siRNA transfection to ensure that virus entry occurred prior to the reduction in cell surface GM1. Efficient infection of the control and knockdown cells was confirmed by T antigen staining and flow cytometry (see Fig. S2 in the supplemental material). At 72 h postinfection, vacuole formation was almost completely abrogated in siRNA-GM3-transfected cells, but not in cells transfected with the siRNA control (Fig. 1C). This result provided further evidence that GM1 is required for SV40-induced vacuolization. In addition, it suggested that the initial interaction between GM1 and VP1 during virus entry at this relatively low MOI is not sufficient for vacuolization.
Similarly, we depleted VP1 synthesis by lentivirus-mediated shRNA transduction during SV40 infection. VP1 levels at late times in infection were depleted to an amount similar to that present in the input virus (Fig. 1D). As shown in Fig. 1E, VP1 knockdown caused a striking inhibition of vacuole formation, demonstrating that VP1 is required for host cell vacuolization. We note that this RNA interference approach inhibits only new VP1 synthesis occurring at late times during SV40 infection. This finding implies that vacuolization is mediated by newly synthesized and not input VP1, and again that the GM1-VP1 interaction during virus entry at a relatively low MOI is not sufficient for vacuolization. Taken together, these results suggest that binding of newly synthesized VP1 to GM1 plays a key role in inducing host cell vacuolization.
Intracellular expression of VP1 does not cause vacuole formation in CV-1 cells. Since VP1 is required for vacuolization, we tested whether overexpression of VP1 per se induces vacuoles in CV-1 cells. Lentivirus-mediated ectopic expression of wild-type SV40 VP1 resulted in VP1 expression levels equal to or higher than those during the late stages of productive SV40 infection, as assessed by immunofluorescence (IF) staining and by flow cytometry ( Fig. 2A and B). As expected, ectopically expressed VP1 was localized largely to the nucleus. Despite this high expression level, Luo et al. ectopic VP1 failed to induce vacuole formation in CV-1 cells (Fig. 2C). Thus, nuclear expression of VP1 fails to cause vacuolization, suggesting that GM1-VP1 binding at the cell surface is required for vacuolization and/or that vacuolization requires virus replication or viral gene products in addition to VP1.
Vacuolization during productive SV40 infection is caused by released progeny viruses that bind to GM1. During SV40 infection at an MOI of 10, vacuole formation occurred at the late stage of infection (60 to 72 h postinfection) (Fig. 3A). Determination of titers of virus released into the cell culture medium at different time points postinfection showed a dramatic increase of released progeny virus at these late times (Fig. 3B). Compared with the initial infection with an input of 10 infectious units (IU)/cell, mas-sive numbers of progeny viruses (~1,000 IU/cell) were released into the medium by 60 h postinfection, approximately the time when large numbers of vacuoles formed. Therefore, we hypothesized that vacuolization during productive infection is caused by progeny viruses binding to GM1.
To test the role of released virus in vacuolization occurring late during infection, we determined whether vacuolization was abrogated by reducing the release of progeny viruses from infected cells. SV40 release is mediated in part by the late viral protein VP4, which forms pores in infected cell membranes (24,25). Although the absence of VP4 inhibits SV40 release, it has little influence on intracellular viral replication or VP1 expression (24,26). We infected CV-1 cells with wild-type and VP4 mutant SV40 at an MOI of 10 and confirmed that the VP4 mutation impaired progeny virus release despite robust intracellular replication of the mutant (see Fig. S3 in the supplemental material). In parallel, we compared the ability of the VP4 mutant and wild-type SV40 viruses to induce vacuole formation. As shown in Fig. 3C, the absence of VP4 caused substantial inhibition of vacuole formation.
As an additional test of the ability of progeny virus to induce vacuoles, we treated CV-1 cells with conditioned medium collected 72 h postinfection of CV-1 cells by wild-type SV40. Following treatment with conditioned medium from infected cells, vacuolization occurred within 4 h (Fig. 3D), a time prior to the initiation of SV40 replication as assessed by the lack of large T antigen expression (see Fig. S4 in the supplemental material). Im-portantly, the acute vacuolization induced by conditioned medium was blocked by neutralizing SV40 antiserum (Fig. 3D), by soluble GM1, which binds to authentic SV40 and neutralizes infection (21) (Fig. 3D), or by knockdown of GM3 synthase in the recipient cells (Fig. 3E). The vacuolization defect in the knockdown cells was rescued by addition of GM1 to the culture medium, which inserts into the plasma membrane of the cells, where it can serve as the SV40 receptor (15) (Fig. 3E). Thus, conditioned medium from SV40-infected cells contains a factor that acutely induces vacuolization in a VP1-and GM1-dependent fashion. Similarly, acute vacuolization occurred when CV-1 cells were infected with wild-type SV40 at a high MOI (100 to 1,000), whereas the LLQ mutant was defective for inducing vacuoles even at an MOI of 1,000 (Fig. 3F). Taken together, these results strongly suggest that vacuolization during SV40 permissive infection is caused by the massive amount of released progeny viruses that bind to GM1.
Vacuolization is triggered by GM1 binding but not virus replication. The experiments described above indicate that SV40 released at late times in infection induces vacuolization in a GM1dependent fashion. Furthermore, the ability of SV40 or progeny virus in conditioned medium to rapidly induce vacuolization in naive CV-1 cells implies that neither virus replication nor a late infected cell milieu is required in the cells undergoing vacuolization. To establish conclusively that virus replication is not required for vacuolization, we infected CV-1 cells with a replicationdefective SV40 pseudovirus named Pava-1. Pava-1 consists of the SV40 capsid surrounding a genome that cannot replicate in CV-1 cells because the SV40 large T antigen gene, which is absolutely required for SV40 DNA replication, has been replaced by a segment of bovine papillomavirus type 1 (BPV) DNA encoding the BPV E2 transcription factor and the E5 oncoprotein (27). As shown in Fig. 4A, at an MOI of 1,000 Pava-1 caused vacuolization in CV-1 cells beginning as early as 4 h postinfection. Vacuoles increased in size with increasing time postinfection, and significant cell death was evident at 72 h. Acute vacuolization by Pava-1 was inhibited by knockdown of GM3 synthase and restored by addition of GM1 to the knockdown cells (Fig. 4B). This result indicates that a large number of Pava-1 particles can cause acute GM1-dependent vacuolization in the absence of viral DNA replication and also that persistent vacuolization can occur in cells that are not undergoing events occurring late in the lytic virus life cycle.
We also tested the vacuolization activity of Pava-1 in Vero cells, an African green monkey kidney cell line with lower cell surface GM1 levels than CV-1 cells (see Fig. S5 in the supplemental material). Pava-1 infection of unmodified Vero cells failed to induce vacuolization, consistent with the low level of endogenous GM1 in these cells, but incorporation of GM1 into the plasma membrane allowed robust vacuolization ( Fig. 4C; see also Fig. S5). Thus, acute vacuolization induced by a replication-defective SV40 pseudovirus is GM1 dependent, as is classic vacuolization occurring late during productive virus replication.
As an independent approach to determine whether GM1 binding is required for acute vacuole formation in the absence of virus replication, we studied the related human polyomavirus BKV. Although the VP1 sequences of SV40 and BKV are very similar and are thought to engage their ganglioside receptors in a similar fashion, wild-type BKV utilizes ganglioside GD1b or GT1b for infection but does not bind to GM1 and fails to induce vacuolization (16,18,28). For the experiments reported here, we used CsCl- purified replication-defective BK pseudoviruses (BKpsV), consisting of a Gluc reporter genome packaged in a capsid composed of BKV VP1 and VP2/3 (16). As expected because of its inability to bind to GM1, wild-type BKpsV did not induce vacuolization in CV-1 cells (Fig. 4D). We previously reported a single amino acid substitution in the ganglioside binding site of BKV VP1, lysine 68 to serine (K68S), which allows the mutant VP1 pentamers to bind to GM1 (16). Strikingly, unlike wild-type BKpsV, the K68S BKpsV mutant induced acute and persistent vacuolization in CV-1 cells, as well as cell death at late times (Fig. 4D). Wild-type and K68S BKpsV infected CV-1 cells to a comparable extent, as assessed by expression of luciferase from the packaged reporter genome (see Fig. S6 in the supplemental material). Collectively, these results demonstrate that vacuolization can be acutely induced by VP1-GM1 binding even in the absence of virus replication and that the inability of BKV to induce vacuolization is due solely to its inability to bind to GM1.
SV40 VP1 pentamers and cholera toxin B induce acute vacuolization. The pseudovirus stocks used in the preceding section contained not only VP1 but also VP2/3, as well as products released from the cells used to package pseudovirus. To determine if VP1 alone is able to induce acute vacuolization in the absence of other viral proteins and mammalian cell constituents, we used VP1 pentamers purified from bacteria. Bacterially produced SV40 VP1 oligomerizes to form pentamers and can assemble into higher-order pentameric structures, including virus-like particles (VLPs) (14,29,30). To determine whether VP1 pentamers are sufficient to induce vacuolization, we purified full-length wildtype SV40 VP1 from Escherichia coli. In parallel, we also purified GM1 binding-defective VP1 (containing the LLQ mutations) and a C-terminal deletion VP1 mutant (⌬C) that forms pentamers but is unable to form higher-order structures because the deletion removes sequences required for interpentameric interaction (18,21). The ⌬C pentamers have intact GM1 binding sites and remain competent for GM1 binding (21). Purified VP1 and its derivative mutants following SDS-PAGE are shown in Fig. S7A in the supplemental material. Electron microscopy (EM) confirmed that both wild-type VP1 and the ⌬C mutant formed pentamers (see Fig. S7B). The full-length wild-type pentamers tended to form higher-order structures, but we did not observe intact VLPs in our preparations. In contrast, as expected, the ⌬C pentamers did not associate with each other and remained individual pentamers. As shown in Fig. 5A, 10 g/ml of full-length wild-type VP1 pentamers acutely induced vacuole formation in CV-1 cells. Vacuoles induced by pentamers tended to be smaller and more uniform in size than vacuoles induced by SV40 or pseudoviruses. Pentamerinduced vacuoles persisted for 24 h but were less evident at later time points (see Fig. S8 in the supplemental material). In contrast, the GM1 binding-defective pentamers did not induce vacuoles, even when tested at higher concentration ( Fig. 5A; see also Fig. S7C). Furthermore, the ability of wild-type VP1 pentamers to induce vacuolization was inhibited by knockdown of GM3 synthase and rescued by addition of GM1 to the cells (Fig. 5B). These results confirm that the GM1-VP1 interaction is responsible for acute vacuolization and that viral replication and other viral or cellular proteins (e.g., VP2/3 or VP4) are not required.
The ⌬C truncated pentamers at 10 g/ml did not induce vacuoles, but vacuoles were induced when 50 g/ml of the truncated pentamers were added to cells (Fig. 5A). To test whether the reduced ability of truncated VP1 to induce vacuole formation is due to its inability to form higher-order structures than pentamers, we used a nonneutralizing VP1 monoclonal antibody and secondary anti-IgG to cross-link ⌬C pentamers prior to incubation with cells. As shown in Fig. 5C, cross-linked ⌬C pentamers induced vacuolization in CV-1 cells as early as 4 h posttreatment, whereas antibody treatment in the absence of pentamers did not induce vacuoles. Pentamers cross-linked with anti-VP1 plus secondary anti-IgG were more effective at inducing vacuoles than pentamers treated with anti-VP1 alone. This result demonstrates that the truncated pentamers do not have an intrinsic vacuolization defect, but rather suggests that higher-order pentameric VP1 structures are more effective than individual pentamers at inducing vacuolization.
Like VP1, CTXB forms a pentameric structure that binds to GM1 (31). X-ray crystallographic studies demonstrated that CTXB and VP1 bind to GM1 in essentially the same conformation, but none of the specific molecular interactions responsible for binding is common to the two (18,31). Figure 5D shows that CTXB also induced vacuolization in CV-1 cells, but at a 4-to 10-fold-higher concentration than that required for wild-type VP1 pentamers. These results show that vacuolization of CV-1 cells can be triggered by different pentameric structures that bind to GM1 and that no virus-specific sequences are required.
VP1 does not accumulate in vacuoles. The experiments described above demonstrate that binding of SV40 VP1 to GM1 triggers the formation of vacuoles. We used three approaches to determine if VP1 accumulates in vacuoles. First, we used transmission electron microscopy to examine SV40-infected CV-1 cells late in infection. As shown in Fig. 6A, although progeny virus particles were present in high numbers in the nuclei of infected cells, we did not observe viruses within the vacuoles. Second, we stained CV-1 cells for intracellular VP1 late during productive infection with wild-type SV40 (Fig. 6B). Although nuclear and cytoplasmic VP1 was readily detectable, VP1 was not present within the vacuoles. Finally, we stained CV-1 cells for intracellular VP1 4 h after high-multiplicity infection with Pava-1, at a time when cytoplasmic vacuoles were present. The immunofluorescent images in Fig. 6C show that input VP1 was readily apparent at the cell periphery, far from the vacuoles themselves. Thus, although VP1 triggers vacuole formation, it does not accumulate within the vacuoles either late during productive SV40 infection or acutely following treatment of cells with SV40 pseudovirus.

DISCUSSION
Vacuolization, a hallmark of productive SV40 infection in African green monkey cells, is a mysterious phenotype that led to the dis-covery of the virus more than half a century ago. In this report, we show that vacuolization is triggered by binding of newly synthesized and released VP1 to cell surface GM1 at the late stages of SV40 infection. In addition, we show that vacuolization does not require active virus replication or viral gene products other than VP1. Our study not only identifies the trigger for the vacuolization phenotype characteristic of this historically important virus, but This study identifies the viral and cellular components required for SV40-induced vacuolization. Oligomeric SV40 VP1 is the only viral protein required for vacuolization, and virus replication or late viral events in the cells undergoing vacuole formation are not required. The cellular ganglioside GM1 is also re-quired for vacuolization. Our data further show that a direct interaction between VP1 pentamers and GM1 is required for vacuoles to form. This conclusion is supported by the observations that point mutations in the GM1 binding site of SV40 VP1 that prevent GM1 binding also abolish vacuole formation, that knockdown of ganglioside synthesis inhibits vacuolization (which is restored by treating the knockdown cells with GM1), and that Vero cells that are naturally resistant to vacuolization can be converted into susceptible cells by incorporation of GM1 into the plasma membrane. Most strikingly, a single amino acid substitution in the ganglioside binding site of BKV VP1 that generates GM1 binding also generates vacuolization activity. This result demonstrates that a nonvacuolating polyomavirus can be converted into a vacuolating one simply by acquiring the ability to bind to GM1. Thus, the VP1-GM1 interaction is essential for two distinct features of the virus life cycle, virus binding during cell entry and vacuole formation at late times.
Several pieces of data indicate that the GM1-VP1 interaction plays a direct role in vacuole formation and not that this interaction merely delivers the virus or VP1 into cells where it induces vacuoles in a GM1-independent fashion. The SV40 VP1 mutants defective for GM1 binding are also defective for vacuolization even though they efficiently infect and replicate in CV-1 cells.
These viable mutants were selected for their ability to undergo productive replication resistant to GM1-mediated neutralization and rely on other gangliosides for entry (21). Second, vacuolization is inhibited by siRNA-mediated depletion of GM1 after infection is established. In addition, wild-type BKpsV does not induce vacuoles in CV-1 cells even though it efficiently enters these cells, and the ability of BKpsV K68S to induce vacuoles is not due to more efficient entry. Finally, the B subunit of cholera toxin, whose only known biochemical activity is binding to GM1 and has no other biological similarity to VP1, can also induce vacuoles.
Our experiments also shed light on the relationship between classic vacuolization occurring late during infection and acute vacuolization induced by a high multiplicity of infection. It was initially suggested that these were two different phenomena because of the timing and durability of vacuole formation and differences in vacuole size (13). Our results provide strong evidence VP1-GM1 Binding Triggers SV40-Induced Vacuolization that early and late vacuoles result from the same biological process. Most notably, both early and late vacuoles require binding of VP1 to GM1. In addition, although vacuoles acutely induced by VP1 pentamers are small and transient, high-multiplicity infection with SV40 and BKV (K68S) pseudoviruses can result in the rapid formation of large vacuoles that persist for several days. The low numbers of virus typically used in infection studies are not sufficient to induce acute vacuolization. Rather, several lines of evidence indicate that vacuole formation at late times after infection at a standard MOI occurs in response to massive amplification and release of the virus. First, vacuole formation is coincident with virus release at late times. Second, newly synthesized VP1 is required for vacuolization. Third, late vacuolization is inhibited by a VP4 mutation that blocks virus release. In addition, expression of intracellular VP1 is not sufficient for vacuole formation. Finally, released progeny virus in the culture medium is sufficient to rapidly induce vacuole formation through a VP1-GM1 interaction. These results indicate that VP1 must be released from the infected cells and bind to GM1 on the cell surface to trigger vacuolization. Thus, vacuoles formed late in infection are in fact an acute response to the binding of recently released virus to GM1.
Pentamerization of VP1 is required to generate the ganglioside binding sites (18), but higher-order structures appear to be more active in inducing vacuolization. SV40 capsids (or GM1 binding K68S BPV capsids) are most active, whereas purified SV40 pentamers (which, although they can aggregate into higher-order structures, do not form VLPs in our hands) induce relatively small and transient vacuoles. In contrast, the ⌬C pentamers, which are unable to form higher-order oligomers but retain GM1 binding, induced vacuolization only at a high concentration. Importantly, these truncated VP1 pentamers acquired robust vacuole-inducing activity after antibody-mediated cross-linking, demonstrating that higher-order structures induce vacuole formation more efficiently than individual pentamers. It is possible that interpentameric contacts generate new structural motifs or a geometry that facilitates vacuole formation, or that the increased number of close-proximity contacts due to large assemblages of VP1 may contribute avidity effects important for vacuole formation. Although CTXB binds to GM1 with~1,000-fold-higher affinity than VP1 pentamers, it induces vacuoles only at much higher concentrations than VP1 pentamers, perhaps reflecting in part its inability to form higher-order oligomers (18,19).
Neither progeny viruses nor VP1 pentamers accumulate within vacuoles, suggesting that vacuoles do not form from the virus-lined membrane invaginations described by Ewers et al. (20). This conclusion is consistent with the ability of truncated VP1 pentamers to induce invaginations (20), even though they are defective at inducing vacuoles. Rather, our results suggest that vacuole formation is mediated by a downstream signaling pathway(s) triggered by GM1 binding. This signaling may be related to GM1-induced signaling in other systems, such as neuronal cells (32). GM1 signaling leading to vacuole formation appears to be initiated by some specific feature of the VP1-GM1 complex, because binding of other polyomaviruses to their cognate gangliosides does not induce vacuoles. Thus, there must be a difference in the structure or clustering of VP1-bound GM1 itself or the resulting signal(s), compared with other ganglioside-VP1 combinations that do not trigger vacuole formation. In addition, the interaction of polyomaviruses with cells can acutely induce mitogenic signaling that is independent of ganglioside binding (33).
Our results show that GM1 binding plays different roles in different stages of SV40 infection. During virus entry, engagement of VP1 with GM1 is necessary for virus internalization (15,34), most likely by causing stable association of the virion with the cell surface and causing plasma membrane invagination as an initial step in endocytosis (20). In addition, as demonstrated here, late during productive infection VP1-GM1 binding triggers vacuolization, which does not appear to be a direct result of plasma membrane invagination. It is possible that vacuole formation is a consequence of productive SV40 infection without any impact on virus replication, but we have not ruled out the possibility that vacuolization provides a benefit to SV40. We note that BKpsV (K68S) but not wild-type BKpsV induces vacuolization and cell death several days after infection, suggesting that there may be a direct link between vacuole formation and cell death. Such a process may be related to vacuole-associated nonapoptotic cell death induced by some chemicals (35)(36)(37). If SV40-induced vacuolization or the responsible signaling pathway facilitates cell death, this in turn might enhance progeny virus release and spread. This model is consistent with the finding that vacuolization-defective mutants, including LLQ, display a plaque-forming defect (reference 22; T. G. Magaldi and D. DiMaio, unpublished results). It is clear that the role of GM1-VP1 binding and vacuolization in virus replication and cell biology warrants further investigation.

MATERIALS AND METHODS
Reagents. Monosialoganglioside GM1 (containing ␣-5-N-acetylmuraminic acid) from bovine brain and unlabeled cholera toxin B were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Alexa Fluor 488labeled CTXB was purchased from Invitrogen (Carlsbad, CA). Rabbit anti-SV40 VP1 antibody was purchased from Abcam (Cambridge, MA). Monoclonal anti-SV40 VP1 polyclonal antibody (PAb) 597 and monoclonal anti-SV40 large T antigen PAb108 were harvested from cultured hybridoma cells. Neutralizing anti-SV40 horse antiserum was a kind gift of James Pipas, University of Pittsburgh. Lipofectamine RNAi Max reagent, SimplyBlue SafeStain, and SuperSignal West Pico chemiluminescent substrate were purchased from Thermo, Fisher (Grand Island, NY). A DNeasy kit was purchased from Qiagen (Valencia, CA). IScript cDNA synthesis and iQ SYBR green supermix kits were purchased from Bio-Rad (Hercules, CA). TaqMan gene expression master mix was purchased from Applied Biosystems (Foster City, CA).
Cell culture and virus infection. CV-1 cells and Vero cells (both of African green monkey origin) were purchased from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin-streptomycin, 10 mM L-glutamine, and 10 mM HEPES (pH 7.2) in 5% CO 2 at 37°C. An SV40 mutation preventing expression of VP4 was constructed by converting the VP4 start codon to ATA by site-directed mutagenesis (24). SV40 (strain 776), the LLQ mutant (21), and VP4 mutant virus stocks were produced from cloned DNA as previously described (38). Briefly, monolayers of CV-1 or CMT4 cells in T75 flasks were infected at an MOI of 10 with seed virus stock. After 4 to 5 days, when significant cell death was observed, flasks were subjected to multiple rounds of freeze-thawing. Cellular debris was removed by centrifugation at 1,000 rpm for 5 min, and supernatants were filtered through 0.45-m syringe filters, aliquoted, and stored at Ϫ80°C. The VP4 mutant was passaged serially to generate sufficient virus. Virus titers, expressed as the number of infectious units per milliliter, were determined by infecting cells with virus stocks followed by staining for intracellular SV40 large T antigen with antibody PAb108 and flow cytometry at 48 h postinfection. Unless specified otherwise, infection was performed at an MOI of 10 IU per cell. Virus was added directly to the medium of cells at~80% confluence, and the medium containing virus was not replenished for the dura-tion of the experiment. Pava-5=B⌬S pseudovirus (designated here Pava-1) was produced as described above and titers were determined by flow cytometry using the Abcam antibody to stain VP1 in infected CMT4 cells (27,38). BKpsV and BKpsV(K68S) pseudoviruses (Dunlop strain) were produced in 293TT cells from plasmids expressing human codonoptimized BKV virion proteins and purified by using CsCl gradient centrifugation as described elsewhere for JC pseudovirus (17,39), and titers were determined by quantitative PCR for encapsidated genome copies with primers targeting the GLuc gene in the phGluc reporter plasmid: forward primer, 5=-ACGCCCAAGATGAAGAAGTT-3=; reverse primer, 5=-ACCCAGGAATCTCAGGAATG-3=; and iQ SYBR Green Supermix (Bio-Rad). Approximately 10 5 genome copies of BKpsV were added per cell for infection studies.
Microscopy. Phase-contrast images were taken at 40ϫ magnification by using a Canon PowerShot G10 camera directly attached to an Olympus CK30 microscope. Immunofluorescence and bright-field images were taken by using a Leica SP5 confocal microscope at 60ϫ or 100ϫ magnification. Images were merged by using LAS AF (Leica) software.
Flow cytometry. Cell surface GM1 levels were determined by staining with Alexa Fluor 488-conjugated CTXB as previously described (21). To stain intracellular VP1, cells were fixed with methanol, permeabilized, and stained with a 1:200 dilution of VP1 antibody (Abcam) and an anti-rabbit secondary antibody. Stained cells were analyzed on an LSR II cytometer (BD Biosciences) at the Flow Cytometry Core, Yale University. All flow cytometry data were analyzed using FACSDiva and FlowJo software (BD Biosciences).
GM3 synthase knockdown by siRNA transfection. The siRNA oligonucleotide was purchased from Thermo Scientific (Grand Island, NY). The following target sequence was used to knock down expression of the GM3 synthase gene (ST3 beta-galactoside alpha-2,3-sialyltransferase 5 [ST3GAL5]): CAAUGGCGCUGUUAUUUGA. Twenty nanomoles of siRNA was transfected in CV-1 cells by reverse transfection using the Lipofectamine RNAi MAX reagent according to the manufacturer's instructions.
Lentiviral plasmid construction and virus production and use. For SV40 VP1 knockdown, SV40 VP1 shRNA (5=-CCGGGAAGGGACTTCC CAGATATTTCTCGAGAAATATCTGGGAAGTCCCTTCTTTTTG-3=) was inserted into the pLKO.1 shRNA vector (Addgene, Cambridge, MA). Lentivirus was generated and concentrated following the instructions provided by the manufacturer. Cells were incubated with a 1:10 dilution of concentrated lentivirus stock for 48 h at 37°C. For SV40 VP1 expression, the DNA coding sequences for SV40 VP1 were human codon optimized by GenScript USA Inc. (Piscataway, NJ) and inserted into the pLenti-CMV-IRES-GFP-WPRE expression vector, which was a kind gift from Jianming Qiu (Kansas University Medical Center). Infections were performed as described above.
Indirect immunofluorescence. CV-1 cells were seeded on 8-well chamber glass slides (Millipore, MA). At the indicated times after polyomavirus or lentivirus infection, cells were fixed with 3.7% paraformaldehyde (PFA) for 30 min and then permeabilized with 1% saponin for 30 min at room temperature, followed by primary and secondary antibody staining (each at a 1:100 dilution) and confocal microscopy analysis. Primary antibodies used were anti-large T antigen (PAb108) and anti-VP1 (PAb 597).
Experiments with conditioned medium. Conditioned medium was collected 72 h after mock infection or infection of CV-1 cells in a 24-well plate with wild-type SV40 at an MOI of 10. Conditioned medium was centrifuged for 5 min at 1,500 rpm, and the supernatant was added undiluted to~5 ϫ 10 4 uninfected CV-1 cells, which in some cases had been previously transfected as above with siRNA targeting GM3 synthase. Based on flow cytometry of large T antigen expression in infected cells, we estimated that this results in an MOI of~500. Cells were examined by phase-contrast microscopy 4 h later. For some experiments, conditioned medium was treated with 10 M GM1 or a 1:2,000 dilution of neutralizing anti-SV40 antiserum (obtained from James Pipas) for 1 h at 37°C prior to addition to cells.
GM1 supplementation assay. CV-1 and Vero cells were either left untreated or treated with 10 M GM1 overnight in DMEM with 1% FBS. The cells were then washed with regular cell culture medium, followed by one of the various indicated treatments.
Luciferase assay. The Gluc assay was performed using a BioLux Gaussia luciferase assay kit (New England Biolabs, MA) according to the manufacturer's standard assay protocol. Briefly, CV-1 cells seeded on 8-well chamber glass slides were infected with wild-type BKpsV or the K68S mutant at 10 5 genome copies/cell. Four hours later, medium was replaced with fresh medium lacking pseudovirus. At the indicated time postinfection, 20 l of cell culture medium was analyzed for Gluc activity. A Veritas microplate luminometer (Promega, CA) with injectors was used to inject luciferase reaction solution and read the results (relative light units).
For electron microscopy, purified pentamers were diluted in pH 7.2 PBS at 20 g/ml and analyzed after negative staining with 1% uranyl acetate (21) via Tecnai T12 transmission electron microscopy at the Yale Electron Microscopy Core Facility. For transmission electron microscopy of infected cells, 2 ϫ 10 6 CV-1 cells were mock infected or infected with wild-type or A70L SV40 at an MOI of 1.5. At 64 h postinfection, samples were fixed, stained with 2% uranyl acetate, dehydrated, embedded, sectioned, and mounted onto copper grids as described elsewhere (40).
Cross-linking truncated VP1 pentamers with antibody. Aliquots (10 g/ml) of truncated VP1 pentamers in DMEM without serum were incubated with a 1:100 dilution of nonneutralizing anti-VP1 PAb597 or with 1:100 PAb597 plus a 1:100 dilution of anti-mouse IgG for 30 min at 37°C prior to addition to uninfected CV-1 cells. Control cells were treated identically except without pentamer addition. Images were taken 4 h later.
Treatment of cells with cholera toxin. Cholera toxin subunit B (1mg/ml stock solution in H 2 O) was diluted to a final concentration of 40 g/ml and 100 g/ml in culture medium and added to CV-1 cells. Images were obtained 24 h later.