GD1a addition to a cell line lacking functional receptors stimulates Py binding, entry, ER transport, and infection.
In this study, we used A1-1, a murine mammary tumor-derived cell line, which was characterized previously as devoid of ganglioside GD1a, Py's functional receptor, on the cell surface (13
); addition of GD1a to this cell line stimulates Py infection (13
). To test whether supplementation of GD1a to the A1-1 cells affects Py cell surface binding, control and GD1a-supplemented cells, as well as cells supplemented with ganglioside GM1, previously shown not to bind virus in vitro
), were incubated with Py at 4°C (to prevent endocytosis) for 1 h. Unbound virus was removed by washing, the cells were fixed, and a Py VP1-specific antibody was used for immunostaining to detect surface-bound virus. We found that the number of virus particles increased in the GD1a-supplemented cells compared to control and GM1-supplemented cells (Fig. , top panels; quantified in the graph at the bottom of the panels), indicating that GD1a interacts with Py on the plasma membrane. When these cells were heated to 37°C to promote virus entry, we found more Py entering the GD1a-supplemented cells compared to control cells (Fig. , top panels; quantified in the graph at the bottom of the panels). Thus, GD1a promotes Py binding and internalization.
FIG. 1. GD1a addition to a murine cell line lacking functional receptors stimulates Py binding, entry, ER transport, and infection. (A and B) Control cells, GM1-supplemented cells (shown in panel A alone), and GD1a-supplemented A1-1 cells were incubated with (more ...)
Postentry, Py is driven to the ER in a GD1a-dependent manner to cause infection (12
). We tracked transport of Py to the ER in these cells by expressing CFP-HO2 and assessed the extent of Py colocalization with CFP-HO2 at 4.5 h postinfection. As the ER is highly convoluted in these cells, the ER images were filtered as previously described (27
) in order to better define the boundaries of the ER. This effort enables a more accurate analysis of Py-ER colocalization. An example of a filtered image demonstrating colocalization of Py (red) with the ER (green) is shown in Fig. (top panels). Using this method, we found that addition of GD1a increased the number of ER-localized virus particles (Fig. ; see quantification in graph at bottom of the panels), which is consistent with previous observations of other cells (12
). Furthermore, Py infection (as measured by expression of the large T antigen) increased significantly in the GD1a-supplemented cells compared to control cells (Fig. ), as expected (13
). Together, these findings indicate that ganglioside GD1a is an entry receptor, facilitating the binding, entry, ER trafficking, and infectivity of Py.
Py colocalizes with GD1a on the plasma membrane, the late endosomes, and the ER.
Since it is a functional entry receptor, we hypothesized that GD1a engages Py on the plasma membrane, guiding the virus through the endolysosomes en route to the ER. This scenario necessitates colocalization of GD1a with Py on the cell surface, the endolysosomes, and the ER.
To visualize the behavior of GD1a in cells, we used a modified form of GD1a in which the BODIPY fluorophore is conjugated to the ceramide domain of GD1a (BODIPY-GD1a) (3
). When the A1-1 cells were incubated with BODIPY-GD1a at 4°C for 20 min, this lipid localized mostly to the plasma membrane (Fig. , top panels). In contrast, when the cells were heated to 37°C for 30 min, the majority of BODIPY-GD1a localized to vesicular structures (Fig. , bottom panels), indicating that this lipid was internalized.
FIG. 2. Py colocalizes with GD1a on the plasma membrane, the late endosomes, and the ER. (A) A1-1 cells were incubated with BODIPY-GD1a at 4°C for 20 min (top panels) or at 37°C for 30 min (bottom panels). (B) Cells were incubated first with BODIPY-GD1a (more ...)
In addition to BODIPY-GD1a, we also used Py labeled with Alexa Fluor 594 to study colocalization of Py with GD1a. The labeled Py was previously shown to recapitulate the normal cellular transport and infection processes of unlabeled Py (27
). For analysis of Py-GD1a colocalization on the plasma membrane, cells were first incubated with BODIPY-GD1a at 4°C for 15 min, followed by addition of labeled Py at 4°C for 30 min. A typical image of labeled Py (red) colocalizing with BODIPY-GD1a (green) on the plasma membrane is depicted in Fig. . Quantification of the extent of colocalization demonstrated that only a small amount (approximately 12%) of Py colocalized with BODIPY-GD1a on the cell surface (Fig. ). This finding is not surprising, as nonganglioside receptors likely compete with BODIPY-GD1a for Py binding.
To determine the level of Py colocalization with the endolysosomal system, we analyzed cells expressing CFP-Rab7 (a marker of the late endosomes) that were first incubated with BODIPY-GD1a at 4°C for 15 min and then with labeled Py at 37°C for 3 h. An example of an image of labeled Py (red) colocalizing with BODIPY-GD1a (blue) in the Rab7-containing vesicle (green) is shown in Fig. (see inset). Quantification analysis showed that 43% of labeled Py in the late endosomes colocalized with BODIPY-GD1a (Fig. ). Hence, a higher percentage of Py colocalizes with BODIPY-GD1a in the late endosomes than in the plasma membrane.
To assess the extent of Py-GD1a colocalization in the ER, cells expressing CFP-HO2 initially incubated with BODIPY-GD1a at 4°C for 15 min and then with labeled Py at 37°C for 4.5 h were analyzed. A typical image depicting labeled Py (red) colocalizing with BODIPY-GD1a (blue) in the ER (green) is shown in Fig. (see inset). When quantified, approximately 67% of Py in the ER colocalized with BODIPY-GD1a (Fig. ). That the percentage of Py-GD1a colocalization was highest in the ER (67%) compared to the late endosomes (43%) and the plasma membrane (12%) suggests that Py bound to GD1a on the cell surface, upon entry, is preferentially targeted to the ER.
Ligand-induced retrograde transport of gangliosides to the ER.
The observations that addition of GD1a stimulates Py transport to the ER (Fig. ) and that a majority of Py in the ER colocalizes with GD1a (Fig. ) suggest that Py should in turn promote transport of GD1a to the ER.
To test this prediction, we measured the BODIPY-GD1a level in the ER of cells in a virus-dependent manner. A1-1 cells expressing CFP-HO2 were incubated with BODIPY-GD1a at 4°C for 15 min followed by incubation with or without Py at 37°C for 4.5 h. A representative image of an ER-localized BODIPY-GD1a is shown in Fig. . Indeed, our quantification analysis showed a significant increase in ER-localized BODIPY-GD1a levels in cells incubated with Py compared to those with no virus incubation (Fig. ; see quantification in the middle panel). In contrast, the cholera toxin B (CTB) subunit, which binds to ganglioside GM1, did not stimulate ER transport of BODIPY-GD1a (Fig. ; see quantification in the right panel). GM1 normally engages CT on the cell surface, targeting the toxin to the ER to cause intoxication of intestinal epithelial cells (20
). We conclude that binding between Py and ganglioside GD1a triggers the specific retrograde transport of its receptor to the ER.
FIG. 3. Ligand-induced retrograde transport of gangliosides to the ER. (A) A1-1 cells expressing CFP-HO2 were incubated first with BODIPY-GD1a at 4°C for 15 min and were then either incubated with Py at 37°C for 4.5 h or left untreated or incubated (more ...)
To determine whether the observed Py-induced reverse transport of GD1a to the ER is a common mechanism, we assessed whether CTB triggers the ER transport of GM1. NIH 3T3 cells expressing CFP-HO2 were incubated with BODIPY-GM1 at 4°C for 15 min, followed by incubation with or without CTB at 37°C for 1 h or Py at 37°C for 4.5 h. We found that CTB, but not Py, stimulated the transport of GM1 to the ER (Fig. ; see quantification in the left and right graphs). These findings indicate that interaction between a ligand (e.g., virus or toxin) and its respective ganglioside receptor is a general mechanism driving the ganglioside to the ER. In this context, it is interesting that both Py VP1 and CTB are pentamers when assembled into their native structures (20
), suggesting that a multivalent ligand-ganglioside interaction may be important for transportation of this complex to the ER.
GD1a must be added before, but not after, incubation of cells with Py to stimulate infection.
Our finding that addition of GD1a to cells increased the plasma membrane binding and entry of Py (Fig. ) suggests that GD1a functions as the entry receptor. To further strengthen our contention that Py engages GD1a on the plasma membrane prior to entry to cause infection, we asked whether addition of GD1a after virus entry stimulates infection as well. We reasoned that, should addition of GD1a after virus entry promote Py infection, virus interaction with GD1a on the cell surface must not be a prerequisite step for stimulation of infection. In this scenario, it is possible that other sialic acid-galactose-containing receptors such as glycoproteins serve as alternative entry receptors and deliver Py from the cell surface to the endolysosomes. In the endolysosomes, Py is conceivably released from the glycoproteins to bind to GD1a. Alternatively, if addition of GD1a after virus entry fails to stimulate Py infection, Py must therefore interact with GD1a on the cell surface prior to entry to enable infection.
In the A1-1 cells, we found that Py infection was stimulated only when GD1a was added before, but not after, incubation of the cells with the virus (Fig. A). A potential trivial explanation is that GD1a added subsequent to virus entry fails to reach the endolysosomes efficiently, thereby resulting in an inability to bind Py that has been delivered to this compartment via nonganglioside receptors. However, we found that the extents of colocalization between BODIPY-GD1a and labeled Py in the Rab7-positive late endosomes were similar regardless of whether BODIPY-GD1a was added before or after virus incubation (Fig. ; see also Fig. ). Hence, the inability of GD1a to stimulate infection when this glycolipid was added after virus entry cannot be attributed to a deficiency in transport of GD1a to the same vesicles harboring Py. Instead, the simplest explanation of these results is that, to cause infection, Py must bind to GD1a on the cell surface before internalization. The observation that GD1a must be added before but not after incubation with Py to stimulate infection was recapitulated in experiments with NIH 3T3 cells (Fig. ). These data further underscore our view that Py initiates its binding to GD1a on the plasma membrane, and not within an intracellular organelle, to infect cells. The GD1a-Py complex is then internalized and transported through the endolysosomes en route to the ER.
FIG. 4. GD1a must be added before, and not after, incubation of cells with Py to stimulate infection. (A) A1-1 cells were treated with 80 μM GD1a at the indicated time points with respect to addition of cells with Py. At 48 h after incubation of cells (more ...) Removing plasma membrane glycoproteins stimulates Py infection and ER transport.
In addition to glycolipids such as gangliosides, many glycoproteins also contain the sialic acid-galactose moiety (18
), a defining motif for binding to Py (33
). Hence, in principle, glycoproteins displaying the terminal sialic acid-galactose sugars should also engage Py. What then is the functional consequence of such an interaction in controlling Py infection?
To assess the function of glycoproteins in regulating virus infection, NIH 3T3 cells were treated with or without a low concentration of the general protease proteinase K at 4°C for 1 h to remove cell surface proteins. The contents of the media from these cells were precipitated and subjected to SDS-PAGE analysis followed by Coomassie staining. Degraded protein products of various molecular weights were observed in the media derived from cells treated with proteinase K, while a very small amount was found in the media from control cells (Fig. ; compare lanes 2 and 1). This finding demonstrates that the protease effectively removed proteins from the cells.
FIG. 5. Removal of plasma membrane glycoproteins stimulates Py infection and ER transport. (A) NIH 3T3 cells were treated with 4 mg of proteinase K/ml at 4°C for 1 h or left untreated. The contents of media from these cells were precipitated and subjected (more ...)
To test whether the proteinase K treatment affected the integrity of cell surface proteins but not internal proteins, cell lysates from cells treated with the protease or left untreated were subjected to SDS-PAGE followed by immunoblotting with antibodies against the plasma membrane proteins, EGF receptor (EGFR), and transferrin receptor (TfR), as well as an ER membrane protein, Derlin-1. Whereas EGFR and TfR were efficiently degraded, Derlin-1 was not proteolyzed (Fig. ; compare lanes 2 and 1). We conclude that under the conditions used for the protease treatment, plasma membrane proteins were effectively removed without degradation of internal proteins.
We then assessed the ability of Py to interact with these cells and found that removing proteins from the cell surface decreased Py binding (Fig. ). This finding indicates that protein factors engage Py on the plasma membrane, as suggested by our previous finding (27
). Strikingly, Py infection was stimulated in cells treated with proteinase K compared to control cells (Fig. ), suggesting that proteins on the cell surface act to attenuate infection normally. This effect was not due to increased cell surface GD1a expression, as cell surface binding of a quantum dot (Q-dot) conjugated to an antibody against GD1a (GD1a Ab) shown previously to bind to GD1a (27
) did not increase in the proteinase K-treated cells (Fig. ).
Presumably, the increased infection was due to increased Py binding to GD1a. This could be because of the fact that glycoproteins bind to Py normally and their absence allows more Py to engage GD1a. Alternatively, it is also possible that glycoproteins prevent virus access to GD1a simply because they protrude from the membrane. We used fluorescently labeled Py and BODIPY-GD1a to measure their colocalization on the cell surface of A1-1 cells (Fig. ); this cell line lacks endogenous GD1a on the plasma membrane (13
) that would compete with BODIPY-GD1a for virus binding. We found that removing plasma membrane glycoproteins with proteinase K increased cell surface Py-GD1a colocalization in these cells (Fig. ), which is consistent with the hypothesis that the increased infection observed in NIH 3T3 cells in the absence of glycoproteins was due to increased Py-GD1a binding.
As the plasma membrane-to-ER transport pathway constitutes the infectious route, one clear implication of these results is that virus trafficking to the ER should also be enhanced when plasma membrane proteins are removed. Indeed, we found an increase in colocalization of Py with the ER in cells treated with proteinase K compared to nontreated cells (Fig. ). This finding is consistent with the infection data and demonstrates that, in the absence of cell surface proteins, Py is preferentially transported to the ER to promote infection.
As an independent method to support these findings, NIH 3T3 cells were treated with PNGase F, an enzyme that removes the carbohydrate moiety from glycoproteins but not glycolipids, at 37°C for 1 h (PNGase F does not function effectively at 4°C). This set of conditions removed the sugar moiety from the EGFR membrane glycoprotein but not from the Ribo I ER membrane glycoprotein (Fig. ; compare lanes 2 and 1), indicating that PNGase F acted only on cell surface glycoproteins and not on internal glycoproteins. In similarity to cells treated with proteinase K, cells incubated with PNGase F supported more Py infection than the nontreated control cells (Fig. ). Thus, we conclude that glycoproteins normally act to restrict Py infection, likely by binding to and targeting Py on nonproductive pathways.
Overexpression of model glycoprotein receptors decreases Py infection.
In addition to the loss-of-function approach, we investigated whether a gain-of-function strategy in which cells overexpress a model glycoprotein would result in a block in Py infection. EGFR is a classic membrane glycoprotein containing, among other sugars, terminal sialic acid-galactose residues (7
). We first investigated whether EGFR interacts with Py by the use of coimmunoprecipitation analysis. NIH 3T3 cells were incubated with Py at 4°C for 1 h, the unbound virus was removed by washing, and the resulting cell pellet was incubated with the cross-linker dithiobis succinimidyl propionate (DSP) at 4°C for 1 h. Cells were lysed, and the resulting lysate was subjected to immunoprecipitation using either a control Ribo I- or an EGFR-specific antibody. The precipitated sample was subjected to SDS-PAGE followed by immunoblotting with antibodies against Py VP1 and EGFR. Using this approach, we found a low level of Py that coprecipitated with the EGFR but not with Ribo I (Fig. , top panel; compare lanes 2 and 1), demonstrating that the EGFR interacts with Py.
FIG. 6. Overexpression of model glycoprotein receptors decreases Py infection. (A) NIH 3T3 cells were incubated with Py at 4°C for 1 h and washed to remove the unbound virus; the resulting cell pellet was incubated with the cross-linker DSP at 4°C (more ...)
Based on this finding, we hypothesized that overexpression of EGFR competes with ganglioside GD1a in interacting with Py, potentially attenuating infection. To test this hypothesis, cells were transfected with either the control GFP construct or a combination of GFP and a FLAG-tagged human EGFR construct, and the total lysates from these cells were analyzed for EGFR expression. As expected, we found an increase in the EGFR level in cells transfected with GFP and EGFR compared to cells transfected with GFP alone (Fig. ). It should be noted that, as the transfection efficiency in these cells is approximately 20 to 25%, the difference in the EGFR expression levels in cells transfected with or without EGFR is likely to be even more exaggerated than that revealed in immunoblot analysis (which reflects the difference determined from the entire pool of cells).
We then analyzed the extent of Py binding to the cell surface in cells transfected with GFP or in cells cotransfected with GFP and EGFR by incubating the cells with Py at 4°C for 1 h. Only cells expressing GFP were analyzed. Our results indicated that cells cotransfected with GFP and EGFR supported more Py binding than cells transfected with GFP alone (Fig. ). We conclude that EGFR overexpression increases Py plasma membrane binding.
Importantly, we found a decrease in Py infection in cells that were cotransfected with GFP and EGFR compared to cells transfected with GFP alone (Fig. ). Thus, overexpressing EGFR attenuates Py infection. In the A1-1 cell experiments, we found that the extent of cell surface colocalization between labeled Py and BODIPY-GD1a decreased in cells overexpressing CFP and EGFR compared to cells transfected with CFP alone (Fig. ). Thus, the simplest explanation is that, in NIH 3T3 cells, excess EGFR competes with GD1a for Py binding, thereby decreasing infection.
We then asked whether ER transport of Py is similarly decreased in cells overexpressing EGFR. Cells were first transfected with either the ER marker CFP-HO2 alone or a combination of CFP-HO2 and EGFR. The cells were then incubated with Py, and the extent of Py-ER colocalization was assessed by immunofluorescence. Our findings show that the Py-ER colocalization was decreased in cells cotransfected with CFP-HO2 and EGFR compared to cells transfected with CFP-HO2 alone (Fig. ). By shifting the extent of Py binding toward glycoproteins, overexpressing EGFR prevented proper Py trafficking along the ER infection route, consequently blocking infection.
What might be the mechanism by which EGFR overexpression attenuates infection? We found a modest increase in Py colocalization with Rab7 in cells overexpressing EGFR and YFP-Rab7 compared to cells overexpressing YFP-Rab7 alone (Fig. ). This finding suggests that the increased level of EGFR in cells targets more Py to the late endosomes, where the viral particles are trapped and unable to sort further to the ER.
Finally, we found that Py infection markedly decreased in NIH 3T3 cells stably overexpressing the IGF-1 glycoprotein receptor (IGF-1R) compared to control cells (Fig. ). As IGF-1R is known to induce signaling properties different from those induced by the EGFR (35
), the decrease in infection observed when either of the glycoproteins was overexpressed was not likely due to their signaling events. Instead, our results suggest that glycoproteins such as the EGFR or IGF-1R normally function to restrict Py infection by engaging the virus, leading it along nonproductive routes. Thus, glycoproteins execute an function opposite to that of the glycolipid gangliosides that act as functional entry receptors.