Surface-to-ER Traffic of SV40
SV40 has been shown to be internalized by caveolae and then to reach a subdomain of the ER. The molecular machinery and sorting mechanisms by which the virus reaches the compartment are presently unknown.
We first examined the nature of the SV40-containing compartment by using both plastic sections and immunolabeling. Epon sections of cells incubated with SV40 for 21 h revealed the virus in reticular, smooth-membraned areas of the ER connected to ribosome-studded rough ER membranes (Figure , A and B). The membrane is closely apposed to the surface of the viral particles, suggesting that the virus remains bound to the lumenal surface of the membrane. Ultrathin frozen sections of these cells were labeled with antibodies to the virus together with antibodies to a cis-Golgi marker, p23, or to a marker of the ER, PDI. The virus-containing membranes were negative for the cis-Golgi/intermediate compartment labeled by antibodies to p23 (our unpublished data) but were labeled by antibodies to PDI (Figure C). Although the majority of the virus particles was observed in these very prominent enlarged ER domains, virus particles were occasionally observed in proximity to the Golgi complex, and possibly in Golgi-associated membranes (our unpublished data), raising the possibility that virus may transiently associate with these compartments during infection.
Figure 1 Electron microscopic localization of SV40 in PDI-positive subdomain of ER. Vero cells were incubated with SV40 for 18 to 21 h at 37°C before fixation and processing for either epon embedding (A and B) or frozen sectioning (C). (A and B) SV40 accumulates (more ...)
Brefeldin A Inhibits Entry and Postentry Trafficking of SV40
The above-described observations prompted us to further examine the possibility that SV40 passes transiently through the Golgi compartment in a similar manner to many bacterial toxins, which also follow an endocytic route to the ER. Because transient intermediates in infection may be difficult to identify morphologically and the viral infection process is relatively unsynchronized, we chose to use defined membrane trafficking inhibitors together with assays of infection to further delineate the trafficking pathway.
We first examined the possible involvement of the Golgi complex in the SV40 trafficking pathway by using the fungal metabolite BFA. This drug inhibits guanine nucleotide exchange on the small GTPase Arf1 (Jackson and Casanova, 2000
), an adapter protein responsible for recruitment of COPI coats onto endosomal and biosynthetic membranes. The result is an inhibition of anterograde transport from ER to Golgi complex (Misumi et al., 1986
) and a concomitant tubulation and fusion of the Golgi with the ER (Lippincott-Schwartz et al., 1989
; Scheel et al., 1997
). Consequently, BFA has been widely used to inhibit both anterograde and retrograde transport between the Golgi complex and ER. Vero cells were pretreated for 3 h with BFA and then incubated with SV40 plus BFA for 21 h before fixation and immunolabeling for the virus-encoded T-antigen (T-ag). As shown in Figure , BFA is a potent inhibitor of SV40 infection. With concentrations as low as 0.1 μg/ml we observed 99% inhibition of infection (Figure E). These relatively low doses of 0.1 and 0.5 μg/ml BFA were found to be effective in disrupting Golgi morphology within 1 and 3 h of treatment, respectively (Figure , A and B).
Figure 2 BFA sensitivity of early events in SV40 infection. Treatments of 0.1 μg/ml BFA for 3 h (B) were found to disrupt the Golgi complex of Vero cells (cf. untreated cells in A) as revealed by immunolabeling for the Golgi marker giantin. In Vero cells (more ...)
To determine the stage of the infectious entry pathway that was affected by BFA, Vero cells were exposed to a 3-h pulse treatment of 0.1 μg/ml BFA at various time points into SV40 infection at 37°C. Fixation and immunolabeling for T-ag after the 21-h infection period revealed a clear inhibition of SV40 infection when BFA was applied at early stages of viral infection (Figure E). To examine the possibility that BFA blocks viral entry, we made use of a neutralizing anti-SV40 antibody, which inactivates surface virus. Addition of the neutralizing antibody to cells incubated with the virus at 4°C with no warming step to allow internalization caused a complete block of infection (our unpublished data). Vero cells cultured in the presence or absence of 0.5 μg/ml BFA for 1 h at 37°C were incubated with SV40 plus or minus BFA on ice for 1 h and then at 37°C for 2 h to allow infection to occur. The cells were then incubated in SV40 neutralizing antibody for 30 min and washed to remove BFA. A further 46 h of infection at 37°C was followed by fixation and immunofluorescence labeling for T-ag. Quantitation of SV40 infection efficiency revealed a substantially greater sensitivity of SV40 infection to the neutralizing antibody in BFA-treated cells compared with untreated cells, indicating a trapping of the virus at the cell surface by BFA (Figure F). Thus, it was apparent that the first effect of BFA on SV40 infection was the inhibition of initial viral entry. Consistent with this, treatment with 0.3 μg/ml BFA and incubation with the virus for 21 h showed SV40 immunolabeling only at the periphery of cells, unlike the pattern observed in untreated controls (Figure , C and D). Dual immunolabeling for caveolin-1 and SV40 revealed no significant colocalization (our unpublished data). Similar results were obtained in cells transiently transfected with caveolin-1-YFP with no colocalization of peripheral SV40 labeling with caveolin-1-YFP (our unpublished data). Epon sections of these cells revealed virus particles in tight-fitting, surface-connected invaginations indistinguishable from those seen in untreated cells at early time points. In accordance with the immunofluorescence data, few virus particles were observed intracellularly (Figure G).
To investigate whether BFA also inhibits postsurface trafficking steps, Vero cells were incubated with SV40 for 2 h at 37°C in the absence of BFA to allow viral infection to proceed past the initial internalization step. Any virus remaining at the cell surface was then inactivated with neutralizing antibody and the cells incubated in either the presence or absence of 0.5 μg/ml BFA for the remaining 20 h of infection. Quantitation revealed a strong inhibition of infection in BFA-treated cells compared with untreated controls, showing inhibition of an internal step in SV40 infection (Figure H).
We conclude that the initial entry step of SV40 entry is BFA sensitive. In addition, SV40 infection involves a second postentry BFA-sensitive step, possibly involving endosomes and/or the Golgi complex.
SV40 Entry Is Inhibited at 20°C
Although BFA is best known for its inhibitory effect on transport between ER and Golgi complex, it has also been shown to inhibit early endosome-to-Golgi complex traffic (e.g., of endocytosed Shiga toxin; Mallard et al., 1998
). To determine whether SV40 is internalized to early endosomes, we made use of the observation that exit from the early endosome is blocked at 20°C (Griffiths et al., 1988
). Vero cells were infected with SV40 at 20°C for 4 h. Fixation and immunolabeling for the virus revealed a striking loss of staining for the virus (our unpublished data). To confirm this apparent failure of the virus to be internalized at 20°C, we again used the SV40 neutralizing antibody assay. Vero cells were infected with the virus either at 20 or at 37°C for 4 h. Subsequent exposure of the cells to the neutralizing antibody for 30 min was followed by incubation at 37°C for a further 24 h before fixation and immunolabeling for T-ag. Quantitation of infection efficiency revealed a much greater sensitivity of the virus to the neutralizing antibody after a 4-h infection at 20°C than after infection at 37°C (Figure ). We conclude that initial entry of SV40 is sensitive to both BFA and incubation at 20°C.
Figure 3 SV40 internalization is inhibited at 20°C. Vero cells were infected with SV40 for 4 h at either 20 or 37°C before neutralizing surface-exposed virus with SV40 neutralizing antiserum. After subsequent incubation at 37°C for a remaining (more ...)
CT Exit from Early Endosomes Is Inhibited by BFA and by Incubation at 20°C
We next examined the effects of these treatments on a well-characterized retrograde transport pathway. CT is efficiently transported to the ER via the Golgi complex (see INTRODUCTION). We have used either FITC-labeled CT-B (CT-B-FITC), which is transported to the Golgi, or immunolabeling for the holotoxin, which is transported to the ER (Lencer, 2001
). Vero cells allowed to bind and internalize the FITC-labeled B subunit of the toxin at 20°C showed no inhibition of toxin accumulation in early endosomes (Figure , A–C). Similarly, Vero cells pretreated for 1 h with 0.5 μg/ml BFA also internalized the toxin to early endosomes (Figure , D–F). In cells exposed to BFA for a 21-h period comparable with that used in SV40 infection experiments, internalization of CT was still observed, although a greater intensity of plasma membrane fluorescence, possibly indicating a decrease in uptake, was detected (our unpublished data).
Figure 4 Early endosome-to-Golgi transport of CT-B is inhibited at 20°C and by BFA. Incubation of Vero cells in 0.5 μg/ml CT-B-FITC for 1 h at 20°C resulted in accumulation of the toxin in EEA1-positive early endosomes (arrows) and a failure (more ...)
Although BFA and a 20°C incubation had little effect on internalization of CT-B, subsequent transport to the Golgi was completely inhibited by both treatments. Even after a 1-h incubation at 20 or 37°C in the presence of BFA, the toxin failed to reach the Golgi (Figure , A–F). In cells exposed to BFA (Figure , D–I) the toxin accumulated in sorting endosomes and tubulated recycling endosomes identified by colocalization of cointernalized Texas Red-labeled transferrin with CT-B-FITC (Figure , G–I). We conclude that the sensitivity of SV40 entry to both BFA and incubation at 20°C is in contrast to other known endocytic pathways, including that of the putative caveolae marker CT (Lencer et al., 1993
; Nambiar et al., 1993
) and suggests that SV40 uses a novel entry pathway.
Inhibition of SV40 Infection by Microinjected Antibodies to βCOP and by Expression of Arf1 and Sar1 Mutants
The sensitivity of a postsurface SV40 trafficking step to BFA treatment raised the possibility of Golgi complex involvement in the viral entry pathway. Inhibition of retrograde traffic between the Golgi complex and ER has been convincingly demonstrated by microinjection of an antibody to βCOP, a component of the coatomer complex of COPI-coated vesicles. Microinjection of anti-βCOP (EAGE) (Pepperkok et al., 1993
) was found to inhibit COPI-mediated retrograde transport of the KDEL receptor and of ERGIC-53, both molecules that constitutively cycle between the ER and Golgi complex (Girod et al., 1999
). Retrograde traffic of endocytosed CT-A subunit has also been found to be inhibited by anti-βCOP injection (Majoul et al., 1998
; Girod et al., 1999
). Vero cells were microinjected with anti-βCOP antibodies. Three to four hours later the cells were infected with SV40 for 21 h before fixation and immunolabeling for the microinjected antibody and T-ag. The microinjected antibodies showed cytosolic labeling and strong Golgi staining (Figure A). Quantitation of infection efficiency revealed a strong inhibition of viral infection in microinjected cells, compared with surrounding uninjected cells (Figure B), showing that the anti-βCOP antibody is a potent inhibitor of SV40 infection.
Figure 5 Inhibition of SV40 infection by microinjection of a βCOP antibody. Vero cells were microinjected with the βCOP antibody, anti-EAGE, and subjected to SV40 infection. Fixation and dual immunolabeling for the viral nuclear antigen T-ag (red) (more ...)
As a second independent method to inhibit COPI-mediated transport we used overexpression of a GTPase-deficient Arf1 mutant (Q71L). This mutant has previously been shown to inhibit COPI-dependent transport in the early secretory pathway (Dascher and Balch, 1994
) and also to inhibit sorting and concentration of cargo molecules into COPI-coated vesicles in vitro (Lanoix et al., 1999
). Vero cells were microinjected with either a mixture of Arf1(Q71L) and GFP expression plasmids or the GFP plasmid alone (as a control). After 5 h to allow expression of proteins the cells were incubated with SV40. Expression of Arf1(Q71L) in GFP-expressing cells injected with both plasmids was verified by immunolabeling the cells for βCOP. A clear disruption or complete disappearance of the Golgi βCOP labeling pattern in cells showing high levels of GFP expression was observed in cells injected with both plasmids but not in cells injected only with the GFP construct (Figure , A–D). Quantitation of infection efficiency revealed a potent inhibition of SV40 infection in Arf1(Q71L)-expressing cells compared with neighboring nonexpressing cells. No such inhibition was observed in cells expressing GFP alone (Figure E).
Figure 6 Inhibition of SV40 infection by Arf1(Q71L) and Sar1(H79G). Vero cells were injected with either a mixture of Arf1(Q71L) and GFP expression plasmids (A) or the GFP plasmid alone (C) and incubated for 5 h to allow protein expression to occur. Immunolabeling (more ...)
In addition to their effects on COPI-mediated retrograde transport between the Golgi and ER, the dominant negative Arf1 mutant and microinjected βCOP antibody could also be affecting events early in endocytosis because Arf1 and COP proteins have been implicated in endosomal trafficking (Aniento et al., 1996
; Daro et al., 1997
; Gu et al., 1997
; Gu and Gruenberg, 2000
; Jackson and Casanova, 2000
). To determine whether disruption of ER/Golgi transport could be responsible for inhibiting viral infection, we examined the effect of a selective inhibitor of ER/Golgi transport on SV40 infection by expression of the GTP-restricted mutant of Sar1, Sar1(H79G) (Aridor et al., 1995
). We observed that cells coinjected with expression plasmids for GFP and Sar1(H79G) showed significant inhibition of SV40 infection (Figure F). Use of a temperature sensitive form of vesicular stomatitis virus glycoprotein (ts-045-G) confirmed that Sar1(H79G) and also Arf1(Q71L) were potently inhibiting anterograde transport in this system (Figure ). These results show a direct or indirect role for Arf1, Sar1, and COP-dependent trafficking steps in SV40 infection.
Figure 7 Exocytic transport of ts-045-G is inhibited by Sar1(H79G) and Arf1(Q71L). Vero cells were injected with GFP-tagged ts-045-G expression plasmid alone (A), or with a mixture of ts-045-G and either Sar1(H79G) (B) or Arf1(Q71L) (C) expression plasmids. Subsequent (more ...)
Inhibition of CT Transport to Golgi by Arf1(Q71L) and Sar1(H79G)
To further compare the CT and SV40 trafficking pathways, we examined the effect of the above-mentioned inhibitors on trafficking of CT-B to the Golgi complex or of CT holotoxin to the ER. Cells microinjected with plasmids encoding Arf1(Q71L) and Sar1(H79G) were incubated for 5 h and then allowed to bind and internalize CT-B-FITC for 40 min. A clear inhibition of toxin arrival at the Golgi was observed. The toxin was, however, internalized to early endosomes, although an obvious increase in intensity of cell-surface signal indicated a slight inhibition of entry (Figure ). A similar inhibition of CT internalization was elicited by microinjection of the anti-βCOP antibody (EAGE) (our unpublished data).
Figure 8 CT transport to the Golgi complex is inhibited by Arf1(Q71L) and Sar1(H79G). Vero cells were injected with a mixture of GFP and either Arf1(Q71L) (A–D) or Sar1(H79G) (E–H) expression plasmids. After a 5-h period of protein expression, (more ...)
We then examined trafficking of CT holotoxin to the ER. Untreated cells showed that CT reached the ER in 2 h as judged by immunofluorescence (Figure ). Vero cells were injected with the above-mentioned constructs and protein expression was prevented by incubation with cycloheximide for 4 h. The cells were then allowed to bind and internalize CT holotoxin at 20°C for 1 h in the absence of cycloheximide. After subsequent incubation at 37°C for a further 2 h, the cells were fixed and immunolabeled for CT. This protocol allowed initial internalization of CT to endosomes at 20°C in the absence of mutant protein, but subsequent transport to the Golgi and ER at 37°C occurred in the presence of newly synthesized mutant proteins. As shown in Figure , the mutant proteins caused inhibition of transport to the ER with toxin accumulating in early endosomes (identified by colocalization with EEA1; our unpublished data) or perinuclear putative Golgi elements.
Figure 9 CT transport to the ER is inhibited by Arf1(Q71L) and Sar1(H79G). Vero cells were injected with a mixture of GFP and either Arf1(Q71L) (A and B) or Sar1(H79G) (C–F) expression plasmids and incubated in 10 μg/ml cycloheximide for 4 h. The (more ...)
The inhibition of CT transport out of endosomes and to the Golgi by Arf1(Q71L) is in full accord with the previously described effect of BFA and could be explained by an Arf1/COPI-mediated transport step between endosomes and the Golgi complex. However, the similar inhibition by Sar1(H79G) can only be due to either disruption of the Golgi or a reliance of this endocytic pathway on a functional exocytic pathway.
Inhibition of SV40 Infection by Cbz-gly-phe-NH2
To further compare SV40 infectious trafficking with the trafficking of CT to the ER and then cytosol, we investigated the effect of a recently described inhibitor of CT toxicity. The dipeptide benzyloxycarbonyl Cbz-gly-phe-NH2 causes a potent block in the late stages of toxin action and has no effect on the initial toxin entry step (De Wolf, 2000
). This agent thus provides an important tool for comparing late stages in SV40 trafficking with those of CT.
Vero cells were preincubated for 1 h with 2 mM Cbz-gly-phe-NH2 or an inactive anolog, Cbz-gly-gly-NH2. The cells were then incubated for 21 h with SV40 in the continued presence of the same drug. Quantitation of T-ag expression efficiency revealed a potent inhibition of infection by Cbz-gly-phe-NH2 (Figure A). In contrast, the inactive analog Cbz-gly-gly-NH2 had no effect at the same concentration (Figure A). Infection was similarly inhibited in cells allowed to internalize the virus for 4 h before inactivation of surface-exposed SV40 (using the neutralizing antibody) and simultaneous exposure to the inhibitory dipeptide. This confirms an inhibition of the virus postentry (Figure D).
Figure 10 Cbz-gly-phe-NH2 inhibits SV40 infection but not SFV infection. (A) Vero cells were pretreated for 1 h with 2 mM Cbz-gly-phe-NH2 or its inactive analog Cbz-Gly-Gly-NH2, or were untreated. Subsequent infection with SV40 in the continued presence or absence (more ...)
To assess whether the drug is specific for a retrograde trafficking pathway such as that followed by SV40 or CT, we examined its effects on infection by an enveloped virus, SFV. This virus enters cells by clathrin-mediated endocytosis and requires delivery to acidic endosomes for translocation to the cytosol and productive infection (Marsh et al., 1984
). When Vero cells were exposed to recombinant SFV in the presence of the drug, no inhibitory effects on infection and expression of the SFV-encoded protein, caveolin-3 [SFV(cav-3)], were observed (Figure B).
As shown for the inhibition of CT toxicity, the effect of Cbz-gly-phe-NH2 on SV40 infection was found to be quickly reversible. Cells pretreated for 1 h before washing out the drug and infection with SV40 showed no inhibition of infection compared with untreated controls (our unpublished data). This suggests no lasting effects of the 1-h pretreatment. Furthermore, as observed for CT internalization, the block in SV40 infection appeared to be in late steps of the infectious pathway. A 21-h SV40 infection in the presence of the drug was followed by various periods of incubation in the absence of the drug before fixation and immunolabeling for T-ag. The percentage infection of treated cells stayed very low for the first 3 h after washing out the drug but began to recover after 6 h and reached control levels after 9 h (Figure C). This time period is shorter than that required for infection if virus is added to the outside of the cells and shows that the virus accumulates at a late stage in the infectious entry pathway. In keeping with this, electron microscopy of plastic sections revealed no detectable difference in the number of viral particles reaching ER cisternae in inhibitor- and control-treated cells (our unpublished data).
Finally, we examined CT trafficking in Cbz-gly-phe-NH2–treated cells. No inhibition of CT-B-FITC arrival to the Golgi was detected in inhibitor-treated cells, suggesting that only late events in the toxic entry pathway are affected (Figure ).
Figure 11 Post-Golgi inhibition of CT toxicity by Cbz-gly-phe-NH2. Vero cells were preincubated in 2 mM Cbz-gly-phe-NH2 (A and B) or its inactive analog (C and D) before binding and internalization of CT-B-FITC (A and C) for 40 min in the continued presence of (more ...)
In conclusion, we have identified a potent new reversible inhibitor of SV40 infection that acts at a late stage in the infectious entry process. This makes Cbz-gly-phe-NH2 an invaluable tool for detailed characterization of these trafficking pathways.