1 University of St Andrews, School of Biology, Centre for Biomolecular Sciences, Biomolecular Sciences Building, North Haugh, St Andrews KY16 9ST, UK
2 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, UK
Correspondence
Thomas Wileman
thomas.wileman{at}bbsrc.ac.uk
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ABSTRACT |
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Supplementary material supplied in JGV Online.
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INTRODUCTION |
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FMDV, like other members of the family Picornaviridae, is a non-enveloped RNA virus which contains a single-stranded, positive-sense RNA molecule of approximately 8500 nt that serves as a genome (Forss et al., 1984). This (+) RNA, functioning as mRNA, encodes a high molecular mass polyprotein that undergoes co-translational autoproteolysis to yield four structural polypeptides (VP14) which comprise the viral capsid, and nine non-structural proteins of the P2 and P3 regions that control the viral life cycle within host cells (Ryan et al., 1989
). In contrast to poliovirus, the replication complex of FMDV, and its possible association with cellular membranes, has not yet been described in detail. Our recent work using electron microscopy shows that the virus assembly site forms next to the nucleus and contains membranous structures (Monaghan et al., 2004
), yet the origins of the membranes are unknown. In this report, we use immunofluorescence analysis to demonstrate that cytoplasmic structures staining for viral proteins form at perinuclear sites close to a dispersed Golgi apparatus. The viral non-structural protein 2C co-localizes with VP1, 3A and 3D within these structures, but not with proteins associated with organelles of the host cell secretory pathway, including the ER, the ER to Golgi intermediate compartment (ERGIC), Golgi complex, trans-Golgi network (TGN) or lysosomes. The results suggest that these membranes of the secretory pathway are not used by the virus as a platform for replication and assembly, or that the FMDV-induced structures are formed by membrane rearrangements which involve exclusion of organelle-specific protein markers.
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METHODS |
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Antibodies and reagents.
Monoclonal antibodies (mAb) against FMDV non-structural proteins 2C, 3A and 3D were a kind gift from E. Brocchi at The Instituto Zooprofilatico in Brescia, Italy. Mouse mAb B2 against FMDV structural protein VP1, and rabbit antiserum DM10 recognizing FMDV non-structural protein 2C were provided by World Reference Laboratory (IAH, Pirbright, UK). Polyclonal antibodies against -mannosidase II (Moreman et al., 1991
) were purchased from the Dept of Biochemistry and Molecular Biology, University of Georgia, USA. Antibodies recognizing ERp57 were raised in rabbits by using a peptide PIIQEEKPKKKKKAQEDL found at the C terminus of the protein. Rabbit polyclonal antibodies against the peripheral Golgi marker
-COP, and mAb G1/93 against ERGIC p53 were kind gifts from J. Lippincott-Schwartz and Hans-Peter Hauri, respectively (University of Basel, Switzerland). Antibodies recognizing TGN46 (Prescott et al., 1997
) were raised in sheep and kindly provided by V. Ponnambalam (University of Leeds, UK). An antibody specific for CD63 was provided by M. Marsh (University of London, UK). mAb H4B4 against human LAMP-2 was purchased from the Developmental Studies Hybridoma Bank, University of Iowa, USA. FITC- and Alexa dye conjugated secondary antibodies were purchased from Molecular Probes. BFA was purchased from Sigma.
Infections.
Near-confluent cultures growing in 100 mm Petri dishes containing 13 mm glass coverslips were infected at 37 °C with FMDV (m.o.i approximately 5 p.f.u. per cell) in medium containing 5 % fetal calf serum for 30 min. Fresh medium was then added and incubation continued for a further 26 h (depending on cell type) to allow expression of viral proteins. Cells were then either lysed for biochemical experiments or fixed for immunofluorescence analysis (see below).
Labelling of cells, preparation of lysate fractions and immunoprecipitation.
FMDV-infected BHK-21 cells growing in 100 mm Petri dishes were incubated for 10 min at 37 °C in methionine/cysteine-free Eagle's medium and then pulse-labelled for 60 min with 2 MBq of 35S-Promix (Amersham Life Science) per ml of the same medium. Cells were then trypsinized, washed twice in 0·25 M buffered sucrose (50 mM Tris/HCl, 1 mM EDTA, pH 7·4) and resuspended in 200 µl of the same buffer before being homogenized by 15 passages through a 25 gauge needle. Whole cells and nuclei were removed by pelleting at 2000 r.p.m. for 1 min at 4 °C in an Eppendorf 5402 centrifuge and resuspended in immunoprecipitation buffer (10 mM Tris/HCl pH 7·8, 150 mM NaCl, 0·1 % Triton X-100, 1 mM PMSF, 1 mM EDTA and 1 mg ml1 each of leupeptin, pepstatin, chymostatin and antipain) for further analysis by immunoprecipitation (see below). Membrane and cytosolic fractions were then obtained by centrifugation of the post-nuclear supernatant at 14 000 r.p.m. (in an Eppendorf 5415 centrifuge) for 15 min. The cytosolic fraction was retained for immunoprecipitation while the membrane fraction was solubilized in immunoprecipitation buffer and recentrifuged at 14 000 r.p.m. (in an Eppendorf 5415 centrifuge) for 15 min to separate protein aggregates and solubilized membrane protein fractions. Membrane fractions were also resuspended in 1 ml 30 % Nycodenz and layered under Nycodenz step gradients prepared using 5 % (2 ml), 10 % (3 ml) and 17·5 % (5 ml) Nycodenz in buffered sucrose. Gradients were centrifuged at 60 000 g for 90 min in a SW40 Ti rotor at 4 °C. Fractions were collected from the bottom and assayed for ER marker -glucosidase by using 4-methylumbelliferyl-
-D-glucose, for the Golgi marker enzyme galactosyltransferase by monitoring transfer of UDP-6-[3H]galactose (New England Nuclear) to ovalbumin, and for the lysosomal enzyme
-hexosaminidase using 4-methylumbelliferyl-N-acetylglucosamine (Cobbold et al., 1996
). For immunoprecipitation of FMDV 2C from each fraction, polyclonal antibody DM10 bound to protein A-Sepharose beads (Pharmacia) was incubated overnight at 4 °C with precleared lysate. Beads were then washed four times in immunoprecipitation buffer, denatured in SDS gel loading buffer and separated by 12 % SDS-PAGE.
Immunofluorescence.
Infected cells growing on 13 mm coverslips were fixed for 30 min at room temperature in 4 % paraformaldehyde adjusted to pH 7·58·0, washed three times in TBS (150 mM NaCl, 100 mM Tris/HCl, pH 7·5) and permeabilized by incubation in TBS containing 1 % gelatin and 0·5 % NP-40. Samples were incubated with antibodies against viral proteins together with antibodies specific for host cell protein markers diluted in TBS containing 1 % gelatin, 0·5 % NP-40 and 30 % goat serum. Samples were washed with PBS containing 0·1 % Tween-20 and then incubated with secondary antibodies conjugated to Alexa dyes. Finally, washed samples were mounted in Fluoromount G (Southern Biotechnologies).
Digital imaging.
Cell preparations were viewed at x60/1·4 NA with a Nikon E800 inverted microscope fitted with epifluorescent optics, and photographed with a Hammatsu C-4746A charge coupled camera device (CCD). For each cell preparation, nine digital images were collected at 0·2 µm intervals and deconvolved using an Openlab software package (Improvision). After being adjusted for contrast and intensity, images were compiled using Quark Xpress software.
Serial optical sectioning.
In order to enhance resolution and reveal the precise positions of the proteins with respect to each other, several optical sections were taken at 0·2 µm intervals through the cell and digitally deconvolved. To obtain double-label fluorescence images, corresponding deconvolved slices of two different colourized protein signals were superimposed. In such images, red and green pixels represent separate protein localizations, whereas yellow areas indicate co-localization of the two proteins.
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RESULTS |
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Given that approximately half of 2C was membrane-associated, subcellular membrane fractionation was used to test whether 2C associated with membranes of the secretory pathway. The membranes within a post-nuclear membrane fraction prepared from cells infected with FMDV were analysed using Nycodenz gradients. The locations of ER and Golgi membrane markers are shown in Fig. 3(b), and the migration of 2C was determined by immunoprecipitation of gradient fractions. The gradient gave good separation of the ER and Golgi; however, 2C did not co-sediment with the markers tested, and was broadly distributed between fractions 16, suggesting association with membranes of variable density. Even so, the highest levels of 2C were found in fractions slightly heavier than the ER membranes. Since approximately half the 2C signal was found in fractions 35 that also contained ER and Golgi enzymes, these fractions were pooled and separated on a second shallow Nycodenz gradient to resolve ER and Golgi membranes further. The ER marker migrated towards the bottom of the gradient (Supplementary material in JGV Online) and the Golgi marker was found in two peaks. Under these conditions a proportion of the membrane-associated 2C signal co-fractionated with the light Golgi membrane peak, but not ER or heavy Golgi fractions. In summary, the bulk of the 2C signal was recovered from membranes that did not co-sediment with ER or Golgi marker enzymes, suggesting a lack of co-localization with these organelles. Approximately 25 % of membrane-associated 2C did, however, co-fractionate with a light membrane fraction containing the Golgi sugar processing enzyme UDP-galactosyltransferase.
Distribution of pre-Golgi marker proteins in cells infected with FMDV
The above observation led us to speculate that host cell membranes other than those of the Golgi may be the source of membranes in replication complexes. To examine the possibility that pre-Golgi compartments were involved we compared the distribution of 2C with that of the ER-resident protein ERp57. As expected, ERp57 produced a fine reticular staining pattern characteristic of the ER (Fig. 4b). FMDV 2C again localized in a juxtanuclear position (Fig. 4a
). The ERp57 signal was reduced in this region but the reticular pattern was retained in the infected cell. When corresponding deconvolved images were superimposed, the two protein signals remained separate, and yellow areas were not observed, indicating that 2C is not associated with ERp57-positive structures (Fig. 4c
). We next compared the distribution of 2C with a marker protein of the intermediate membrane compartment between the ER and Golgi. This tubulo-vesicular structure is identified by an integral membrane protein, ERGIC53 (Schweizer et al., 1988
), which cycles between the cis-Golgi and the ER. Since staining with the antibody available for ERGIC53 was found to be negative in CHO-K1 cells, we compared the distributions of 2C and ERGIC53 in porcine kidney RS-2 cells, which gave a positive signal for ERGIC53 and were susceptible to FMDV infection. In uninfected cells ERGIC53 was located to a crescent-shaped juxtanuclear area similar to the staining seen for Man II (data not shown). In infected cells, however, ERGIC53 was redistributed into weakly stained spots scattered throughout the cytoplasm (Fig. 4e
). When corresponding deconvolved images of 2C and ERGIC53 were merged, the two protein signals remained completely separate as red and green pixels (Fig. 4f
). These results indicate that, although FMDV caused disruption of ERGIC53 distribution, 2C was not associated with membranes of the ERGIC.
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DISCUSSION |
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The juxtanuclear location of FMDV replication complexes suggested an association with one or more Golgi compartments. In fact, when cells were double-stained with antibodies against 2C and Man II, which is found in the medial-Golgi, both proteins were found in the juxtanuclear region of the cell. The compact crescent-shaped distribution of Man II found in uninfected cells did, however, change during infection, and the protein became dispersed within structures positive for 2C. From these results we reasoned that medial-Golgi membranes may contribute to the formation of replication complexes, or be incorporated into them. Surprisingly, three observations argued against this. Firstly, 2C and Man II remained as separate immunofluorescence signals in merged, deconvolved images of infected cells (Fig. 2), and in the same cells examined by confocal microscopy (data not shown). Secondly, in cells treated with BFA, Man II redistributed to the ER, while 2C retained its juxtanuclear localization, suggesting that the two proteins are associated with different subcellular structures. Thirdly, the bulk of the membrane-associated 2C produced in infected cells failed to co-fractionate with Golgi membranes on density gradients.
The above experiments established that medial-Golgi proteins did not co-localize with viral proteins present in FMDV assembly sites. This stimulated a study of other proteins known to locate to different compartments of the secretory pathway. Recent analysis of cells infected with poliovirus have implicated the ER, particularly COPII coated vesicles, as the major source of virus-induced vesicles (Rust et al., 2001). In our study, however, we were unable to see co-localization of FMDV 2C with ER markers. Similar results were obtained for the ERGIC marker ERGIC53. The staining pattern of ERGIC53 was nonetheless found to be markedly altered in infected cells, with the protein dispersing from a juxtanuclear location into the cytoplasm. The next experiment investigated whether FMDV-induced structures were derived from membranes of post-Golgi compartments. The 2C signal was distributed within TGN46-containing membranes; however, deconvolved images again showed that the two proteins were in separate structures. Likewise, 2C was absent from endocytic vesicles and lysosomes.
In summary, our experiments show that membranes containing FMDV proteins thought to be important for virus replication form at a juxtanuclear sites close to a dispersed Golgi apparatus. Importantly, these membranes do not contain resident Golgi proteins, or protein markers associated with proximal and distal Golgi compartments. The Golgi is not therefore the source of the membranes found at virus assembly sites. This also means that the juxtanuclear location of the assembly site is not maintained by association with the Golgi apparatus, and probably involves and alternative mechanism. This could involve retrograde microtubule transport, as has been reported for cytoplasmic assembly sites formed by other viruses (Heath et al., 2001; Ploubidou et al., 2000
; Sfakianos et al., 2003
). The inability to detect membrane markers in FMDV replication complexes makes it difficult to predict how these structures are formed in cells. One possibility is that they are formed de novo through the action of viral proteins on lipid biosynthesis pathways. A more likely mechanism, given the wealth of evidence from other picornaviruses, is that FMDV gains membranes from the secretory pathway, but somehow excludes host cell membrane proteins. A trivial explanation for our results could be that host proteins are present in replication complexes, but FMDV proteins mask epitopes. A coat of viral proteins cross-linked to the outside of vesicles during fixation could certainly prevent antibodies from binding epitopes present in the lumen of vesicles. Preliminary experiments suggest that epitope masking is unlikely because observation of cells expressing GFP-tagged ER or Golgi markers also show separation of these fluorescence signals from 2C (data not shown). Under these circumstances the natural fluorescence of GFP cannot be masked by viral proteins coating the external surface of the organelle.
A second, and more interesting, explanation is that host proteins are indeed absent because they are removed during the delivery of membranes from the secretory pathway to virus assembly sites. Proteins are sorted to individual compartments of the secretory pathway through the use of coat proteins and membrane vesicles (Guo et al., 2000). COPI and COPII coats, for example, sort proteins within the ER and Golgi apparatus, and clathrin coats regulate the formation of vesicles within the endocytic pathway. In each case the protein coat aids the separation of resident proteins from cargo proteins destined for other membrane compartments. It is possible that picornavirus proteins disrupt coat proteins and upset the normal sorting of membrane proteins in cells. Interestingly, Gazina et al. (2002)
show that in cells infected with parechovirus 1 the Golgi COPI coat component,
-COP, is dispersed in the cytoplasm. This demonstrates that parechovirus 1 can displace coat proteins important for protein sorting, and this may be a mechanism used to prevent Golgi proteins from reaching, or remaining in, virus-induced membranes. It would be interesting to see if, as observed here for FMDV, other membrane markers are absent from replication sites formed by parechovirus 1. An inhibition of protein sorting has also been proposed for poliovirus by Rust et al. (2001)
where the poliovirus 2BC protein is thought be incorporated into COPII coated vesicles formed at the ER. The 2BC protein may prevent fusion of the ER-derived transport vesicles with the Golgi apparatus, thereby providing a source of vesicles for use in virus replication, which again exclude Golgi markers. In our study we could not find co-localization of FMDV proteins with COPII coats labelled with msec13 (data not shown). This means that FMDV either does not use COPII coats, or has a means of removing them before formation of the replication site. Recent experiments by Suhy et al. (2000)
looked at the exclusion of host proteins from poliovirus replication complexes in some detail. They proposed a complex mechanism where poliovirus proteins 2BC and 3A induce the ER to produce vesicles containing two membranes, in a manner similar to autophagy. Again, the coating of vesicles by viral proteins is thought to exclude host proteins during vesicle maturation.
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ACKNOWLEDGEMENTS |
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Received 20 April 2004;
accepted 30 November 2004.
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