Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK
Correspondence
Geoffrey L. Smith
glsmith{at}imperial.ac.uk
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ABSTRACT |
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These authors contributed equally to this work.
A figure depicting genome analysis of recombinant viruses, and two movies showing intracellular enveloped virus movement in cells are available as supplementary material in JGV Online.
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INTRODUCTION |
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Two VACV proteins, F12L (Zhang et al., 2000; van Eijl et al., 2002
) and A36R (van Eijl et al., 2000
), are associated with IEV, but not IMV, CEV or EEV, and each has been implicated in IEV transport. Viruses lacking either protein form a small plaque and are highly attenuated in vivo (Parkinson & Smith, 1994
; Zhang et al., 2000
). F12L is a 6570 kDa protein that is conserved in chordopoxviruses (Zhang et al., 2000
). Confocal and electron microscopy has shown that, in the absence of F12L, VACV morphogenesis halts after the formation of IEV particles, and these are not transported to the cell periphery (van Eijl et al., 2002
). Although the role of F12L in IEV egress is unknown, its requirement for IEV transport makes it a prime candidate for direct or indirect interactions with microtubules (van Eijl et al., 2002
).
A36R is a 45 kDa protein (Parkinson & Smith, 1994) with type 1b membrane topology, and with a 195 aa cytosolic domain that is located beneath CEV on the cytosolic face of the plasma membrane (Röttger et al., 1999
; van Eijl et al., 2000
). Deletion of A36R does not prevent IEV and CEV production, but inhibits actin tail formation from the cell surface (Sanderson et al., 1998
; Wolffe et al., 1998
; Röttger et al., 1999
; van Eijl et al., 2000
). An interaction between the cytoplasmic domain of A36R and the N-terminal tetratricopeptide repeat region of the microtubule motor kinesin was demonstrated using the yeast two-hybrid system, and GST (glutathione S-transferase) pull-down assays (Ward & Moss, 2004
). This implies an additional function of A36R in the recruitment of kinesin to IEV. However, CEV is still produced in viruses lacking this gene (Wolffe et al., 1998
; Sanderson et al., 2000
; van Eijl et al., 2000
; Hollinshead et al., 2001
). Two previous reports concluded that IEV lacking A36R exhibited little (Ward & Moss, 2001a
) or no (Rietdorf et al., 2001
) movement.
Recombinant VACVs were described, in which the enhanced green fluorescent protein (EGFP) was fused to VACV core protein A5L (Carter et al., 2003), and the IEV proteins F13L (Ward & Moss, 2000
; Geada et al., 2001
; Husain & Moss, 2001
; Rietdorf et al., 2001
) and B5R (Hollinshead et al., 2001
; Ward & Moss, 2001b
; Rodger & Smith, 2002
). These viruses have been used to follow the movement of viral cores and whole virions in cells. To investigate the egress of VACV IEV, viruses lacking A36R or F12L (Parkinson & Smith, 1994
; Zhang et al., 2000
) were each labelled with EGFP on A5L to make v
A36R-EGFPA5L and v
F12L-EGFPA5L, respectively. In addition, v
A36R was labelled with EGFP fused to B5R (v
A36R-EGFPB5R) to investigate IEV localization and movement by confocal, and time-lapse fluorescent microscopy. Data presented here show that IEV lacking the A36R protein co-localize with microtubules, move at speeds consistent with microtubule transport and this movement is inhibited reversibly by microtubule-depolymerizing drugs. Therefore, IEV must attach to microtubule motors using at least one virally encoded protein other than A36R, and IEV are transported on microtubules without A36R.
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METHODS |
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Recombinant virus construction.
VACV strain Western Reserve (WR) and recombinant viruses derived from WR were used. VACV vA36R (Parkinson & Smith, 1994
), v
F12L (Zhang et al., 2000
), vEGFPB5R (Rodger & Smith, 2002
) and vEGFPA5L (Carter et al., 2003
) were described previously. Recombinant VACV v
A36R-EGFPA5L, v
F12L-EGFPA5L and v
A36R-EGFPB5R were constructed by transient dominant selection (Falkner & Moss, 1990
) using the Escherichia coli guanine xanthine phosphoribosyltransferase (Ecogpt)-encoding gene as the transiently selectable marker. CV-1 cells were infected with v
A36R or v
F12L at 0·05 p.f.u. per cell, and transfected 4 h later with pEGFPA5L (Carter et al., 2003
) or pEGFPB5R (Rodger & Smith, 2002
) in the presence of Lipofectin (Gibco-BRL), following the manufacturer's recommendations. Recombinant virus expressing Ecogpt was selected by three rounds of plaque purification in the presence of mycophenolic acid. The mycophenolic-acid-resistant virus was then plaque purified once on D98R cells in the presence of 6-thioguanine (Sigma) to select against virus expressing the Ecogpt gene, followed by four rounds of plaque purification on BS-C-1 cells to isolate a pure population of virus. Ecogpt-negative virus containing either EGFPA5L or EGFPB5R was identified by PCR, and stocks were prepared and titrated by plaque assay on BS-C-1 cells.
Genome analysis by PCR.
Genomes of VACV containing the EGFPA5L gene were analysed by PCR using oligonucleotide primers that either flanked the A5L gene (1, A5LFlankF 5'-CTCCGTTGAATTCGATGACTATAGGACAAGAACCCTCCTC-3' and 2, A5LFlankR 5'-CGTACTCCAAGCTTGTGTAGATGCTACTTCGTCGATGG-3'), amplified the EGFP gene (3, EGFPF 5'-GTGAGCAAGGGCGAG-3' and 4, EGFPR 5'-CTTGTACAGCTC-3'), or amplified EGFPA5L only (oligonucleotides 3 and 2). These reactions produced DNA fragments of 2·3, 0·7 and 2·0 kb, respectively (see Supplementary Fig. S1a available in JGV Online) for vA36R-EGFPA5L and v
F12L-EGFPA5L viruses, confirming that the EGFPA5L gene had been inserted into the A5L locus.
The genome of vA36R-EGFPB5R was analysed by PCR using oligonucleotide primers that either flanked the B5R gene (5, B5RFlankF 5'-TCATTTAAGCTTCCTTCTTTCGTGAAATGC-3' and 6, B5RFlankR 5'-GTACTCAAGCTTGCTTACAGAAACATCGCGTT-3'), amplified the short consensus repeat (SCR) domains 24 of B5R (7, SCR2-4F 5'-TGTGCACAGTTTCTGATTACAT-3' and 8, SCR2-4R 5'-TCGTACACATATTGGGAGTAC-3'), or amplified EGFPB5R only (oligonucleotides 3 and 6). These reactions gave DNA fragments of 1·7 and 1·6 kb using primers 5 and 6, and 3 and 6, respectively, but no product was obtained using primers 7 and 8 for the SCR 24 domains, confirming that the EGFPB5R gene had been inserted into the B5R locus, and that no wild-type B5R remained (see Supplementary Fig. S1b available in JGV Online).
Confocal microscopy.
To measure the formation of CEV, Ptk2 cells were seeded onto 22 mm glass coverslips, grown to 50 % confluence, infected with vEGFPA5L, vA36R-EGFPA5L or v
F12L-EGFPA5L at 1 p.f.u. per cell, and incubated for 0, 6, 8, 10 or 24 h at 37 °C. For the time-zero sample, the virus inoculum was added for 30 min on ice before processing for immunostaining and fixation. Live cells were stained on their surface with anti-B5R rat mAb 19C2 (diluted 1 : 50) for 1 h at room temperature, and were then blocked and permeabilized with 0·1 % saponin (Sigma). For fixation, cells were washed with ice-cold PBS, incubated with 4 % and then 8 % paraformaldehyde in 250 mM HEPES (10 min on ice and 20 min at room temperature, respectively), and quenched with 50 mM ammonium chloride for 10 min at room temperature. Bound antibody was detected with donkey anti-rat TRITC (tetramethylrhodamine B isothiocyanate)-conjugated antibodies (Jackson Immunoresearch Laboratories) diluted 1 : 250.
To investigate the effect of microtubule depolymerization on CEV production, cells were infected with 1 p.f.u. vEGFPA5L or vA36R-EGFPA5L per cell, and incubated for 1 h. Then 10 µg N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH) ml1 (kindly provided by Riccardo Wittek, University of Lausanne, Lausanne, Switzerland) was added for 11 h. At 12 h post-infection (p.i.) IMCBH was washed away and replaced with medium containing either fresh IMCBH (10 µg ml1), nocodazole (33 µM), colchicine (250 µM) or cytochalasin D (1 µM), and incubated for 90 min at 37 °C. At 12·5 h p.i., CEV were identified as above. To improve the accuracy of CEV quantification, images of x6·5 zoomed areas of the cell surface from the x63 magnified optical field were acquired for each cell, and the number of CEV in this area was counted.
To study the localization of vEGFPB5R and vA36R-EGFPB5R virions, Ptk2 cells were infected with 1 p.f.u. per cell for 12 h, and fixed and permeabilized as above. Mouse mAb B-5-1-2 (diluted 1 : 2000; Abcam) that detects
-tubulin was added for 1 h at room temperature, and bound antibodies were detected with donkey anti-mouse TRITC-conjugated antibodies (diluted 1 : 250). All samples were mounted in Vectashield (Vectorlabs) containing DAPI (4,6-diamidino-2-phenylindole), and were analysed using a Zeiss 510 Meta or LSM5 Pascal confocal microscope.
Time-lapse microscopy.
Ptk2 cells were grown to 3040 % confluence on 38 mm coverslips (PeCon), and were infected with 5 p.f.u. vEGFPB5R or vA36R-EGFPB5R per cell for 10 h. The coverslips were then transferred to a microscope stage preheated to 37 °C and the cells were observed on a Zeiss 510 Meta confocal microscope using Zeiss time-lapse software. Images were acquired at 0·98 s intervals for 180250 frames at 1014 h p.i.
Statistical analysis.
Student's t-test was used to test for the significance of the results (P<0·05, unless stated otherwise).
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RESULTS |
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Location of virions in WT, vA36R- and v
F12L-infected cells
Previous reports had indicated that the location of virions in cells infected by viruses lacking A36R or F12L was qualitatively different (van Eijl et al., 2000, 2002
). This was reassessed in cells infected with viruses lacking these genes and expressing EGFP fused to A5L. Fig. 1
shows that at 10 h p.i. the distribution of virions in cells infected with these viruses differ. F12L-negative virions are not widely dispersed in the cell, whereas the A36R-negative virions are distributed throughout the cell similar to vEGFPA5L (Fig. 1
). To test whether CEV is formed in the absence of F12L or A36R, live cells were stained with anti-B5R mAb, and the B5R signal was merged with the direct fluorescence from EGFP (Fig. 1
merged images). Double-positive structures represent cell-surface virions, CEVs. These data show that without A36R, virions are transported to the cell periphery and CEVs are present on the cell surface, whereas without F12L, virions remain in the central region of the cell and are not widely dispersed. F12L-negative IEV also failed to disperse even at later time points (data not shown). This observation does not fit with previous reports that without A36R IEV particles show little or no movement within infected cells (Rietdorf et al., 2001
; Ward & Moss, 2001a
).
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The relative locations of microtubules and IEV were examined in Ptk2 cells infected with vA36R-EGFPB5R and vEGFPB5R (Fig. 4
) using confocal microscopy. Microtubules were stained with a mAb to
-tubulin, and IEV was identified by virtue of its EGFP fluorescence. When
-tubulin and EGFP images were merged, the vast majority of IEV virions from both virus infections were coincident with microtubules. To quantify the proportions of IEV coincident with
-tubulin for each virus, and to determine whether these proportions differed between the viruses, images from 10 cells for each virus were acquired as z sections (0·36 µm slices) and a 300 µm2 area was analysed for each cell. Overall, 155/167 (94 %) and 157/159 (97 %) of IEV for vEGFPB5R- and v
A36R-EGFPB5R-infected cells, respectively, was coincident with
-tubulin. There was no statistically significant difference between the two viruses (P 0·17, t-test). A similar result was obtained using BS-C-1 cells (data not shown). These data show that IEV are associated with microtubules in the absence of A36R.
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DISCUSSION |
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Analysis of the time at which CEVs were present on the cell surface was followed after infection with viruses that lacked A36R or F12L, and compared with wild type. Without F12L, CEV production was reduced by >99 % at all time points tested, whereas without A36R, 65 and 54 % fewer CEV were found than in the control at 8 and 10 h p.i., but by 24 h p.i. this difference was reduced to only 38 % and was not significantly different. Only at the earliest time measured (6 h) was the production of CEV reduced greatly (>90 %) compared to the control. These data show that vA36R IEV egress was delayed compared to wild-type, but that the production of CEV did not require A36R.
The next question addressed was whether IEV egress in the absence of A36R required microtubules. It was found that CEV formation was inhibited by microtubule-depolymerizing drugs both in the presence and absence of the A36R protein. This indicated that microtubules are needed for IEV egress in the absence of the A36R protein.
The relative locations of microtubules and IEV were then examined using viruses with EGFP fused to B5R with or without A36R. IEV particles were visualized by virtue of their EGFP fluorescence, and microtubules were stained with anti--tubulin mAb. When the EGFP and
-tubulin images were merged, the vast majority of IEV particles from both viruses were coincident with microtubules in all cells examined (Fig. 4
), providing evidence for co-localization of IEV particles and microtubules in the absence of A36R.
The movement of IEV made by viruses that did or did not express A36R (vEGFPB5R and vA36R-EGFPB5R) was followed by time-lapse confocal microscopy from 10 to 14 h p.i. IEV from each virus were found to move at speeds consistent with movement on microtubules. However, the mean length of time of uninterrupted movement was significantly shorter for v
A36R-EGFPB5R IEV (Fig. 5
). Either this shorter movement, or the fact that CEV appearance was delayed without A36R, might explain why IEV movement was not found (Rietdorf et al., 2001
) or appeared greatly restricted (Ward & Moss, 2001a
) in earlier reports.
In summary, data are presented that show that the A36R protein is not required for transport of IEV to the cell surface, and that this transport is dependent on microtubules. This indicates that another protein(s) enables the interaction of IEV particles with microtubules. A candidate for this is the F12L protein, without which IEVs are not transported to the cell periphery and CEVs are not formed.
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ACKNOWLEDGEMENTS |
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Received 13 June 2005;
accepted 21 July 2005.
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