1 Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK
2 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
Correspondence
Geoffrey L. Smith
(at Imperial College)
glsmith{at}imperial.ac.uk
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ABSTRACT |
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Present address: Division of Virology, IBLS, University of Glasgow, Church Street, Glasgow G11 5JR, UK.
Present address: Dorfstrasse 28, 64720 Michelstadt, Germany.
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INTRODUCTION |
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The transport of a large virus such as VV with dimensions of 250 by 350 nm would be an inefficient process in the cytosol without specific transport mechanisms and it has been estimated that diffusion of VV across 10 µm of cytosol would take 5·7 h (Sodeik, 2000). Therefore, VV has evolved to exploit host cell transport machinery. IMV particles are transported on microtubules from virus factories to near the MTOC. This process requires the A27L protein, because if expression of the A27L gene is repressed, IMV are not transported away from factories (Sanderson et al., 2000
). Once IMV are wrapped to form IEV, the IEV particles are transported on microtubules to the cell surface (Geada et al., 2001
; Hollinshead et al., 2001
; Rietdorf et al., 2001
; Ward & Moss, 2001b
). Two VV proteins have been implicated in this transport: F12L and A36R. In the absence of F12L, IEV are formed but not transported to the cell surface (Zhang et al., 2000
; van Eijl et al., 2002
). In the absence of A36R, both IEV and CEV are formed (Sanderson et al., 1998
; Wolffe et al., 1998
; van Eijl et al., 2000
, 2002
; Hollinshead et al., 2001
), indicating virion transport, although it has been reported that the A36R protein affects IEV transport (Rietdorf et al., 2001
). However, the A36R protein is not essential for transport because viruses lacking A36R and with mutations in either the A33R or B5R proteins release enhanced levels of EEV (Katz et al., 2002
).
Once CEV are formed on the cell surface, actin filaments polymerize on the cytosolic face of the plasma membrane beneath CEV particles (van Eijl et al., 2000; Hollinshead et al., 2001
; Ward & Moss, 2001b
). Originally, these actin tails were proposed to form on IEV particles (Cudmore et al., 1995
, 1996a
; Frischknecht et al., 1999
) but they appear only after CEV formation and their role is to drive the CEV particles into surrounding cells. The A36R protein is essential for actin tail formation (Sanderson et al., 1998
; Wolffe et al., 1998
; Frischknecht et al., 1999
), although mutations in other EEV proteins (A33R, A34R, F13L and B5R) also cause a reduction in actin tail formation; for a recent review see Smith et al. (2002)
. The importance of actin tail formation in cell-to-cell spread was illustrated by a side-by-side comparison of mutants lacking each EEV or IEV specific protein (Law et al., 2002
). In all cases where the production of actin tails was inhibited the virus had a small plaque phenotype.
The binding and entry of VV into uninfected cells have been controversial and different models have been proposed. When considering VV entry it is necessary to define which form of virus is being described. The IMV and EEV forms are both infectious, although the EEV form has a higher specific infectivity (Vanderplasschen & Smith, 1997), but these virions are surrounded by different numbers of membranes and so they face different problems during entry. The number of membranes around an IMV particle has been disputed; some authors have claimed a single membrane (Dales & Siminovitch, 1961
; Morgan, 1976
; Hollinshead et al., 1999
) whereas others have claimed at least two (Sodeik et al., 1993
; Griffiths et al., 2001a
, b
; Risco et al., 2002
). If IMV has a single membrane then EEV has two; if IMV has two then EEV has three. Before transcription of the virus genome can commence in the cytosol all membranes surrounding the core must be shed or permeabilized. Previously, several IMV proteins were reported to be lost during virus entry (Vanderplasschen et al., 1998
; Pedersen et al., 2000
) and some cores were found to co-localize with microtubules (Mallardo et al., 2001
).
Virus replication occurs in factories in the perinuclear region of the cytoplasm. The transport of virus cores to this site is addressed here. Several recombinant VVs have been described in which the green fluorescent protein (GFP) or enhanced GFP (EGFP) was fused to a protein in the EEV envelope. Either the B5R (Hollinshead et al., 2001; Ward & Moss, 2001a
; Rodger & Smith, 2002
) or F13L (Ward & Moss, 2000
; Geada et al., 2001
; Husain & Moss, 2001
; Rietdorf et al., 2001
) proteins have been used. These viruses have been used to follow the location and movement of individual proteins or whole virions in the cell. To investigate the movement of virus cores, it was necessary to have EGFP fused to a core protein. We selected gene A5L from VV strain Western Reserve (WR) for this purpose. This gene is called A4L in VV Copenhagen (Goebel et al., 1990
) and the gene product has also been called p39 (Maa & Esteban, 1987
; Demkowicz et al., 1992
; Cudmore et al., 1996b
). A5L is present on the surface of the core of IMV (Cudmore et al., 1996b
) and of cores released into the cytosol during infection (Pedersen et al., 2000
). The protein is essential for the formation of infectious IMV (Williams et al., 1999
). Despite the requirement for A5L, we were able to replace the wild-type A5L gene with A5L fused to EGFP at either terminus. IMV derived from vA5L-EGFP-N was used to follow the movement of cores after infection of new cells by using time-lapse fluorescent microscopy. Data presented show that cores move inward from the cell periphery using microtubules. Thus VV uses microtubules for multiple stages of its life-cycle.
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METHODS |
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Plasmid construction.
Fusion of EGFP to the N or C terminus of A5L was achieved by PCR splicing by overlap extension (Horton et al., 1989) using VV strain WR genomic DNA as template. For the C-terminal fusion, oligonucleotides (1) 5'-CTCCGTTGAATTCGATGACTATAGGACAAGAACCCTCCTC-3' and (2) 5'-CAGCTCCTCGCCCTTGCTCACCTTTTGGAATCGTTCAAAACC-3' were used to generate a 1303 bp fragment containing the A5L ORF and 433 bp upstream. Oligonucleotides (3) 5'-GGCATGGACGAGCTGTACAAGTAATAAGATTGGATATTAAAATCACGCTTTCGAG-3' and (4) 5'-CGTACTCCAAGCTTGTGTAGATGCTACTTCGTCGATGG were used to generate a PCR fragment 375 bp downstream of the A5L ORF. The EGFP ORF was amplified using pEGFPC1 (Clontech) as template and oligonucleotides (5) 5'-GTGAGCAAGGGCGAG-3' and (6) 5'-CTTGTACAGCTC-3' generating a 714 bp fragment. Oligonucleotides (1) and (4) introduced EcoRI and HindIII restriction sites, respectively (underlined), while oligonucleotides (2) and (3) contained EGFP sequences enabling the individual fragments to be assembled into a single 2392 bp gene. This was digested with EcoRI and HindIII and cloned into EcoRI- and HindIII-digested pSJH7 (Hughes et al., 1991
) to form pA5L-EGFP-C.
For the N-terminal fusion, oligonucleotides (1) and (7) 5'-CAGCTCCTCGCCCTTGCTCACCATTTAAGGCTTTAAAATTGAATTGCG-3' were used to generate a fragment encoding 463 bp upstream of the A5L ORF. Oligonucleotides (8) 5'-GGCATGGACGAGCTGTACAAGGACTTCTTTAACAAGTTCTCACAGGGG-3' and (4) were used to generate a 1215 bp fragment containing the A5L ORF and 346 bp downstream of the A5L ORF. The EGFP ORF was amplified as described above. Oligonucleotides (7) and (8) contained EGFP sequences enabling the individual fragments to be assembled into a single 2392 bp gene. This was digested with EcoRI and HindIII and cloned into EcoRI- and HindIII-digested pSJH7 to form pA5L-EGFP-N.
Revertant viruses, wherein the WT A5L gene was reinserted into vA5L-EGFP-C and vA5L-EGFP-N to replace the EGFP-fusion sequences, were constructed using a plasmid containing the WT A5L gene. This gene was amplified by oligonucleotides (1) and (4) and cloned into pSJH7 to form pA5L-rev. The fidelity of all cloned PCR products was confirmed by sequencing.
Recombinant virus construction.
vA5L-EGFP and vA5L-EGFP-REV were constructed by transient dominant selection (Falkner & Moss, 1990) using the E. coli guanine : xanthine phosphoribosyltransferase (Ecogpt) gene. CV-1 cells were infected with VV WR at 0·1 p.f.u. per cell and transfected with either pA5L-EGFP-C or pA5L-EGFP-N using Lipofectin (Gibco BRL). For vA5L-EGFP-C-rev and vA5L-EGFP-N-rev, CV-1 cells were infected with either vA5L-EGFP-C or vA5L-EGFP-N and transfected with pA5L-rev. Recombinant virus expressing Ecogpt was selected by three rounds of plaque purification on BS-C-1 cells in the presence of mycophenolic acid (MPA). The MPA-resistant virus was then plaque-purified three times on D98R cells with 6-thioguanine to select against virus expressing Ecogpt. Ecogpt-negative viruses containing A5L-EGFP-C/N or A5L-EGFP-C/N-rev were identified by PCR and stocks were prepared and titrated by plaque assay on BS-C-1 cells.
Immunoblotting.
IMV was purified from infected RK13 cells by sucrose density-gradient centrifugation as described (Mackett et al., 1985). Proteins were analysed by SDS-PAGE and immunoblotting (Towbin et al., 1979
). A5L, EGFP and D8L were detected by rabbit
-core antibody (Cudmore et al., 1996b
; Vanderplasschen et al., 1998
) (diluted 1 : 2000), mouse mAb JL-8 (Clontech) (diluted 1 : 1000) and mouse mAb AB1.1 (Parkinson & Smith, 1994
; Vanderplasschen et al., 1998
) (diluted 1 : 1000), respectively. Specific signals were visualized using the ECL Western blotting detection kit (Amersham).
Electron microscopy.
RK13 cells were infected at 1 p.f.u. per cell for 8 h and were processed for thin-section transmission microscopy as described (Hollinshead et al., 1999; Krauss et al., 2002
). For immunoelectron microscopy, ultrathin cryosections were labelled with anti-GFP (Clontech) (diluted 1 : 10). All digital images were captured with the integrated SIS image analysis package and processed using Adobe Photoshop software.
Confocal microscopy.
Cells on glass coverslips were infected and at the indicated times thereafter were processed as described previously (Hollinshead et al., 1999). Tetramethylrhodamine B isothiocyanate (TRITC)phalloidin (Sigma) was used to stain F-actin and 4,6-diamine-2-phenylindole (DAPI) was added to the mounting medium to stain DNA.
For co-localization of VV cores and microtubules, PtK2 cells on glass coverslips were infected with vA5L-EGFP-N at 0·25 p.f.u. per cell for 1 h at 37 °C. Cells were fixed with PFA at 37 °C for 10 min and permeabilized with either 0·1 % saponin or 0·1 % Triton X-100. MAbs AB1.1 (mouse, 1 : 500) and YL1/2 (rat, 1 : 1000), which detect the VV D8L protein and -tubulin, respectively, were added for 1 h at room temperature. Bound antibodies were detected using donkey anti-mouse Cy5-conjugated and donkey anti-rat TRITC-conjugated antibodies (Jackson Immunoresearch Laboratories) diluted 1 : 100 or 1 : 400, respectively. Coverslips were mounted in Mowiol containing DAPI and were analysed using a Zeiss LSM 510 Meta confocal microscope. Images were collected and processed using LSM 510 and Adobe Photoshop software.
Time-lapse microscopy.
PtK2 cells grown to 50 % confluence on coverslips (PeCon) were infected at 0·25 p.f.u. per cell on ice for 30 min with purified vA5L-EGFP-N IMV. Where applicable, 33 µM nocodazole (Calbiochem) or 250 µM colchicine (Sigma) was added at 37 °C for 30 min before infection, or 1 µM cytochalasin D (Calbiochem) was added during infection. After binding, cells were washed with ice-cold medium and transferred to a microscope stage preheated to 37 °C. Cells were observed on the Zeiss 510 Meta confocal microscope using Zeiss time-lapse software. Images were acquired at 3 s intervals for 100 frames at different times between 0 and 90 min post-heating.
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RESULTS |
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Growth properties of recombinant viruses
The isolation of each recombinant virus indicated that the fusion of EGFP to the A5L protein did not prevent virus replication. To determine if there was a growth difference between WR, A5L-EGFP-N, vA5L-EGFP-C and the revertant viruses, the yield of infectious intracellular and extracellular virus was investigated 24 h post-infection (p. i.) of BS-C-1 cells infected at 10 p.f.u. per cell. Intracellular and extracellular vA5L-EGFP-N and vA5L-EGFP-C were each reduced about 3-fold compared to WR (data not shown). Similarly, the proportion of virus released into the supernatant that was resistant to neutralization by mAb 2D5, which neutralizes IMV (Ichihashi, 1996), was indistinguishable between these viruses and WR (data not shown).
The plaque phenotype of the WR, vA5L-EGFP-C, vA5L-EGFP-N and revertant viruses was investigated under liquid or semi-solid overlay in BS-C-1 cells (Fig. 1a). Under semi-solid medium, the plaques formed by vA5L-EGFP-C were slightly smaller than WR and the revertant control, whereas plaques formed by vA5L-EGFP-N were similar to WR and revertant. With a liquid overlay virus can spread as EEV and form comets. Under these conditions, WR and the revertant viruses formed comets, but vA5L-EGFP-N and vA5L-EGFP-C did not. This result was consistent with the 3-fold reduction in EEV produced by the latter viruses.
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Location of EGFP-A5L fusion proteins in infected cells
Fig. 1(b) suggested that the A5L-EGFP-C and A5L-EGFP-N proteins were incorporated into VV particles. This was investigated further by confocal microscopy to analyse the distribution of these fusion proteins in infected cells (Fig. 2
). In cells infected with vA5L-EGFP-C, direct visualization of EGFP showed punctate structures distributed throughout the cell (panel a). If the cell was also stained with DAPI for DNA (panel b) and the image merged (inset), it was evident that these EGFP-positive structures contained DNA and therefore are very likely to be virions. Analysis with vA5L-EGFP-N gave similar data (data not shown). In addition, to investigate if cells infected with vA5L-EGFP-N formed actin tails, cells were stained with phalloidin (panel d) and merged with an image of EGFP fluorescence (panel c). Panel (d) shows the presence of many actin tails and the inset shows a merged image demonstrating that the actin tails each have a virus particle at their tip.
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Movement of EGFP-cores observed by time-lapse microscopy
Purified IMV from vA5L-EGFP-N was used to investigate how virus cores move within a newly infected cell. IMV was bound on ice to PtK2 cells, which have a large flat morphology and so are useful for time-lapse microscopy. After binding, cells were warmed to 37 °C and analysed at different times post-warming by confocal microscopy with a series of 100 images captured every 3 s. A representative experiment is shown in Fig. 6. In panel (a), the direct fluorescence deriving from EGFP in a single focal plane at all 100 time-points is shown. Part of this image is shown at higher magnification in panel (b). It is evident that many of the virus particles exhibit little movement as their images remain reasonably stationary over the 300 s period. This may be because they have already entered and moved, or they may not have entered the cell and may move at a later stage. However, several particles showed clear movement. This is illustrated in the remaining images. Arrowheads in panel (c) show the position of a single particle between frames 13 to 22. In panels (e), (g), (h), (f) and (d) the position of this particle at 6 s intervals is illustrated by an arrowhead. Particles were seen moving in a saltatory manner both towards and away from the perinuclear region. Time-lapse analyses of multiple particles (n=20) showed particles moved with speeds ranging from 27 to 80 µm min-1 (average 51·8 µm min-1, SEM±3·9 µm min-1). These types of movement and speeds are consistent with movement on microtubules.
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DISCUSSION |
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Previously, recombinant VVs were described in which the F13L or B5R proteins were fused to GFP or EGFP and these viruses were useful for studying the movement of IEV particles during virus egress. However, these viruses could not be used for studying movement of IMV particles during morphogenesis, or virus cores during re-infection, because the virus particles only acquired the EGFP-tagged protein after formation of IEV during morphogenesis and the EGFP tag was lost during re-entry because it was present in the EEV outer envelope. To overcome this problem we sought a core protein that might be tagged with EGFP without inhibiting function and selected the A5L gene for study. Although this gene is essential for virus morphogenesis and the morphogenesis of a mutant virus in which expression of the gene is repressed arrests prior to the formation of IMV, we were able to replace the wild-type A5L gene with A5L fused to EGFP at either the N or C terminus and recover infectious virus. Moreover, immunoblotting of proteins extracted from purified IMV showed the protein was present in IMV particles, and cryo-immunoelectron microscopy showed that the A5L-EGFP fusion proteins were incorporated into the virus core. These observations suggest that interactions of the A5L protein via either the N or C terminus is not critical for functions such as packaging into virions or interaction with other proteins.
Electron microscopy showed that recombinant viruses expressing the A5L-EGFP fusion proteins underwent normal morphogenesis and induced the formation of virus-tipped actin tails at the cell surface. Consistent with this observation, the plaque size of the recombinant viruses was similar to or indistinguishable from wild-type and revertant virus controls. The virus with the EGFP fused to the C terminus formed a slightly smaller plaque than the wild-type.
Immunofluorescent microscopy showed that IMV particles bound to the cell surface retained the D8L IMV surface protein, but were not recognized by the anti-core antibody. Conversely, particles that had entered the cell were recognized by core antibody but not antibody to D8L. Both observations are in accord with a previous report (Vanderplasschen et al., 1998) and are relevant to the entry mechanism for IMV. It was proposed that IMV uncoats outside the cell followed by transport of cores across the cell membrane without membrane fusion (Krijnse Locker et al., 2000
). The failure to detect cores on the cell surface is inconsistent with this proposal, unless cores somehow remain inaccessible to anti-core antibody once released from the IMV membrane. Particles that had entered the cell moved from the cell periphery towards the cell interior and co-localized with
-tubulin. This co-localization of virus cores with microtubules is consistent with a previous report (Mallardo et al., 2001
) that also noted co-localization of virus mRNAs and microtubules.
The movement of cores derived from IMV was followed by time-lapse confocal microscopy after infection of new cells, and cores were found to move with speeds consistent with transport on microtubules. Moreover, addition of either colchicine or nocodazole inhibited core movement, and the washout of nocodazole restored core movement. These observations too are consistent with movement on microtubules. In contrast, addition of cytochalasin D, an inhibitor of actin function, did not prevent movement of cores. However, cytochalasin D did reduce the number of cores (25 % less) moving within cells, although the rate of movement was not altered. This reduction in intracellular cores in the presence of cytochalasin D is less than reported previously (81 %) (Vanderplasschen et al., 1998) because of the lower drug concentration and shorter incubation period with drug in PtK2 cells. The role of actin in IMV entry reported by Vanderplasschen et al. (1998)
was later confirmed and extended by Krijnse Locker et al. (2000)
who also reported the formation of cell surface projections upon binding of IMV to cells.
The use of microtubules for movement of (i) IMV particles from virus factories to sites of wrapping to form IEV, (ii) IEV particles to the cell surface and (iii) virus cores from the periphery to the cell interior illustrates how VV exploits the cell transport processes for its intracellular transport. The proteins that interact with microtubules are not defined but are likely to be different between IMV, IEV and cores. Candidate proteins include the IMV surface protein A27L and the IEV surface protein F12L. In the absence of A27L IMV are formed but not transported (Rodriguez & Smith, 1990); similarly, in the absence of F12L IEV are formed but not transported (van Eijl et al., 2002
). The A36R protein has also been reported to have a role in movement of IEV particles on microtubules (Rietdorf et al., 2001
) but EEV and CEV are still formed by viruses lacking A36R (Introduction). For incoming virus cores, the A27L and F12L proteins are absent and so other VV proteins are likely to be involved. In vitro microtubule-binding assays have suggested an interaction between L4R and A10L and microtubules (Ploubidou et al., 2000
), but these proteins are considered to be within the core rather than on its surface, and so it is uncertain how such an interaction would be mediated.
Lastly, VV is another virus shown to use microtubules during virus entry. Others include herpes simplex virus (Sodeik et al., 1997), adenovirus (Suomalainen et al., 1999
) and HIV-1 (McDonald et al., 2002
). It is probable that many viruses including those described to date (Sodeik, 2000
) will utilize aspects of the cell transport machinery to facilitate entry of incoming cores or capsids.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cudmore, S., Cossart, P., Griffiths, G. & Way, M. (1995). Actin-based motility of vaccinia virus. Nature 378, 636638.[CrossRef][Medline]
Cudmore, S., Reckmann, I., Griffiths, G. & Way, M. (1996a). Vaccinia virus: a model system for actinmembrane interactions. J Cell Sci 109, 17391747.
Cudmore, S., Blasco, R., Vincentelli, R., Esteban, M., Sodeik, B., Griffiths, G. & Krijnse Locker, J. (1996b). A vaccinia virus core protein, p39, is membrane associated. J Virol 70, 69096921.[Abstract]
Dales, S. & Siminovitch, L. (1961). The development of vaccinia virus in Earles L strain cells as examined by electron microscopy. J Biophys Biochem Cytol 10, 475503.
Demkowicz, W. E., Maa, J. S. & Esteban, M. (1992). Identification and characterization of vaccinia virus genes encoding proteins that are highly antigenic in animals and are immunodominant in vaccinated humans. J Virol 66, 386398.[Abstract]
Falkner, F. G. & Moss, B. (1990). Transient dominant selection of recombinant vaccinia viruses. J Virol 64, 31083111.[Medline]
Frischknecht, F., Moreau, V., Röttger, S., Gonfloni, S., Rechmann, I., Superti-Furga, G. & Way, M. (1999). Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. Nature 401, 926929.[CrossRef][Medline]
Geada, M. M., Galindo, I., Lorenzo, M. M., Perdiguero, B. & Blasco, R. (2001). Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein. J Gen Virol 82, 27472760.
Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. P. & Paoletti, E. (1990). The complete DNA sequence of vaccinia virus. Virology 179, 247266.[Medline]
Griffiths, G., Roos, N., Schleich, S. & Krijnse Locker, J. (2001a). Structure and assembly of intracellular mature vaccinia virus: thin-section analyses. J Virol 75, 1105611070.
Griffiths, G., Wepf, R., Wendt, T., Krijnse Locker, J., Cyrklaff, M. & Roos, N. (2001b). Structure and assembly of intracellular mature vaccinia virus: isolated-particle analysis. J Virol 75, 1103411055.
Hollinshead, M., Vanderplasschen, A., Smith, G. L. & Vaux, D. J. (1999). Vaccinia virus intracellular mature virions contain only one lipid membrane. J Virol 73, 15031517.
Hollinshead, M., Rodger, G., van Eijl, H., Hollinshead, R., Law, M., Vaux, D. T. & Smith, G. L. (2001). Vaccinia virus utilizes microtubules for movement to the cell surface. J Cell Biol 154, 389402.
Horton, R. M., Cai, Z. L., Ho, S. N. & Pease, L. R. (1989). Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8, 528535.
Hughes, S. J., Johnston, L. H., de Carlos, A. & Smith, G. L. (1991). Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae. J Biol Chem 266, 2010320109.
Husain, M. & Moss, B. (2001). Vaccinia virus F13L protein with a conserved phospholipase catalytic motif induces colocalization of the B5R envelope glycoprotein in post-Golgi vesicles. J Virol 75, 75287542.
Ichihashi, Y. (1996). Extracellular enveloped vaccinia virus escapes neutralization. Virology 217, 478485.[CrossRef][Medline]
Katz, E., Wolffe, E. & Moss, B. (2002). Identification of second-site mutations that enhance release and spread of vaccinia virus. J Virol 76, 1163711644.
Krauss, O., Hollinshead, R., Hollinshead, M. & Smith, G. L. (2002). An investigation of the incorporation of cellular antigens in vaccinia virus particles. J Gen Virol 83, 23472359.
Krijnse Locker, J., Kuehn, A., Schleich, S., Rutter, G., Hohenberg, H., Wepf, R. & Griffiths, G. (2000). Entry of the two infectious forms of vaccinia virus at the plasma membrane is signaling-dependent for the IMV but not the EEV. Mol Biol Cell 11, 24972511.
Law, M., Hollinshead, R. & Smith, G. L. (2002). Antibody-sensitive and antibody-resistant cell-to-cell spread of vaccinia virus: role of the A33R protein in antibody-resistant spread. J Gen Virol 83, 209222.
Maa, J. S. & Esteban, M. (1987). Structural and functional studies of a 39,000-Mr immunodominant protein of vaccinia virus. J Virol 61, 39103919.[Medline]
Mackett, M., Smith, G. L. & Moss, B. (1985). The construction and characterization of vaccinia virus recombinants expressing foreign genes. In DNA Cloning: a Practical Approach, pp. 191211. Edited by D. M. Glover. Oxford: IRL Press.
McDonald, D., Vodicka, M. A., Lucero, G., Svitkina, T. M., Borisy, G. G., Emerman, M. & Hope, T. J. (2002). Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159, 441452.
Mallardo, M., Schleich, S. & Krijnse Locker, J. (2001). Microtubule-dependent organization of vaccinia virus core-derived early mRNAs into distinct cytoplasmic structures. Mol Biol Cell 12, 38753891.
Morgan, C. (1976). Vaccinia virus reexamined: development and release. Virology 73, 4358.[Medline]
Moss, B. (2001). Poxviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 28492883. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & WIlkins.
Parkinson, J. E. & Smith, G. L. (1994). Vaccinia virus gene A36R encodes a Mr 4350 K protein on the surface of extracellular enveloped virus. Virology 204, 376390.[CrossRef][Medline]
Pedersen, K., Snijder, E. J., Schleich, S., Roos, N., Griffiths, G. & Krijnse Locker, J. (2000). Characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure. J Virol 74, 35253536.
Ploubidou, A., Moreau, V., Ashman, K., Reckmann, I., Gonzalez, C. & Way, M. (2000). Vaccinia virus infection disrupts microtubule organization and centrosome function. EMBO J 19, 39323944.
Rietdorf, J., Ploubidou, A., Reckmann, I., Holmstrom, A., Frischknecht, F., Zettl, M., Zimmermann, T. & Way, M. (2001). Kinesin-dependent movement on microtubules precedes actin-based motility of vaccinia virus. Nat Cell Biol 3, 9921000.[CrossRef][Medline]
Risco, C., Rodriguez, J. R., Lopez-Iglesias, C., Carrascosa, J. L., Esteban, M. & Rodriguez, D. (2002). Endoplasmic reticulum-Golgi intermediate compartment membranes and vimentin filaments participate in vaccinia virus assembly. J Virol 76, 18391855.
Rodger, G. & Smith, G. L. (2002). Replacing the SCR domains of vaccinia virus protein B5R with EGFP causes a reduction in plaque size and actin tail formation but enveloped virions are still transported to the cell surface. J Gen Virol 83, 323332.
Rodriguez, J. F. & Smith, G. L. (1990). IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress. Nucleic Acids Res 18, 53475351.[Abstract]
Sanderson, C. M., Frischknecht, F., Way, M., Hollinshead, M. & Smith, G. L. (1998). Roles of vaccinia virus EEV-specific proteins in intracellular actin tail formation and low pH-induced cellcell fusion. J Gen Virol 79, 14151425.[Abstract]
Sanderson, C. M., Hollinshead, M. & Smith, G. L. (2000). The vaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particles. J Gen Virol 81, 4758.
Smith, G. L., Vanderplasschen, A. & Law, M. (2002). The formation and function of extracellular enveloped vaccinia virus. J Gen Virol 83, 29152931.
Sodeik, B. (2000). Mechanisms of viral transport in the cytoplasm. Trends Microbiol 8, 465472.[CrossRef][Medline]
Sodeik, B., Doms, R. W., Ericsson, M., Hiller, G., Machamer, C. E., van't Hof, W., van Meer, G., Moss, B. & Griffiths, G. (1993). Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks. J Cell Biol 121, 521541.[Abstract]
Sodeik, B., Ebersold, M. W. & Helenius, A. (1997). Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol 136, 10071021.
Suomalainen, M., Nakano, M., Keller, S., Boucke, K., Stidwill, R. P. & Greber, U. F. (1999). Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus. J Cell Biol 144, 657672.
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 43504354.[Abstract]
Vanderplasschen, A. & Smith, G. L. (1997). A novel virus binding assay using confocal microscopy: demonstration that the intracellular and extracellular vaccinia virions bind to different cellular receptors. J Virol 71, 40324041.[Abstract]
Vanderplasschen, A., Hollinshead, M. & Smith, G. L. (1998). Intracellular and extracellular vaccinia virions enter cells by different mechanisms. J Gen Virol 79, 877887.[Abstract]
van Eijl, H., Hollinshead, M. & Smith, G. L. (2000). The vaccinia virus A36R protein is a type Ib membrane protein present on intracellular but not extracellular enveloped particles. Virology 271, 2636.[CrossRef][Medline]
van Eijl, H., Hollinshead, M., Rodger, G., Zhang, W.-H. & Smith, G. L. (2002). The vaccinia virus F12L is associated with intracellular enveloped virus particles and is required for their egress to the cell surface. J Gen Virol 83, 195207.
Ward, B. M. & Moss, B. (2000). Golgi network targeting and plasma membrane internalization signals in vaccinia virus B5R envelope protein. J Virol 74, 37713780.
Ward, B. M. & Moss, B. (2001a). Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera. J Virol 75, 48024813.
Ward, B. M. & Moss, B. (2001b). Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails. J Virol 75, 1165111663.
Williams, O., Wolffe, E. J., Weisberg, A. S. & Merchlinsky, M. (1999). Vaccinia virus WR gene A5L is required for morphogenesis of mature virions. J Virol 73, 45904599.
Wolffe, E. J., Weisberg, A. S. & Moss, B. (1998). Role for the vaccinia virus A36R outer envelope protein in the formation of virus-tipped actin-containing microvilli and cell-to-cell virus spread. Virology 244, 2026.[CrossRef][Medline]
Zhang, W.-H., Wilcock, D. & Smith, G. L. (2000). The vaccinia virus F12L protein is required for actin tail formation, normal plaque size and virulence. J Virol 74, 1166311670.
Received 3 April 2003;
accepted 6 June 2003.