1 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
2 Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK
Correspondence
Geoffrey L. Smith
(at Imperial College)
glsmith{at}ic.ac.uk
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ABSTRACT |
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Present address: Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia.
Present address: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20852, USA.
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INTRODUCTION |
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A feature of VV infection in cell culture is the induction of cytopathic effect (CPE). This CPE is the result of numerous changes within the cell and starts with the induction of actin-containing protrusions in the plasma membrane (Locker et al., 2000) upon intracellular mature virus (IMV) binding. After virus entry, microtubules transport viral cores (Carter et al., 2003
) and then early mRNAs (Mallardo et al., 2001
). With the onset of early gene expression, host cell macromolecular synthesis is shut down (Buller & Palumbo, 1991
). Also at early times post-infection (p.i.) cellcell dissociation and cell rounding begin (Bablanian et al., 1978
) and cell motility is enhanced (Sanderson et al., 1998
). Viral DNA replication and late gene expression bring further changes to the cytoskeleton: the normal aster configuration of microtubules is lost (Ploubidou et al., 2000
) and actin stress fibres are disrupted. Surface microvilli (Hiller et al., 1979
) and long cellular projections (Sanderson et al., 1998
; Ploubidou et al., 2000
) are produced as microtubules are organized into long, stable bundles. In addition, cell/extracellular matrix (ECM) adhesion changes from a Ca2+-dependent to a Ca2+-independent type, possibly mediated by integrins (Sanderson & Smith, 1998
).
The VV morphogenic pathway utilizes both microtubules and actin as it produces multiple forms of infectious progeny (for review see Smith et al., 2002). First IMV (Sanderson et al., 2000
) and then intracellular enveloped virus (IEV) (Hollinshead et al., 2001
; Rietdorf et al., 2001
; Ward & Moss, 2001
) are transported within cells on microtubules. Cell-associated enveloped virus (CEV) particles are propelled away from the infected cell by polymerization of actin (Cudmore et al., 1995
; van Eijl et al., 2002
) and are important for cell-to-cell spread since mutants deficient in their formation form small plaques (Law et al., 2002
; Rodger & Smith, 2002
) (and references therein). Extracellular enveloped virus (EEV) mediates long-range dissemination in vitro and in vivo (Appleyard et al., 1971
; Payne, 1980
; Smith et al., 2002
).
VV genes C2L, F3L and A55R are non-essential for growth in culture but otherwise remain uncharacterized (Kotwal & Moss, 1988; Perkus et al., 1991
). These genes are located either at the left (C2L and F3L) or right (A55R) ends of the genome, and whilst having low overall amino acid identity with each other, they share structural motifs (Fig. 1
a) and belong to the kelch superfamily of proteins.
|
VV WR gene C2L is conserved (>97 % amino acid similarity) in several orthopoxviruses, including VV strains Copenhagen, Tian Tan and Rabbitpox-Utrecht, Camelpox virus strains CM-S and M-96, Ectromelia virus strains Moscow and Naval.Cam and Cowpox virus strains GRI-90 and Brighton Red (sequences are available through www.poxvirus.org/data.asp). However, the C2L gene is absent from modified vaccinia Ankara (MVA) and only fragments of kelch genes are found in Variola virus (Shchelkunov et al., 2002). C2L has no closely related counterparts outside the Orthopoxvirus genus. Given this background, it seemed likely that the VV POZ-kelch proteins may have differing roles, and might affect cell function. To test this, a VV mutant lacking C2L was constructed and characterized. We report here that the C2L protein contributes to the formation of VV-induced cellular projections and the Ca2+-independent adhesion of cells to the ECM and also affects the outcome of infection in a murine dermal model.
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METHODS |
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Plasmid construction.
Plasmid pC2L was assembled as follows. Two DNA fragments that contained DNA from either the 5' or 3' ends of the C2L gene and flanking regions were amplified by PCR (using VV strain WR genome DNA as template). The 5' fragment was generated with oligonucleotides 5'-AAAGAATTCGATTCGTACAGAACAAGAGCC-3' (C2L2R), containing an EcoRI restriction site (underlined), and 5'-TTCTCTAGAGATAGAAAATATCACGCTTTCC-3' (C2L2F), containing an XbaI restriction site (underlined). The 3' fragment was amplified using oligonucleotides 5'-AAATCTAGACTACAATAGCGAACATATATGGGC-3' (C2L1R), containing an XbaI restriction site (underlined), and 5'-TTGAAGCTTGAGTGTCCATCCAGTACCTG-3' (C2L1F), containing a HindIII restriction site (underlined). The 3' fragment was digested with HindIII and XbaI and ligated into HindIII- and XbaI-cut pSJH7 (Hughes et al., 1991
) to form pSJH7A. The 5' fragment was digested with XbaI and EcoRI and ligated into XbaI- and EcoRI-cut pSJH7A to form p
C2L. This plasmid lacked 98 % of the C2L gene corresponding to the region between nucleotides 12258 and 10404 (codons 9510) (numbering according to Kotwal & Moss, 1988
). Plasmid pC2Lrev was used to construct the C2L revertant virus and was generated as follows. A complete version of the C2L gene plus 5' and 3' flanking regions was generated by PCR, using VV strain WR genome DNA as template, and oligonucleotide primers C2L1F and C2L2R. The PCR product was digested with HindIII and ligated into HindIII- and SmaI-cut pSJH7 to form pC2Lrev. The fidelity of the PCR-derived regions from all plasmids was verified by DNA sequencing. Compared to the published sequence (Kotwal & Moss, 1988
), there was one nucleotide difference in the C2L 5' flanking region (G to A at position -247 relative to the first nucleotide of the C2L gene). This change is within gene C1L, but did not alter its amino acid composition (glycine-171 encoded by GGA instead of GGG). This alteration was found in several clones obtained from independent PCRs.
Plasmid pGS61C2LFlag was used to insert the C2L gene with the Flag epitope (N-DYKDDDDK-C) attached to the 3' end into the VV thymidine kinase (TK) locus of the VV mutant lacking C2L. The C2L gene with a C-terminal Flag sequence was obtained by PCR using VV DNA as template and oligonucleotide 5'-TATGGATCCTCAAGAATGGAAAGCGTG-3' (C2LFlag1), containing a BamHI restriction site (underlined) and the C2L start codon (in bold), and oligonucleotide 5'-TTTAAGCTTCTActtgtcatcgtcgtccttgtagtcTTGTAGAAATTGTTTTTCACAGTTGC-3' (C2LFlag2), containing a HindIII restriction site (underlined), the stop codon (complement of) of the C2L gene (in bold) and the sequence for the Flag epitope (in lower-case). The PCR fragment was digested with BamHI and HindIII and ligated into BamHI- and HindIII-cut pSJH7 to form pSJH7C2LFlag. The PCR fragment was excised from pSJH7C2LFlag using BamHI and HindIII and cloned into pGS61 (Smith et al., 1987) that had been cut with the same enzymes, to form pGS61C2LFlag.
Recombinant virus construction.
A deletion mutant lacking 98 % of gene C2L was constructed by transient dominant selection (Falkner & Moss, 1990) using plasmid p
C2L and VV strain WR as described previously (Ng et al., 2001
). The deletion mutant, v
C2L, and a plaque-purified wild-type virus were obtained from the same intermediate virus in parallel. A revertant virus (vC2L-rev) in which the C2L locus was restored to wild-type was constructed by transfecting plasmid pC2Lrev into cells infected with v
C2L.
A C2LFlag gene was inserted into the TK locus of the vC2L using plasmid pGS61C2LFlag as described previously (Smith, 1995
). A recombinant virus (vC2LFlag) was distinguished from spontaneous TK- isolates by screening infected cell extracts by immunoblotting with an anti-Flag mAb (see below).
Immunoblotting.
Samples for immunoblotting were prepared and treated as described previously (Parkinson & Smith, 1994).
Virus growth curves.
For one-step growth curves, BS-C-1 cells were infected with 10 p.f.u. per cell for 90 min, washed with PBS to remove unbound virus and incubated in DMEM/2·5 % FBS. The culture supernatant was removed at 24 h p.i., centrifuged at 800 g at 4 °C for 10 min to pellet detached cells and the supernatant was retained as the EEV fraction. Cells were scraped into DMEM/2·5 % FBS, added to the pelleted cells from above, frozen and thawed three times, and sonicated to obtain the cell-associated virus. For multi-step growth curves, BS-C-1 cells were infected at 0·01 p.f.u. per cell as above. At the indicated times, cells were scraped into the medium, frozen and thawed three times and sonicated to obtain total virus. Virus titres were determined by plaque assay on duplicate BS-C-1 cell monolayers.
Immunocytochemistry.
Cells growing on glass coverslips (Cover glass, BDH) were infected with 1 or 5 p.f.u. per cell for 1 h on ice. After adsorption, non-adherent viruses were washed away and cells were incubated in DMEM/2·5 % FBS at 37 °C. At the indicated times p.i., cells were washed with PBS and with BRB80 buffer (80 mM PIPES pH 6·8, 1 mM MgCl2, 1 mM EGTA) before being fixed in 4 % paraformaldehyde (PFA) in BRB80 buffer for 20 min at room temperature. Cells were incubated for 15 min at room temperature with 20 mM glycine in PBS, permeabilized with 0·1 % Triton X-100 in PBS and blocked with PBS/10 % FBS. For indirect immunofluorescence mouse mAbs anti--tubulin (B-5-1-2, Sigma), anti-vinculin (hVIN-1, Sigma) and anti-paxillin (349, Transduction Laboratories) were diluted (1 : 100, 1 : 100 and 1 : 500, respectively) in PBS/10 % FBS and added to cells for 1 h at room temperature. Primary mAbs were detected with a donkey anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Jackson Laboratories, dilution 1/50) for 30 min at room temperature. For detection of C2LFlag, goat anti-DDDDK tag antibody (Abcam, Cambridge), which recognizes the Flag epitope, was diluted (1 : 1000) and added to fixed and permeabilized cells for 1 h at room temperature. Bound antibodies were detected by adding donkey anti-goat FITC-conjugated antibody (Jackson Laboratories, dilution 1/100) for 30 min at room temperature. Live staining was done as described previously (Bartlett et al., 2002
). For simultaneous staining of filamentous actin, tetramethylrhodamine B isothiocyanate (TRITC)phalloidin (Sigma, diluted 1 : 100) was added with the secondary antibody. Images were acquired with a Zeiss confocal laser-scanning microscope (LSM 510) and processed with Adobe Photoshop software.
Quantification of cellular projections.
BS-C-1 cells were seeded onto glass coverslips to give well-isolated cells, and were infected with 5 p.f.u. per cell. Cells were processed for immunocytochemistry as described above and stained with TRITCphalloidin. For each coverslip, 200 cells were analysed by fluorescent microscopy and scored positive for VV-induced cell projections when either two or more projections were observed, or a single ramified projection was observed.
Depletion of extracellular Ca2+ and quantification of Ca2+-independent adhesion.
Depletion of extracellular Ca2+ was performed as described (Sanderson & Smith, 1998). Confluent monolayers of BS-C-1 cells were mock-infected or infected with 3 p.f.u. per cell and incubated for 18 h at 37 °C. After three washes with PBS, cells were incubated with PBS/1 mM EGTA at room temperature. The morphology of cells was recorded by phase-contrast microscopy before and after depletion of extracellular Ca2+. Three random areas of the monolayer (
150 cells per field) were photographed under a 10x objective and the number of round or adherent cells was scored from projected images.
Mouse intradermal model.
Groups of 8- to 10-week old female BALB/c mice were injected intradermally in their left ear pinnae with 104 p.f.u. in 10 µl of PBS as described (Tscharke & Smith, 1999). The diameter of the lesions was estimated daily to the nearest 0·5 mm using a micrometer. The titres of infectious virus in the ears was determined by plaque assay using extracts from the ears prepared by grinding in a glass homogenizer, followed by three freezethaw cycles and sonication.
Analysis of cell populations in infected ears by fluorescence activated flow cytometry (FC).
Groups of 6- to 8-week old female C57BL/6 mice were injected intradermally in both ears with 104 p.f.u. At 14 days p.i. mice were sacrificed and both ears were removed, washed in 70 % ethanol and dried. The ventral and dorsal leaflets of each ear were separated and placed internal side down onto RPMI 1640 medium (Gibco) containing 50 IU of penicillin and streptomycin (Gibco), 10 % FBS and 2·5 mM HEPES, pH 7·4 in a plastic bacterial plate. Samples were incubated at 37 °C for 8 h to allow cells to migrate from the ear. Non-adherent cells were collected and adherent cells were washed with PBS and removed from the plate by incubation in PBS (Ca2+ and Mg2+-free) with 2 mg glucose ml-1 for 20 min at 37 °C. Adherent and non-adherent cells were pooled, washed once with RPMI/10 % FBS, once with Tris/NH4Cl buffer (0·14 M NH4Cl in 17 mM Tris, pH 7·2) and twice with FC buffer (0·1 % BSA, 0·1 % NaN3 in PBS). The number of viable cells was determined by trypan blue exclusion.
About 106 cells (corresponding to pooled cells from six ears per infection group) in FC buffer were incubated for 10 min on ice with 10 % rat serum and 0·5 µg rat anti-mouse CD16/CD36 (BD Pharmingen) to block Fc receptors on cells. Cells were then double or triple stained with rat mAbs conjugated to FITC, phycoerythrin (PE) or tricolor (TC), or incubated with the appropriate isotype controls, for 30 min on ice. Cells were washed with FC buffer and with PBS before being fixed in PBS/2 % PFA for 30 min at room temperature. Fixed cells were collected, resuspended in PBS and analysed in a fluorescence activated cell sorter (FACScalibur, Becton Dickinson) using CellQuest software.
The dermal cells were identified by characteristic size (forward scatter) and granularity (side scatter) combined with two-colour analysis. CD4+ and CD3+ T lymphocytes were identified by their small size, low granularity and bright CD3 (PE anti-CD3, Caltag Laboratories) and CD4 (TC-anti-CD4, Caltag Laboratories) or CD8 (FITCanti-CD8, Sigma) staining, respectively. Neutrophils were identified by their small size, high granularity and bright Ly6-G (PE anti-Ly6-G, Caltag Laboratories) staining. Macrophages and dendritic cells were identified by their large size and granularity, and bright F4/80 (PE-anti-F4/80, Serotech) or DEC205 (FITCanti-DEC205, Serotech) staining, respectively.
Statistical analysis.
Student's t-test (two tailed, unpaired) was used to test for the significance of the results.
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RESULTS |
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The location of the C2Lprotein was analysed by immunofluorescence (Fig. 1c). Cells were infected with vC2L-wt, v
C2L, or vC2LFlag or mock-infected and were processed for immunofluorescence. Cells infected with vC2LFlag showed a diffuse cytoplasmic fluorescence that was absent from cells infected with vC2L-wt or v
C2L or from mock-infected cells. If the anti-flag mAb was added to live cells infected with vC2LFlag no fluorescence was detected indicating that the C2L protein was intracellular (data not shown).
The C2L gene does not affect VV replication in vitro
To examine the function of C2L, a deletion mutant virus lacking 98 % of the C2L gene (vC2L), a plaque-purified wild-type virus (vC2L-wt) and a revertant virus (vC2L-rev) were constructed (see Methods). The virus genomes were analysed by restriction digestion, PCR and Southern blotting, using DNA extracted from virus cores (Esposito et al., 1981
). These analyses confirmed the deletion of C2L in v
C2L and the absence of gross genomic alterations or changes in the regions surrounding C2L in the recombinant viruses (data not shown).
The isolation of vC2L confirmed previous observations (Kotwal & Moss, 1988
) that the C2L gene is non-essential for virus replication in vitro. Nevertheless, it was possible that there were differences in virus replication and therefore the growth properties of v
C2L were analysed. After infection of BS-C-1 cells at 10 p.f.u. per cell for 24 h, the virus yields of the vC2L-wt, v
C2L and vC2L-rev in the cell or supernatant [see supplementary data (a) at JGV Online: http://vir.sgmjournals.org)] were indistinguishable. Similarly, there were no differences in the virus yields after infection of BS-C-1 cells at 0·01 p.f.u. per cell [supplementary data (b)].
Plaque phenotype
Although VV replication in vitro was unaffected by loss of C2L, it was evident that the plaques formed by vC2L in BS-C-1 cells had a different morphology to those formed by vC2L-wt and vC2L-rev (Fig. 2
). The plaques formed by v
C2L in BS-C-1 cells (Fig. 2b
) were similar to those of VV strain WR mutant 6/2 (Fig. 2d
), which has a large deletion in the left end of the genome including C2L (Kotwal & Moss, 1988
). Despite the fact that plaques from v
C2L appeared smaller than those from the control viruses, measurements of the diameter of the plaques from the outer edge of virus-induced CPE showed there were no differences (Fig. 2a
c). The different plaque morphology of v
C2L compared to controls is seen more clearly under higher magnification of the edge (Fig. 2e, f, g
) or centre (Fig. 2h, i, j
) of plaques.
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DISCUSSION |
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Previously, it was suggested that the transition to Ca2+-independent adhesion might be necessary for the formation of cellular projections because: (i) the Ca2+-independent adhesion phenotype precedes projection formation; (ii) VV-induced projections adhere to the ECM in a Ca2+-independent manner and their formation was supported by the same ECM components that support the Ca2+-independent adhesion; and (iii) both phenotypes required late gene expression (Sanderson & Smith, 1998). Therefore, the disruption of C2L could have affected the Ca2+-independent adhesion directly and thereby reducing the formation of cellular projections.
The mechanism of VV-induced Ca2+-independent cellECM adhesion is probably mediated by integrins (Sanderson & Smith, 1998). Integrins are heterodimeric glycoproteins formed by an
and
subunit, and constitute the major transmembrane components of the focal adhesions (Hynes, 1992
). Their extracellular domains participate in binding to ECM components, whereas their short cytoplasmic tails interact with a multimolecular complex of proteins (Petit & Thiery, 2000
; Geiger et al., 2001
). Most integrins require extracellular divalent cations for function (Mould, 1996
) but there are a few exceptions (Lallier & Bronner-Fraser, 1992
; Sanderson & Smith, 1998
). Possibly, late in VV infection the class of integrins expressed does not require extracellular Ca2+ for function. Alternatively, since the conformation of integrins can be regulated and binding to the ECM is influenced by conformation (Mould, 1996
), it is possible that during VV infection the requirement for extracellular Ca2+ for integrin conformation, and therefore function, could be reduced. Like other POZ-KREP proteins, C2L is intracellular and therefore might interfere with integrin function by interacting directly or indirectly with the integrin cytoplasmic tail. Alternatively, C2L could interact directly or indirectly with other focal adhesion transmembrane proteins, which influence integrin binding to the ECM (Petit & Thiery, 2000
).
Although loss of C2L had no effect on VV virulence in an intranasal mouse model, in an intradermal mouse model the lesions produced by vC2L remained at their maximum size longer and healed more slowly than lesions produced by the control viruses. This was not due to a difference in virus titres, suggesting that the larger lesions reflect increased immunopathology. The latter hypothesis was supported by the increased cellular infiltrate in the v
C2L-infected ears. This represented a general increase in the quantity of all cell types analysed (except CD4+ T-cells). Therefore, in this model C2L reduces the cell infiltrate in VV-infected ears and reduces pathology associated with VV infection, promoting healing of lesions.
The finding that an intracellular kelch-like protein reduces the general cell infiltrate in VV-infected ears is intriguing. As the larger lesions caused by vC2L were observed at later stages of infection, C2L may affect the complex process of healing. The C2L protein may alter the balance of cytokines and chemokines produced by infected leukocytes or dermal cells, thereby influencing the recruitment of leukocytes. C2L might also influence the migratory properties of immune cell types or epidermal cells, thereby influencing the lesion resolution, or co-operate with other intracellular VV proteins that halt the immune response to VV infection. The mechanism of C2L action in this regard is not clear, but we note that inflammation and wound healing involve integrin-mediated adhesion, providing a potential link with the in vitro findings and possible role of C2L discussed above.
In summary, VV C2L is an intracellular 56 kDa protein that contributes to the formation of VV-induced cellular projections and to the development of the Ca2+-independent adhesion of infected cells to the ECM. In addition, C2L reduces the pathology of VV infection in an intradermal mouse model.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmad, K. F., Engel, C. K. & Prive, G. G. (1998). Crystal structure of the BTB domain from PLZF. Proc Natl Acad Sci U S A 95, 1212312128.
Appleyard, G., Hapel, A. J. & Boulter, E. A. (1971). An antigenic difference between intracellular and extracellular rabbitpox virus. J Gen Virol 13, 917.[Medline]
Bablanian, R., Baxt, B., Sonnabend, J. A. & Esteban, M. (1978). Studies on the mechanisms of vaccinia virus cytopathic effects. II. Early cell rounding is associated with virus polypeptide synthesis. J Gen Virol 39, 403413.[Abstract]
Bardwell, V. J. & Treisman, R. (1994). The POZ domain: a conserved proteinprotein interaction motif. Genes Dev 8, 16641677.[Abstract]
Bartlett, N., Symons, J. A., Tscharke, D. C. & Smith, G. L. (2002). The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence. J Gen Virol 83, 19651976.
Bork, P. & Doolittle, R. F. (1994). Drosophila kelch motif is derived from a common enzyme fold. J Mol Biol 236, 12771282.[Medline]
Buller, R. M. & Palumbo, G. J. (1991). Poxvirus pathogenesis. Microbiol Rev 55, 80122.[Medline]
Carter, G., Rodger, G., Murphy, B. J., Law, M., Krauss, O., Hollinshead, M. & Smith, G. L. (2003). Vaccinia virus cores are transported on microtubules. J Gen Virol 84, 24432458.
Cudmore, S., Cossart, P., Griffiths, G. & Way, M. (1995). Actin-based motility of vaccinia virus. Nature 378, 636638.[CrossRef][Medline]
Esposito, J., Condit, R. & Obijeski, J. (1981). The preparation of orthopoxvirus DNA. J Virol Methods 2, 175179.[CrossRef][Medline]
Falkner, F. G. & Moss, B. (1990). Transient dominant selection of recombinant vaccinia viruses. J Virol 64, 31083111.[Medline]
Geiger, B., Bershadsky, A., Pankov, R. & Yamada, K. M. (2001). Transmembrane crosstalk between the extracellular matrix and the cytoskeleton. Nat Rev Mol Cell Biol 2, 793805.[CrossRef][Medline]
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, 517563.
Hiller, G., Weber, K., Schneider, L., Parajsz, C. & Jungwirth, C. (1979). Interaction of assembled progeny pox viruses with the cellular cytoskeleton. Virology 98, 142153.[Medline]
Hollinshead, M., Rodger, G., Van Eijl, H., Law, M., Hollinshead, R., Vaux, D. J. & Smith, G. L. (2001). Vaccinia virus utilizes microtubules for movement to the cell surface. J Cell Biol 154, 389402.
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.
Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 1125.[Medline]
Kotwal, G. J. & Moss, B. (1988). Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant. Virology 167, 524537.[CrossRef][Medline]
Lallier, T. & Bronner-Fraser, M. (1992). Alpha 1 beta 1 integrin on neural crest cells recognizes some laminin substrata in a Ca2+-independent manner. J Cell Biol 119, 13351345.[Abstract]
Law, M., Hollinshead, R. & Smith, G. L. (2002). Antibody-sensitive and antibody-resistant cell-to-cell spread by vaccinia virus: role of the A33R protein in antibody-resistant spread. J Gen Virol 83, 209222.
Locker, J. K., 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.
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.
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.
Mould, A. P. (1996). Getting integrins into shape: recent insights into how integrin activity is regulated by conformational changes. J Cell Sci 109, 26132618.
Ng, A., Tscharke, D. C., Reading, P. C. & Smith, G. L. (2001). The vaccinia virus A41L protein is a soluble 30 kDa glycoprotein that affects virus virulence. J Gen Virol 82, 20952105.
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]
Payne, L. G. (1980). Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia. J Gen Virol 50, 89100.[Abstract]
Perkus, M. E., Goebel, S. J., Davis, S. W., Johnson, G. P., Norton, E. K. & Paoletti, E. (1991). Deletion of 55 open reading frames from the termini of vaccinia virus. Virology 180, 406410.[Medline]
Petit, V. & Thiery, J. P. (2000). Focal adhesions: structure and dynamics. Biol Cell 92, 477494.[CrossRef][Medline]
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]
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.
Sanderson, C. M. & Smith, G. L. (1998). Vaccinia virus induces Ca2+-independent cell-matrix adhesion during the motile phase of infection. J Virol 72, 99249933.
Sanderson, C. M., Way, M. & Smith, G. L. (1998). Virus-induced cell motility. J Virol 72, 12351243.
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.
Shchelkunov, S., Totmenin, A. & Kolosova, I. (2002). Species-specific differences in organization of orthopoxvirus kelch-like proteins. Virus Genes 24, 157162.[CrossRef][Medline]
Smith, G. L. (1995). Expression of genes by vaccinia virus vectors. In Molecular Virology: a Practical Approach, pp. 257283. Edited by A. J. Davison & R. Elliott. Oxford: Oxford University Press.
Smith, G. L., Levin, J. Z., Palese, P. & Moss, B. (1987). Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses. Virology 160, 336345; erratum 163, 259.
Smith, G. L., Vanderplasschen, A. & Law, M. (2002). The formation and function of extracellular enveloped vaccinia virus. J Gen Virol 83, 29152931.
Tscharke, D. C. & Smith, G. L. (1999). A model for vaccinia virus pathogenesis and immunity based on intradermal injection of mouse ear pinnae. J Gen Virol 80, 27512755.
Tscharke, D. C., Reading, P. C. & Smith, G. L. (2002). Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes. J Gen Virol 83, 19771986.
van Eijl, H., Hollinshead, M., Rodger, G., Zhang, W. H. & Smith, G. L. (2002). The vaccinia virus F12L protein 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. (2001). Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails. J Virol 75, 1165111663.
Xue, F. & Cooley, L. (1993). Kelch encodes a component of intercellular bridges in Drosophila egg chambers. Cell 72, 681693.[Medline]
Received 12 April 2003;
accepted 5 June 2003.