Departments of Molecular Biology1 and EIectron Microscopy2, Institute for Animal Health, Compton Laboratory, Newbury, Berkshire RG20 7NN, UK
Author for correspondence: Denise Boulanger. Present address: GSF-Institute for Virology/TUM, Trogerstraße 4b, D-81675 München, Germany. Fax +49 89 4140 7444. e-mail boulanger{at}gsf.de
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Abstract |
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Introduction |
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Little is known about the morphogenesis of FWPV, compared to the extensively studied VV maturation process. VV replication occurs entirely in the cytoplasm of infected cells. The first viral structures detectable by electron microscopy are crescent-shaped structures formed around the viroplasm in the virus factories. These structures develop into spherical immature virions (IV), which subsequently mature into characteristic brick-shaped particles, called intracellular mature virions (IMV), which are infectious. The membranes of these IMVs were first thought to be synthesized de novo (Dales & Mosbach, 1968 ) but it was later suggested that they were derived from the intermediate compartment between the endoplasmic reticulum and the cis-Golgi cisternae and consisted of two lipid bilayers (Sodeik et al., 1993
). A recent study using high resolution electron microscopy showed, however, that the crescents, the IV and IMV particles contain only a single lipid bilayer that does not show continuity with any pre-existing cellular membrane (Hollinshead et al., 1999
). The mechanism of formation of these membranes is, however, still unknown. The fate of the IMV seems to vary depending on the strain of virus, the type of cells and the time post-infection (Ichihashi et al., 1971
; Payne & Kristenson, 1979
). They can either: (i) remain in the cell until cellular lysis, (ii) bud through the plasma membrane (Tsutsui, 1983
; Tsutsui et al., 1983
) or (iii) acquire an additional double membrane by wrapping (Dales, 1963
; Ichihashi et al., 1971
; Morgan, 1976
; Tooze et al., 1993
). The two additional membranes acquired by wrapping derive either from the tubular early endosomes (Tooze et al., 1993
) or from the trans-Golgi network (TGN) (Schmelz et al., 1994
). This unusual mechanism of envelopment is used not only by poxviruses but also by herpesviruses (Tooze et al., 1993
). The intracellular enveloped virions (IEV), formed after wrapping, migrate to the cell surface where the external membrane of the virus fuses with the plasma membrane, releasing extracellular infectious enveloped virions, EEV (Dales, 1963
; Ichihashi et al., 1971
; Morgan, 1976
; Payne & Kristenson, 1979
). This migration can be facilitated by formation of actin tails, propelling IEV particles at the tip of microvilli (Cudmore et al., 1995
; Hiller et al., 1979
; Ichihashi et al., 1971
; Stokes, 1976
; Tsutsui et al., 1983
), increasing the efficiency of cell-to-cell spread (Roper et al., 1998
; Sanderson et al., 1998
). A proportion of virions leaving the cell, referred to as cell-associated enveloped virus (CEV), remain associated with the outside of the plasma membrane (Blasco & Moss, 1992
; Payne & Kristensson, 1982a
). By electron microscopy, FWPV IMV morphogenesis is similar to that of VV (Arhelger & Randall, 1964
). The mechanism of production of extracellular particles, antigenically different to intracellular particles (Maiti et al., 1991
), is less clear.
EEV particles acquire a number of viral proteins specifically associated with the extra envelope. VV genes encoding six of them have been identified: A33R (Roper et al., 1996 ), A34R (Duncan & Smith, 1992
), A36R (Parkinson & Smith, 1994
), A56R (Shida, 1986
), B5R (Engelstad et al., 1992
; Isaacs et al., 1992
) and F13L (Hirt et al., 1986
). Deleting the genes encoding these proteins, followed by characterization of the mutants, has been used for studying the role these proteins play in egress. Four EEV proteins, B5R (Engelstad & Smith, 1993
; Wolffe et al., 1993
), F13L (Blasco & Moss, 1991
) and the A33R (Roper et al., 1998
) and A34R (Wolffe et al., 1997
) gene products, as well as the 14 kDa protein (A27L) present on the IMV envelope (Rodriguez & Smith, 1990
), have so far been shown to be essential for efficient wrapping and production of IEV. However, the few IEV produced by B5R and F13L deletion mutants are able to induce actin tails, while A33R and A34R deletion mutants are unable to do so (Rottger et al., 1999
). The other two EEV proteins, A56R and A36R, are both non-essential for IEV production (Sanderson et al., 1998
; Wolffe et al., 1998
) but, whereas A56R is also non-essential for actin tail formation, A36R is essential and seems to be critical for actin tail formation, alone or in association with A34R (Rottger et al., 1999
; Sanderson et al., 1998
; Wolffe et al., 1998
). The efficiency of formation of IEV does not correlate necessarily with the amount of EEV produced, as the A33R (Roper et al., 1998
) and A34R (McIntosh & Smith, 1996
) deletion mutants produce even more EEV than the wild-type virus.
The A34R gene product, predicted to be a type II membrane protein with homology to C-type animal lectins (Smith et al., 1991 ), is apparently involved in regulating the release of progeny virions from the surface of infected cells, perhaps by binding to a carbohydrate component (Blasco et al., 1993
).
An FWPV homologue (p43K) of the F13L gene has been reported (Calvert et al., 1992 ), deletion of which also affects the release of EEV (Calvert et al., 1992
). Interestingly, disruption of the FWPV F12L homologue also results in reduced EEV production (Ogawa et al., 1993
), but the F12L gene product has not yet been studied in VV and it is not known whether it is present in the EEV envelope. No homologues of other EEV proteins have so far been reported in FWPV. Because of this lack of markers, we investigated the release of FWPV by another approach used previously with VV. This involved the use of different chemicals that specifically block the production of EEV, either by inhibiting the wrapping process or by interfering with the release of the enveloped virus. N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH) has been shown to inhibit selectively the release of EEV particles, intracellular virus production being only slightly reduced (Kato et al., 1969
). This effect was shown to depend on the virus strain and the host-cell type (Kato et al., 1969
) and to result from a blockage in wrapping (Payne & Kristenson, 1979
). The mechanism of action of IMCBH is not yet understood. Other drugs have also been shown to inhibit wrapping, such as monensin, brefeldin A and inhibitors of glycosylation such as 2-deoxy-D-glucose and glucosamine. The last two were shown to reduce the production of infectious virus, affecting EEV production rather than IMV production, with glucosamine being more efficient than 2-deoxy-D-glucose (Payne & Kristensson, 1982b
; Weintraub et al., 1977
). Monensin, a carboxylic ionophore known to perturb the Golgi causing a dilation of the cisternae, blocking the transport of secreted proteins and O-glycosylation, had the same effect (Payne & Kristensson, 1982a
). Brefeldin A, an inhibitor of vesicular transport between the ER and the cis-Golgi, has been shown to inhibit specifically the formation of EEV, also by inhibiting wrapping (Ulaeto et al., 1995
). Cytochalasin D, a potent inhibitor of actin microfilament formation, was shown to prevent EEV release from the cell surface (Payne & Kristensson, 1982a
), suggesting that actin filaments are essential for the final release of EEV from the cell surface.
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Methods |
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WR-VTN, a WR VV recombinant containing the porcine transmissible gastroenteritis virus nucleoprotein inserted into the thymidine kinase gene (Pulford & Britton, 1990 ), was grown under the same conditions as FWPV and was kindly provided by Paul Britton (IAH, Compton, UK).
Drugs.
IMCBH was kindly provided by R. Wittek (Switzerland), as a 10 mg/ml stock in DMSO. Monensin, glucosamine, brefeldin A and cytochalasin D were purchased from Sigma. Monensin (0·1 M in methanol), cytochalasin D (10 mg/ml in DMSO) and brefeldin A (5 mg/ml in 75% ethanol) stock solutions were stored at -20 °C. Glucosamine solutions were prepared freshly.
Electron microscopy.
CEFs were infected with FP9 (1 p.f.u. per cell) and fixed in situ with 2·5% 0·1 M phosphate-buffered glutaraldehyde at different times post-infection (p.i.). Samples were then processed for transmission electron microscopy as described previously (Boulanger et al., 1998 ).
Production of cell-associated virus (CAV) and extracellular virus (EV) in the presence of inhibitors.
CEFs were inoculated in triplicate with FWPV FP9, or with VV recombinant WR-VTN as a control, at an m.o.i. of 0·01 or 1 p.f.u. in M199 medium supplemented with 2% NBCS and containing 0, 5, 10 or 20 µg/ml IMCBH. Three days p.i., or 24 h p.i. depending on the m.o.i. used, the supernatant was removed, clarified by centrifugation at 1000 g for 10 min and stored at -70 °C until titration. The monolayers were covered with fresh medium and freezethawed once before titration.
Experiments using other drugs were performed by infecting CEFs with FWPV or with VV at an m.o.i. of 1 p.f.u. per cell in M199 medium supplemented with 2% NBCS. Three hours p.i. the medium was removed and replaced by fresh medium containing 2% NBCS and supplemented with 0·1 or 10 µM monensin; 0, 5 or 20 mM glucosamine; 0, 0·1 or 1 µg/ml cytochalasin D (in triplicate). Brefeldin A, at a concentration of 0, 50 or 500 ng/ml, was added 12 h p.i. in medium containing 4% foetal calf serum (FCS). Twenty-four hours p.i., the supernatant was removed, clarified and stored at -70 °C until titration. The monolayers were covered with fresh medium and freezethawed once before titration.
In all cases, the triplicate samples were titrated on CEFs cultured in six-well plates.
Virus purification.
Confluent CEFs were infected with FP9 at an m.o.i. of 1 p.f.u.. Forty-eight hours p.i., the supernatant was harvested (the EEV fraction), the monolayers washed once with PBS and dispersed by addition of trypsin. The cell suspension was spun 10 min at 1000 g after neutralizing the trypsin with 10% NBCS. The supernatant was harvested (the CEV fraction) and the cell pellet resuspended in PBS before being submitted to one cycle of freezethawing (the IMV fraction). All fractions were clarified for 30 min at 1000 g. Supernatants were harvested and the virions were pelleted by centrifugation at 40000 g for 1 h. The pellets were resuspended in TMN buffer (10 mM Tris pH 7·5, 1·5 mM MgCl2, 10 mM NaCl) and centrifuged 2 h at 160000 g through a 25% (w/w) sucrose cushion in TMN buffer. Pellets were resuspended in 1 ml TMN buffer and sonicated mildly before being layered over a preformed CsCl gradient (2·5 ml of 1·3 g/ml, 3·75 ml of 1·25 g/ml, 5 ml of 1·2 g/ml) and centrifuged at 182000 g for 1 h. Fractions (0·5 ml) were collected from the bottom of the tube. The absorbance at 280 nm and the density of each fraction were measured.
Immunofluorescence microscopy.
CEFs, seeded in chamber slides (Nunc) at 2x105 cells/ml of M199 medium supplemented with 10% FCS, were infected with FWPV FP9 (m.o.i. of 2). After various periods of incubation the cells were fixed in acetone. Cells were treated with RNase A (3 mg/ml in PBS) for 1 h at 37 °C. After three washes in PBS the DNA was labelled overnight at 4 °C with propidium iodide (PI; 50 µg/ml in PBS). After three washes with PBS the cells were blocked for 1 h at 4 °C with BSA (10 mg/ml in PBS). Actin was detected with a specific MAb (Sigma) and a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse antibody (DAKO). Slides were mounted under DAKO Fluorescent Mounting Medium. Immunofluorescence was observed and recorded using a Leika TCS NT confocal microscope. The resulting images were processed with the Adobe Photoshop program.
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Results |
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Concentrations of brefeldin A higher than 0·1 µg/ml were toxic for CEFs when they were incubated in its presence for 24 h. The cells lost their characteristic morphology, became rounded and then detached. Brefeldin A was, therefore, added only 12 h p.i. like monensin. In these conditions neither CAV nor EV production was affected in the presence of up to 500 ng/ml brefeldin A (Fig. 5) (P=0·04 and 0·74 respectively with an rdf=6). VV was also not affected by brefeldin A under these conditions (Table 1
).
Effect of cytochalasin D on virus production
In the presence of cytochalasin D at a concentration of 1 µg/ml the titres of FWPV and VV EV were slightly reduced (both down to about 20%; see Table 1), whereas the CAV titres were not affected (P=0·82 and 0·004, respectively, for FWPV CAV and EV titres, with an rdf=6).
Cytochalasin D reduces the release of CEV from infected cells
To check whether the reduction of the EV titre was due to an inhibition of release of EEV particles from the cell surface as described for VV (Payne & Kristensson, 1982a ) or due to an inhibition of the production or migration to the cell surface of IEV particles, FWPV was grown on CEFs in the presence of 1 µg/ml cytochalasin D (or in the absence of the drug as a control) and the different types of particles produced were analysed by purification on CsCl gradients. In the presence of cytochalasin D, the EEV peak was reduced whereas the CEV peak was increased (Fig. 6C
F
). Each peak showed the characteristic density for each type of particle. The intracellular fraction did not seem to contain an increased level of IEV (Fig. 6B
).
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Discussion |
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As FWPV replicates only in avian cells, we chose to study FWPV production on CEFs and used, as a control, a VV WR recombinant virus, a low producer of EEV, grown on the same cell type. VV EEV production had been shown to be affected by several drugs inhibiting wrapping IMCBH, glucosamine, monensin and brefeldin A but none of them affected specifically FWPV EV production. IMCBH had been shown to inhibit specifically the production of VV EEV particles. This effect is characterized by its variable efficacy depending on the virus strain and the host cell type (Kato et al., 1969 ; Payne & Kristenson, 1979
). We showed that VV WR, a poor EEV producer, when grown on CEFs, is also sensitive to IMCBH whereas FWPV FP9 strain is not, even in a highly sensitive low m.o.i. experiment. A single amino acid in the VV F13L gene product has been shown to be critical for the effect of IMCBH. This residue is not conserved in FWPV, possibly explaining the lack of effect of IMCBH on FWPV EV production (unless FWPV is not sensitive to this drug because it uses predominantly budding instead of wrapping). However, the degree of homology between VV and FWPV F13L is relatively low and other residue substitutions or a different conformation of the protein could be responsible for the observed resistance. Further studies involving mutagenesis would need to be performed to address that question.
Glucosamine also did not affect FWPV production, whereas VV production was reduced in the presence of 20 mM glucosamine (CAV production being affected as much as EV production). This is in contrast with the results published by Payne & Kristensson (1982b) , who used the VV IHD-J strain in RK-13 cells, and showed that the production of infectious intracellular virus was less sensitive to the presence of glycosylation inhibitors than was the production of infectious EV.
Despite its overall effect on the cell metabolism, monensin did not affect either FWPV nor VV EV production, suggesting that neither O-glycosylation nor the three-dimensional structure of the Golgi compartment are essential for FWPV and VV egress in CEFs. The lack of effect of monensin on VV production is also in contrast with the results published by Payne & Kristensson (1982a) . This discrepancy between our results and previously published data could be explained by the use of another VV strain or more likely of another cell type.
Brefeldin A did not have any effect either on FWPV or VV EV production in CEFs. These results with VV are also in contradiction with previously published data (Ulaeto et al., 1995 ), which showed a 3 log10 reduction in the EEV titre of IHD-J, grown for 24 h on RK-13 cells in the presence of 100 ng/ml of brefeldin A. However, in our system (CEFs), brefeldin A at a concentration higher than 10 ng/ml was cytotoxic. Uninfected cell morphology changed after 8 h incubation with brefeldin A, finally resulting in a CPE. Tooze et al. (1993)
investigated, by electron microscopy, the wrapping of both human cytomegalovirus (HCMV) and VV WR strain, showing that wrapping was not inhibited by brefeldin A. As brefeldin A affects the Golgi but not the tubular compartment, the authors concluded that the wrapping membranes do not originate from the TGN but from the tubular endosomal compartment. However, wrapping of HCMV was affected by the drug after 3 or 4 h, resulting in an accumulation of unenveloped cytoplasmic nucleocapsids, probably due to a depletion of the pool of endosomal membranes and proteins that usually transit through the Golgi. This supports both the toxic effect of brefeldin A to CEFs in our experiments and could explain the effect on VV EEV production observed by Ulaeto et al. (1995)
after a 24 h incubation in the presence of the drug.
In summary, none of the wrapping inhibitors tested, with the exception of IMCBH, had any specific effect on VV WR EV production in CEFs. This shows how important the virus strain and cell type are for this kind of study, a consideration which also applies to this study, as only one strain of FWPV has been considered and only in CEFs. The lack of specific effect of the drugs (glucosamine, monensin and brefeldin A) on the VV control in our experimental conditions also rules out the use of these inhibitors to study the FWPV egress mechanism in CEFs as the results do not allow us to compare both systems and to draw any definite conclusion on FWPV exit mechanism. We can only conclude that those drugs have no effect on the morphogenesis and exit of the FP9 strain of FWPV in CEFs.
The effect of cytochalasin D on the release of VV EEV particles (Payne & Kristensson, 1982a ) has been confirmed and extended to FWPV EEV production. Cytochalasin D inhibited the release of CEV particles from the surface of the cell, suggesting that actin is essential for this last step in the release of extracellular particles. The co-localization of actin with viral particles (Fig. 7C
) and the presence of filaments underneath budding particles (Fig. 2B
) argues in favour of this hypothesis. These structures are, however, quite short and differ from the long actin tails observed in VV-infected cells. Several viruses have been shown to bud through the plasma membrane by a mechanism involving actin microfilaments. The iridivirus frog virus 3 induces the formation of actin-containing projections from which the virions bud (Murti & Goorha, 1983
). Cytochalasin B and D inhibit the release of EV and lead to an accumulation of intracellular virus just beneath the cell surface (Murti et al., 1985
). Cytochalasin also inhibits the release of measles virus, which buds through the plasma membrane in close association with actin filaments (Bohn et al., 1986
), leading to an accumulation of nucleocapsids in the cytoplasm, and of mammalian B- and C-type retroviruses which bud from the cell on the tip of an actin-rich microvillus [reviewed in Sanders & Theriot (1996)
and Cudmore et al. (1997)
]. In the case of FWPV, cytochalasin D does not inhibit budding, as CEV particles are produced, as for VV. Actin filaments seem therefore to be involved in the exit of FWPV and of VV, probably by two different mechanisms, but, in both cases, cytochalasin D only inhibits the release of CEV from the cell surface and does not inhibit the formation of these particles. This effect of cytochalasin D had been interpreted by Payne & Kristensson (1982 a)
as the requirement for cell movement to liberate CEV particles from the cell surface. The A34R protein, a homologue of animal C type lectins, has been shown to be involved in the retention of VV CEV particles on the cell surface (Blasco et al., 1993
; McIntosh & Smith, 1996
); it is not known, however, whether the A34R protein is involved directly or indirectly in this process. Actin has been shown to be linked through the ERM (ezrin, radixin, moesin) protein family to the integral membrane protein CD44 (Tsukita et al., 1994
), which is highly glycosylated and, therefore, could be a potential ligand for A34R if this protein is really a lectin. Another hypothesis for the effect of cytochalasin D would be that this drug would disrupt the interaction between actin and its membrane linker or ligand, destabilizing the interaction between the virus particles and the cell surface. The liberation of CEV particles by trypsin also argues in favour of a proteinprotein interaction.
In addition to its role in the release of FWPV particles from the cell, our results indicate that actin may play an earlier role in FWPV morphogenesis, not observed in VV-infected cells (Meyer et al., 1981 ), as it co-localizes with the FWPV virus factories.
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Acknowledgments |
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Footnotes |
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References |
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Blasco, R. & Moss, B. (1991). Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein. Journal of Virology 65, 5910-5920.[Medline]
Blasco, R. & Moss, B. (1992). Role of cell-associated enveloped vaccinia virus in cell-to-cell spread. Journal of Virology 66, 4170-4179.[Abstract]
Blasco, R., Sisler, J. R. & Moss, B. (1993). Dissociation of progeny vaccinia virus from the cell-membrane is regulated by a viral envelope glycoprotein: effect of a point mutation in the lectin homology domain of the A34R gene. Journal of Virology 67, 3319-3325.[Abstract]
Bohn, W., Rutter, G., Hohenberg, H., Mannweiler, K. & Nobis, P. (1986). Involvement of actin filaments in budding of measles virus: studies on cytoskeletons of infected cells. Virology 149, 91-106.[Medline]
Boulanger, D., Green, P., Smith, T., Czerny, C. P. & Skinner, M. A. (1998). The 131-amino-acid repeat region of the essential 39-kilodalton core protein of fowlpox virus FP9, equivalent to vaccinia virus A4L protein, is nonessential and highly immunogenic. Journal of Virology 72, 170-179.
Calvert, J. G., Ogawa, R., Yanagida, N. & Nazerian, K. (1992). Identification and functional analysis of the fowlpox virus homolog of the vaccinia virus p37K major envelope antigen gene. Virology 191, 783-792.[Medline]
Cudmore, S., Cossart, P., Griffiths, G. & Way, M. (1995). Actin-based motility of vaccinia virus. Nature 378, 636-638.[Medline]
Cudmore, S., Reckmann, I. & Way, M. (1997). Viral manipulations of the actin cytoskeleton. Trends in Microbiology 5, 142-148.[Medline]
Dales, S. (1963). The uptake and development of vaccinia virus in strain L cells followed with labeled viral deoxyribonucleic acid. Journal of Cell Biology 18, 51-72.
Dales, S. & Mosbach, E. H. (1968). Vaccinia as a model for membrane biogenesis. Virology 35, 564-583.[Medline]
Duncan, S. A. & Smith, G. L. (1992). Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress. Journal of Virology 66, 1610-1621.[Abstract]
Engelstad, M. & Smith, G. L. (1993). The vaccinia virus 42-kDa envelope protein is required for the envelopment and egress of extracellular virus and for virus virulence. Virology 194, 627-637.[Medline]
Engelstad, M., Howard, S. T. & Smith, G. L. (1992). A constitutively expressed vaccinia gene encodes a 42-kDa glycoprotein related to complement control factors that forms part of the extracellular virus envelope. Virology 188, 801-810.[Medline]
Hiller, G., Weber, K., Schneider, L., Parajsz, C. & Jungwirth, C. (1979). Interaction of assembled progeny pox viruses with the cellular cytoskeleton. Virology 98, 142-153.[Medline]
Hirt, P., Hiller, G. & Wittek, R. (1986). Localization and fine structure of a vaccinia virus gene encoding an envelope antigen. Journal of Virology 58, 757-764.[Medline]
Hollinshead, M., Vanderplasschen, A., Smith, G. L. & Vaux, D. J. (1999). Vaccinia virus intracellular mature virions contain only one lipid membrane. Journal of Virology 73, 1503-1517.
Ichihashi, Y., Matsumoto, S. & Dales, S. (1971). Biogenesis of poxviruses: role of A-type inclusions and host cell membranes in virus dissemination. Virology 46, 507-532.[Medline]
Isaacs, S. N., Wolffe, E. J., Payne, L. G. & Moss, B. (1992). Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope. Journal of Virology 66, 7217-7224.[Abstract]
Kato, N., Eggers, H. J. & Rolly, H. (1969). Inhibition of release of vaccinia virus by N1-isonicotinoly-N23-methyl-4-chlorobenzoylhydrazine. Journal of Experimental Medicine 129, 795-808.[Medline]
Limbach, K. J. & Paoletti, E. (1996). Non-replicating expression vectors: applications in vaccine development and gene therapy. Epidemiology and Infection 116, 241-256.[Medline]
McIntosh, A. A. & Smith, G. L. (1996). Vaccinia virus glycoprotein A34R is required for infectivity of extracellular enveloped virus. Journal of Virology 70, 272-281.[Abstract]
Maiti, N. K., Oberoi, M. S. & Sharma, S. N. (1991). Comparative-studies on the antigenicity of extracellular and intracellular viruses of fowlpox. Comparative Immunology Microbiology and Infectious Diseases 14, 59-62.[Medline]
Meyer, R. K., Burger, M. M., Tschannen, R. & Schafer, R. (1981). Actin filament bundles in vaccinia virus infected fibroblasts. Archives of Virology 67, 11-18.[Medline]
Morgan, C. (1976). Vaccinia virus reexamined: development and release. Virology 73, 43-58.[Medline]
Murti, K. G. & Goorha, R. (1983). Interaction of frog virus-3 with the cytoskeleton. I. Altered organization of microtubules, intermediate filaments, and microfilaments. Journal of Cell Biology 96, 1248-1257.[Abstract]
Murti, K. G., Chen, M. & Goorha, R. (1985). Interaction of frog virus 3 with the cytomatrix. III. Role of microfilaments in virus release. Virology 142, 317-325.[Medline]
Ogawa, R., Calvert, J. G., Yanagida, N. & Nazerian, K. (1993). Insertional inactivation of a fowlpox virus homolog of the vaccinia virus F12L gene inhibits the release of enveloped virions. Journal of General Virology 74, 55-64.[Abstract]
Parkinson, J. E. & Smith, G. L. (1994). Vaccinia virus gene A36R encodes a M(r) 4350 K protein on the surface of extracellular enveloped virus. Virology 204, 376-390.[Medline]
Payne, L. G. & Kristenson, K. (1979). Mechanism of vaccinia virus release and its specific inhibition by N1-isonicotinoyl-N23-methyl-4-chlorobenzoylhydrazine. Journal of Virology 32, 614-622.[Medline]
Payne, L. G. & Kristensson, K. (1982a). The effect of cytochalasin-D and monensin on enveloped vaccinia virus release. Archives of Virology 74, 11-20.[Medline]
Payne, L. G. & Kristensson, K. (1982b). Effect of glycosylation inhibitors on the release of enveloped vaccinia virus. Journal of Virology 41, 367-375.[Medline]
Pulford, D. J. & Britton, P. (1990). Expression and cellular localisation of porcine transmissible gastroenteritis virus N and M proteins by recombinant vaccinia viruses. Virus Research 18, 203-218.
Rodriguez, J. F. & Smith, G. L. (1990). Inducible gene-expression from vaccinia virus vectors. Virology 177, 239-250.[Medline]
Roper, R. L., Payne, L. G. & Moss, B. (1996). Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene. Journal of Virology 70, 3753-3762.[Abstract]
Roper, R. L., Wolffe, E. J., Weisberg, A. & Moss, B. (1998). The envelope protein encoded by the A33R gene is required for formation of actin-containing microvilli and efficient cell-to-cell spread of vaccinia virus. Journal of Virology 72, 4192-4204.
Rottger, S., Frischknecht, F., Reckmann, I., Smith, G. L. & Way, M. (1999). Interactions between vaccinia virus IEV membrane proteins and their roles in IEV assembly and actin tail formation. Journal of Virology 73, 2863-2875.
Sanders, M. C. & Theriot, J. A. (1996). Tails from the hall of infection: actin-based motility of pathogens. Trends in Microbiology 4, 211-213.[Medline]
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 cell-cell fusion. Journal of General Virology 79, 1415-1425.[Abstract]
Schmelz, M., Sodeik, B., Ericsson, M., Wolffe, E. J., Shida, H., Hiller, G. & Griffiths, G. (1994). Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network. Journal of Virology 68, 130-147.[Abstract]
Schmutz, C., Payne, L. G., Gubser, J. & Wittek, R. (1991). A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release. Journal of Virology 65, 3435-3442.[Medline]
Shida, H. (1986). Nucleotide sequence of the vaccinia virus hemagglutinin gene. Virology 150, 451-462.[Medline]
Smith, G. L., Chan, Y. S. & Howard, S. T. (1991). Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat. Journal of General Virology 72, 1349-1376.[Abstract]
Sodeik, B., Doms, R. W., Ericsson, M., Hiller, G., Machamer, C. E., Vanthof, W., Vanmeer, G., Moss, B. & Griffiths, G. (1993). Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks. Journal of Cell Biology 121, 521-541.[Abstract]
Stokes, G. V. (1976). High-voltage electron microscope study of the release of vaccinia virus from whole cells. Journal of Virology 18, 636-643.[Medline]
Tooze, J., Hollinshead, M., Reis, B., Radsak, K. & Kern, H. (1993). Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes. European Journal of Cell Biology 60, 163-178.[Medline]
Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A. & Tsukita, S. (1994). ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. Journal of Cell Biology 126, 391-401.[Abstract]
Tsutsui, K. (1983). Release of vaccinia virus from FL cells infected with the IHD-W strain. Journal of Electron Microscopy 32, 125-140.[Medline]
Tsutsui, K., Uno, F., Akatsuka, K. & Nii, S. (1983). Electron microscopic study on vaccinia virus release. Archives of Virology 75, 213-218.[Medline]
Ulaeto, D., Grosenbach, D. & Hruby, D. E. (1995). Brefeldin A inhibits vaccinia virus envelopment but does not prevent normal processing and localization of the putative envelopment receptor p37. Journal of General Virology 76, 103-111.[Abstract]
Weintraub, S., Stern, W. & Dales, S. (1977). Biogenesis of vaccinia. Effects of inhibitors of glycosylation on virus-mediated activities. Virology 78, 315-322.[Medline]
Wolffe, E. J., Isaacs, S. N. & Moss, B. (1993). Deletion of the vaccinia virus B5R gene encoding a 42-kilodalton membrane glycoprotein inhibits extracellular virus envelope formation and dissemination. Journal of Virology 67, 4732-4741.[Abstract]
Wolffe, E. J., Katz, E., Weisberg, A. & Moss, B. (1997). The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus. Journal of Virology 71, 3904-3915.[Abstract]
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, 20-26.[Medline]
Received 24 June 1999;
accepted 12 November 1999.