Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1
Author for correspondence: Geoffrey L. Smith. Fax +44 1865 275521. e-mail glsmith{at}molbiol.ox.ac.uk
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Abstract |
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Introduction |
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As morphogenesis progresses, virus crescents extend to form spherical immature virus (IV) particles that in turn condense to form infectious intracellular mature virus (IMV). To date, 11 VV proteins are known to be associated with the IMV membrane (Takahashi et al., 1994 ; Jensen et al., 1996
). Of these, the protein (D13L) that is sensitive to rifampicin (Tartaglia et al., 1986
; Baldick & Moss, 1987
; Zhang & Moss, 1992
), A17L (Rodriguez et al., 1995
, 1997
; Wolffe et al., 1996
) and A14L (Rodriguez et al., 1997
) are required for the formation of IMV particles. In addition to facilitating IMV assembly, the A17L protein also functions to bind A27L protein on the IMV surface (Rodriguez et al., 1993
). Although most IMV particles remain within the cytoplasm until cell death, some IMV particles become enveloped by membranes of the trans-Golgi network (TGN) (Hiller & Weber, 1985
; Schmelz et al., 1994
) or tubular endosomes (Tooze et al., 1993
) to form intracellular enveloped virus (IEV).
Components within the outer membrane of IEV particles promote the polymerization of actin tails that are thought to assist IEV particle movement and enhance infection of neighbouring cells (Cudmore et al., 1995 , 1996
). When IEV particles reach the cell surface their outer membrane fuses with the plasma membrane, exposing infectious cell-associated enveloped virus (CEV) on the surface of the cell. In the case of the Western Reserve (WR) strain of VV, the majority of enveloped virus particles remain attached to the cell surface as CEV, and only a small percentage of enveloped virions are released from the cell as extracellular enveloped virus (EEV). The outer membrane of CEV or EEV particles contains six virus-encoded proteins that are not found in IMV particles. These are A56R [haemagglutinin (HA) gp86] (Payne & Norrby, 1976
; Shida, 1986
), F13L (p37) (Hirt et al., 1986
), B5R (gp42) (Engelstad et al., 1992
; Isaacs et al., 1992
), A34R (gp2224) (Duncan & Smith, 1992
), A36R (p4550) (Parkinson & Smith, 1994
) and A33R (gp2328) (Roper et al., 1996
). Deletion of the F13L or B5R genes or repression of A27L gene expression reduces dramatically the formation of IEV particles (Rodriguez & Smith, 1990a
; Blasco & Moss, 1991
; Engelstad et al., 1992
).
The A27L protein (p14) is a multifunctional protein that has been implicated in virus attachment (Chung et al., 1998 ), viruscell fusion (Doms et al., 1990
), cellcell fusion (Gong et al., 1990
), plaque size (Dallo et al., 1987
; Rodriguez & Smith, 1990a
) and the formation of enveloped virions (Rodriguez & Smith, 1990a
). In this study, two closely related recombinant viruses, WR32-7/14K (Rodriguez & Smith, 1990a
) and WR32-7/Ind 14K (Rodriguez & Smith, 1990b
), have been used to characterize further the role of A27L in VV morphogenesis. Both viruses were derived from a variant form of WR (WR32-7) that was isolated from persistently infected Friend erythroleukaemia cells (Dallo & Esteban, 1987
). Analyses of the WR32-7 virus identified an 8 MDa deletion in the left terminus of the virus genome, and alterations in the size of three structural proteins with molecular masses of 39, 21 and 14 kDa (Paez et al., 1987
). In particular, the protein encoded by gene A27L migrated with an apparent electrophoretic mobility of 15·5 kDa and not 14 kDa as observed for the parental WR virus. Phenotypically, WR32-7 produced smaller plaques and fewer enveloped virions (IEV, CEV and EEV) than the parental WR virus. Genetic analyses of the WR32-7 A27L gene identified a single base change (C to A) resulting in the substitution of Ala-25 by Asp, which was shown to be responsible for the observed changes in both plaque size and electrophoretic mobility (Gong et al., 1989
).
After characterization of VV WR32-7, a recombinant virus was generated (WR32-7/14K) in which a wild-type (WT) A27L ORF was inserted into the thymidine kinase (TK) locus of the WR32-7 virus genome under the transcriptional control of the VV late p4b promoter and repressed by the E. coliLacI protein and lac operator (Rodriguez & Smith, 1990a ). Consequently, the WT A27L protein was expressed only in the presence of IPTG. To generate a virus that encoded only the 14 kDa A27L protein, the mutated A27L gene contained within the endogenous locus of WR32-7/14K was deleted leaving only the inducible WT A27L ORF within the VV TK locus (Rodriguez & Smith, 1990b
). Ultrastructural analysis of cells infected with WR32-7/Ind 14K in the absence of IPTG showed that although IMV particles were formed, IEV particles were not (Rodriguez & Smith, 1990b
). As such, both WR32-7/14K and WR32-7/Ind 14K exhibit a small plaque phenotype in the absence of IPTG and are defective in the formation of enveloped virions. In this report we show that WR32-7/14K and WR32-7/Ind 14K are defective at different stages of VV morphogenesis, demonstrating that A27L plays a multifunctional role in VV assembly. In addition, we show that intracellular movement of mature IMV particles is dependent upon microtubules and A27L gene expression.
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Methods |
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Fluorescent microscopy.
VV was adsorbed onto cells on ice for 1 h using 1 p.f.u. per cell. After adsorption, non-adherent viruses were removed by washing repeatedly and cells were incubated in MEM containing 2·5% FBS at 37 °C. At the indicated times, cells were fixed in 4% formaldehydePBS for 15 min at room temperature and immunocytochemistry was performed as described (Herzog et al., 1994 ). Monoclonal antibody (MAb) AB1.1, which recognizes the VV D8L protein (Parkinson & Smith, 1994
), was used (diluted 1:300) for the identification of VV particles, while MAb 19C2 (Schmelz et al., 1994
) was used to identify the VV B5R protein (hybridoma culture supernatants were used at a dilution of 1:8). A27L was identified using MAb C3 (Rodriguez et al., 1987
) at a dilution of 1:50, while F-actin was visualized with tetramethylrhodamine B isothiocyanate (TRITC)phalloidin (Sigma). Images were recorded using a Bio-Rad MRC 1024 confocal laser scanning microscope and processed using Adobe Photoshop software.
Electron microscopy.
WR32-7/Ind 14K virus (1 p.f.u. per cell) was adsorbed onto BS-C-1 cells on ice for 1 h. Unbound virus was then washed away and cells were incubated at 37 °C in MEM containing 2·5% FBS for 12 h before being washed in ice-cold PBS and fixed in 0·5% glutaraldehyde in 200 mM sodium cacodylate (pH 7·4) for 30 min at room temperature. After fixation, cells were washed in water and post-fixed in 1% osmium tetroxide and 1·5% potassium ferrocyanide for 60 min at room temperature. Samples were then washed in water, incubated overnight at 4 °C in Mg2+-uranyl acetate, washed in sodium cacodylate, dehydrated in ethanol and flat-embedded in Epon. Sections were cut parallel to the surface of the dish, lead citrate was added as a contrast agent, and the sections were examined on a Zeiss Omega EM 912 electron microscope.
Kinetic analyses of the number of cytoplasmic virions.
BS-C-1 cells were infected with the indicated virus at 0·1 p.f.u. per cell so that each productive infection should result from the entry of a single infectious particle. In each case viruses were adsorbed onto cells for 1 h at 4 °C and unbound viruses were removed by washing repeatedly with PBS (4 °C) before addition of MEM at 37 °C. At the indicated times, cells were fixed in 4% formaldehydePBS for 30 min at room temperature. After fixation, cells were processed for immunofluorescence using MAb AB1.1 to identify virus particles. For each virus, projected reconstructions of five infected cells were prepared and for each cell the number of cytoplasmic virions was counted manually from images compiled in Adobe Photoshop. For each time-point the standard deviation of the mean was calculated form data derived from five cells.
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Results |
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Discussion |
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Early work on the involvement of cytoskeletal components in VV assembly and dissemination concluded that both actin-containing microfilaments and microtubules were required for the formation of extracellular enveloped virus (Stokes, 1976 ; Hiller et al., 1979
). Although the role of actin in movement of enveloped virus has been studied (Hiller et al., 1979
, 1981
; Hiller & Weber, 1982
; Cudmore et al., 1996
) and the requirement for several VV proteins defined (Wolffe et al., 1997
, 1998
; Mathew et al., 1998
; Roper et al., 1998
; Sanderson et al., 1998
), the role of microtubules in VV assembly remains unclear. Neither the microtubule-dependent stage of VV assembly nor the VV proteins required were known. Data presented in this report show that movement of IMV particles from the periphery of virus factories to the site of IEV formation is microtubule-dependent. As such, this aspect of VV assembly is similar to that described for African swine fever virus (ASFV), which uses microtubules to facilitate intracellular movement of nascent virus particles (Carvalho et al., 1988
; Alves de Matos & Carvalho, 1993
).
Although our data show that both A27L and microtubules are required for the efficient cytoplasmic transport of IMV particles, the precise role of each in IMV movement remains unclear. One interpretation of these data would be that A27L mediates microtubuleIMV association directly. However, other explanations are possible. Firstly, the lack of A27L on the surface of IMV particles may prevent recruitment of other proteins (virus or host) that in turn mediate microtubule association or microtubule-dependent movement. Secondly, as a reduction in A27L expression induces inappropriate association between virus crescents and IMV particles (Fig. 5), it is possible that A27L may function to block deleterious interactions occurring between IMV particles or IMV particles and virus crescents. For example, A27L recruitment may block the interaction of A17L and A14L proteins in/on adjacent membranes of juxtaposed virus structures. Consequently, intracellular movement of IMV particles may be impeded by physical aggregation of particles when A27L expression is repressed. Induction of A27L expression at 11 h p.i. resulted in dispersal of IMV particles that were clustered at the periphery of virus factories. This suggests that any inter-particle interactions that occur are disrupted competitively in the presence of A27L.
As both A27L (p14) and F13L (p37) are required for IEV formation (Rodriguez & Smith, 1990b ; Blasco & Moss, 1991
) there has been speculation (Rodriguez & Smith, 1990b
) that there may be direct interaction between A27L on the surface of IMV particles and F13L on the cytoplasmic surface of wrapping membranes (Hiller & Weber, 1985
). Although no experimental evidence exists to support a direct A27LF13L interaction, it is interesting to note that defects in the dissemination of IMV particles have also been reported when the F13L gene contains mutations which prevent palmitoylation (Grosenbach & Hruby, 1998
) or when cells are infected with VV in the presence of the drug N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH) (Hiller et al., 1981
). Also, non-palmitoylated forms of F13L have been found in association with IMV particles (Grosenbach & Hruby, 1998
). Given the similarities in the two phenotypes it is again tempting to speculate that accumulation of IMV particles results from incorrect A27LF13L interaction. However, our data show that the sub-cellular dispersal of IMV particles was restricted more severely when cells were infected with WR32-7/Ind 14K (-IPTG) than when infected with v
F13L or WR virus in the presence of IMCBH. This observation was also true in CEF cells where IMCBH-induced clustering of IMV particles had been observed previously (Hiller et al., 1981
). Consequently, it is possible that defects resulting from the lack of A27L and F13L expression may be similar but not identical. Alternatively, the observed differences in IMV distribution in the absence of either A27L and F13L could reflect other genetic variations in the genomes of WR and WR 32-7/Ind 14K viruses.
In conclusion, data presented show that the A27L protein is required to prevent deleterious interaction between IMV particles and virus membranes within virus factories, to facilitate efficient intracellular transport of IMV particles from virus factories to the site of IEV formation and for the targeting and/or envelopment of IMV particles during IEV formation. In addition, we have shown that movement of IMV particles from virus factories is dependent upon microtubules as reported for ASFV.
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Acknowledgments |
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Footnotes |
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References |
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Received 21 June 1999;
accepted 6 September 1999.