Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1
Department of Infectious Diseases, Division of Investigative Science, Faculty of Medicine, Imperial College, St Marys Campus, Norfolk Place, London W2 1PG, UK2
Author for correspondence: Geoffrey L. Smith (at Imperial College). Fax +44 207 594 3973. e-mail glsmith{at}ic.ac.uk
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Abstract |
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Introduction |
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The B5R gene encodes a 42 kDa type I membrane glycoprotein that is present on the surface of cells and EEV particles (Engelstad et al., 1992 ; Isaacs et al., 1992
), and a 35 kDa form of unknown function that is present in the cell culture supernatant (Martinez-Pomares et al., 1993
). The B5R extracellular domain contains four 5070 amino acid short consensus repeats (SCRs) similar to those found in complement control proteins. Deletion of the B5R protein produced a small plaque and attenuation in vivo (Engelstad & Smith, 1993
; Wolffe et al., 1993
). Electron and confocal microscopy showed that morphogenesis of the B5R deletion mutant virus (v
B5R) was arrested after the formation of IMV particles, so that few IEV, CEV and EEV particles or actin tails were formed (Engelstad & Smith, 1993
; Wolffe et al., 1993
; Sanderson et al., 1998
; Röttger et al., 1999
).
Several studies have reported mutagenesis of the B5R protein to investigate the function of particular domains. Deletion of one or more of the SCRs caused inhibition of actin tail formation, a small plaque phenotype and enhanced levels of EEV (Mathew et al., 1998 ). A similar study, in which all four SCR domains were deleted, reported that the plaque size was normal although actin tail formation was inhibited (Herrera et al., 1998
). The transmembrane region of B5R was sufficient for targeting the protein to the trans-Golgi network (Lorenzo et al., 1999
; Ward & Moss, 2000
) and the transmembrane and cytoplasmic tail of B5R was sufficient to direct incorporation of human immunodeficiency virus gp120 into EEV (Katz et al., 1997
). Consistent with this, targeting of B5R to the endoplasmic reticulum (ER) resulted in virions deficient in B5R with a phenotype similar to v
B5R infection (Mathew et al., 1999
). Further analysis showed that the cytoplasmic tail was dispensable for virus replication in cell culture (Lorenzo et al., 1999
; Mathew et al., 2001
) but affected the rate of B5R transport to the cell surface and the level of B5R on the surface (Mathew et al., 2001
). Additionally, the cytoplasmic tail was reported to mediate retrograde transport of B5R from the plasma membrane to the trans-Golgi network (Ward & Moss, 2000
). Recently, the enhanced green fluorescent protein (EGFP) from Aequorea victoria has been fused to the VV B5R protein so that processes such as viral morphogenesis can be monitored in real time (Hollinshead et al., 2001
; Ward & Moss, 2001
). These studies showed that IEV particles move along microtubules at rates of 4098 µm/min rather than being transported by actin polymerization as is seen with intracellular bacteria (Cudmore et al., 1995
, 1996
). Here a further characterization of vB5REGFP, a recombinant VV in which the B5R SCR domains have been replaced with the EGFP, is reported. We demonstrate that vB5REGFP expresses and incorporates a 40 kDa chimaeric B5REGFP protein into the membranes used to wrap IMV to form IEV and into virus particles wrapped by those membranes. The vB5REGFP virus does not make actin tails but nonetheless virus particles are present on the cell surface, indicating actin-independent transport. Infection with vB5REGFP produced a plaque size intermediate between that formed by wild-type (WT) virus and v
B5R, and gave reduced levels of EEV. A virus lacking all four SCR domains (Herrera et al., 1998
) also showed a reduced plaque size, contrary to a previous report, but consistent with the reduced actin tail formation by that virus.
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Methods |
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Plasmid construction.
Construction of an in-frame mutation in which the four SCR domains of the B5R ORF were replaced with the EGFP ORF was achieved by splicing by overlap extension (Horton et al., 1989 ) and gene cloning. pSTH2 (Engelstad et al., 1992
) contains the entire B5R gene and flanking sequences cloned into pUC13 and was used as a template for a PCR to generate two fragments, which included the 5' and 3' ends of the B5R ORF. For details of oligonucleotides used see Hollinshead et al. (2001)
. Briefly, a 387 bp fragment corresponding to the 5' end of the B5R gene and including the B5R signal peptide, and a 579 bp fragment encoding the 3' end of the B5R protein including the stalk region, transmembrane domain and the cytoplasmic tail were fused to the EGFP ORF (Clontech) and assembled into a single gene in which the EGFP ORF replaced the SCR domains of B5R. This was then cloned into pSJH7 (Hughes et al., 1991
) to form pB5REGFP. The fidelity of the chimaeric gene was confirmed by sequencing.
Recombinant virus construction.
The recombinant VV vB5REGFP was constructed by transient dominant selection (Falkner & Moss, 1990 ) using the E. coli guanine xanthine phosphoribosyltransferase (Ecogpt) gene as the transient selectable marker. CV-1 cells were infected with VV strain WR at 0·1 p.f.u. per cell and transfected with pB5REGFP in the presence of Lipofectin (Gibco BRL) as recommended by the manufacturer. Recombinant virus expressing Ecogpt was selected by three rounds of plaque purification in BS-C-1 cells in the presence of mycophenolic acid (MPA). The MPA-resistant virus was then plaque purified three times on D98R cells in the presence of 6-thioguanine to select against virus expressing the Ecogpt gene. Ecogpt-negative virus containing B5REGFP was identified by PCR, and stocks were prepared and titrated by plaque assay on BS-C-1 cells.
Immunoblotting.
RK13 cells were infected at 10 p.f.u. per cell and cell extracts were prepared 24 h post-infection (p.i.) as described previously (Parkinson & Smith, 1994 ). Purified IMV was prepared from Dounce-homogenized infected cells by sucrose density-gradient centrifugation as described by Mackett et al. (1985)
. EEV was collected from the supernatants of infected cells by centrifugation as described by Mathew et al. (1998)
. After resolution by SDSPAGE (10% gel), proteins were transferred to nitrocellulose membranes (Towbin et al., 1979
) and detected by incubation with specific antibodies. To detect B5R or EGFP the membrane was incubated with rabbit
-B5R (Galmiche et al., 1999
) diluted 1:2000 or mouse mAb JL-8 (Clontech) diluted 1:1000. To confirm that the cells had been infected equally by each of the viruses the membrane was incubated with mouse mAb AB1.1 directed to the VV D8L gene product (Parkinson & Smith, 1994
). Bound antibodies were detected by anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (Amersham) diluted 1:1000. Membranes were incubated with the ECL Western blotting detection kit (Amersham) as per the manufacturers instructions and exposed to X-ray film (Kodak). Images were collected and processed using Precision Pro Scan HP and Adobe Photoshop software.
Plaque phenotype.
The plaque phenotype was examined on BS-C-1 cells under semi-solid (MEM/1·5% carboxymethylcellulose/2·5% FBS) or liquid (MEM/2·5% FBS) overlays. At 3 or 4 days p.i. the medium was removed and the cell monolayers were stained with 0·1% crystal violet in 15% ethanol. Images were collected and processed using Precision Scan Pro HP and Adobe Photoshop software.
Infectivity assays.
RK13 cells were infected at 10 p.f.u. per cell and the culture supernatant and infected cells were harvested at 24 h p.i. The titre of infectious virus in the culture supernatant was measured by clarifying the supernatant fraction via low speed centrifugation (2000 r.p.m., 10 min; Beckman GPR bench top centrifuge) followed by plaque assay on BS-C-1 cells in the presence or absence of mAb 2D5 (Ichihashi, 1996 ) to neutralize IMV as described previously (Law & Smith, 2001
). Virus infectivity present in cells was measured by scraping the monolayer into PBS, combining this with the pellets obtained from clarifying the culture supernatant (see above), followed by three cycles of freezethawing before plaque assay on BS-C-1 cells.
Immunofluorescence.
Cells growing on glass coverslips (Chance Proper) were infected at 1 or 10 p.f.u. per cell. At the indicated time p.i. cells were fixed in 4% paraformaldehyde (PFA) in 250 mM HEPES for 20 min on ice. The cells were then blocked and permeabilized in PBS containing 0·1% Triton X-100 and 10% FBS for 30 min. MAbs 15B6 (Hiller & Weber, 1985 ) and AB1.1, which recognize the F13L and D8L proteins, respectively, were used to detect virus particles. Bound mAbs were detected by either a fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG antibody, diluted 1:50, or a tetramethylrhodamine B isothiocyanate (TRITC)-conjugated donkey anti-mouse IgG antibody, diluted 1:100 (both from Jackson Immunoresearch Laboratories). Alternatively, cells were incubated with rabbit
-B5R (Galmiche et al., 1999
), diluted 1:200, or mouse mAb JL-8, diluted 1:100, at 37 °C for 1 h prior to fixation in 4% PFA. Fixed cells were then blocked and permeabilized in PBS containing 0·1% saponin and 10% FBS for 30 min. Bound mAbs were detected by either a TRITC-conjugated donkey anti-rabbit IgG antibody, diluted 1:100, or TRITC-conjugated donkey anti-mouse IgG antibody, diluted 1:100, (both from Jackson Immunoresearch Laboratories). TRITCphalloidin (Sigma) was used to stain for F-actin. 4,6'-Diamine-2-phenylindole (DAPI) was added to the mounting medium to stain for DNA. Cells were analysed using a Zeiss LSM 510 confocal laser-scanning microscope. Images were collected and processed using LSM 510 acquisition and Adobe Photoshop software.
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Results |
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BS-C-1 cells were infected with 10 p.f.u. per cell and the levels of intracellular and extracellular virus were determined at 24 h p.i. Under these conditions, the infectious virus titre in cells infected with vB5REGFP was reduced approximately 2-fold compared to WR, and the titre of virus in the supernatant was reduced 3-fold compared to WR (Fig. 7a). Therefore, the proportion of total infectivity that was released into the culture medium was similar for WR (0·21%) and vB5REGFP (0·14%). In comparison, only 0·026% of total infectivity was released from cells infected by v
B5R, consistent with the previous report that the titre of extracellular virus produced by v
B5R was reduced approximately 10-fold compared to WT (Engelstad & Smith, 1993
). The result with vB5REGFP contrasted with other mutants in which the SCR domains had been deleted, where there was a 10- to 50-fold increase in EEV (Herrera et al., 1998
; Mathew et al., 1998
), but was in accord with another report where the extracellular domain of the B5R protein was replaced with coding sequences from the VV haemagglutinin and that resulted in decreased levels of EEV (Mathew et al., 2001
).
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Discussion |
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Katz et al. (1997) showed that fusion of the B5R transmembrane and the cytoplasmic tail to HIV gp120 was sufficient to localize the protein to the wrapping membranes and EEV particles. Consistent with this, immunofluorescence showed that the B5REGFP protein co-localizes with particulate intracellular structures that were shown to be enveloped virus particles because they contained DNA and the F13L protein. Like WT B5R, B5REGFP is also present in the Golgi and on the cell surface. Together, this demonstrates that B5REGFP is incorporated into the wrapping membranes and subsequently into IEV, CEV and EEV particles. The incorporation of B5REGFP into virus particles has been investigated by immunoelectron microscopy (Hollinshead et al., 2001
).
Actin tail formation by VV is inhibited in mutants in which one or more of the SCR domains of B5R are missing (Herrera et al., 1998 ; Mathew et al., 1998
). In agreement with this observation, actin tail formation by vB5REGFP was reduced 86% compared with WR. Nonetheless, CEV particles were detected on the surface of infected cells by immunofluorescence (here) and electron microscopy (Hollinshead et al., 2001
). This supports the view that IEV particles utilize microtubules (Hollinshead et al., 2001
; Ward & Moss, 2001
) rather than the polymerization of actin (Cudmore et al., 1995
) for their transport from the site of wrapping to the cell surface. Consistent with the reduction in actin tail formation, the cell-to-cell spread of vB5REGFP was diminished resulting in a smaller plaque phenotype than those formed by WT VV. Another B5R mutant virus in which all the SCR domains were deleted, W-B5R
SCR14, was reported to form a plaque phenotype comparable to WT (Herrera et al., 1998
). However, in this study, we found that W-B5R
SCR14 had a small plaque phenotype more similar to that of vB5REGFP than WT. This reinforces the view that the SCR domains are required for actin tail formation (Mathew et al., 1998
) and W-B5R
SCR14 now fits with the phenotype of all other VV mutants that have diminished actin tail formation in having a reduced plaque size.
The titres of IMV and EEV made by vB5REGFP were reduced 2- and 3-fold compared to WT, but the proportion of virus released into the supernatant was similar. This contrasted with some other SCR mutants (Herrera et al., 1998 ; Mathew et al., 1998
) that formed 10- to 50-fold more EEV. However, unlike those mutants where B5R sequences were just deleted, the vB5REGFP mutant has B5R sequences fused to a foreign protein sequence, and previously where the SCR domains of the B5R protein were fused to the extracellular domain of the VV haemagglutinin, the formation of EEV was reduced (Mathew et al., 2001
).
In summary, infection with vB5REGFP, a virus in which the SCR domains have been replaced with EGFP, resulted in a significant decrease in actin tail formation and consequently a small plaque phenotype. Furthermore, infection with W-B5RSCR14, a VV mutant lacking the SCR domains of B5R, also gives a small plaque phenotype, contrary to a previous report. These observations reinforce the view that the ability of VV to spread efficiently from cell to cell is dependent on the formation of actin tails. Lastly, the vB5REGFP virus is a useful tool for the study of VV morphogenesis, egress, re-entry and spread. This mutant has been used previously to show that IEV particles move to the cell surface associated with microtubules (Hollinshead et al., 2001
). In the future, this mutant may be useful to study the interactions between EEV and the host cell during virus binding and re-entry in vitro, and virus infection in vivo, as has been described recently for the spatial-temporal imaging of bacterial infections (Zhao et al., 2001
).
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Acknowledgments |
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References |
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Received 27 August 2001;
accepted 20 October 2001.