1 Institute for Medical Microbiology, Infectious and Epidemic Diseases, Ludwig-Maximilians-Universität München, Veterinärstr. 13, D-80539 Munich, Germany
2 Institute for Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstraße 5, D-80802 Munich, Germany
3 Boehringer Ingelheim Vetmedica GmbH, D-55216 Ingelheim, Germany
Correspondence
Antonie Neubauer
toni.neubauer{at}micro.vetmed.uni-muenchen.de
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ABSTRACT |
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Present address: MSD Sharp&Dohme GmbH; D-85440 Haar, Germany.
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INTRODUCTION |
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Amidst a large number of herpesviral glycoproteins, gM is remarkable because it is conserved through all herpesvirus subfamilies but not essential for replication of most alphaherpesviruses in cell culture e.g. herpes simplex virus 1 (HSV-1), pseudorabies virus (PRV) or EHV-1 (Baines & Roizman, 1991; Dijkstra et al., 1996
; Osterrieder et al., 1996
). However, merely deleting the respective sequences of Marek's disease virus (MDV), a strictly cell associated alphaherpesvirus, abolishes virus growth in vitro (Tischer et al., 2002
).
EHV-1 gM has been studied in some detail. It is thus known that the product of gene 52 is translated at late times post-infection (p.i.) into a 44 kDa precursor protein that is co- and post-translationally processed into a 5055 kDa protein (Osterrieder et al., 1997). The glycosylated type III transmembrane protein is predicted to cross membranes eight times with the 95 aa C-terminal tail of the polypeptide passing into the cytoplasm of infected cells (Telford et al., 1992
; Pilling et al., 1994
; Seyboldt et al., 2000
). The most prominent epitope(s) are apparently located within this tail-region (Osterrieder et al., 1996
; Day, 1999
). gM-oligomers are incorporated into virions and the protein physically interacts with the EHV-1 UL49·5 homologue (gene 10). The meaning of this interaction is not yet fully understood but it is important for the transport of gM into the trans-Golgi-network, and only the mature gM is fully functional (Rudolph et al., 2002
). Similar complex formations between gM and the respective UL49·5 products have been described for PRV, bovine herpesvirus 1 (BoHV-1), EpsteinBarr virus and human cytomegalovirus (Jöns et al., 1998
; Lake et al., 1998
; Wu et al., 1998
; Mach et al., 2000
). In most herpesviruses analysed to date, deleting gM does not result in a marked phenotype in vitro, causing its specific function still to be a matter of discussion. Studying a gM, gE and gI triple deletion mutant in PRV, however, revealed a clear defect in secondary envelopment of particles (Brack et al., 1999
). The interruption of gM sequences in the EHV-1 low passage strain RacL11 leads to a reduction in plaque sizes of about 50 % and to a 50100-fold decrease in extracellular virus titres. In contrast, deletion of gM in its highly passaged cell culture derivative, the modified live vaccine strain RacH, has only a minor influence on plaque sizes (reduction of about 10 %) or production of infectious virus progeny (about 10-fold decrease; Osterrieder et al., 1996
; Neubauer et al., 1997b
; Seyboldt, 2000
; Rudolph & Osterrieder, 2002
). Again, removing gM from a virus background lacking expression of gE and gI resulted in a major impact on virus release and plaque phenotype. A block in secondary envelopment of virions at Golgi vesicles was reported, suggesting that gM or rather the gM/UL49·5 complex and the gE/gI complex play additive or partially overlapping roles in EHV-1 virus egress (Seyboldt et al., 2000
). Based on its structure, formation of an ion channel by gM was discussed. However, EHV-1 gM by itself does not constitute an ion channel as assessed after transient transfection into Xenopus laevis oocytes (Osterrieder et al., 1997
). Interestingly, comparing the protective potential of a gM-negative RacH to that of RacH itself in a mouse model revealed an increased immunogenicity of the recombinant virus, indicating another role of EHV-1 gM. It is hypothesized that the multiple membrane spanning protein might also serve as a ligand for molecules modulating immune responses (Osterrieder et al., 2001
).
The aim of this study was to investigate the function of EHV-4 gM in cell culture. Using an anti-EHV-1 gM serum, EHV-4 gM was identified and its role assessed after deleting a major part of gM-sequences in a wild-type EHV-4 strain. A major decrease in plaque sizes and a substantial impact on the ability to replicate in non-complementing cells was noted. These data together with an analysis of infected cells by electron microscopy allowed the conclusion that the deletion of gM strongly affects mechanisms influencing virus egress and also cell-to-cell spread. This study also represents the first step towards understanding the differences in the replication cycle of EHV-1 and EHV-4.
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METHODS |
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Cells and viruses.
Vero cell clone C1008, Edmin337 cells and Rk13 cells were maintained as previously described (Neubauer et al., 1997a). Recombinant cell line Vero-gM was generated by Effectene (Qiagen) mediated co-transfection of plasmids pCgM4 and pSV2neo (conferring resistance to G418; Neubauer et al., 1997a
) into Vero cells. G418 (Calbiochem) resistant cell clones were selected for trans-complementation of a gM-negative EHV-1 (KyA
gM; Seyboldt et al., 2000
).
The EHV-4 strain used had been isolated from a horse with rhinitis from the USA. It was initially characterized and then passaged in Vero cells. The eleventh to thirteenth cell culture passages were used in this study. EHV-1 strain RacH and its recombinant derivative HgMGFP+ were propagated on Rk13 cells (Hübert et al., 1996
; Seyboldt, 2000
).
Purification of virions.
Cells were infected at a m.o.i. of 0·5, harvested when the cytopathic effect was complete, and subjected to two rounds of freeze-thawing. Cell detritus was then removed by low-speed centrifugation. The virus suspension was carefully layered onto a 30 % sucrose cushion and centrifuged for 3 h at 23 000 r.p.m. in a Beckman SW 28 rotor. This step was repeated once.
Generation of recombinant viruses.
Initial experiments had shown that the GFP-marker was easier to handle when selecting for EHV-4 recombinants than the lacZ-marker. Homologous recombination into EHV-4 was therefore achieved by calcium phosphate mediated co-transfection of Vero-gM cells with plasmid pgM4GFP+ (see Fig. 2b) and EHV-4 DNA. Recombining sequences of plasmid pgM4w with DNA of the generated gM-negative EHV-4, E4
gM-GFP, resulted in another mutant virus, E4
gM-w (see Fig. 2c
). Finally, the gM-repaired EHV-4, E4RgM (see Fig. 2a
), was isolated after co-transfection of plasmid pgM4R with DNA of E4
gM-GFP into Vero-cells. Supernatants of transfected cells were plated on the respective cells and GFP-positive plaques were detected under a methocellulose overlay using an inverted fluorescence microscope (Axiovert; Zeiss).
Southern blot and control sequence analysis.
Viral DNA was prepared from Vero or Vero-gM cells infected with the respective viruses by using standard protocols. Agarose gel electrophoresis and Southern blotting were done as previously described (Osterrieder et al., 1996). GFP-sequences, released from the vector pEGFP-C1 (Clontech) and the viral fragment of plasmid pgM4R (see Fig. 2a
) were used as probes. The region encompassing the manipulated sequences within recombinant virus E4
gM-w was PCR amplified and the nucleotide sequence of the PCR product determined using primers depicted in Fig. 2(c)
.
Western blotting and antibodies.
Cells were infected with the indicated viruses (m.o.i. of 1) and lysates prepared at the stated time points p.i. To inhibit synthesis of viral DNA, cells were infected and incubated for 24 h in the presence of phosphonoacetic acid (PAA; 0·5 or 1·0 mg ml1). To digest N-linked carbohydrates, virions suspended in deglycosylation buffer (Klupp et al., 1998) were incubated for 16 h in the presence or absence of PNGase-F (2 U; Roche Molecular Biochemicals). Samples were mixed with buffer containing 5 % 2-mercaptoethanol (Sambrook et al., 1989
) and then either heated to 99 °C for 5 min or kept on ice. Proteins were separated by using either SDS-10 % Tris/glycine PAGE or SDS-16·5 % Tris/tricine PAGE (UL11) and blotted onto nitrocellulose filters. The monoclonal anti-EHV-1 gB antibody (3F6; Allen & Yeargan, 1987
), anti-EHV-1 UL11 (Schimmer & Neubauer, 2003
) and gM polyclonal rabbit antibodies (Seyboldt et al., 2000
), and polyclonal anti-EHV-4 gE and gI mouse sera (Damiani et al., 2000a
) were used in this study. Antibody binding was visualized using anti-species immunoglobulin G peroxidase conjugates (Sigma) followed by ECL detection (Pharmacia-Amersham).
Virus growth kinetics and plaque size measurements.
Viruses were allowed to adsorb to Vero, Vero-gM or Edmin337 cells in 24- or 6-well plates for 90 min at 4 °C. After another 90 min of virus penetration at 37 °C, extracellular infectivity was inactivated using a citrate buffer (pH 3·0). At the indicated times parallel samples of supernatants and infected cells were harvested. Cell samples were treated with citrate buffer to exclude contamination with extracellular infectivity, while supernatants were cleared of cellular debris by low-speed centrifugation. Intracellular and extracellular virus titres were compared by plaque titration on Rk13, Vero or Vero-gM cells, respectively.
Plaque sizes were determined on cells in 6-well plates infected with 50 p.f.u. per well of the respective viruses and incubated for 4 days under a methocellulose overlay. Plaques were screened, immunofluorescently labelled as described elsewhere (Schimmer & Neubauer, 2003) and digitally documented in an Axiovert microscope (Zeiss) or stained with crystal violet and measured. For each virus, maximum diameters of 150 randomly selected plaques were determined using a magnifying glass with a metric scale and mean sizes calculated. Measurements were compared with those of parental plaques that were set to 100 %.
Penetration assays.
Penetration assays were performed as previously described (Neubauer et al., 1997a). Briefly, 100 p.f.u. of the respective viruses were allowed to adsorb for 90 min to Vero cells. The assay was started by shifting the incubation temperature to 37 °C and replacing supernatants with fresh medium. At different times after this shift, extracellular virus was inactivated with citrate buffer (pH 3·0) and parallel control samples were washed with PBS. The penetration efficiency was taken as the percentage of plaques present after citrate treatment relative to the number of plaques present after control treatment.
Electron microscopy.
Vero or Edmin337 cells were infected at an m.o.i. of 1 with the different viruses. Cells were fixed at the indicated times p.i. for 2 h in 5 % glutaraldehyde, 4 % formaldehyde, buffered with a 0·1 M sodium phosphate buffer to pH 7·4 and then washed with 0·1 M sodium phosphate buffer. Cells were post-fixed with 1 % OsO4, 0·8 % K3Fe(CN)6 in 0·1 M sodium phosphate buffer for 2 h. All samples were stained with 2 % aqueous uranyl acetate for 90 min, dehydrated in graded ethanol and finally embedded in ERL 4206 (Spurr, 1969). The blocks were sectioned on an Ultracut microtome, stained with uranyl acetate and lead citrate (Plattner & Zingsheim, 1987
) and examined with a Philips CM10 transmission electron microscope at 80 kV.
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RESULTS |
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EHV-4 gM is important for cell-to-cell spread
To compare expression of selected proteins, Vero cells infected with EHV-4, E4RgM, E4gM-w or E4
gM-GFP were analysed by Western blotting or indirect immunofluorescence. In Fig. 1(b)
, it was shown that expression of gM was found in EHV-4- and E4RgM-infected cells only. Moreover, the deletion of gM-sequences did not detectably influence the production of gB, gE, gI or of the UL11-protein (Figs 1b, 3b
), indicating not only that expression of other early-late EHV-4-proteins was not altered, but also specifically that the expression of the adjacent UL11-ORF was unaffected.
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Deletion of gM severely impairs EHV-4 egress
To compare virus growth kinetics of the various mutant viruses, Vero cells in 24-well plates were infected at an m.o.i. of 2 and extracellular, and intracellular virus titres were determined separately at different times p.i. (Fig. 4a). Growth properties of the repaired virus E4RgM resembled those of EHV-4, whereas E4
gM-w and E4
gM-GFP presented with a marked growth defect. Within these experiments extracellular infectivity could not be detected before 24 h p.i. Even at 30 h p.i. only extremely low extracellular titres were observed, although corresponding cells clearly showed cytopathic effect. The reduction in intracellular infectivity was substantial as well. However, it never reached 100-fold (maximal 84-fold between EHV-4 and E4
gM-w at 24 h p.i.) and detection was only delayed by one time point. EHV-4 and E4RgM as well as E4
gM-GFP and E4
gM-w behaved virtually indistinguishably, respectively; therefore only the data for EHV-4 and E4
gM-GFP are depicted in the following experiments. To prove the direct link between this growth defect and gM expression, a similar kinetic was generated on Vero-gM cells, showing that the gM-expressing cells were significantly able to rescue virus growth (Fig. 4a
). More experiments were dedicated to the question of whether this effect on virus replication was cell type- or virus-specific. Therefore, virus growth was also addressed on equine dermal cell line Edmin337 in 6-well plates. However, as shown at 24 h p.i., intracellular titres of gM deletion viruses were again reduced by about 100-fold, and no extracellular infectious virus progeny was detectable (Fig. 4b
). In addition, although EHV-1 replication was clearly ineffective on Vero cells, the influence of deleting gM in EHV-1 (Seyboldt, 2000
) was minor relative to the effect in EHV-4 (Fig. 4c
, shown at 24 h p.i.; m.o.i. of 0·02). In the absence of gM the ability of EHV-4 to directly infect adjacent cells was markedly reduced, as shown in Fig. 3
. Therefore to analyse the effect of cell-to-cell spread on EHV-4 growth kinetics, replication was assayed after inoculation with different m.o.i. values. It was assumed that the effect of cell-to-cell spread on growth kinetics should increase with lower m.o.i. and longer incubation. To this aim, Vero cells in 6-well plates were infected with m.o.i. values of 0·5, 0·02 or 0·001 and resulting virus titres were measured at 24 and 48 h p.i. Representative data are given in Fig. 4(d)
. As the observed relative reduction (given as the factor of difference between titres) remained similar, no matter what the input m.o.i. had been, it could be concluded that the replication defect of gM-negative EHV-4 detected in these kinetics (Fig. 4a
) was not appreciably related to the efficiency of cell-to-cell spread. Taken together, the presented series of experiments allowed us to conclude that the marked defect in EHV-4 replication was a specific consequence of the deletion of EHV-4 gM and that it was not Vero-cell-specific.
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DISCUSSION |
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The primary focus of this study was to elucidate the function of EHV-4 gM. The gM deletion virus isolated was unexpectedly growth deficient, and to avoid an effect of the GFP-expression another recombinant EHV-4 without GFP-marker sequence was generated. In further experiments a set of four viruses was thus compared and it was readily observed that neither GFP-expression nor undetected mutations were of particular influence. These conclusions were sustained by demonstration of good complementation on Vero-gM cells, which constitutively express EHV-4 gM. Moreover, it was shown that expression of the UL11 product, which is encoded adjacent to gM (UL10), was unaffected as well as that of other structural proteins. If the expression of the second adjacent ORF, UL9, had been reduced, a general reduction in expression of late proteins would have been expected, as the HSV-1 UL9 homologue is known to be the essential DNA origin binding-protein (Roizman & Knipe, 2001).
Taking all data presented in this study into consideration, it could be concluded that EHV-4 gM is important for both virus egress and cell-to-cell spread. As the deletion only marginally influenced virus penetration and as expression of the protein depended on synthesis of viral DNA, newly synthesized gM could only function late in infection. Plaque sizes, which are generally taken as a surrogate marker for efficiency of cell-to-cell spread, were drastically diminished in the absence of gM, and irrespective of cell-to-cell spread an important function in assembly and egress was demonstrated. Assuming that similar to replication of other alphaherpesviruses in vitro (Johnson & Huber, 2002), the processes of EHV-4 cell-to-cell spread and virus egress are at least partially distinct in Vero cells, these data suggest that the replication defect in gM deletion mutants in cell culture involves a mechanism necessary for both pathways, or completely different mechanisms exerted by a multifunctional gM, or an early step in assembly influencing both processes. The latter appears to be less probable as one would then have expected to note a morphological correlate in ultra-thin sections of infected cells.
Deleting gM had a more pronounced effect on EHV-4 replication than reported for EHV-1. The question of whether this observation really was EHV-4-specific was addressed by using two different cell lines, Vero and Edmin337, in various experiments, thereby showing that the growth behaviour of gM-deleted EHV-4 was independent of the cell line used. Also, the growth disadvantage of the gM-deleted EHV-1, HgMGFP+, was similar on Vero cells to what had been reported on Rk13 cells (Seyboldt, 2000
), again corroborating the virus specificity of the observations in this study.
Apparently, EHV-4 gM is more important for a carefully balanced set of functional and structural interactions than the respective homologues in most other herpesviruses. Only the gM-homologue of the strictly cell associated MDV is essential for virus replication (Tischer et al., 2002). Therefore, it could be hypothesized that EHV-4 might be more cell-associated than EHV-1. Further experiments will be necessary to address such speculations. In some alphaherpesviruses, including EHV-1, the simultaneous deletion of gM, gE and gI increases the observed defects in plaque formation and virus growth, demonstrating that these three proteins fulfil somewhat overlapping functions in secondary envelopment (Brack et al., 1999
; Seyboldt et al., 2000
), but none is essential by itself. One of the reasons to initiate electron microscopic analysis was the idea that gM alone might be so important for secondary envelopment in EHV-4 that an accumulation of particles in the Golgi area might occur. This was not the case within the settings of our study. With the exception of a complete absence of extracellular particles, no distinct phenotype could be discerned in sections of cells infected with the gM-mutants. EHV-4 gM must therefore be important for more than just secondary envelopment. As gM probably spans membranes several times it might thereby influence or stabilize them, and it is tempting to assume a function in all steps of virus replication involving fusion of virus envelopes and cellular membranes. Although exact mechanisms are still unknown, fusion is certainly important in virus penetration, assembly of particles, egress and also cell-to-cell spread. EHV-1 and PRV gM have been shown to prevent syncytia formation efficiently in a PRV membrane fusion assay (Klupp et al., 2000
) and thus directly to affect fusion of membranes, and both the EHV-1- and EHV-4-homologues have been shown to, at least slightly, facilitate virus penetration. As electron microscopic analyses can only give pictures of selected moments in replication, the stability, efficiency or even directionality of membrane fusion events might not be reflected in these analyses. In summary, a surprisingly marked replication defect was shown after deleting EHV-4 gM, strongly influencing the efficiency in cell-to-cell spread and virus egress. In combination with analyses of sections of infected cells by electron microscopy, it was demonstrated that the functions of EHV-4 gM are even more complex than had been assumed.
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ACKNOWLEDGEMENTS |
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Received 23 June 2004;
accepted 15 September 2004.
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