1 Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, D-17498 Insel Riems, Germany
2 Institute for Diagnostic Virology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, D-17498 Insel Riems, Germany
Correspondence
Martin Beer
beer{at}rie.bfav.de
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ABSTRACT |
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MAIN TEXT |
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The mutant viruses were syngeneic and based on wild-type BHV-1, subtype 2 strain Schönböken (Matheka & Straub, 1972; Engelhardt & Keil, 1996
), which was cloned as a BAC. Using this clone it could be demonstrated that gG and gE or the gEgI complex probably act in different steps of ctcs, because additive effects were observed after deletion of both gE and gG, and because deletion of gE resulted in a much greater reduction of virus plaque sizes than a deletion of gG.
BHV-1 strain Schönböken was propagated as described previously in MadinDarby bovine kidney (MDBK, ATCC CCL-22), cells, which were grown in Dulbecco's modified essential medium (DMEM) supplemented with 10 % foetal calf serum (FCS) (Engelhardt & Keil, 1996). To obtain a BHV-1 BAC, viral DNA was prepared from infected cells and co-transfected with recombinant plasmid p
gE-pHA2 by the calcium phosphate precipitation method exactly as described by Rudolph et al. (2002)
. Recombinant plasmid p
gE-pHA2 was generated by cloning of PCR fragments flanking the gE deletion (Fig. 1
A) and insertion of the PacI fragment of plasmid pHA2 (Adler et al., 2000
) into plasmid pTZ18R. The resulting construct contains an F origin of replication, the enhanced green fluorescent protein (GFP) open reading frame (ORF), and the Escherichia coli (E. coli) guanosine phosphoribosyl transferase (gpt) gene for selection in mammalian cells using mycophenolic acid, xanthine and hypoxanthine (Schumacher et al., 2000
; Adler et al., 2000
; Rudolph & Osterrieder, 2002
). Progeny fluorescing virus resulting from the co-transfections of viral and p
gE-pHA2 DNA was purified to homogeneity and checked for the presence of pHA2 sequences by Southern blot analysis (data not shown). Virus DNA was prepared 6 h after infection and used to electroporate E. coli DH10B cells, which were spread on chloramphenicol (cam)-containing agar plates (Schumacher et al., 2000
; Rudolph & Osterrieder, 2002
). Resistant colonies were grown in liquid medium and DNA was prepared by affinity chromatography (Qiagen). After primary testing of several BAC-containing colonies, one clone was chosen for further analysis and termed pBHV-1
gE. Restriction enzyme digestion and Southern blotting using pHA2 as a probe demonstrated that pBHV-1
gE exhibited the expected banding pattern and that mini F plasmid sequences had been inserted instead of a portion of the gE gene (Fig. 1B
). Subsequently, bovine kidney cells [PT11; RIE11; collection of cell lines in veterinary medicine at the Federal Research Centre for Virus Diseases of Animals (CCLV), Insel Riems] were transfected with 1 µg of pBHV-1
gE DNA isolated from E. coli. GFP-expressing cells which developed into BHV-1-specific plaques (Fig. 2
) were already seen 24 h after transfection. These results confirmed that the BHV-1 genome was successfully cloned as an infectious BAC in E. coli.
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In the next series of experiments, the growth properties of the generated mutant viruses were compared to those of the parental strain Schönböken. Single-step growth kinetics were determined on MDBK cells by infecting the cells at an m.o.i. of 1 for 1 h. Input virus was then inactivated by low-pH treatment using a citrate buffer (Highlander et al., 1987), and at the indicated times after infection, intra- and extracellular virus titres were determined. It could be shown that both intra- and extracellular virus titres remained virtually unaffected by deletion of gE, gG or both gE and gG from BHV-1 (Fig. 2A
), because no marked differences between the titres induced by the various viruses at any time post-infection could be detected. These results strongly suggested that the deleted US glycoproteins did not have a prominent effect on secondary envelopment or virus egress, even if a deletion of gE and gG was introduced into the BHV-1 genome. Previous studies had suggested that both gE and gG are involved in direct spread of infectivity from an infected to a neighbouring uninfected cell and that mislocalization of gE is responsible for impaired ctcs of gG deletion mutants (Nakamichi et al., 2000
). To examine the effect of a gE/gG double deletion on BHV-1 ctcs, MDBK cells were seeded in six-well plates (Nunc), and 200 p.f.u. of the virus mutants was used to infect 1 xtimes; 106 cells. At 2 days after infection under a 0·25 % methylcellulose overlay (Neubauer et al., 1997
), plaque diameters of at least 150 plaques of each virus were determined and mean diameters and standard errors were calculated. Values for parental strain Schönböken were set to 100 % and the plaque diameters observed for the mutant viruses were expressed relative to this value. It could be shown that deletion of gE resulted in a 45 % reduction in plaque diameters, whereas the single deletion of gG did not result in reductions of plaque diameters exceeding 19 % (Fig. 2B
). Simultaneous deletion of gE and gG resulted in virus plaques exhibiting a 55 % reduction in diameters (Fig. 2B
). These findings strongly suggested that gE and gG, which are both involved in direct ctcs, act in different and non-overlapping steps of this membrane fusion process.
The salient findings presented in this communication are that the entire genome of the wild-type BHV-1 strain Schönböken was cloned as an infectious BAC and that the absence of both gE and gG had an additive effect on direct ctcs of this Alphaherpesvirus. Construction of a BAC was a good basis for these experiments, because adaptation of double mutants, which is frequently observed by cell culture isolation of mutant herpesviruses, is unlikely using this novel technology. Previous studies had suggested that gE and gG function in ctcs and it was speculated that both glycoproteins may interact or act even synergistically in this membrane fusion event, because distribution of gE in infected cells was altered in the absence of gG expression (Nakamichi et al., 2002). The results presented here using gE and gG single or double deletion mutants, which were based on an infectious BHV-1 clone and included the analysis of relevant revertant viruses, clearly demonstrated that the absence of gG did not over-proportionally influence the growth properties of a gE-negative BHV-1 mutant. In addition, the observed additive effect strongly argues against the possibility that the reduced ctcs capabilities of gG-negative BHV-1 are caused by mislocalization of gE (Nakamichi et al., 2002
). Whereas gE was confirmed as a major player in BHV-1 ctcs, gG appears to play a minor role in a step of ctcs that is clearly independent from that mediated by gE. It is important to note that no significant reductions in intra- or extracellular virus titres were observed in any of the generated BHV-1 mutants, although virus plaque sizes were significantly reduced in the case of gE- and gE/gG-negative BHV-1 (>50 %). Mahony et al. (2002)
speculated that genetic differences between BHV-1 subtypes 1 and 2 are the reason for the absence of virus plaque formation, which they observed after deletion of gE. Since the BHV-1 BAC reported here was also derived from a subtype 2 strain (Schönböken; Matheka & Straub, 1972
), and plaque formation was clearly evident using methylcellulose overlays, other reasons may be responsible for these differing observations. Strain-specific properties, an influence of TK deletion on virus growth (Mahony et al., 2002
) or technical details, e.g. the use of different overlays for plaque size determinations, may be the reason for the differing interpretations of an effect of deletion of gE.
Taken together, our observations strongly support the hypothesis that Alphaherpesvirus secondary envelopment and egress on the one hand, and direct cell-to-cell spread on the other, are independent from each other, although fusion of membranes containing viral proteins is required for both processes. Future work will concentrate on the systematic analysis of BHV-1 tegument and membrane (glyco)proteins to contribute to the elucidation of the general principles of Alphaherpesvirus egress and direct cell-to-cell spread.
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ACKNOWLEDGEMENTS |
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Received 2 July 2002;
accepted 26 August 2002.