Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, D-17498 Insel Riems, Germany1
Max von Pettenkofer-Institut, Genzentrum, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany2
Author for correspondence: Nikolaus Osterrieder. Fax +49 38351 7151. e-mail klaus.osterrieder{at}rie.bfav.de
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Abstract |
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The MDV-1 genome harbours the alphaherpesvirus-specific set of glycoprotein (g) genes, except for a gG gene (Lee et al., 2000 ; Tulman et al., 2000
). Expression of gB, gC, gE, gH, gI, gK, gL and gM has been shown (Binns & Ross, 1989
; Osterrieder, 1999
; Ren et al., 1994
; Ross et al., 1989
; Tan et al., 2001
; Wu et al., 1999
; Yoshida et al., 1994
). A differential expression of gC could be demonstrated. The secreted A antigen (gC) is expressed from virulent MDV-1 strains but serial passage of MDV-1 in chicken embryo fibroblast (CEF) cells resulted in abrogation of gC expression, which was correlated with a loss of virulence (Churchill et al., 1969
; Wilson et al., 1994
). Recent studies have shown that gB, as well gE and gI, are indispensable for the ability of MDV-1 to spread from cell to cell (Schumacher et al., 2000
, 2001
). These results were in contrast to those obtained for other members of the subfamily Alphaherpesvirinae, for which gE and gI are nonessential, with the notable exception of VZV (Balan et al., 1994
; Mijnes et al., 1996
; Yoshitake et al., 1997
; Zuckermann et al., 1988
). VZV mutants lacking gI exhibited cell type-specific growth properties, whereas deletion of gE resulted in a virus that was unable to replicate in the investigated cell lines (Cohen & Nguyen, 1997
; Mallory et al., 1997
, 1998
).
gM and its complex partner, the UL49.5 gene product, which is glycosylated and therefore designated gN in the case of suid herpesvirus type 1 (pseudorabies virus, PRV) and EpsteinBarr virus (EBV), are conserved throughout all three subfamilies of the Herpesviridae. Despite this conservation, deletion of one of the respective open reading frames (ORF) in herpes simplex virus type 1 (HSV-1), bovine herpesvirus type 1, equine herpesvirus type 1 (EHV-1) or PRV resulted in viable virus progeny (Baines & Roizman, 1991 ; Liang et al., 1993
; Rudolph et al., 2002
; Osterrieder et al., 1996
; Dijkstra et al., 1996
; Jöns et al., 1998
), but 10- to 100-fold reduced virus titres and reductions in virus plaque sizes were observed. Deletion of the EBV gM or gN ORF also resulted in viable progeny (Lake et al., 1998
; Lake & Hutt-Fletcher, 2000
); however, analysis of a library of human cytomegalovirus (HCMV) mutants suggested that gM is essential for growth of this betaherpesvirus (Hobom et al., 2000
). Although the EHV-1 and PRV gM and UL49.5 products are nonessential, concomitant deletion of gM and gEgI led to significantly reduced virus titres and plaque sizes, which were caused by an inefficient secondary envelopment (Brack et al., 1999
; Seyboldt et al., 2000
). The same observation of reduced growth properties caused by inefficient secondary envelopment was made if the UL49.5 gene product of EHV-1 was absent (Rudolph et al., 2002
). These results suggested similar, albeit not strictly overlapping, functions of two glycoprotein complexes of the Alphaherpesvirinae (Brack et al., 1999
, 2000
; Seyboldt et al., 2000
; Rudolph et al., 2002
). The essential function of both gE and gI for MDV-1 and of at least gE in its closest relative, VZV, demonstrated that this interpretation does not hold for highly cell-associated members of the subfamily Alphaherpesvirinae. In the case of both MDV-1 and VZV, the function of the gEgI complex cannot be compensated for by the putative UL10UL49.5 complex (Schumacher et al., 2001
; Cohen & Nguyen, 1997
; Mallory et al., 1997
).
In an effort to systematically analyse the complex action of membrane and tegument proteins in MDV-1 cell-to-cell spread, the aim of this study was to explore the effect of the deletion of UL10 (gM) and UL49.5. Virus mutants were constructed using an infectious MDV-1 bacterial artificial chromosome (BAC) clone and recE/T-based mutagenesis (Muyrers et al., 1999 ; Narayanan et al., 1999
; Zhang et al., 1998
). Both, UL10- and UL49.5-negative MDV-1 were unable to grow in cultured cells; however, growth of the mutants was restored after co-transfection of an expression plasmid harbouring the respective gene. The results demonstrate that both gM and the UL49.5 gene product are indispensable for MDV-1 replication.
UL10 and UL49.5 deletion mutants were generated from the infectious MDV-1 BAC20 clone (Schumacher et al., 2000 , 2001
; Dorange et al., 2002
). The UL10 (20
gM) and the UL49.5 (20
49.5) deletion mutants were generated by recE/T cloning in Escherichia coli DH10B cells, essentially as described previously (Muyrers et al., 1999
; Narayanan et al., 1999
; Schumacher et al., 2000
; Zhang et al., 1998
). The respective ORF was replaced with the kanamycin resistance gene (kan) amplified from plasmid pACYC177 (MBI Fermentas) by PCR using appropriate primers (Table 1
). For recombination of the linear fragment into BAC20, 300 ng of the purified PCR product was electroporated using standard parameters (1·25 kV/cm, 200
and 25 µF). Cells were grown in 1 ml LB for 90 min and plated onto LB agar plates containing 30 µg/ml chloramphenicol and 30 µg/ml kanamycin. Extrachromosomal DNA of double-resistant colonies was prepared by column chromatography (Qiagen). Mutant BAC DNA was analysed by restriction enzyme digestion using BamHI or HindIII, Southern blot analysis and cycle sequencing of the recombination sites (Schumacher et al., 2000
, 2001
). The results of these investigations demonstrated that the respective ORF was deleted successfully from clones 20
gM and 20
49.5 (Fig. 1
).
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The results of the transfection experiments using mutant MDV-1 BAC20 DNA and the generation of revertant viruses, in which plaque formation in CEF cells was restored, indicated that growth of MDV-1 in cultured cells required both the UL10 and the UL49.5 gene products. To confirm this assumption further, mutant 20gM and 20
49.5 DNA was co-transfected with 10 µg of the expression plasmids pcMgM or pcM49.5, respectively (Table 1
). The plasmids contained the respective ORFs cloned into vector pcDNA3 (Invitrogen), where expression is controlled by the HCMV immediate early promoter/enhancer (Osterrieder, 1999
). Co-transfection of 20
gM with pcMgM, which provided the deleted gM gene in trans, resulted in the formation of MDV-1-specific plaques, as visualized by the anti-pp38 mAb, H19. Similarly, co-transfection of mutant 20
49.5 BAC DNA with pcM49.5 also resulted in MDV-1-specific plaques (Fig. 2C
). The results of the co-transfection experiments eventually demonstrated that cell-to-cell spread of MDV-1 requires the presence of both the UL10-encoded gM and the UL49.5 gene product.
The results presented here demonstrate that growth of MDV-1, i.e. the formation of virus plaques by direct cell-to-cell spread, requires both the UL10 and the UL49.5 gene products. Being a strictly cell-associated virus, MDV-1 appears to depend on a different set of (glyco)proteins for growth in cultured cells when compared to other members of the Alphaherpesvirinae. Previously, we demonstrated that both gE and gI are essential for the growth of MDV-1. These results indicated that the partially redundant functions of the gEgI complex and the gMUL49.5 complex, as was described for EHV-1 and PRV, could not be confirmed in the case of MDV-1 (Schumacher et al., 2001 ). The findings of the essential nature of MDV-1 gM and the UL49.5 gene product, or the putative complex between these transmembrane proteins, indicate that their action cannot be substituted for by another virus protein. Therefore, the gEgI and the putative gMUL49.5 complexes of MDV-1 act in different and probably nonoverlapping steps of cell-to-cell spread. Hence, the mechanism of MDV-1 cell-to-cell spread appears to be different from that of other members of the subfamily Alphaherpesvirinae. These observations are not entirely surprising, because MDV-1 expresses neither gD nor gG in cultured cells and is unable to produce free infectious virus (Lee et al., 2000
; Tan et al., 2001
; Tulman et al., 2000
; Biggs, 2001
). It is conceivable that in the absence of gD, which is essentially involved in cell-to-cell spread of most members of the subfamily Alphaherpesvirinae, the presence of other players involved in this process is more important. This interpretation is supported by the fact that the expression of gE and gI is required for the efficient growth of VZV, which also does not encode a gD homologue and is highly cell associated (Cohen & Nguyen, 1997
; Mallory et al., 1997
, 1998
). The high cell association in cultured cells and most target cells in vivo of both MDV-1 and VZV, which produce free infectious virus only in the feather follicle epithelium (MDV-1) and in epithelial cell pustules in the acute rash of chicken pox or recurrent infections (VZV), may be reflected by a different evolution of egress and cell-to-cell spread mechanisms when compared to other members of the Alphaherpesvirinae, which efficiently release cell-free infectious virus.
In light of the obviously different requirements for virus spread, it is important to note also that a different set of tegument proteins is necessary for the growth of MDV-1 when compared to other members of the subfamily Alphaherpesvirinae. Recently, the essential nature of the MDV-1 VP22 (UL49) homologous protein, which is dispensable for the growth of HSV-1 and PRV, was demonstrated. Conversely, the MDV-1 VP16 homologue is nonessential for virus assembly, as had been reported for HSV-1, and the expression of the UL48 gene product, VP16, was barely detectable in MDV-1-infected cells (Dorange et al., 2000 ). Further studies will therefore concentrate on the interaction between MDV-1 gMUL49.5 and gEgI complexes with tegument proteins. In addition, the nature of the putative gMUL49.5 complex will be analysed by the generation of specific antibodies.
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References |
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Received 1 November 2001;
accepted 21 December 2001.