1991, 1993), is glycosylated in HSV-1 (Baines & Roizman,. 1993), equine herpesvirus 1 (EHV-1; Osterrieder et al., 1996) and PrV (Dijkstra et al., 1996). However ...
Journal of General Virology (1998), 79, 851–854. Printed in Great Britain ..........................................................................................................................................................................................................
SHORT COMMUNICATION
Different point mutations within the conserved N-glycosylation motif of pseudorabies virus glycoprotein M result in expression of a nonglycosylated form of the protein Johannes M. Dijkstra, Alexandra Brack, Alice Jo$ ns, Barbara G. Klupp and Thomas C. Mettenleiter Institute of Molecular and Cellular Virology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany
Glycoprotein M (gM) constitutes one of the rare examples of a nonessential glycoprotein conserved throughout all herpesvirus subfamilies. Whereas gM in wild-type pseudorabies virus (PrV) strains carries an N-glycan, gM of the attenuated strain Bartha is not glycosylated due to a point mutation in the N-glycosylation motif. Since PrV Bartha lacks glycoproteins E and I and carries a mutated gC, we analysed glycosylation of gM in isogenic PrV glycoprotein deletion mutants. Whereas gM was glycosylated normally in most mutants, two independent gC deletion mutants and a gI mutant expressed a nonglycosylated form of gM. DNA sequence analyses revealed the presence of point mutations in the N-glycosylation consensus motif. Surprisingly, mutations in strain Bartha, the two gCdeletion mutants and the gI mutant proved to be different, although all affected the N-glycosylation motif. Thus, our data show that different, apparently independent point mutations cause expression of nonglycosylated gM.
In pseudorabies virus (PrV), a member of the alphaherpesvirus subfamily of the herpesviruses, 11 glycoproteins have been described up to now (reviewed in Mettenleiter, 1994). They include homologues of the essential gB, gH and gL, and the nonessential gM and gN, which are found in all herpesvirus subfamilies. Conservation of glycoproteins apparently not required for productive replication of the virus, at least in cell culture, appears to be rare and most nonessential glycoproteins detected in alphaherpesviruses are not present in beta- or gammaherpesviruses. We recently identified the product of the UL49.5 open reading frame of PrV as an Oglycosylated virion envelope component designated gN (Jo$ ns Author for correspondence : Thomas C. Mettenleiter. Fax 49 38351 7151. e-mail mettenleiter!rie.bfav.de
0001-5283 # 1998 SGM
& Mettenleiter, 1996). Surprisingly, the homologous proteins in herpes simplex virus type 1 (HSV-1 ; R. Adams, C. Cunningham, M. D. Davison, C. A. MacLean & A. J. Davison, personal communication) and bovine herpesvirus 1 (BHV-1 ; Liang et al., 1996) are not glycosylated which raises the question of importance of glycosylation for function of the protein (Paulson, 1989). gM, the product of the nonessential UL10 gene (Baines & Roizman, 1991, 1993 ; MacLean et al., 1991, 1993), is glycosylated in HSV-1 (Baines & Roizman, 1993), equine herpesvirus 1 (EHV-1 ; Osterrieder et al., 1996) and PrV (Dijkstra et al., 1996). However, the attenuated PrV strain Bartha (PrV-Ba) expresses a nonglycosylated gM (Dijkstra et al., 1997) due to a point mutation in the DNA sequence specifying the sole conserved consensus motif for Nglycan addition in gM. Absence of this N-glycan has only a minimal effect on replication of PrV in cultured cells (Dijkstra et al., 1997). PrV strain Bartha carries different mutations which all contribute to its avirulence, i.e. a deletion of the gE and gI genes, a mutation in the gC gene affecting the signal sequence and point mutations in UL21. Repair of all three defects is required for restoration of the virulent phenotype (reviewed in Mettenleiter, 1994). To analyse a possible influence of the glycoprotein mutations on gM expression, we analysed gM of isogenic PrV mutants based on strain Kaplan (PrV-Ka ; Kaplan & Vatter, 1959), each unable to express one of the known PrV glycoproteins, i.e. gB (Rauh et al., 1991), gC (PrV-321 ; T. C. Mettenleiter, unpublished), gD (Rauh & Mettenleiter, 1991), gE (Mettenleiter et al., 1987), gG (Mettenleiter et al., 1990), gH (Babic et al., 1996), gI (Zuckermann et al., 1988), gK (Klupp et al., 1998), gL (Klupp et al., 1997) or gN (Jo$ ns et al., 1998). As shown in Fig. 1, the glycosylated mature 45 kDa gM is present in lysates of cells infected by PrV-Ka or mutants deleted in gB, gD, gE, gG, gH, gI, gK, gL and gN. In gN− PrV infected cells, only a small amount of 45 kDa mature gM was present and the 35 kDa glycosylated precursor form was more prominent. Most interestingly, in cells infected with the gC− deletion mutant PrV-321 the gM pattern paralleled that found in PrVBa infected cells. Only the nonglycosylated 33 kDa precursor form was present. In cells infected with the gI− PrV, besides
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J. M. Dijkstra and others
Fig. 1. Analysis of gM expression in different PrV glycoprotein mutants. Lysates of RK 13 cells infected with wild-type PrV-Ka, PrV-Ba and the indicated glycoprotein mutants were separated in an SDS–10 % polyacrylamide gel, transferred to nylon membrane and probed with a gMspecific rabbit antiserum (Dijkstra et al., 1996). Bound antibody was visualized after incubation with a peroxidase-conjugated secondary antibody with enhanced chemiluminescence (ECL, Amersham) and recorded on X-ray film. Molecular mass markers are indicated on the left. Arrows point to the location of the 33 kDa nonglycosylated and 35 kDa glycosylated precursor forms as well as 45 kDa mature gM.
mature 45 kDa gM, the nonglycosylated precursor could also be demonstrated. PrV-321 contains an 1±4 kb deletion removing most of the gC and part of the upstream UL43 gene (Mettenleiter et al., 1988) and an insertion of a gG–βgalactosidase expression cassette into the unique BamHI site which remains at the 3«-end of the gC gene. It was isolated after cotransfection of PrV Ka-DNA and plasmid TT-321 (T. C. Mettenleiter, unpublished). A second gC− PrV mutant, PrV8411 (Karger et al., 1995), which contains the same deletion in the gC gene but carries the gG–β-galactosidase cassette in the gG gene locus, was obtained independently from PrV-321. Surprisingly, it also exclusively expressed nonglycosylated gM (data not shown). Since our gI− PrV stock appeared to consist of a heterogeneous population of viruses, the majority expressing glycosylated gM and a minority expressing nonglycosylated gM, single plaques were picked and reanalysed. Out of 17 single plaque isolates, two expressed
nonglycosylated gM. The higher molecular mass forms of 65, 70 and 90 kDa present in some of the lysates represent probable dimers (Dijkstra et al., 1996 ; Osterrieder et al., 1997). Also, a certain variation between the relative amounts of the 33 and 35 kDa proteins was regularly observed (Dijkstra et al., 1996). To pinpoint the reason for this lack of glycosylation the DNA sequence of a portion of the UL10 gene, encompassing codons specifying the only consensus sequence for Nglycosylation (Kornfeld & Kornfeld, 1985) present in PrV gM, was determined for both gC mutants and one of the two single plaque isolates of gI− PrV expressing nonglycosylated gM, PrV gI− 1612. As shown in Fig. 2, both gC− mutants contained the same point mutation resulting in an alteration of the amino acid sequence NDT to NDP, thus destroying the consensus motif. Interestingly, this mutation was different from those previously detected in strain Bartha (Dijkstra et al., 1997), and also differed from the one present in PrV gI− 1612, which resulted in an alteration of NDT to SDT. Thus, three different, apparently independent mutational events in PrV-Ba, both gC− PrV mutants and the gI− PrV mutant all resulted in destruction of the consensus motif for addition of N-glycans. We further analysed whether restoration of gC expression would lead to reversion of the gM mutation. To this end, the gC gene of mutant PrV-321 was repaired and rescuants were analysed by Western blot (data not shown) and DNA sequencing (Fig. 2 A). Results demonstrated the continuing presence of the point mutation and thus continuing expression of nonglycosylated gM. This shows that restoration of gC expression does not result in concomitant repair of the gM defect. To assay whether expression of nonglycosylated gM can be selected for in cell culture, two novel independent gC− mutants identical to PrV-321 were isolated after cotransfection of PrV-Ka DNA and plasmid TT-321 (T. C. Mettenleiter, unpublished). Virus progeny was screened for lack of gC expression and purified to homogeneity. Two independent single plaque isolates, PrV-gC− 111 and PrV-gC− 272, were picked and passaged in MDBK and PK15 cells. However, even
Fig. 2. Comparison of the nucleic acid sequence of part of the UL10 gene (A) and the deduced amino acid sequence (B) of strains PrV-Ka, PrV-Ba, gC− mutants 321 and 8411, gC-rescuant gCR42 and gI− mutant 1612. Numbers in (A) indicate nucleotide positions within the UL10 gene ; numbers in (B) denote amino acid position in the deduced protein sequence.
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Non-glycosylated PrV M protein
after 20 passages at either high or low m.o.i. no alteration in glycosylation of gM was observed. In addition, gC was deleted from a PrV-Ka recombinant carrying the PrV-Ba UL10 gene. Extensive in vitro analyses of this mutant did not reveal any difference in growth as compared to PrV gC− 111 or 272 (data not shown). Thus, we have been unable so far to explain the emergence of nonglycosylated gM or pinpoint any biological effect of expression of nonglycosylated gM. Although we cannot completely rule out the presence of gM glycosylation mutants in our PrV-Ka stock, analysis of several single plaque isolates of PrV-Ka as well as several PrVKa virus stocks did not reveal any difference in glycosylation of gM (see also Fig. 1). Thus, the observation that nonglycosylated gM is expressed by two independent gC deletion mutants suggests a correlation between the gC and gM mutations. In summary, we show that the single conserved Nglycosylation motif within herpesviral gM homologues can be abolished by different mutations in pseudorabies viruses with defects in expression of gC and}or gI. gM has recently been shown to form a complex with gN (Jo$ ns et al., 1998), which could explain the effect of the absence of gN on maturation of gM (Fig. 1). In contrast, gI forms a complex with gE, which is thought to represent the functional unit (Zuckermann et al., 1988). However, a similar lack of glycosylation of gM as detected in the gI− mutant was not observed in the gE− mutant, which might indicate an effect of gI on gM glycosylation independent of the gE}gI complex. No interaction between gM, and gC and}or gI has so far been demonstrated which could explain the correlation between mutations in gC and}or gI and the mutations in the Nglycosylation motif within gM. This study was supported by grants from the EU (ERB-CHRX-CT920029) and the DFG (Me 854}3).
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Pseudorabies virus glycoprotein L is necessary for virus infectivity but dispensable for virion localization of glycoprotein H. Journal of Virology 71, 7687–7695. Klupp, B., Baumeister, J., Dietz, P., Granzow, H. & Mettenleiter, T. C. (1998). Pseudorabies virus glycoprotein gK is a structural component of
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