Institute for Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, UK
Correspondence
Michael D. Baron
michael.baron{at}bbsrc.ac.uk
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ABSTRACT |
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We have established rescue systems for both the Plowright vaccine strain (Plowright & Ferris, 1962) of RPV (RBOK) and the virulent parent from which it was derived, Kabete O (KO) (Baron & Barrett, 1997
; Baron et al., 2005
). These rescue systems allow us to recover recombinant viruses with gene additions or modifications (Baron & Barrett, 2000
; Baron et al., 1999
; Walsh et al., 2000
). For the virulent KO strain of RPV, the rescue uses only the lymphoblastoid line B95a (Kobune et al., 1991
). We have found that wild-type virus grows in these cells without prior adaptation and without apparent loss of virulence. We have used this system to create a number of chimaeric viruses in which specific genes from the RBOK strain have been replaced with the corresponding gene from the KO strain and vice versa. All of these viruses were rescued in B95a cells and stocks were grown and titrated in the same cell line.
One of the major differences between the growth of vaccine and virulent strains of RPV in vivo is the much greater replication of the latter (Wohlsein et al., 1995). When I tried to study the growth of the KO strain in a bovine cell line, MadinDarby bovine kidney (MDBK) cells, I was surprised to find that it grew very poorly (<1 %) relative to the vaccine virus, as measured by virus yield 48 h post-infection (p.i.) with virus at an m.o.i. of 0·04 (Fig. 1a
). Immunofluorescence studies showed that exposure of MDBK cells to RBOK at this m.o.i. led to widespread infection, whereas, after exposure to the same amount of KO, very few cells were infected (data not shown). This suggested that the low recovery of virus was due to a defect in entry or replication. Altered cell tropism in morbilliviruses has been found to be due to the viral H gene (Moeller et al., 2001
; Ohgimoto et al., 2001
; von Messling et al., 2001
) or the P gene (Miyajima et al., 2004
). To try to identify which gene was responsible for the relative defect in infectivity of KO in MDBK cells, I tested the growth of recombinant chimaeric viruses in which the N, P or H gene had been exchanged between KO and RBOK strains. KO viruses containing the RBOK N or P gene (KO-NR, KO-PR) were still defective in growth in MDBK cells, whereas KO with the RBOK H gene (KO-HR) grew as well as RBOK (Fig. 1a
). Corresponding results were found with the modified RBOK viruses, in that replacing the RBOK H gene with that of KO (RBOK-KOH) resulted in greatly decreased virus yield, whilst exchanging the N or P genes had no effect (Fig. 1a
). Immunofluorescence studies showed, as expected, that KO-HR infected MDBK cells readily, whereas RBOK-KOH showed very low levels of infection; similar results were also found with primary bovine skin fibroblasts (data not shown).
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Cell-culture adaptation has, for a number of viruses, involved the selection of mutants that can utilize heparan sulphate (HS) as a receptor (Bose & Banerjee, 2002; Goodfellow et al., 2001
; Jackson et al., 1996
; Klimstra et al., 1998
; Mandl et al., 2001
; Summerford & Samulski, 1998
). To see whether this was also true of RPV, CHO cells were treated with heparinase I (Sigma; 10 U ml1 for 90 min at 37 °C) and the ability of RBOK or KO-KRH (the minimal KO mutant that showed infection of ordinary CHO cells) to infect the cells was determined. As shown in Fig. 3(b)
, heparinase treatment of the cells greatly reduced the subsequent infection by RPV. In addition, I assessed the ability of RPV to infect the cell line pgsD-677 (Lidholt et al., 1992
) (a gift of Dr J. D. Esko, UCSD, CA, USA), a mutant CHO cell that does not make HS. Neither RBOK nor KO-KRH could infect pgsD-677 cells (Fig. 3c
). Transient transfection of pgsD-677 cells with a plasmid expressing bovine SLAM (cloned by PCR from Tp4/9 cells) did allow RPV to infect and replicate in them (data not shown), confirming that there was no underlying defect in the ability of the cells to support RPV replication. These data showed that the ability of the RBOK strain of RPV to infect so many tissue-culture cell lines is due to its use of this almost ubiquitously expressed glycosaminoglycan.
Comparison of the sequences of KO and the minimal HS-using mutant KO-KRH showed just four changes to the protein sequence (T496I, R556K, I586V and E603K) (the sequences of the H genes of all of the viruses used in these studies were checked to ensure that no mutations were acquired during rescue and stock amplification). Previous studies with Foot-and-mouth disease virus (Baranowski et al., 1998; Fry et al., 1999
), Sindbis virus (Klimstra et al., 1998
) and Tick-borne encephalitis virus (Mandl et al., 2001
) have indicated that adaptations to use HS as a receptor are mutations that increase the positive charge in positions at the surface of the virus. Assuming that the RPV H protein structure is similar to that modelled for the MV H protein (Massé et al., 2004
), the E603K mutation, which changes the charge at that point on the surface from negative to positive, is situated in a very flexible region on the surface of the globular head, close to the putative CD46-binding site on the MV H protein, and therefore presumably in a suitable position to mediate virus binding to a host-cell surface protein. Interestingly, this residue is also lysine in the H protein of RPV KO grown in bovine kidney cells (Yamanaka et al., 1988
), which is therefore presumably able to enter cells in a SLAM-independent manner. The arginine at 556 is also in a surface-exposed position, close to the CD46-binding site. The sequence around and including R556 is conserved in morbillivirus H proteins, suggesting that it plays an important part in the structure, although it does not appear to be a part of the SLAM-binding domain (Massé et al., 2004
). The R556K mutation will not change the surface positive charge, but may alter internal hydrophobic interactions or hydrogen bonds, allowing greater flexibility of the side chain. Interestingly, this position is also lysine in the bovine kidney cell-adapted KO virus (Yamanaka et al., 1988
). Further mutations would be needed to identify the exact mutation(s) that allows RPV H to bind to HS; it would be interesting to see whether a similar mutation would allow MV to use the same receptor.
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ACKNOWLEDGEMENTS |
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Received 16 December 2004;
accepted 10 February 2005.
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