Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes: G and F proteins bind heparin

Axel Karger1, Ulrike Schmidt1 and Ursula J. Buchholz1

Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, D-17498 Insel Riems, Germany1

Author for correspondence: Ursula J. Buchholz. Fax +49 38351 7275. e-mail buchholz{at}rie.bfav.de


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Bovine respiratory syncytial virus (BRSV) encodes three transmembrane envelope glycoproteins, namely the small hydrophobic (SH) protein, the attachment glycoprotein (G) and the fusion glycoprotein (F). The BRSV reverse genetics system has been used to generate viable recombinant BRSV lacking either the G gene or the SH gene or both genes. The deletion mutants were fully competent for multicycle growth in cell culture, proving that, of the BRSV glycoprotein genes, the SH and G genes are non-essential. Virus morphogenesis was not impaired by either of the deletions. The deletion mutants were used to study the role of the F glycoprotein and the contributions of SH and G with respect to virus attachment. Attachment mediated by the F protein alone could be blocked by soluble heparin, but not by chondroitin sulphate. Heparin affinity chromatography revealed that both the BRSV G and F glycoproteins have heparin-binding activity, with the affinity of the F glycoprotein being significantly lower than that of G. Therefore, the roles of the BRSV glycoproteins in virus attachment and receptor binding have to be reconsidered.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Bovine respiratory syncytial virus (BRSV) represents the major virus aetiological agent of respiratory disease of calves (Van der Poel et al., 1994 ). Together with human respiratory syncytial virus (HRSV) and pneumonia virus of mice, BRSV belongs to the genus Pneumovirus of the family Paramyxoviridae, order Mononegavirales (Collins et al., 1996b ). The BRSV genome is 15140 nt in length (strain ATue51908) (Buchholz et al., 1999 ) and contains ten monocistronic genes in the order 3'-NS1–NS2–N–P–M–SH–G–F–M2–L-5'. As for all members of the order Mononegavirales, the RSV RNA is tightly encapsidated by the nucleoprotein (N), which, together with the phosphoprotein (P) and the polymerase (L), forms the ribonucleoprotein (RNP) complex, the minimal replication unit. Transcription of full-length mRNAs additionally requires the virus transcription elongation factor, M2-1 (Collins et al., 1996a ; Fearns & Collins, 1999 ; Hardy & Wertz, 1998 ). RSV transcription initiates at the 3' genome end and proceeds in a sequential start–stop mechanism, directed by the highly conserved gene-start and gene-end signals that flank each gene (Kuo et al., 1996 ; Zamora & Samal, 1992 ).

The matrix protein (M) is thought to form the bridge between the RNP complex and the envelope (Teng & Collins, 1998 ). RSV encodes two non-structural proteins (NS1 and NS2) that are synthesized in infected cells (Collins et al., 1996b ). The M2-2 protein, which is a virus regulatory factor for the balance of RSV transcription and replication (Bermingham & Collins, 1999 ), is translated from a second open reading frame in the M2 gene by an internal translation initiation at a methionine codon contained in the M2 mRNA (Ahmadian et al., 2000 ).

Finally, RSV encodes three envelope glycoproteins, namely the major attachment glycoprotein (G) (Levine et al., 1987 ), the fusion glycoprotein (F) (Walsh & Hruska, 1983 ) and the small hydrophobic (SH) protein (Olmsted & Collins, 1989 ; Samal & Zamora, 1991 ), which has no known function. The major RSV attachment glycoprotein, G, is a heavily glycosylated, type II membrane protein that does not share any sequence or structural homologies with attachment proteins of other paramyxoviruses (Satake et al., 1985 ; Wertz et al., 1985 ) and lacks haemagglutinating and neuraminidase activities. The RSV G glycoproteins exhibit a high degree of species- and strain-specific genetic and antigenic divergence (Furze et al., 1994 ; Lerch et al., 1990 ; Mallipeddi & Samal, 1993 ). However, there are some conserved features, such as a central globular hydrophobic region, which has been discussed as a receptor-binding domain (Johnson et al., 1987 ; Langedijk et al., 1996 ). The fusion glycoprotein, F, which is conserved to a higher degree between the RSV species, mediates penetration and syncytium formation. It is synthesized as an F0 precursor, which is cleaved post-translationally by endoproteases into F1 and F2 subunits (Collins & Mottet, 1991 ) that remain linked by disulphide bonds. There is evidence that the HRSV F protein exists as an oligomer (Collins & Mottet, 1991 ). Among the Paramyxoviridae, the third envelope-associated glycoprotein that is present in RSV, the SH protein, is present only in members of the subfamily Pneumovirinae and in simian virus 5 (SV5) and mumps virus. To date, the SH proteins have no known function. It has been shown that the SH gene of SV5 can be deleted without causing a recognizable phenotype (He et al., 1998 ). Also, a naturally occurring mumps strain has been described that does not express the SH protein (Takeuchi et al., 1996 ).

Recently, RSV deletion mutants lacking individual genes have been generated from cDNA by using reverse genetics systems (Buchholz et al., 1999 ; Collins et al., 1995 ). For BRSV, the NS1 and NS2 genes are non-essential; cell-culture growth of BRSV deletion mutants lacking NS2 or NS1 or both is reduced (Buchholz et al., 1999 ; U. J. Buchholz, unpublished results). It has been shown that the HRSV NS2 (Teng & Collins, 1999 ), SH (Bukreyev et al., 1997 ) and M2-2 (Bermingham & Collins, 1999 ; Jin et al., 2000 ) genes can be deleted; of these, the SH deletion caused the smallest phenotypic change. Moreover, a non-recombinant, cold-passaged, attenuated HRSV subgroup B mutant (B1 cp-52) was shown to replicate to high titres in tissue culture despite the absence of functional SH and G proteins (Karron et al., 1997 ).

Heparin and heparan sulphate belong to a class of carbohydrates designated as glycosaminoglycans (GAGs), which are unbranched polymers of repeating disaccharide units. Covalently linked to membrane proteins, they are found as proteoglycans on the cell surface of most mammalian cells. The most prominent physico-chemical property of GAGs is a large and varying number of negative charges that, in the case of most GAG types, is conferred on the molecule by sulphate residues. The HRSV attachment protein (G) binds cell-surface heparan sulphate (Feldman et al., 1999 ; Krusat & Streckert, 1997 ) but, only recently, in vitro heparin-binding capacity was also demonstrated for the HRSV F protein (Feldman et al., 2000 ). Here, we used the BRSV reverse genetics system to generate isogenic deletion mutants lacking the SH and/or the G gene. These recombinant viruses, which differ only in the respective gene deletions, were used to study virus attachment and heparin binding in cell culture. The in vitro heparin-binding capacity of G and F was characterized further by heparin-affinity chromatography (HAC).


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Molecular cloning.
The plasmid containing the complete antigenomic sequence of BRSV strain ATue51908 (GenBank accession no. AF092942), described previously (Buchholz et al., 1999 ), was modified to contain restriction sites in the BRSV SH/G (SalI; ATue51908 nt 4673) and G/F (SphI; ATue51908 nt 5539) intergenic regions (Buchholz et al., 2000 ) (Fig. 1). The SalI and SphI restriction sites were used to excise an 862 bp fragment comprising the complete BRSV G gene. After Klenow treatment and religation, the SalI site was reconstituted. In a second full-length BRSV construct, a second SalI site was created in the M/SH intergenic region (ATue51908 nt 4165). This plasmid was used to construct an SH gene deletion mutant or a double-deletion mutant lacking both the SH and G genes, as depicted in Fig. 1.



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Fig. 1. Construction of BRSV glycoprotein deletion mutants. The BRSV genome is drawn to scale. ORFs are depicted as shaded rectangles. Enlargements show the SH and G gene deletions, with the gene-start signals shown as triangles and the gene-end signals represented by bars. Locations of synthetic restriction sites are indicated. The SalI site located in the M/SH intergenic region (in parentheses) is present only in the plasmid used for generation of the SH and SH/G deletion mutants. The genome lengths of the recombinant viruses are indicated to the left.

 
{blacksquare} Generation and characterization of recombinant BRSV (rBRSV).
rBRSVs were generated as described previously (Buchholz et al., 2000 ). Briefly, 32 mm dishes of subconfluent BHK T7/5 cells stably expressing T7 RNA polymerase were transfected with 5 µg of the respective full-length plasmid (pBRSV, pBRSV{Delta}SH, pBRSV{Delta}G or pBRSV{Delta}SHG) and a set of four support plasmids (2 µg pN, 2 µg pP, 1 µg pM2 and 1 µg pL), from which the N, P, M2 and L proteins are expressed. All cDNA constructs were under the control of a T7 promoter. Every 2–3 days, the transfected cells were divided. When the CPE was extensive, cells were frozen and thawed three times and the clarified supernatants were used for production of virus stocks on MDBK cells, as described previously (Buchholz et al., 2000 ).

The identity of the recombinant viruses was verified by RT–PCR. Total RNA was prepared from 32 mm dishes of MDBK cells 96 h after infection at an m.o.i. of 0·1. First-strand cDNA was generated from 1 µg RNA by using a primer complementary to the M gene (primer Mc2b; ATue51908 nt 3612–3635). Second-strand cDNA was synthesized by using primer Mc2b and a reverse primer BFr, hybridizing to the F gene (ATue51908 nt 5964–5941). The purified RT–PCR products were subjected to restriction digestion and analysed on agarose gels.

{blacksquare} Viruses and cells.
rBRSV was propagated on MDBK cells. Ninety min after infection at an m.o.i. of 0·1, the inoculum was removed and cells were incubated at 37 °C in MEM supplemented with 3% FCS in a 5% CO2 atmosphere. Eight days post-infection, when an extensive CPE could be observed, the medium was adjusted to 100 mM MgSO4 and 50 mM HEPES (pH 7·5) and the highly cell-associated virus was released by freezing and thawing. Growth analyses of rBRSV, rBRSV{Delta}SH, rBRSV{Delta}G and rBRSV{Delta}SHG were done on MDBK cells as described previously (Buchholz et al., 2000 ). Duplicate titrations were carried out in microwell plates by the limiting dilution method. To 0·1 ml of serial tenfold virus dilutions per well, 104 BSR T7/5 cells were added in a 0·1 ml volume. After 48 h, cells were fixed in 80% acetone. An indirect immunofluorescence assay using a bovine serum specific for BRSV was performed and foci of infected cells were counted.

{blacksquare} Indirect immunofluorescence assay.
MDBK cells were infected with rBRSV at an m.o.i. of 0·1, incubated for 42 h, fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100. After incubation with monoclonal antibody (MAb) F9, directed to BRSV F (Taylor et al., 1984 ), or with MAb G66, directed to BRSV G (Furze et al., 1994 ), cells were stained with an Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes). The nuclei of cells were stained with propidium iodide. Immunofluorescence was examined by confocal laserscan microscopy.

{blacksquare} Serum neutralization.
Aliquots of 0·05 ml of serial twofold dilutions of heat-inactivated rabbit anti-BRSV serum in MEM were incubated for 30 min at room temperature with an equal volume containing 100 p.f.u. rBRSV. Subsequently, 104 BSR T7/5 cells were added in a 0·1 ml volume. After 4 days, the ND50 was determined. Control experiments were performed with negative sera collected prior to immunization of the rabbits.

{blacksquare} Northern blot.
Northern blots were done as described previously (Buchholz et al., 1999 ). Total RNA of MDBK cells infected with the recombinant viruses was analysed by denaturing agarose gel electrophoresis, blotted onto nylon membranes, UV cross-linked and hybridized with DNA probes. PCR-generated fragments of the BRSV N (ATue51908 nt 1429–2277), SH (ATue51908 nt 4268–4534), G (ATue51908 nt 4690–5431) or F (ATue51908 nt 6233–7459) genes were labelled with [{alpha}-32P]dCTP (3000 Ci/mmol; ICN) (Nick translation kit, Amersham).

{blacksquare} Sodium chlorate treatment.
MDBK cells were divided three times at a ratio of 1:5 at intervals of 48 h and reseeded in standard medium supplemented with 30 mM NaClO3 (Keller et al., 1989 ). For control titrations, MDBK cells were similarly passaged in standard medium. Suppression of GAG synthesis by chlorate treatment was verified by titration of pseudorabies virus, which shows an approximately tenfold decrease in titre on chlorate-treated cells, whereas the titre of a gC-negative mutant is unimpaired by chlorate treatment (A. Karger, unpublished data).

{blacksquare} Attachment assays.
Ninety-six well plates of subconfluent untreated or sodium chlorate-treated MDBK cells were incubated for 90 min at 4 °C with tenfold serial virus dilutions in MEM containing the indicated concentrations of heparin (Sigma cat. no. H3393) or chondroitin sulphate C (Sigma cat. no. C4384). Subsequently, cells were washed three times. After 3 days of incubation at 37 °C, foci of infected cells were counted.

{blacksquare} Preparation of virus extracts.
One 75 cm2 cell culture flask with MDBK cells was infected with recombinant virus at an m.o.i. of 0·1. At 96 h post-infection, infected cells were subjected to three freeze–thaw cycles in 2 ml cell culture medium. After removal of whole cells by low-speed centrifugation, membrane proteins were solubilized by addition of 1 vol 2x extraction buffer (20 mM sodium phosphate, pH 7·0, 2% Zwittergent 3-12; Boehringer Mannheim) and incubation on ice for 2 h. Insoluble material was removed by ultracentrifugation (TLA 45 rotor, Beckman; 45000 r.p.m., 45 min, 4 °C). The final supernatants (‘virus extracts’) were collected and used for HAC.

{blacksquare} Heparin-affinity chromatography (HAC).
A 1 ml HiTrap heparin column packed with heparin–Sepharose High Performance (cat. no. 17-0406-01, Amersham Pharmacia) connected to an HPLC system (BT 9200 Titan pump, BT 9520 IN UV monitor, WinChrom v1.21 software, Eppendorf Biotronik; conductivity monitor 18-1500-00, Pharmacia Biotech) was used for HAC. All solvents were filtered and degassed in line and all reagents were of analytical grade. The column was washed with 10 column vols (CVs) buffer A (sodium phosphate 20 mM, pH 7·4, 1% Zwittergent 3-12) and 10 CVs buffer B (2 M NaCl in buffer A) and equilibrated in buffer A. One ml of virus extract was diluted 1:2 in buffer A and applied to the column at 0·5 ml/min. The flow-through was collected for further analysis. The column was washed with buffer A at 1 ml/min until baseline levels of A280 were obtained. A non-linear NaCl gradient was applied to the column (0–50% buffer B in 10 min, curvature 1; 50–100% buffer B in 1 min, curvature 1; 1 ml/min) and elution of proteins was monitored at 280 nm. The fraction size was 0·5 ml. The salt concentration in the eluate was registered by a conductivity monitor. The column was regenerated for the next chromatography run by a 10 CV wash with buffer B and equilibration in buffer A at 1 ml/min. Data were analysed with the SuperCompare program of the WinChrom software.

{blacksquare} SDS–PAGE and immunoblotting.
For analysis of the heparin-binding proteins from the virus extracts by immunoblotting, 100 µl samples of the fractions were precipitated with 300 µl ice-cold acetone after addition of 2 µg BSA as carrier. After 2 h incubation at -20 °C, the precipitate was collected by centrifugation, air-dried and solubilized in SDS–PAGE sample buffer at 56 °C for 5 min. Under these conditions, the F-specific antibody 47F yields a specific signal at approximately 200 kDa, which probably represents the F protein trimer. SDS–PAGE (Laemmli, 1970 ) was carried out using a Mini Protean II unit (Bio-Rad) and proteins were electrotransferred to nitrocellulose membranes (Schleicher & Schuell) (Towbin et al., 1979 ). The antibody G66 (Furze et al., 1994 ), specific to the G protein, was a gift from Geraldine Taylor (IAH, Compton, UK). The F protein-specific antibody 47F (García-Barreno et al., 1989 ) was obtained from José Antonio Meléro (Instituto de Salud Carlos III, Madrid, Spain).

{blacksquare} Production of rabbit hyperimmune sera.
Two rabbits were immunized with 105 p.f.u. rBRSV or rBRSV{Delta}SHG in Freund’s complete adjuvant. The rabbits were reimmunized twice at 4 week intervals with the same amount of the respective virus in Freund’s incomplete adjuvant. Sera were collected 10 days later.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Construction and recovery of BRSV deletion mutants
A T7 RNA polymerase-driven system that allowed the generation of rBRSV from cDNA has been described previously (Buchholz et al., 1999 ). In order to generate BRSV lacking the SH or G gene or both genes, regions spanning the complete SH gene or G gene, including the gene-start and -end signals, or the SH and G genes from the SH gene-start signal to G gene-end signal were deleted from a full-length antigenome plasmid. The BRSV antigenomes expressed by the respective plasmids are 14632 (rBRSV{Delta}SH), 14278 (rBRSV{Delta}G) or 13770 nt (rBRSV{Delta}SHG) in length, compared with the 15140 nt of the parental rBRSV (Fig. 1). After co-transfection of BSR T7/5 cells (Buchholz et al., 1999 ) with the respective full-length plasmid and four support plasmids encoding BRSV N, P, L and M2, viable rBRSVs were recovered from all of the cDNA constructs. About 7 days after transfection, several foci exhibiting the typical BRSV CPE of large syncytia could be observed in all transfected cells, with no difference between the various recombinant viruses. The deletion mutants and the parental recombinant virus were propagated by passage on MDBK cells. Five days after infection of MDBK cells at an m.o.i. of 0·1, all virus isolates produced a comparably extensive CPE.

The identities of the recombinant viruses were verified by RT–PCR 4 days after infection. As shown in Fig. 2, RT–PCR with primers specific for the BRSV M gene and F gene yielded products encompassing deletions of the expected sizes of 2353 (rBRSV), 1845 (rBRSV{Delta}SH), 1491 (rBRSV{Delta}G) or 983 bp (rBRSV{Delta}SHG). The synthetic marker restriction sites were present in the respective RT–PCR products, as shown in Fig. 2.



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Fig. 2. Demonstration of marker restriction sites in the genomes of rBRSV and of the deletion mutants. RT–PCR was performed on total RNA of infected cells. PCR products were subjected to restriction digestion and analysed on a 3% agarose gel. M, Marker DNA ladder (Life Technologies), with the sizes of selected fragments indicated. The RT–PCR products were consistent with the predicted sizes of 2353 (rBRSV), 1845 (rBRSV{Delta}SH), 1491 (rBRSV{Delta}G) or 983 bp (rBRSV{Delta}SHG). Digestion with SalI yielded the expected fragments of 1291 and 1062 bp (rBRSV), 1291 and 554 bp (rBRSV{Delta}SH), 429 and 1062 bp (rBRSV{Delta}G) or 554 and 429 bp (rBRSV{Delta}SHG); digestion with SphI yielded fragments of 1928 and 425 bp (rBRSV) and 1420 and 425 bp (rBRSV{Delta}SH). As expected, the rBRSV{Delta}G and rBRSV{Delta}SHG PCR fragments were not cleaved by SphI.

 
Transcription analysis of the recombinant viruses
The expression of viral RNA and of mRNA was verified in Northern blots performed on total RNA from infected cells (Fig. 3). Replica blots were incubated with probes hybridizing to the N, SH, G or F genes. With an SH-specific probe, a strong signal from bicistronic M/SH mRNA was observed, with an intensity comparable to the monocistronic SH mRNA, which reflects the incomplete transcription termination at the BRSV M gene-end signal. Consequently, the deletion mutants that lacked the SH gene or the SH and G genes were characterized by high levels of readthrough mRNAs of the M gene and the respective downstream gene (Fig. 3). The Northern blots confirmed the absence of the deleted genes in the genomes of the deletion mutants.



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Fig. 3. Demonstration of transcripts by Northern hybridization. Total RNA of MDBK cells infected with the viruses indicated was isolated 96 h after infection and analysed on a 2% agarose gel under denaturing conditions. Replica blots were incubated with probes hybridizing to the BRSV N, SH, G or F gene. Transcripts corresponding to the respective mRNAs and bicistronic mRNAs are indicated.

 
Expression of glycoproteins
An indirect immunofluorescence assay was performed on MDBK cells 42 h post-infection with rBRSV or with the deletion mutants. Immunostaining with antibody F9, directed to BRSV F protein, yielded a typical fluorescent staining, with infected cells surrounded by an intensely stained ‘corona’ of long, filamentous, budding particles (Fig. 4). The deletion mutants were indistinguishable from rBRSV with respect to intensity of staining and to budding of particles. Thus, particle formation and incorporation of F glycoprotein are apparently not affected by deletion of the G or SH proteins. As expected, MAb G66, directed to BRSV G, did not react with cells infected with rBRSV{Delta}G or rBRSV{Delta}SHG (not shown). In MDBK cells, BRSV spreads to neighbouring cells, producing typical foci of infected cells. The sizes of foci produced by the deletion mutants were comparable to the size of foci produced by rBRSV (Fig. 4).



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Fig. 4. Confocal laserscan analyses of MDBK cells infected with rBRSV (a), rBRSV{Delta}SH (b), rBRSV{Delta}G (c) or rBRSV{Delta}SHG (d). Cells were fixed with paraformaldehyde 42 h post-infection, permeabilized, reacted with F protein-specific MAb F9 and counterstained with propidium iodide. Infected cells are surrounded by a ‘corona’ of budding filamentous particles, proving that budding of virus particles is not affected by deletion of the SH and/or G glycoprotein(s).

 
Growth of the deletion mutants was reduced slightly in cell culture
The recombinant viruses were fully viable in tissue culture with respect to multicycle growth competence (Fig. 5). Final titres of the BRSV deletion mutants were reduced only slightly, but reproducibly, compared with parental rBRSV. Thus, the deletion mutants lacking the G protein readily attach to and penetrate cells, even though they lack the putative major attachment glycoprotein. Obviously, the BRSV F glycoprotein is functional in the BRSV envelope with respect to fusion, but also with respect to virus attachment, independent of the presence of G and/or SH.



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Fig. 5. Multicycle growth of rBRSV ({blacksquare}), rBRSV{Delta}SH ({diamondsuit}), rBRSV{Delta}G ({blacktriangleup}) and rBRSV{Delta}SHG ({bullet}) in MDBK cells. Duplicate cell monolayers in 24-well dishes were infected with the indicated virus at an m.o.i. of 0·1. Monolayers were harvested at the times indicated, stored at -70 °C and titrated later in duplicate. Each value is the mean titre of material from two wells.

 
Attachment of BRSV to target cells is dependent on heparin even in the absence of the G and SH proteins
Attachment assays were carried out to characterize the contributions of the BRSV glycoproteins with respect to interaction with cell surface proteoglycans. In a first set of experiments, titres of rBRSV were determined on MDBK cells that had been made GAG deficient by chlorate treatment and compared with titres on untreated cells. As shown in Fig. 6(a), titres of all recombinant viruses decreased by approximately 95% after chlorate treatment of the target cells. In order to test the specificity of this interaction, inhibition experiments were performed for the two major species of glycosaminoglycans in MDBK cells (Karger et al., 1998 ), chondroitin sulphate and heparan sulphate, which is antagonized effectively by the structurally very closely related carbohydrate heparin. For this purpose, cells were inoculated with an appropriate dilution of the respective virus in the presence or absence of heparin or chondroitin sulphate at 4 °C for 90 min. After a thorough wash, cells were incubated further at 37 °C and plaques were counted after 72 h.



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Fig. 6. BRSV attaches to target cells by interaction with cell-surface heparan sulphate. (a) Inhibition of cellular GAG synthesis by chlorate treatment reduced attachment of all recombinant BRSV mutants by approximately 95% compared with attachment to untreated cells, indicating that cellular GAGs are involved in the attachment of BRSV. (b)–(c) Attachment of rBRSV was inhibited specifically by addition of heparin in a dose-dependent manner (b), whereas addition of chondroitin sulphate had no effect over the concentration range tested (c). All recombinants were equally sensitive to suppression of GAG synthesis or heparin inhibition, showing that heparin-binding activity of BRSV is not dependent on the presence of G or SH. Titres of rBRSV{Delta}SH (hatched bars), rBRSV{Delta}G (open bars) and rBRSV{Delta}SHG (shaded bars) were expressed as percentages of titres of rBRSV (filled bars) on untreated cells (a) or without inhibitor (b, c).

 
Titres of all recombinant viruses decreased upon the addition of heparin, in a dose-dependent manner. Inhibition by heparin was detectable at a concentration of 0·1 µg/ml and a maximum inhibition of approximately 95% could be achieved (Fig. 6b). No inhibition of any recombinant virus could be detected with chondroitin sulphate up to a concentration of 10 µg/ml (Fig. 6c). Suppression of titres after chlorate treatment did not exceed inhibition by heparin, indicating that GAGs other than heparan sulphate play only a minor role, if any, in attachment of BRSV.

Heparin-binding affinity of virus envelope glycoproteins G and F
In vitro heparin binding of solubilized G and F protein was assessed by HAC. Viral glycoproteins were extracted from MDBK cells infected with the recombinant viruses and the extracts were passed over a heparin column. Bound proteins were eluted by a non-linear NaCl gradient (Fig. 7a). G and F proteins were identified in the resulting fractions by immunoblot by using G- or F-specific MAbs (Fig. 7b). As expected, the G protein eluted at high NaCl concentrations, with the peak fractions, 12–14, corresponding to a salt concentration of 640–820 mM. A small amount of F protein was detected in the G protein-rich fractions 12 and 13 of the rBRSV extract, indicating the possibility of formation of a complex between F and G proteins. Surprisingly, like the G protein, the F protein was adsorbed completely on the heparin matrix under the conditions employed. In the presence or absence of G and/or SH protein, the F protein eluted mainly in fractions 5–9 (210–430 mM salt), with the peak fraction, 7, corresponding to a salt concentration of 310 mM. This demonstrates a heparin-binding potential of the F protein that is independent of the presence of the SH and G proteins. Thus, the sensitivity of G protein-negative recombinant viruses rBRSV{Delta}G and rBRSV{Delta}SHG to inhibition by heparin (Fig. 6b) can be explained by the heparin-binding activity of the F protein.



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Fig. 7. HAC of virus extracts. Extracts of rBRSV, rBRSV{Delta}SH, rBRSV{Delta}G and rBRSV{Delta}SHG were fractionated on a HiTrap heparin column and the resulting fractions were analysed for the presence of F and G protein by Western blotting. (a) Elution profile of an rBRSV extract, which was indistinguishable from that of the other recombinants. Line A represents the A280 and line C represents the salt concentration (mM) in the eluates, which was corrected for the 20 mM sodium phosphate in buffer A. Every second fraction is indicated at the bottom of the panel. (b) In the resulting immunoblots, arrows indicate F-specific (approximately 200 kDa) and G-specific (approximately 90 kDa) bands. Neither F nor G protein was found in the flow-through (not shown). F protein eluted at low salt concentrations, whereas higher salt concentrations were required to elute the G protein, which bound the heparin matrix more strongly.

 
Antibodies induced by the deletion mutants show neutralizing activity directed to standard rBRSV
As a first test of whether deletion mutants lacking the SH and G proteins were able to induce neutralizing antibodies against rBRSV, antisera directed to rBRSV and rBRSV{Delta}SHG were produced in rabbits. Sera collected after the third immunization were examined in a serum neutralization assay. The sera were able to neutralize standard rBRSV. The serum of the animal immunized with rBRSV{Delta}SHG had an ND50 titre against rBRSV of 5·0 log2, compared to an ND50 titre of 5·6 log2 induced by immunization with standard rBRSV. Pre-immune sera were devoid of neutralizing activity against BRSV. Thus, neutralizing antibodies induced by a recombinant BRSV that carries only the fusion glycoprotein F, but neither the attachment protein G nor the SH protein, of the envelope-associated proteins are sufficient to neutralize wild-type virus. In immunoblots, the sera reacted with the BRSV F glycoprotein and with the N protein (not shown).


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The BRSV reverse genetics system was used to generate isogenic mutants that carried deletions of the SH or the G glycoprotein gene or of the SH and G genes together in an identical genomic background. The phenotypic changes that were caused by deletion of the G and/or SH gene(s) were surprisingly small, even though the major BRSV attachment protein G was absent in two of the deletion mutants. Thus, we conclude that, in cell culture, the BRSV F protein alone is sufficient to mediate attachment and fusion in the absence of SH and G. Also, budding of progeny virus does not require G or SH. To obtain further insight into the functions of the BRSV glycoproteins, we have made use of rBRSV deletion mutants to characterize the contributions of the individual BRSV glycoproteins to virus attachment.

All three deletion mutants were only slightly less efficient in growth in vitro compared with parental rBRSV. It was not surprising that the BRSV SH gene could be deleted, since an SH deletion mutant that grows even more efficiently in cell culture than its full-length parent (Bukreyev et al., 1997 ) and exhibits only a slightly attenuated phenotype in vivo (Whitehead et al., 1999 ) has been described previously for the closely related HRSV. Apart from members of the genus Pneumovirus, there are only a few paramyxoviruses that contain an SH gene, namely mumps virus and SV5. An SV5 deletion mutant lacking the SH gene could be recovered from cDNA (He et al., 1998 ). In the case of mumps virus, a naturally occurring SH deletion mutant has been characterized (Takeuchi et al., 1996 ). To date, there is no evidence for a function of the SH proteins. It has been reported that expression of HRSV SH in E. coli led to alterations in membrane permeability (Perez et al., 1997 ). However, the significance of this finding remains unclear, since entry of RSV occurs by a pH-independent fusion pathway.

There are reports for both HRSV and BRSV (Heminway et al., 1994 ; Pastey & Samal, 1997 ) that co-expression of all three membrane-associated glycoproteins in a vaccinia virus-based expression system is needed for efficient fusion. However, the cell culture growth characteristics of the rBRSV deletion mutants prove that, in a helper virus-free system in the background of the homologous virus, the functionality of F is not affected by the absence of SH or G or both. F protein-mediated fusion is essential for virus entry and cell-to-cell spread. We have observed here that virus entry and cell-to-cell spread, as well as syncytium formation (U. Buchholz, unpublished data), can be accomplished by the BRSV F protein alone. The isolation of the naturally derived HRSV subgroup B mutant B1 cp-52, which bears a deletion of most of the SH and G genes, leading to a truncated SH/G transcript devoid of an open reading frame (Karron et al., 1997 ), provides evidence that HRSV G and SH are not essential. However, HRSV B1 cp-52 is cold-passaged and contains additional mutations, including an amino acid change in the F protein that might represent a compensating mutation acquired during passage. We show here that the native F protein alone is able to mediate attachment of BRSV by interaction with cell surface heparan sulphate, independent of the presence of other envelope proteins.

In a first attempt to find out whether rBRSV{Delta}SHG, which carries the F glycoprotein but not SH or G, would induce neutralizing antibodies against parental rBRSV, rabbit hyperimmune sera were prepared. rBRSV{Delta}SHG did induce neutralizing antibodies against rBRSV, showing that the epitopes that neutralizing antibodies are directed against are present in the appropriate conformation. Vaccinia virus recombinants expressing HRSV G or F are able to induce neutralizing antibodies to HRSV in animals, with vaccinia recombinants expressing HRSV F being superior to those expressing HRSV G (Olmsted et al., 1986 ). However, in chimpanzees, which are permissive for HRSV to a degree comparable to that in humans, the efficiency as a vaccine was poor, due to the lack of local immunity in the respiratory tract (Collins et al., 1990 ). Thus, it will be interesting to test whether rBRSVs that lack the SH and/or G gene(s) are able to induce protective immunity after mucosal application to calves, the natural host for BRSV.

Interaction with heavily charged cell-surface GAGs is quite a common mechanism of attachment to target cells for enveloped and non-enveloped viruses and also for other intracellular parasites (Rostand & Esko, 1997 ). We have found that attachment of BRSV is heparin dependent, as it is for the closely related HRSV. Heparin binding by HRSV has been attributed to its attachment protein, G (Krusat & Streckert, 1997 ). The G proteins of HRSV and BRSV are structurally and genetically related (Lerch et al., 1990 ) and share an amino acid identity of about 30%. Surprisingly, BRSV G-deletion mutants were sensitive to inhibition by heparin and to pre-treatment of target cells with chlorate to the same degree as rBRSV. Since the only remaining envelope glycoprotein in rBRSV{Delta}SHG is the F protein, we have analysed this protein for its heparin-binding potential in vitro. As shown by HAC, F protein binds heparin in vitro, although with lower affinity than the G protein. We have found that F protein elutes from the heparin–Sepharose matrix at a salt concentration of approximately 310 mM, which is well above physiological salt concentrations of approximately 150 mM. Moreover, the heparin-binding potential of the F protein as an attachment protein localized in the virus membrane is probably underestimated by HAC. In our experiments, a high concentration of Zwittergent 3-12 was chosen in order to obtain highly dispersed glycoprotein solutions necessary for high-resolution chromatography. This situation differs from that in the attachment process, where the virus envelope opposes the cell surface with a multitude of potential ligands and receptors in close proximity. Stable attachment of the virion can thus be established by a multitude of weak co-operative interactions of F protein molecules with abundant heparan sulphate residues on the cell surface. Recently, the F protein of HRSV has been shown to bind heparin, and replication of the G protein-negative HRSV strain B1 cp-52 can be inhibited by addition of heparin (Feldman et al., 2000 ). In the latter paper, elution of heparin-binding proteins from the heparin-modified matrix was carried out by a batch process, so no data on the affinity of the viral proteins were obtained. We have chromatographed HRSV extracts under our conditions and found essentially the same distribution of F and G protein as for rBRSV (data not shown). Different affinities for heparin might indicate different functions of F and G proteins in the heparin-dependent attachment process or differential binding to different heparin/heparan sulphate species or to different domains within the same heparin/heparan sulphate species. As in the HRSV F protein, candidate sequences for heparin-binding domains can be found in the BRSV F protein, but the functional heparin-binding domain has not yet been mapped.

As a consequence of our results, the functions of the F and G proteins in BRSV have to be reconsidered. Our data suggest (i) that the F protein is multifunctional and plays a role in attachment, in addition to its function in fusion, and (ii) that heparin-dependent attachment of BRSV virions can be established by the F and/or G protein, with the high-affinity interaction of the G protein with heparin being non-essential for a productive infection in cell culture. The function of the SH protein is still unclear, but a significant contribution to the attachment and entry process can be excluded.


   Acknowledgments
 
We thank Geraldine Taylor (IAH, Compton, UK) and J. A. Meléro (Instituto de Salud Carlos III, Madrid, Spain) for their gifts of monoclonal antibodies. We also thank Kathrin Schuldt for perfect technical assistance and Thomas C. Mettenleiter for helpful comments on the manuscript. This work was supported by Intervet International BV, The Netherlands.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 3 October 2000; accepted 27 November 2000.