Effect of heparin on infection with BRSV-∆G/GFP and BRSV-GFP mutants

5.2 A NALYSIS OF THE BRSV MUTANTS

5.2.3 Effect of heparin on infection with BRSV-∆G/GFP and BRSV-GFP mutants

To analyze the influence of the basic amino acids in the F2 putative heparin-binding domain on the interaction with soluble heparin, the infection of Vero cells by BRSV-GFP and BRSV-∆G/GFP mutants with replaced basic residues was analyzed.

In a competition assay, heparin was chosen as competitor as this GAG showed the highest inhibition activity towards to BRSV-∆G/GFP.

Figure 13. Inhibition of BRSV-∆G/GFP mutants infectivity by soluble heparin.

Mean values calculated from 3 separate experiments are shown.

BRSV-∆G/GFP parental virus infectivity was reduced by more than 50 % at a concentration of 2.5 µl/ml compared with the untreated control. The infectivity of parental and mutant viruses was slightly reduced in the presence of 0.25 µg/ml soluble heparin. All BRSV-∆G/GFP mutants with point mutations in the F2 subunit showed a higher sensitivity to inhibition with soluble heparin than the parental virus (Fig. 13).

The highest sensitivity towards heparin was found with

BRSV-∆G/GFP(K63/66) and BRSV-∆G/GFP(K63/66/70). BRSV-∆G/GFP(K85N) showed the 0.25 µg/ml 2.5 µg/ml 25 µg/ml 250 µg/ml

lowest sensitivity to inhibition compared with all other mutants. None of the viruses showed complete inhibition of infection even at a concentration of 250 µg/ml suggesting an alternative pathway that led to productive infection.

Soluble heparin differentially affected the infectivity of the mutants depending on the presence of the G protein. All mutants expressing the G protein showed a lower sensitivity to inhibition compared with the corresponding mutants lacking the G gene. This indicates that the decline in heparin binding of the recombinant viruses containing point mutation in F2 could be compensated to some extent from the heparin binding activity of the G protein. The degree of compensation differs between different mutants (Fig. 14).

Figure 14. Inhibition of recombinant BRSV viruses by soluble heparin.

2.5µg/ml 25µg/ml 250µg/ml Heparin concentration

0 10 20 30 40 50

2.5µg/ml 25µg/ml 250µg/ml

Infectivity (% of control)

GFP K63/66/70N

∆G/GFP K63/66/70N

GFP parental

∆G/GFP parental

Figure 14. Inhibition of recombinant BRSV viruses by soluble heparin.

2.5µg/ml 25µg/ml 250µg/ml

Figure 14. Inhibition of recombinant BRSV viruses by soluble heparin.

Heparin concentration

5.2.4 Analysis of mutations at positions K75 and K77

A reverse genetics system is not feasible if mutations affect the viability of the recombinant virus. Attempts to generate recombinant viruses containing asparagine at positions K75 and K77 were not successful. To elucidate how the exchange of these amino acids may affect F protein function, a plasmid driven expression system was used. The open reading frame of the parental F protein and F protein containing mutations at the positions 75, 77, and 80 were cloned into the pTM1 vector to give bF parental, bF(K75N), bF(K77N), bF(K75/77N), or pTM1-bF(K75/77780N), respectively. pTM plasmid vector was constructed for expression of genes under control of T7 promoter and contains an internal ribosomal entry site from encephalomyocarditis virus to allow cap-independent translation of the transcripts (Moss et al., 1990). BSR-T7/5 cells stably expressing the T7-RNA polymerase (Buchholz et al., 1999) were infected with vaccinia virus containing the T7-RNA polymerase gene before transfection with the respective pTM1 constructs in order to enhance the expression level of the F protein. Transfected cells expressing the modified F proteins were analyzed by fluorescence microscopy, flow cytometry and surface biotinylation. The parental and the mutant proteins F(K75N), F(K77N), F(K75/77N) and F(K75/77/80N) were characterized with respect to proteolytic cleavage, cell surface transport and fusion activity.

5.2.5 Analysis of F protein cell surface transport

To determine if the mutants F(K75N), F(K77), F(K75/77N), and F(K75/77/80N) are transported to the cell surface, indirect immunofluorescence of transfected BSRT7/5 cells was performed. Transfected cells were labeled with F-specific monoclonal antibodies. Figure 15 shows that F(K75N) and F(K77N) as well as the double mutant F(K75/77N) were detected at the cell surface like the parental F protein. Only the F(K75/77/80N) triple mutant showed a reduced cell surface expression compared to parental F protein.

Figure 15. Indirect immunofluorescence of BSR-T7/5 cells expressing modified F protein. The cells were examined at 400X magnification.

BRSV F parental BRSV F K75N

BRSV F K77N BRSV F K75/77N

BRSV F K75/77/80N

Cell surface expression of the F protein mutants was also quantitatively determined by flow cytometry. Transfected cells were detached without trypsin treatment and were stained with a FITC-conjugated bovine anti-BRSV serum. As a negative control, cells were transfected with non-recombinant pTM1 plasmid and stained in the same way. Table 5 summarizes the results of three independent transfection experiments. The F(K75N) and F(K77N) mutants as well as the double mutant F(K75/77N) showed similar percent of fluorescent cells as the parental BRSV F protein whereas the triple mutant F(K7/77/780N) showed a 75 % reduction, suggesting that the combination of three point mutations has an effect on cell surface transport.

pTm-F mutant % fluorescent cells X mean

pTM F wt 11.2 45

pTM F K75N 10.4 47

pTM F K77N 10.6 46

pTM F K75/77N 9.0 40

pTM F K75/77/80N 2.5 48

Table 5. Flow-cytometric analysis of BSR-T7/5 cells expressing modified BRSV F proteins.

5.2.6 Fusion activity of F mutants

The ability of the mutant F protein to induce formation of syncytia in transfected BSRT7/5 cells was analyzed. Transfected BSR-T7/5 cells with pTM1-F protein mutants were examined at 400X magnification. Only the mutant F(K77N) protein induced formation of syncytia the size of which was comparable to those formed by the parental F protein. F(K75N) as well as F(K75/77N) and F(K75/77/80N) showed F protein expression in single cells but no syncytia formation was detected (Fig. 15).

5.2.6.1 Biotinylation and immunoprecipitation of the mutant F proteins

To analyze whether F protein cleavage is affected by the exchange of the lysines at positions 75 and 77 in the F2 subunit, transfected BSR-T7/5 cells were labeled with sulfo-NHS-biotin at 4 °C. This reagent does not penetrate the plasma membrane and therefore reacts only with proteins at the cell surface (Le Bivic et al., 1989). The labeled cells were immunoprecipitated from the cell lysates by a monoclonal antibody directed against the F1 subunit of the fusion protein. The immunoprecipitates were separated by SDS-PAGE under reducing conditions in the presence of dithiothreitol in order to reduce disulfide bonds. The samples were transferred to nitrocellulose membrane and probed with streptavidin-peroxidase (Fig.

16). All F proteins appeared as single bands of 50 kDa representing the F1 subunit suggesting that the point mutations at positions K75, K77 and K75/77 did not affect furin mediated cleavage of the F0 precursor. Densitometric analysis of the bands revealed that three of the samples showed very similar densities as the parental F protein. In contrast, the triple mutant was detected in lower amounts at the cell surface accounting for about 20%-40% of the amount of the parental F protein.

bF wt bF K75N bF K77N bF K75/77N bF K75/77/80N

Figure 16. Surface biotinylation of the F protein mutants.

Taken together, indirect immunofluorescence, flow cytometry and cell surface biotinylation provide evidence that three of the mutants, namely K75N, K77N and K75/77N of the BRSV F protein are efficiently cleaved and transported to the cell surface, with only slight differences in the rate of transport. The third mutant with three point mutations, F(K75/77/80N), showed a reduced cell surface transport.

F1 (50 KDa) F0 (70 KDa)

5.3 Generation of MBP-F2 hybrids

As an alternative approach for characterization of the F protein heparin-binding activity, a chimeric protein composed of the F2 subunit and the maltose-binding protein (MBP) was generated. cDNA encoding the F2 subunit of HRSV, BRSV and PVM fusion proteins was cloned into the pMaL-c2 plasmid in frame with the maleE gene which encodes the maltose-binding protein of E. coli. The chimeric proteins (MBP-F2) were expressed in E.coli and purified by affinity chromatography on immobilized amylose. Three different approaches were used to analyze the binding capacity of the chimeric proteins: flow cytometry, ELISA using biotin-heparin conjugates bound to streptavidin-coated microtiter plates, and a pull-down assay using heparin-agarose. With neither approach a binding activity of MBP-F2 was detected suggesting that the presence of the F1 subunit might be necessary to obtain a functional molecule. Besides, N-glycosylation which is absent from MBP-F2 might be important to allow the protein to adopt a proper conformation.

6 Discussion

Growing evidence suggests that RSV F protein, apart from its fusion activity has also receptor binding activities. A first indication for this additional activity of the F protein came from the isolation of an RSV mutant lacking SH and G genes, cp-52 (Karron et al., 1997). This mutant grew to relatively high titers in cultured cells, but was poorly infectious in mice and humans. Therefore, not only the fusion but also an attachment function was attributed to the F protein. RSV deletion mutants lacking G and/or SH genes have also been generated by reverse genetics (Techaarpornkul et al., 2001;

Karger et al., 2001). Using these mutants the heparin-binding activities of the F protein were demonstrated for HRSV and BRSV strains (Feldman et al., 2000; Karger et al., 2001). In contrast to the G protein, the sequence responsible for interaction of the F protein with heparin-like molecules has not been identified so far.

Several characteristics of the F2 subunit make this part of the BRSV fusion protein a promising candidate for a domain which can bind cell surface GAGs:

1) It has been shown that the F2 subunit of the F protein accounts for the species specificity of RSV infection (Schlender et al., 2003). This property suggests the presence of a specific receptor binding site in the F2 subunit of HRSV and BRSV, respectively.

2) Comparison of different HRSV and BRSV strains revealed extensive sequence variation in the F2 subunit (Fig. 17). In contrast, the F1 subunit is highly conserved with 90% identity between different RSV strains.

3) Within the F2 subunit there exists a cluster of basic amino acids. This region of basic residues between amino acids positions 62 and 86 is a promising candidate for a heparin-binding domain.

63 66 70 75 77 80 85

BRSV (A Tue51908) ...SKIQKNVCKSTDSKVKLIKQELERYNN...

HRSV (Long) ...SKIKENKCNGTDAKVKLIKQELDKYKN...

Figure 17. Partial amino acid sequence of the F2 subunit of HRSV and BRSV. The basic amino acids of the putative heparin-binding domain are shown in bold letters.

6.1 MBP-basic amino acid epitope in F2 subunit is not sufficient for interaction with heparin

In a first attempt to analyze the effect of point mutations in the putative heparin-binding domain only the small F2 subunit of the BRSV F protein was used. A chimeric protein composed of the F2 subunit and the maltose binding protein (MBP) from E.coli was generated. Using these chimeric constructs an interaction with glycosaminoglycans could not be detected suggesting that the MBP-basic amino acids epitope is not sufficient for interaction with heparin. Furthermore, N-glycosylation which is absent from the MBP-F2 chimeric protein expressed in bacterial cells might be important to allow the protein to adopt a conformation which allows interaction with GAGs. Therefore this approach was not suitable to characterize the basic amino acids of the F2 subunit.

6.2 Most of the point mutations in the putative binding domain of the F protein do not affect virus viability

Recently, reverse genetics has become a powerful tool for studying the function of individual viral proteins in the context of an RSV infection. RSV deletion mutants lacking individual genes have been generated from cDNA by this system (Collins et al., 1995; Buchholz et al., 1999).

Reverse genetics was used in this work for the generation of mutant BRSV containing point mutations in the putative heparin-binding domain of the F2. To exclude the heparin-binding activity of the G protein, deletion mutants were generated by replacing the G gene with the gene encoding the green fluorescent protein (GFP), BRSV-∆G/GFP. Point mutations were introduced also into a second BRSV genome containing the GFP gene as an additional transcription unit, BRSV-GFP. BRSV-GFP and BRSV-∆G/GFP viruses containing the parental F protein were successfully recovered and showed formation of syncytia with similar size. Both mutants were similar in their replication kinetics in accordance with previously published data (Karger et al., 2001). They showed that recombinant BRSV containing the F protein as the only

glycoprotein replicates well in cell culture.

6.3 Effect of different GAGs as inhibitors of recombinant BRSV infection

The analysis of the interaction of BRSV with GAG has shown that compounds containing iduronic acid are more efficient inhibitors than are those containing glucuronic acid. In this respect BRSV is very similar to HRSV which also has been reported to be more sensitive to inhibition by heparin and dermatan sulfate compared to glucuronic acid containing GAGs (Hallak et al., 2000a).

6.4 Role of K80N and R85N mutations

K80N mutants were rescued with high efficiency and propagated on Vero cells to high titers. Though this lysine is conserved in both HRSV and BRSV, replacement of this amino acid with asparagine did not reduce the viability of the mutant in contrast to the conserved residues K75 and K77 the replacement of which had detrimental effects on receptor binding and/or fusion activity of the F protein. The recovered K80N mutant virus was more sensitive to inhibition by heparin and thus – like K63 and K66 – appears to have a modulating effect on the interaction of the F protein with GAG.

The mutation at residue R85 had a more dramatic effect. Amino acid 85 of F2 is a basic residue conserved in both bovine and human strains. In HRSV, this position is lysine, in BRSV it is arginine, suggesting that the presence of a positively charged amino acid may have some functional importance. Mutant R85N could be recovered. However, it grew only to very low titers. The ability of this virus to induce syncytia infected Vero cells was strongly reduced. Therefore, we conclude that the arginine residue at position 85 plays an important role for the fusion activity of BRSV.

6.5 Lysines at positions 75 and 77 of the F2 subunit play an essential role for F protein function

Attempts to recover recombinant viruses containing point mutations at highly conserved residues K75 and K77 were not successful, neither with the BRSV-∆G/GFP nor with the BRSV-GFP backbone. The failure to recover the K75N mutant may be explained by an impaired fusion activity. Upon plasmid-driven expression, the mutant protein was unable to induce syncytia formation. As the protein was transported to the cell surface, proteolytically cleaved into subunits, and detected by a monoclonal antibody we assume that the mutation did not affect the conformation. Therefore, K75 appears to be directly involved in the fusion activity of the F protein. Like K75N, no virus could be recovered for mutant K77N. The reason for this is unclear. The respective mutant protein not only was transported to the cell surface it also effectively induced the formation of syncytia. Thus, this protein had retained its fusion activity. Furthermore it is not known whether replacement of the basic residues by amino acids other than asparagine will show the same effect. Further studies are needed to understand the importance of lysine77. It should be interesting to know whether mutation of this basic amino acid in the HRSV F protein has a similar effect as observed for the BRSV fusion protein.

6.6 Exchange of lysines 63 and 66 has a modulating effect on BRSV infectivity

Lysines located at positions 63 and 66 in the BRSV fusion protein are positioned at residues 65 and 66 in the corresponding protein of HRSV. The different location may reflect the different importance of these basic amino acids for BRSV. The viability of BRSV was not affected when either or both residues were replaced by asparagines. In fact, mutant K66N grew to higher titers than the parental virus, while lower titers were determined for mutant K63N. These difference may be connected with the differences observed in the syncytia formation induced by both mutant viruses. Syncytia induced by

the K63N mutant were somewhat larger compared to the parental virus, whereas infection by the K66N mutant resulted in somewhat smaller syncytia. An inverse relation between the extent of syncytium formation and the titer of infectious virus released into the supernatant has also been reported for other paramyxoviruses. An explanation of the difference in syncytia formation is difficult as the three-dimensional structure of the fusion protein has not yet been elucidated. The inhibition studies indicate that both mutants are more sensitive to heparin inhibition compared with parental virus, but no significant difference between the two mutants was detected. Maybe the two lysines contribute in a similar way to the binding of the F protein to GAG, but differ in their contribution to the transition to the next step, the fusion reaction. As both lysines are conserved, mutations at residues 63 or 66 obviously have no selection advantage in nature. So far the mutants have been analysed only for growth on cell cultures. In the future, virus growth should be determined also on differentiated airway epithelial cells.

This cell system closer to the natural situation of an RSV infection. Maybe, under such conditions, residues 63 and 66 are more important for virus growth compared to infection of conventional cell cultures.

6.7 G protein may compensate for point mutations in the F2 subunit

Some of the recombinant viruses containing point mutations in the F2 subunit and expressing G protein were less affected by soluble heparin than viruses containing the same mutations but lacking the G gene. This indicates that the heparin binding activity of the G protein, compensates to some extent for the decline of heparin binding due to the mutations in the F2 subunit.

6.8 RSV attachment to the cell surface involves additional cellular receptor(s)

As has been shown for parental HRSV and BRSV, we also found for the mutants that infectivity was sensitive to inhibition by heparin. In the inhibition experiments even higher concentrations of soluble heparin did not completely abolish virus infection. The residual infectivity may be explained by the interaction with a ligand different from GAG.

The existence of a so far unidentified receptor has also been suggested by results obtained with chimeric BRSV. Be analysing chimeric F proteins, the F2 subunit was shown to be responsible for the species-specific interaction of RSV with differentiated airway epithelial cells (Schlender et al., 2002). As appears difficult to explain species-specific differences in the binding acitivity by binding to GAG, it has been suggested that the F2 subunit not only harbours residues for the interaction with GAG, but also a binding site for a protein receptor (Schlender et al., 2003).

6.9 Conclusions

Viral fusion proteins that have been studied in more detail, e.g. the HA protein of influenza A virus or the gp120/gp41 of HIV, are known to undergo a conformational change prior to the fusion reaction (Skehel and Wiley, 2000; Weissenhorn et al., 1999).

This intermolecular rearrangement that makes the protein fusion-active is induced in influenza virus by the low pH encountered within endosomes upon internalisation of the virus. HIV does not require endocytotic uptake. Following attachment to CD4 receptors the interaction with members of the family of chemokine receptors triggers the conformational change that makes gp41 fusion-active (Furuta et al., 1998) . Sequential interaction with cell surface molecules in the initiation of infection has also been reported for members of the herpesvirus family, herpes simplex-1 (HSV-1) and pseudorabies.

With these viruses, attachment is mediated by binding of the viral surface glycoprotein gC to cell surface heparan sulphate proteoglycans (WuDunn and Spear, 1989). Virus entry, i.e. fusion of the viral membrane with the plasma membrane, requires the interaction of the viral glycoprotein gD with a member of the nectin family or an

alternative cell surface receptor (Krummenacher et al., 1998). A conformational change is also expected to render the F protein of RSV fusion active. In theory, interaction with GAG may provide such a stimulus. However, it is also possible that as in the case of herpesviruses, interaction with GAG only mediates attachment and that subsequent interaction with a protein receptor is required to induce the fusion of the viral membrane with the plasma membrane.

7 Summary

Characterization of the heparin-binding activity of the bovine respiratory syncytial virus fusion protein

Diana Panayotova

Cell surface glycosaminoglycans (GAGs) are a major factor for respiratory syncytial virus (RSV) attachment to cultured cells leading to infection. The viral glycoprotein G binds to GAGs and was thought to be the only viral attachment protein.

Im Dokument Characterization of the heparin-binding activity of the bovine respiratory syncytial virus fusion protein (Seite 64-0)