Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, 17493 Greifswald-Insel Riems, Germany
Correspondence
Günther M. Keil
Guenther.M.Keil{at}rie.bfav.de
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ABSTRACT |
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Present address: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-8007, USA.
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INTRODUCTION |
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A so far unique feature of both HRSV and BRSV F proteins is the cleavage of the respective F0 proteins at two furin recognition sites after the Arg109 and Arg136 residues, resulting in the release of a 27 aa intervening peptide (Gonzáles-Reyes et al., 2001; Zimmer et al., 2001
), which is N-glycosylated and has been named pep27 (Zimmer et al., 2001
).
Recently it was shown that cleavage of the BRSV F protein after Arg109 and the presence of pep27 are dispensable for BRSV replication in cell culture (Zimmer et al., 2002). Recent studies by Zimmer et al. (2003)
demonstrated that pep27 is further modified after cleavage, resulting in conversion to a bioactive peptide of the tachykinin family, named virokinin. Virokinin acts on specific G protein-coupled receptors, and it has been suggested that virokinin plays a role in the pathogenesis of BRSV, for example by assisting escape from the host immune response and increasing virus survival in the host (Zimmer et al., 2003
).
We report here that the sequence of the intervening peptide influences intracellular transport, maturation and biological activity of the F protein and show that bovine cytokines inserted into F0 in place of pep27 are secreted into the medium of transfected and recombinant BRSV (rBRSV)-infected cells.
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METHODS |
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Construction of plasmids.
All cloning procedures were carried out according to standard methods (Sambrook et al., 1989). An artificial open reading frame (ORF) encoding the BRSV field strain Stormond F protein was assembled from synthetic oligonucleotides using the codon preference of glycoprotein D of bovine herpesvirus type 1 (BHV-1) (P. König & G. M. Keil, unpublished data). This ORF permits expression of the F protein via the nucleus. The synthetic ORF, named Fsyn, was integrated into plasmid vector pSP73, resulting in pspFsyn. In pspFsyn, the codons for the furin cleavage sites were flanked by recognition sequences for the restriction enzymes SmaI and AvrII to facilitate mutagenesis of this region (see Fig. 1
for details). For the construction of the ORF encoding Fpep27, two oligonucleotides (5'-GGGCGAAGCGCTTC-3' and 5'-CTAGGAAGCGCTTCGCCC-3') were hybridized and the resulting fragment was used to replace the SmaIAvrII fragment encompassing the codons for pep27 (Fig. 1
), resulting in plasmid pspFpep27. For replacement of pep27 by the Kat peptide oligonucleotides, Kat+ (5'-GGGCGAAGCGCGGCAAGGCCACGCGCATCAACATCAGCACGCCCGAGCGCTTCGAGAAGACGAAGAAGCGCAAGCGCCGCTTC-3') and Kat (5'-CTAGGAAGCGGCCTTGGCTTTCGTCTTCTCGAAGCGCTCGGGCGTGCTGATGTTGATGCGCGTGGCCTTGCCGCGCTTCGCCC-3') were hybridized and plasmid pspFKat was obtained as described above. Kat stands for the first three amino acids of the Kat peptide Lys-Ala-Thr in one-letter code.
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Amplification products were cleaved with SmaI and AvrII (recognition sites shown in bold) and used to replace the SmaIAvrII fragment encompassing the codons for pep27 (Fig. 1), resulting in plasmids pspFboIL2, pspFboIL4 and pspFboIFN-
.
Correct introduction of the envisaged modifications into the Fsyn ORF was verified by sequence analysis. For transient expression experiments, the ORFs encoding wild-type (wt) and mutated F proteins were cleaved out of the respective plasmids with BglII and inserted into the BglII-cleaved expression vector pe1cas. Plasmid pe1cas, a derivative of plasmid pROMe (Kühnle et al., 1996) from which the glycoprotein D (gD) expression unit has been removed, contains the murine cytomegalovirus (MCMV) early 1 promoter (Bühler et al., 1990
) followed by a polylinker sequence and the polyadenylation signal of the BHV-1 gD gene. To direct expression in transient experiments, the MCMV early 1 promoter requires transactivation by the MCMV immediate-early protein pp89, which is provided by co-transfection with plasmid pAMB25 (Koszinowski et al., 1986
).
The mutated F ORFs described above were used to generate modified BRSV F genes by addition of BRSV transcription start and transcription end/polyadenylation signals and transferred into a BRSV full-length plasmid containing the complete BRSV anti-genome sequence, so that the original BRSV F sequence was replaced by the synthetic genes. In the first step, the respective F ORFs were transferred by restriction with NcoI, a restriction recognition site that is present at and comprises the translation initiation codons, and EcoRI, located downstream of the translation stop codons, into a pBluescript SK vector with a modified polylinker. The polylinker sequence shown below contained a rBRSV adapter with, in 5' to 3' order, an SphI site (italics), the BRSV transcription start site (underlined), an NcoI and an EcoRI site (bold, lower case), a BRSV transcription end/polyadenylation signal (underlined) and a ClaI site (italics): 5'-GCATGCTTCACTAATTAAAACTGGGGCAAATAAccatggCGAgaattcCATGTTGATAGTTATATAAAAATATTATATTATGTCTCGAGGAAAGGAATCGAT-3'. The modified F genes were subsequently transferred into a BRSV full-length plasmid described previously (Buchholz et al., 2000) using the single restriction sites SphI and ClaI, which are present in the rBRSV G/F and F/M2 intergenic regions, resulting in BRSV full-length plasmids pBRSV-Fsyn, pBRSV-Fpep27, pBRSV-FKat, pBRSV-FboIL2, pBRSV-FboIL4 and pBRSV-FboIFN-
.
Transfection experiments.
For transient expression, KOP/R cells were co-transfected with 2·5 µg of the respective expression plasmid and 0·5 µg pAMB25 DNA per 5x105 cells using the Superfect reagent (Qiagen) as recommended by the supplier. For recovery of rBRSV from cloned DNA, subconfluent BSR T7/5 cells stably expressing phage T7 RNA polymerase were transfected as described previously (Buchholz et al., 1999) with 5·5 µg of the full-length plasmid pBRSV-Fsyn, pBRSV-Fpep27, pBRSV-FKat, pBRSV-FboIL2, pBRSV-FboIL4 or pBRSV-FboIFN-
and a set of four support plasmids (2 µg pN, 2 µg pP, 1 µg pM2 and 1 µg pL), which encode the BRSV N, P, M2 and L proteins, respectively. Cells were split every 34 days and recombinant virus replication was monitored by an indirect immunofluorescence assay. Culture medium was adjusted to 100 mM MgSO4 and 50 mM HEPES (pH 7·5), harvested and stored at 70 °C when cytopathic effect was advanced.
Immunoprecipitation.
Transfected or infected cells were incubated with cell culture medium lacking methionine and cysteine for 2 h before addition of [35S]methionine (500 µCi ml1, 18·5 MBq ml1) and [35S]cysteine (250 µCi ml1, 9·25 MBq ml1). Lysis of cells and immunoprecipitation of proteins from cell lysates and culture supernatants were performed as described (Keil et al., 1985) using mono-specific rabbit sera directed against boIL2 and boIL4 (Kühnle et al., 1996
) or a BRSV F1-specific rabbit serum raised against a bacterially expressed F protein containing the entire amino acid sequence of the F1 subunit. Precipitated proteins were visualized by fluorography after 12·5 or 7·5 % SDS-PAGE.
Determination of boIFN- activity.
Secretion of biologically active boIFN- into the cell culture medium of transfected KOP/R cells was analysed by a vesicular stomatitis virus (VSV) plaque reduction assay. Supernatants from KOP/R cells expressing FboIFN-
or Fpep27 as control were harvested 2 days post-transfection (p.t.). The supernatants were serially diluted in normal cell culture medium and added to KOP/R cells in 24-well plates. Cultures were incubated for 24 h at 37 °C and then infected with approximately 100 p.f.u. VSV. Supernatants were removed 1 h post-infection (p.i.) and semi-solid methylcellulose-containing medium was added. Plaques were counted after 24 h incubation at 37 °C.
Indirect immunofluorescence assays.
Cells were fixed with 3 % paraformaldehyde in PBS for 20 min, subjected to membrane permeabilization with 0·2 % Triton X-100 and sequentially incubated with F-specific mAb 19 (kindly provided by Geraldine Taylor, Compton, UK) and 5-[(4,6-dichlorotriazin-2-yl)amino]fluorescein hydrochloride (DTAF)-conjugated rabbit anti-mouse IgG (Dianova).
Analysis of cell culture characteristics.
For multi-cycle growth curves, MDBK cultures were infected with 0·1 p.f.u. per cell. At 6 h p.i., cells were incubated for 2 min with low-pH citrate buffer (40 mM citric acid, 10 mM KCl, 135 mM NaCl, pH 3·0) to inactivate extracellular virions. Cells were washed twice with cell culture medium and incubated for the times indicated, when supernatants and cells were harvested together and stored at 70 °C. Serial dilutions were titrated on MDBK cells and cultures were incubated under semi-solid medium containing methylcellulose for 6 days. Plaques were counted after immunostaining with mAb 19 and DTAF-conjugated anti-mouse IgG under a fluorescence microscope.
Determination of plaque diameters.
MDBK cells were infected with diluted virus stocks and incubated under semi-solid medium containing methylcellulose for 6 days. Diameters of 100 randomly selected plaques were determined after immunostaining with mAb 19 and DTAF-conjugated anti-mouse IgG under a fluorescence microscope using a graduated ocular.
Penetration kinetics.
MDBK cells were pre-cooled at 4 °C for 30 min and further incubated at 4 °C for 2 h after the addition of approximately 200 p.f.u. virus to allow adsorption. Cultures were then shifted to 37 °C and extracellular virions were inactivated at the indicated times by incubation of the monolayers with low-pH citrate buffer for 2 min. Cells were washed twice with cell culture medium and incubated under semi-solid medium containing methylcellulose for 6 days. Plaques were counted after immunostaining with mAb 19 and DTAF-conjugated anti-mouse IgG under a fluorescence microscope. The plaque count of untreated cultures was set as 100 % penetration.
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RESULTS |
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The ORFs encoding wt and mutated F proteins were integrated into pe1cas, which enables protein expression under control of the MCMV early 1 promoter in the presence of the MCMV major immediate-early protein pp89, encoded by plasmid pAMB25 (Bühler et al., 1990; Koszinowski et al., 1986
). Plasmid pe1cas also contains the polyadenylation signal from the BHV-1 gD gene for efficient polyadenylation of transcripts (Kühnle et al., 1996
). Transient expression of the F variants was achieved by co-transfection of the respective pe1cas plasmid together with pAMB25 into KOP/R cells. Cultures expressing wt F protein were fixed 28 h p.t. due to an early cytopathic effect, whereas the mutant F-expressing cells were fixed at 44 h p.t. Expression of the F proteins was monitored by indirect immunofluorescence using F-specific mAb 19 (Fig. 2
). Wt F protein induced large syncytia, which started to detach from the culture dishes at about 30 h p.t. Syncytia were also found in cultures expressing Fpep27. However, the size of the fused cells and the number of nuclei within the syncytia were significantly reduced. Cultures expressing FKat and FboIL4 contained mainly single F-positive cells and the few detected syncytia contained only three to five nuclei. No fused cells were found in cells expressing FboIL2 and FboIFN-
, even when cultures were fixed at 72 h p.t. These results showed that pep27 is not essential but is beneficial for F-mediated syncytium formation and that the cell fusion activity is influenced by the amino acid sequence between the furin cleavage sites.
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The fate of boIL2 and boIL4 after transport by F0boIL2 and F0boIL4 to the trans-Golgi network and cleavage was analysed by immunoprecipitation of cell-associated and secreted proteins using rabbit sera against boIL2 and boIL4 (Kühnle et al., 1996) and supernatants from KOP/R cells infected with boIL2- and boIL4-expressing BHV-1 recombinants BHV-1/ieIL-2 or BHV-1/ieIL-4 (Kühnle et al., 1996
) as controls. From the culture medium of cells transfected with pFboIL2, the anti-boIL2 serum precipitated the 22 kDa boIL2 protein, which migrated at a distance comparable with mature boIL2 secreted into the supernatant of KOP/R cells infected with BHV-1/ieIL-2 (Fig. 3c
, lanes 1 and 3), whereas F0boIL2 (Fig. 3b
, lane 4), but not mature boIL2, could be precipitated from lysed cells (not shown). An equivalent result was obtained after transfection with pFboIL4 followed by immunoprecipitations in which the anti-boIL4 serum precipitated the 20 kDa boIL4 protein, which co-migrated with BHV-1/ieIL-4-expressed boIL4. Again, boIL4 was detected only in the transfected cell culture medium (Fig. 3c
, lanes 2 and 4), but not in cell lysates, from which the anti-boIL4 serum exclusively precipitated the uncleaved F0boIL4 (Fig. 3b
, lane 5). Thus, boIL2 and boIL4 were efficiently secreted from the transfected cells after cleavage of their respective precursors. The additional bands seen in all lanes in Fig. 3(c)
were due to non-specifically precipitated proteins, and faster-migrating polypeptides present in Fig. 3(c, lanes 1 and 3) probably represented breakdown products of boIL2 (Kühnle et al., 1996
).
Secretion of boIFN- was not directly monitored due to the lack of specific antibodies. Evidence that biologically active boIFN-
was released from FboIFN-
-expressing cells was obtained by the results of VSV plaque reduction assays, which revealed that supernatants from cells transfected with pFboIFN-
contained 1020 U IFN-
(ml medium)1 (data not shown). These results demonstrated that the BRSV F protein can be used as a vehicle to transport proteins into the extracellular space.
To study the effect of the mutations on the function of the F protein for productive virus replication, plasmids expressing wt or modified anti-genomic RNAs and support plasmids encoding the BRSV N, P, L and M2 proteins were co-transfected into BSR T7/5 cells (Buchholz et al., 1999). Infectious rBRSV was efficiently recovered from transfections with the pBRSV plasmids containing the ORFs encoding wt F, Fpep27, FKat, FboIL2 and FboIL4, whereas attempts to rescue infectious virus expressing FboIFN-
were repeatedly unsuccessful, suggesting that FboIFN-
interferes with a vital function of BRSV. It appears, however, unlikely that the anti-viral activity of boIFN-
precluded isolation of BRSV-FboIFN-
, since recombinant human RSV, which expresses IFN-
classically from a gene cassette, could be recovered from plasmids (Bukreyev et al., 1999
).
Viral stocks of wt F-expressing BRSV-Fsyn and mutant F-expressing BRSV-Fpep27, BRSV-FKat, BRSV-FboIL2 and BRSV-FboIL4 were prepared on MDBK cells for further characterization. Infection of KOP/R cells with the isolates revealed that all induced syncytium formation, irrespective of the ability of the F proteins to mediate cell fusion in transient expression experiments (not shown). Direct sequencing of RT-PCR fragments of the respective F ORFs using RNA from partially purified virions as templates revealed that the ORFs encoding wt F, Fpep27 and FKat were as expected, whereas the ORFs encoding FboIL2 and FboIL4 carried mutations resulting in an exchange of Val152 to Glu within the F protein sequence of BRSV-FboIL2 and of Gly69 to Arg within the boIL4 protein sequence of BRSV-FboIL4. Whether these mutations were beneficial for the viability and/or cell fusion activity mediated by BRSV-FboIL2 and BRSV-FboIL4 needs to be clarified.
Cell culture characteristics of the isolates were analysed on MDBK cells. For multi-cycle growth curves, cells were infected with 0·1 p.f.u. per cell, non-penetrated virions were inactivated by low-pH treatment at 6 h p.i. and cultures were stored at the times indicated in Fig. 5(a) at 70 °C until titration. BRSV-Fsyn and BRSV-FboIL4 replicated almost identically, whereas reproduction of BRSV-Fpep27 and BRSV-FKat was initially delayed but reached wt titres by 48 h p.i. (Fig. 5a
). In contrast, infectious BRSV-FboIL2 was detected only at 48 h p.i. and final titres reached by 144 h p.i. were approximately 100-fold lower than the titres obtained for the other isolates. Thus, an intervening peptide is not required for infectious replication of BRSV and its amino acid sequence can influence virus yield, which, with regard to pep27, is in accordance with results published by Zimmer et al. (2002)
. To test for the influence of the intervening peptide on direct cell-to-cell spread of BRSV, MDBK cultures were infected with appropriate dilutions of virus stocks and incubated under methylcellulose-containing semi-solid medium. At day 6 p.i., cells were fixed and immunostained using F-specific mAb 19 and DTAF-conjugated anti-mouse IgG. The diameters of 100 plaques of each isolate were measured using a fluorescence microscope and a graduated ocular. Fig. 5(b)
shows that direct spreading of BRSV was not affected by the deletion of pep27, whereas its replacement by the Kat peptide or the bovine cytokines resulted in reduced cell-to-cell spread.
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DISCUSSION |
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That the intervening peptide plays a role in intracellular transport and maturation of the F protein was indicated by the results of pulsechase experiments. Wt F0 protein was readily cleaved, and the F0Kat and F0boIL4 were transported and cleaved with a comparably slight delay; however, processing of F0pep27, F0boIL2 and F0boIFN- was clearly delayed. A possible explanation for these drastic effects could be that the amino acid sequence between the furin cleavage site influences cleavability through conformational changes of the F0 precursor. Two-dimensional gel analysis of the F variants, however, revealed no evidence for significant differences in charge and overall structure (data not shown). In addition, the pulsechase experiments indicated the formation of
, a form of F0 that, to our knowledge, has not been described previously. We assume that
represents the Golgi-associated F protein precursor, which contains complex-type N-glycans and also O-linked carbohydrates and which is subsequently cleaved by furin in the trans-Golgi network.
Analysis of the fate of the bovine cytokines present in the precursor molecules of FboIL2, FboIL4 and FboIFN- showed that boIL2 and boIL4 were secreted into the culture medium of transfected cells and that they migrated at a distance comparable with recombinant BHV-1-expressed mature boIL2 and mature boIL4, respectively, indicating that these cytokines were properly glycosylated. Secretion of boIFN-
could not be analysed directly. However, the presence of IFN-
activity in the culture medium of cells expressing FboIFN-
provided good evidence that boIFN-
was also correctly processed and cleaved from the precursor molecules.
The role of pep27 and the intervening peptides for the replication of BRSV was analysed after generation of rBRSVs expressing the synthetic F ORFs in place of the parental F protein. Recombinant viruses expressing wt F, Fpep27, FKat, FboIL2 and FboIL4 were isolated repeatedly, whereas generation of rBRSV containing the FboIFN- ORF always failed, suggesting that insertion of the amino acids for boIFN-
into the F protein was not compatible with BRSV replication. Isolation of rBRSV lacking the entire pep27 is in accordance with results published by Zimmer et al. (2002)
, who reported that removal of aa 106130 from the F protein had no significant effect on rBRSV-F(
106130) growth. They observed, however, that the deletion mutant showed a drastically reduced syncytium formation activity in Vero cells and concluded that the mutations introduced at the C terminus of the F2 subunit might interfere with cell-to-cell fusion. Our findings have shown that deletion of pep27 in presence of the authentic F2 sequence had no effect on cell-to-cell spread. Expression of FKat, FboIL2 and FboIL4 by BRSV, however, negatively influenced not only direct spreading of the recombinants by cellcell fusion, but also virus entry, as these viruses penetrated significantly more slowly into their target cells, suggesting that F protein-mediated membrane fusion processes involved in entry and direct spreading share mechanistic requirements.
In summary, our results have demonstrated that the amino acid sequence between the furin cleavage sites of the BRSV F protein influences intracellular transport, maturation and F protein-mediated syncytium formation, and affects the membrane fusion activity of recombinant virions. They further indicate that, in principle, the F protein expressed by rBRSV can be used as a transporter for expression and secretion of cytokines and probably other physiologically important heterologous polypeptides. This may be of particular interest for the development of novel vaccines, especially if the assumption that pep27 assists BRSV to escape the immune system and thus increases virus survival in the host (Zimmer et al., 2003) proves to be correct.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 February 2004;
accepted 9 March 2004.