Department of Microbiology, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan1
Department of Microbiology, Suzuka University of Medical Science and Technology, 1001-1 Kishioka-Cho, Suzuka, Mie 510-0226, Japan2
Author for correspondence: Masato Tsurudome. Fax +81 59 231 5008. e-mail turudome{at}doc.medic.mie-u.ac.jp
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Abstract |
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Introduction |
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It is known that the F protein of strain W3A of the paramyxovirus simian virus 5 (SV-5) mediates cell fusion when expressed alone in CV-1 cells using the SV-40 vector system (Horvath et al., 1992 ; Paterson et al., 1985
). Intriguingly, however, when expressed in CV-1 cells by using recombinant vaccinia virus vector system or the vaccinia virus T7 system, the W3A F protein cannot mediate cell fusion by itself and requires co-expression of homologous haemagglutininneuraminidase (HN) protein, similarly to other paramyxovirus F proteins (Heminway et al., 1994
; Horvath et al., 1992
). Recently, by using a plasmid expression system in BHK cells, we have confirmed that the W3A F protein exhibits a remarkable fusing activity on its own, independently of SV-5 HN protein and SV-40 infection (Ito et al., 1997
). In contrast, the F protein of another SV-5 strain (WR) required co-expression of the HN protein in order to induce cell fusion. By mutational analysis of the three amino acids which were not conserved between the F proteins of strains W3A and WR, a critical amino acid (Pro-22) was identified in W3A F2 as being responsible for the HN-independent fusing activity (Ito et al., 1997
). Accordingly, a mutant F protein (L22P), in which a leucine residue at position 22 of the WR F protein was replaced with proline, induced extensive cell fusion in the absence of the HN protein. It seems very likely, however, that there are other important amino acids involved that were not studied in our previous investigation since we evaluated only three (unconserved) amino acids.
In order to tackle this problem, we used an SV-5 strain (T1) isolated from a dog with kennel cough complex (Azetaka & Konishi, 1988 ). On the basis of the difference in the predicted amino acid sequences between the T1 F protein and the mutant L22P, chimeric and mutational analyses were performed, identifying additional amino acids important for the HN-independent fusing activity of the SV-5 F protein.
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Methods |
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Antipeptide antibody.
A synthetic peptide, 5F2, which corresponded to the C-terminal sequence (KLLQPIGENLETIRNQLIPT) of the WR F2 subunit was prepared by Sawady Technology. Rabbit antiserum specific for 5F2 was also provided by Sawady Technology.
SDSPAGE and Western blot analyses.
BHK cells were infected with virus strains WR (m.o.i.=0·001) or T1 (m.o.i.=0·1) and incubated for 26 or 72 h, respectively. Infected cells were then lysed with lysis buffer (1% Triton X-100, 137 mM NaCl, 3 mM -glycerophosphate, 3 mM EDTA, 1 mM PMSF, 25 mM HEPES, pH 7·6) on ice for 20 min and clarified by centrifugation (13000 g for 5 min). Aliquots of the lysates were subjected to SDSPAGE using a Tris/Tricine buffer system (Schägger & von Jagow, 1987
) under non-reducing or reducing conditions and blotted onto a PVDF membrane (Hybond-P; Amersham Pharmacia Biotech). The membrane was treated successively with anti-5F2 rabbit serum (diluted 1:50 in PBS), biotinylated horse immunoglobulin to rabbit IgG (heavy and light chains; Vector Laboratories) and avidinbiotinperoxidase complex (Vector Laboratories) as described previously (Tsurudome et al., 1989
). The membrane was then treated with ECL Western blotting detection reagents (Amersham Pharmacia Biotech) and exposed to an X-ray film (Konica).
Recombinant plasmids.
To obtain a full-length cDNA clone for the F gene of strain T1, synthetic oligonucleotide primers were prepared on the basis of the sequence data of the W3A F gene (Paterson et al., 1984 ). Poly-A(+) RNA was purified from virus-infected Vero cells with the aid of QuickPrep Micro mRNA Purification kit (Amersham Pharmacia Biotech), and cDNA was synthesized by using a First-strand cDNA Synthesis kit (Amersham Pharmacia Biotech). Then, the cDNA fragment was amplified by PCR and cloned in the plasmid expression vector pcDL-SR
296 (SR
), in which the expression is under control of the SV-40 early promoter and/or R-U5 sequence of human T-lymphotropic virus-1 LTR (Takebe et al., 1988
). The nucleotide sequence of amino acid-coding regions was directly determined by using a set of synthetic oligonucleotide primers. The recombinant SR
plasmid harbouring the L22P cDNA or W3A HN cDNA was reported previously (Ito et al., 1997
). To create chimeric recombinant plasmids, common restriction sites (SpeI, PvuII, ScaI and VspI) between the T1 F and L22P cDNAs were utilized. The chimeric structures of recombinant plasmids were confirmed by direct nucleotide sequencing using an ABI PRISM 310 genetic analyser (Applied Biosystems Division; Perkin Elmer).
Site-directed mutagenesis.
Introduction of mutation-generating synthetic oligonucleotide to the target recombinant plasmid was performed by using the U. S. E. Mutagenesis kit (Amersham Pharmacia Biotech) according to the manufacturers instruction. Since an FspI site was present in the SR plasmid (in the ampicillin-resistance gene) but absent in the cDNAs for T1 F and L22P genes, its elimination was a useful marker for the selection of the mutant plasmids in our present study. Thus, an oligonucleotide primer was arranged so that the FspI site was eliminated with a silent mutation in the ampicillin-resistance gene.
Induction of cell fusion in BHK cells by transfection with recombinant plasmids.
BHK cells were seeded at 5x105 cells per well in 6-well culture plates (Costar) and incubated at 37 °C for 24 h in MEM containing 10% FCS. Each recombinant plasmid (2 µg) was added onto subconfluent BHK cells by the calcium phosphate method. After 4 h incubation at 37 °C, the cells were treated with 15% glycerol in HEPES-buffered saline (0·75 mM sodium phosphate, 140 mM NaCl, 50 mM HEPES) at room temperature for 3 min. After 24 h incubation at 37 °C in MEM supplemented with 10% FCS, the cells were quickly dried, fixed with methanol, stained with Giemsas solution and observed with an inverted microscope (Olympus).
Quantification of cell surface expression of F proteins.
The amount of F protein expressed on the cell surface was measured by flow cytometric analysis as described previously (Ito et al., 1997 ; Tabata et al., 1994
; Tsurudome et al., 1995
). Briefly, BHK cells transfected with 2 µg recombinant plasmid encoding each F protein were suspended in 0·02% EDTA in PBS after 12 h incubation at 37 °C. Cells were then immunostained with anti-5F2 rabbit serum (1:100) and fluorescein-conjugated goat anti-rabbit immunoglobulins (1:800) (Cappel Laboratories). Then, the mean fluorescence intensity of 5x104 cells in each sample was measured on a FACScan (Becton Dickinson); the fluorescence intensity value of control cells transfected with SR
plasmid was subtracted from this value and it was normalized by the value given by the T1 F protein. The normalized mean fluorescence intensity was regarded as the relative surface expression.
Quantification of cell fusion.
Cell fusion was quantified as described previously (Tsurudome et al., 1995 , 1998
). Briefly, subconfluent cultures of BHK cells in 6-well culture plates were transfected with 2 µg of each recombinant plasmid by the calcium phosphate method and treated with glycerol as described above. After incubation at 37 °C for 24 h, cells were stained with Giemsas solution and observed by using an inverted microscope (Olympus). Then, photomicrographs were subjected to morphometric measurement of cell fusion, and the average fusion index (%) and SD were calculated (Tsurudome et al., 1995
).
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Results |
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Important amino acids for HN-independent fusing activity
To identify such amino acid(s) which could be involved in the HN-independent fusing activity, we created additional chimeras CF76135 and CF76:92 but they were not expressed on the cell surface (data not shown). As shown in Fig. 8, however, combining the L22P-derived cytoplasmic domain with these chimeras resulted in creation of functional chimeric proteins CF76135:529 and CF76:92:529; they were successfully expressed on the cell surface and induced cell fusion upon co-expression with the W3A HN protein. Furthermore, CF76135:529 mediated cell fusion by itself whereas CF76:92:529 did not. Thus, it was suspected that amino acid(s) at residues 132 and/or 135 in the HR1 domain of L22P played an important role in the HN-independent fusing activity. Therefore, these amino acids were further evaluated by mutational analysis of CF76:92:529. As shown in Fig. 8
, the resulting mutant CF76:92:132:529 mediated cell fusion by itself (5·8%) but the other mutant CF76:92:135:529 did not, whereas these mutant proteins induced cell fusion upon co-expression with the W3A HN protein to similar extents (15·7% and 15·4%, respectively). This result suggested that the L22P-derived Glu-132 was involved in the HN-independent fusing activity. On the other hand, it was also shown that the HN-independent fusing activity of CF76135:529 (3·9%) was apparently lower than that of CF76310:529 (11·9%), whereas the extents of cell fusion with the W3A HN protein were similar (23·1% and 27·3%, respectively). Since the mutant CF76290:529 exhibited even higher HN-independent fusing activity than CF76310:529 (13·2% vs 11·9%) with lower fusing activity with the W3A HN protein (22·6% vs 27·3%), the L22P-derived Met-310 did not seem to be significantly involved. Taken together, it could be postulated that L22P-derived Ala-290 contributed to the HN-independent-fusing activity to some extent.
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Discussion |
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Recent reports have suggested that, in the course of fusion induction, the SV-5 F protein undergoes conformational changes in which the HR1 domain of the F trimer forms a triple-stranded coil and three HR2 domains stably associate to the coil in an antiparallel orientation (Baker et al., 1999 ; Dutch et al., 1999
). The possible interaction between the HR1 domain and F2 subunit may thus somehow interfere with the conformational change of the F protein in the fusion process. The putative tight HR1F2 interaction could be liberated through interaction with the HN protein or destabilized depending on the amino acids at residues 22 and 132 as discussed above.
The HR3 domain seems not to firmly associate with the above HR1HR2 complex (Dutch et al., 1999 ). However, our observation that Ala-290 in the HR3 domain partly contributes to the HN-independent fusing activity suggests a subtle but significant role of the HR3 domain played in the course of fusion induction.
On the other hand, our present study does not exclude the possibility that some chimeric F proteins, showing no HN-independent fusion activity, may be efficiently cleaved only when co-expressed with the HN protein which may result in exhibition of HN-dependent cell fusing activity, since our Western blot analysis hardly detected recombinantly expressed F proteins (not shown). However, to our knowledge, so far there has been no study supporting this possibility (HN-dependent cleavage of the F protein). It should be pointed out, in this context, that the WR F protein was efficiently cleaved and cell surface-localized, but did not exhibit HN-independent fusing activity (Ito et al., 1997 ).
Our present study also proved that the cytoplasmic tail of the dog-derived T1 F protein was longer than that of monkey-derived WR F or W3A F protein. No difference was found between the F proteins in the number of potential glycosylation sites (Fig. 5, and data not shown). Thus, the observation that the F1+2 of strain T1 migrated slower than that of strain WR should mainly reflect the difference in the number of amino acids in the cytoplasmic tails of their F1 subunits. Previously, Randall et al. (1987)
reported that the F1 subunit of a canine isolate of SV-5 migrated slower than that of a simian isolate. Intriguingly, the F1 of four human isolates migrated to the same position as that of the canine isolate, whereas the F1 of another human isolate co-migrated with that of the simian isolate (Randall et al., 1987
). Although the primary structures of these F proteins are not known, this observation may reflect a possible difference in the length of the cytoplasmic tails. Therefore, given this and the previous assumption that the natural host of SV-5 is the dog, from which human and then monkey were successively infected (Baty et al., 1991
), it is conceivable that the prototype, dog SV-5, possessed an F protein with a long cytoplasmic tail. After infecting humans, the cytoplasmic tail of the F proteins of some SV-5 populations might become short, whereas those of others might not. Monkeys might then be infected only with the SV-5 population that has the short-tailed F protein. To certify this hypothesis, a phylogenetic investigation of the F proteins derived from a number of SV-5 isolates is required.
Whether the length of the tail has some relevance to the cytopathogenicity remains an open question, although our present study suggests that the long cytoplasmic tail of the T1 F protein is partly involved in the non-fusion phenotype of strain T1 in vitro. It should be pointed out, in this context, that the length of the cytoplasmic tail of the T1 F protein is comparable to that of the SV-41 F protein which is the longest among the paramyxovirus F proteins (Tsurudome et al., 1991 ; unpublished data) but SV-41 F protein was able to induce cell fusion in the presence of the HN protein (Tsurudome et al., 1995
).
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Acknowledgments |
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References |
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Received 16 August 1999;
accepted 5 November 1999.