INTRODUCTION

Top Abstract Introduction Materials and methods Results Discussion References

Canine distemper virus (CDV) is a negative-strand RNA virus which belongs to the morbillivirus group of the paramyxovirus family. CDV is closely related to measles virus and causes, in dogs and other carnivores, a demyelinating disease which is considered to be an animal model for multiple sclerosis in humans (22). In the infected host, the virus establishes a persistent infection in the central nervous system which is poorly understood (6) but which appears to be associated with defects in virus assembly (described below).

Field isolates of CDV readily replicate in dog or ferret macrophages (8, 19) as well as in primary dog brain cell cultures (DBCC) (26). In contrast, cell lines such as Vero cells do not allow the propagation of field isolates (12). This is very different from the situation observed with cell culture-adapted CDV, such as the Onderstepoort (OP-CDV) vaccine strain, which is able to replicate in all cell lines tested.

Virulent CDV may be adapted to multiply in cell lines, which, however, requires several passages and is associated with major changes in its biological properties. Perhaps the most striking difference from wild-type virus is the failure of cell culture-adapted CDV to cause distemper in experimental animals (12).

Strain A75/17 may be considered as the prototype virulent CDV. In DBCC, this virus produces a noncytolytic, persistent infection with very selective virus spread via microfusions between cell processes (25), characteristic of the in vivo situation in the central nervous system (23). In sharp contrast, OP-CDV produces a highly cytolytic infection in DBCC and other cells, characterized by the formation of giant syncytia. Furthermore, whereas in OP-CDV massive amounts of progeny virus are released from infected cells by budding from the cell membrane, budding appears to be a rare event in strain A75/17, the infectious progeny virions being barely detectable in the culture medium (25). This is consistent with electron microscopic studies which showed major differences in spike formation and alignment between the two CDV strains (24a). In A75/17 CDV, spike formation is very rare, whereas in OP-CDV, large amounts of spikes are seen in the infected cell membrane.

At least some of the biological differences between virulent CDV field isolates and cell culture-adapted CDV may be attributed to differences in the two envelope glycoproteins, the fusion (F) and attachment (H) proteins, due to mutations that have occurred upon serial passage of the virus in cell culture. Thus, loss of host cell specificity in OP-CDV is likely to be associated with changes in the attachment protein, whereas increased formation of syncytia in this virus is likely to be due to changes in the fusion protein.

The fusion proteins of morbilliviruses are essential for virus penetration and cell-to-cell spread (18). The mature protein is formed by cleavage of a large F0 precursor molecule into the F1 and F2 subunits, which are linked by disulfide bridges (11). The precise cleavage site in CDV F0 has been determined by amino acid sequence analysis of the N terminus of the F1 subunit (24). In measles virus, N-linked oligosaccharides are important for proteolytic cleavage, stability, and biological activity of the F protein (1, 2), which, most likely, is also the case for CDV F.

In OP-CDV, the F open reading frame potentially encodes a protein of 662 amino acids (4). However, translation initiation does not appear to occur at the first in-frame AUG. Based on deletion analysis and in analogy to other morbilliviruses, the fourth AUG, which in fact is the third in-frame AUG, has been proposed as a translation initiation codon (10). Translation from this AUG would reduce the size of the resulting F2 subunit to half of the size it would be if the first AUG was used for translation initiation.

The CDV attachment protein H is the equivalent of the measles virus hemagglutinin, but has no hemagglutinating activity. The protein is extensively glycosylated, but does not undergo proteolytic cleavage. After synthesis, the H protein forms disulfide-bound dimers (13). Tetramers and larger structures have also been seen in cross-linking studies (17).

The OP-CDV H protein consists of 604 amino acids (9). The H proteins of field isolates of CDV have 607 amino acids, which is due to three additional residues at the C terminus of the protein (7, 14). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the H proteins of CDV field isolates have a higher apparent molecular mass, which is due to differences in glycosylation (14).

In this study, we have investigated the fine structure and expression of the H and F proteins of CDV strain A75/17.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Cell cultures and viruses. CV1 African green monkey kidney cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C in the presence of 5% CO₂. Virulent CDV strain A75/17 was obtained from M. Appel, Cornell University, Ithaca, N.Y. The OP-CDV strain was derived from the so-called Green's distemperoid virus, which had been isolated from a natural case of distemper and serially passaged in ferrets. The ferret-passaged virus was then adapted to chicken eggs, after which it was called OP-CDV (3). The strain was obtained from the Swiss Federal Vaccine Institute, where it had been propagated in Vero cells. The vaccinia virus strain Western Reserve (WR) was used for transient expression studies (see below) and was obtained from B. Moss, National Institutes of Health, Bethesda, Md.

Isolation of H and F genes of A75/17 CDV and OP-CDV. Total RNA was extracted from the thymus of a dog infected with A75/17 CDV or from Vero cells infected with OP-CDV by using the RNeasy Midi kit from Qiagen. For first-strand cDNA synthesis, the Advantage RT-for-PCR kit from Clontech was used according to the instructions of the supplier. Reactions were performed with 10 µg of total RNA and oligo(dT) at a 20 µM final concentration as a primer.

Cloning and sequencing of H and F genes. The PCR-amplified strain A75/17 H gene was cleaved with KpnI and SalI, gel purified, and cloned into the KpnI and SalI sites of plasmid pCI (Promega, Madison, Wis.). The insert was then excised, the ends were rendered blunt, and the fragment was cloned into the blunt-ended BamHI site of plasmid pHGS-1 (5), which contains a strong vaccinia virus late promoter. The PCR-amplified H gene of OP-CDV was cleaved with BamHI, gel purified, and cloned into the BamHI site of pHGS-1. The PCR-amplified F gene of strain A75/17 was cleaved with EcoRI, gel purified, and cloned into the EcoRI site of pHGS-1. The PCR-amplified F gene of OP-CDV was cleaved with BamHI, gel purified, and cloned into the BamHI site of pHGS-1. All inserts were sequenced by the dideoxynucleotide chain-termination method (20).

Antibodies. The complete H gene of A75/17 CDV was cloned into the pQE16 bacterial expression plasmid (Qiagen). Expression from this plasmid results in a recombinant protein containing a six-histidine tag, allowing purification on an Ni-nitrilotriacetic acid agarose column (Qiagen). The purified, denatured protein was injected into New Zealand White rabbits. A total of five injections were performed with 300 µg of purified protein. The sera were used at a 1/1,000 dilution for the Western blotting.

Mutagenesis. Site-directed mutagenesis of AUG codons was performed with the Quick Change Site Direct mutagenesis kit (Stratagene). Briefly, plasmids were amplified by PCR using complementary primers containing the desired mutation (UUG instead of AUG). Then, parental, naturally Dam-methylated plasmids were digested with DpnI restriction enzyme, and Escherichia coli cells were transformed with the newly synthesized, nonmethylated and thus DpnI-resistant plasmids. The appropriate regions of all plasmids were sequenced to confirm that the desired mutation had been introduced.

Protein expression and tunicamycin treatment. H and F proteins of A75/17 and OP-CDV were expressed by using the vaccinia virus transient expression system. Briefly, CV1 cells grown in 3.5-cm-diameter dishes were infected with vaccinia virus at a multiplicity of infection of 5 in 400 µl of medium and left for 1 h at room temperature. Then, 2 ml of medium was added, and the cells were incubated for another hour at 37°C. The cells were then transfected with 2.5 µg of vaccinia virus expression plasmids by using the SuperFect reagent (Qiagen). Medium was changed 3 h after transfection, and the cells were incubated for 15 h at 37°C with or without 20 µg of tunicamycin per ml (Fluka). The cells were then lysed in a buffer containing 20% glycerol, 2% sodium dodecyl sulfate, 0.2% bromophenol blue, 100 mM Tris-HCl (pH 6.8), and 5% -mercaptoethanol.

Western blotting. Protein samples were fractionated on sodium dodecyl sulfate-8% polyacrylamide gels under denaturing conditions. Separated proteins were transferred to nitrocellulose membranes by electroblotting, and the membranes were then soaked in Tris-buffered saline (TBS)-Tween (25 mM Tris-HCl [pH 7.5], 137 mM NaCl, 3 mM KCl, 0.1% Tween 20) containing 5% nonfat dry milk. The membranes were then incubated with either of the polyclonal rabbit antisera at the appropriate dilution. After one wash for 20 min and two washes for 5 min with TBS-Tween, the membranes were incubated for 1 h at room temperature with a 1:3,000 dilution in blocking buffer of a rabbit anti-immunoglobulin G antibody conjugated with horseradish peroxidase (Sigma). After three washes with TBS-Tween, the bound antibodies were detected with the enhanced chemiluminescence kit (Amersham) according to the manufacturer's instructions.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Sequence analysis of the H and F protein-coding regions. To avoid introducing mutations by cell culture adaptation of the virus, the protein-coding sequences of the two envelope glycoproteins of CDV strain A75/17 were isolated directly from infected dog lymphoid tissue and sequenced. For comparison, we also isolated and sequenced the H and F protein-coding sequences of OP-CDV propagated in cell culture.

View larger version (91K): [in this window] [in a new window]   FIG. 1.   Comparison of the H (A) and F (B) proteins of A75/17 (top line) and OP-CDV (bottom line). Differences in the OP-CDV genes with respect to the published sequences (4, 9) are indicated in parentheses.

Expression of the H protein. The H proteins of CDV field isolates were reported to have a reduced electrophoretic mobility in polyacryamide gels compared to that of OP-CDV H, which is due to differences in glycosylation (14). We therefore tested whether this is also the case for the H protein of the A75/17 strain. To facilitate detection of the proteins by Western blotting, we used a vaccinia virus transient expression system in which the corresponding genes are contained in a plasmid under the control of a strong vaccinia virus promoter. These plasmids were then transfected into cells previously infected with vaccinia virus, providing the transcription machinery. An additional advantage of this system is that the RNAs are transcribed in the cytoplasm of the cell, as in the case of a CDV infection. The risk of aberrant splicing of RNAs normally not in contact with the RNA processing machinery can thus be avoided by using this system, in contrast to expression from transfected plasmids in the nucleus. On the other hand, proteins expressed in the transient expression system may not necessarily represent functional proteins.

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Expression of H proteins of A75/17 CDV and OP-CDV. Extracts from vaccinia virus-infected CV1 cells transfected with empty vector or plasmids containing the respective genes were analyzed by Western blotting. H proteins were also expressed in the presence of tunicamycin, as indicated. The positions and molecular masses (kilodaltons) of markers are shown to the left.

Expression of A75/17 and OP-CDV F proteins. As noted above, the AUG of the OP-CDV F protein, which has been proposed to be the translation initiation codon, is an AUA codon in strain A75/17. We therefore investigated which AUG is used for translation initiation in strain A75/17. Site-directed mutagenesis was used to change all five in-frame AUGs in the F2 portion of the F protein into UUG codons. The mutated plasmids were transfected into vaccinia virus-infected CV1 cells. Cell extracts were analyzed by Western blotting with a polyclonal rabbit antiserum directed against the F0 precursor (Fig. 3A).

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Expression of A75/17 CDV and OP-CDV F genes containing individually mutated AUGs. (Top) Positions of AUGs in the 5' regions of the RNAs encoding the F2 portion of the F protein of A75/17 CDV and OP-CDV. Note that, in A75/17 CDV, only in-frame AUGs are shown, whereas in OP-CDV, the numbering of AUGs is according to that of Evans et al. (10), which takes into account the third AUG, which is not in frame. (A and B) Effect of mutations of individual AUGs on expression of A75/17 (A) and OP-CDV (B) F proteins. Plasmids containing the wild-type (wt) F coding sequences or genes carrying mutations in individual AUGs as indicated at the top were transfected into vaccinia virus-infected cells. Cell extracts were analyzed by Western blotting. Extracts from cells transfected with empty vector are shown as controls. The positions of F0 precursors and of the F1 proteins are indicated. A band of unknown origin (x) is marked (see text). The positions of molecular mass markers and their sizes in kilodaltons are shown to the left.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Expression of A75/17 CDV and OP-CDV F genes containing multiple mutations of in-frame AUGs. Plasmids containing the wild-type F genes from A75/17 CDV (A) or from OP-CDV (B) or genes carrying mutations in increasing numbers of AUGs, as indicated at the top, were transfected into vaccinia virus-infected cells. Cell extracts were analyzed by Western blotting. The positions of F0 precursors and of the F1 proteins are indicated. A band of unknown origin (x) is marked (see text). The positions of molecular mass markers and their sizes in kilodaltons are shown to the left.

View larger version (57K): [in this window] [in a new window]   FIG. 5.   Expression of OP-CDV or A75/17 F proteins in the absence or presence of tunicamycin. Plasmids carrying wild-type (wt) F genes or plasmids in which the first or second AUG of the F gene had been mutated were transfected into vaccinia virus-infected cells. After incubation in the absence or presence of tunicamycin, cell extracts were analyzed by Western blotting. As a control (shown in lane 1), empty plasmid was transfected. A band of unknown origin (x) is indicated (see text). The positions of molecular mass markers and their sizes in kilodaltons are shown to the left.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

In this study, we have investigated the fine structure and expression of the CDV H and F surface glycoproteins. We chose these two proteins because they are likely to be involved in determining some of the biological differences between highly attenuated, cell culture-adapted vaccine strain Onderstepoort and strain A75/17, the prototype virulent CDV field isolate.

In the attachment protein H, we found 57 amino acid differences between OP-CDV and strain A75/17. The A75/17 protein has six potential N-linked glycosylation sites in the extraviral domain, three more than OP-CDV H (9). Other field isolates have either seven (7) or eight potential N-linked glycosylation sites in the extraviral domain (14).

With respect to OP-CDV, strain A75/17 H contains three additional amino acids at the C terminus of the protein, like the H protein of the Convac strain (15) and many field isolates (7, 14). Interestingly, these amino acids create an additional potential N glycosylation site.

When OP-CDV and A75/17 H were expressed in cells treated with tunicamycin, significant reductions in the apparent molecular masses of both proteins were observed, indicating that many of the potential glycosylation sites are used. In Japanese field isolates (14), the H proteins comigrated in polyacryamide gels with OP-CDV H following treatment with the glycosylation inhibitor. Significantly, in our study, the A75/17 protein still had a higher apparent molecular mass than OP-CDV H when expressed in the presence of tunicamycin. We are unable to explain this difference at the present time.

The most significant difference between the fusion proteins of CDV strain A75/17 and OP-CDV was the absence in A75/17 of the AUG that has been proposed as the translation initiation codon in OP-CDV (10). This has been designated the fourth AUG, since a third AUG, which is not in frame, has also been counted. The absence of this AUG in strain A75/17 led us to examine which AUG was used for translation initiation in this strain. To our surprise, site-directed mutagenesis of individual AUGs had little effect on the F1 protein, although, as expected, F0 precursors initiated at the first or second AUG disappeared when the corresponding AUG was mutated. This clearly indicated that, in A75/17, at least the first and second AUGs are used for translation initiation and that mutation of single AUGs is compensated for by more extensive use of the remaining ones. Therefore, to identify which AUGs contribute most to the production of F1, we mutated increasing numbers of AUGs in the same construct. This analysis clearly showed that the first two AUGs in strain A75/17 are by far the most important translation initiation codons of the F protein.

These results led us to reexamine the utilization of various AUGs in OP-CDV. As in A75/17, mutations of individual AUGs, including the fourth one, had little effect on the amount of F1 produced, except for a drastic reduction when the fifth AUG was mutated. Thus, this AUG is either the major translation initiation site in OP-CDV F, or, alternatively, precursors initiated from AUGs located further upstream are not readily cleaved when they contain a Leu instead of a Met residue at the position corresponding to the fifth AUG in the mRNA. The results obtained by mutation of several AUGs in the same construct are in favor of the latter hypothesis. Indeed, mutation of both the first and second AUGs in the same construct greatly reduced the amount of F1, albeit not to the extent as in A75/17. We therefore conclude that, in OP-CDV, the first two AUGs in the F open reading frame are also the major translation initiation sites. The observation that mutation of both of these two AUGs still allowed production of some F1 is best explained by assuming that in OP-CDV, AUGs located further downstream can be more readily used for translation initiation than those in A75/17. Indeed, such small F0 molecules initiated at these AUGs were present in substantial amounts (Fig. 4B), but are apparently not efficiently cleaved, since in these constructs, the F1 band is rather faint. In contrast, F0 precursor molecules initiated at the first and second AUGs appear to be more easily cleaved in OP-CDV F than in strain A75/17 F, since these molecules are either not detectable (Fig. 3B) or are present in small amounts (Fig. 4B), despite the fact that they contribute most to the generation of F1.

The efficiency with which individual F0 precursors are cleaved is most likely determined by their secondary structure, which depends on the amino acid sequence and the AUG used for translation initiation. In this context, it is interesting to note that we have isolated from infected dog lymphoid tissue an A75/17 strain F protein coding sequence which yielded an F protein which was not cleaved (data not shown). Sequence analysis revealed only two amino acid differences with respect to the cleavable version, both of which were located far away from the F1-F2 cleavage site. In view of the large number of amino acid differences between the F proteins of strain A75/17 and OP-CDV, it is not surprising that their F0 precursors are cleaved with different efficiencies. Structural differences between individual F0 precursors initiated at different AUGs presumably also account for the fact that some of them are not readily cleaved into the mature proteins.

An intriguing observation in the mutation analysis was the presence of a band, designated x, which was more intense in OP-CDV than in strain A75/17 and which did not disappear even when all in-frame AUGs were mutated. We therefore also mutated all AUGs which are not in frame (data not shown); however, this band was still present. It is possible that this protein, which does not appear to be a precursor of the mature F proteins, is initiated at another codon than an AUG. Work is in progress to determine the origin of this protein. Preliminary deletion analysis has identified a 75-nucleotide sequence which is required for its synthesis.

Mutation analysis clearly showed that both in strain A75/17 and in OP-CDV, the first and second AUGs are the most important translation initiation sites (described above). To determine their relative usage, we performed expression studies in the presence of tunicamycin. In the presence of this glycosylation inhibitor, F0 precursors are not cleaved, and their abundance should therefore directly reflect the usage of the corresponding AUGs. This analysis showed that in A75/17, the first AUG is the major translation initiation site which is used about three times more efficiently than the second one (Fig. 5). In OP-CDV, the opposite is true. Here, the second AUG appears to be used about three times more frequently than the first one. These results were confirmed with recombinant vaccinia viruses expressing the F proteins under control of the 7.5-kDa early promoter. With this expression system, the same differences in the usage of the AUGs were observed, but the preferences were much more pronounced (data not shown). Interestingly, the first AUG (underlined) is in the same, not ideal, sequence context in both viruses (CCCAUGC). The second (underlined), however, is in a more favorable context in OP-CDV (ACCAUGA) than in A75/17 CDV (AUCAUGA), which may explain why translation initiation occurs more frequently at this AUG in OP-CDV. (Residue differences are in boldface.)

Our study has revealed major differences in the structure and expression of the H and F proteins between strain A75/17 and OP-CDV. These differences are likely to account for at least some of the biological differences between the two CDV strains, one of which establishes a persistent infection and one of which produces a lytic infection. Transport to the cell surface, spike formation, fusion activity, and ability to interact with other viral structural elements in the virus assembly process all depend on structural and biological properties of the viral surface glycoproteins. By studying these properties at a more functional level, we believe that we will learn more about why CDV strain A75/17, but not OP-CDV, establishes a persistent infection in the host.