Polypeptide Synthesis in MDCK Cells Infected with Human and Pig Influenza C Viruses

Transcription

1 ,J. gen. Virol. (1984), 65, Printed in Great Brita& 1873 Key words: influenza C viruses/pigs/polypeptide analysis Polypeptide Synthesis in MDCK Cells Infected with Human and Pig Influenza C Viruses By RICHARD M. ELLIOTT, 1. GUO YUANJI 2,3 AND ULRICH DESSELBERGER 2 1MRC Virology Unit and 2Department of Virology, University of Glasgow, Church Street, Glasgow G l l 5JR, U.K. and 3Institute of Virology, China National Centre for Preventive Medicine, Beijing, China (Accepted 16 July 1984) SUMMARY MDCK cells were infected with six human influenza C virus strains (isolated between 1947 and 1981) and seven pig influenza C virus strains (isolated in 1981 and 1982) and the virus-specific polypeptides were compared by SDS-polyacrylamide gel electrophoresis and ohe-dimensional peptide mapping. The major structural polypeptides, i.e. glycoprotein (gp88), nucleoprotein (NP), and membrane protein (M), and one non-structural polypeptide were identified in all strains by radiolabelling infected cells with [35S]methionine. No differences in the electrophoretic migration of the M proteins or NS proteins were observed. The two earliest human isolates, C/Taylor/1233/47 and C/Great Lakes/1167/54, had faster migrating NP proteins, and another human strain, C/Georgia/I/69, displayed a faster migrating gp88. Minor differences in the onedimensional peptide maps produced by partial digestion of the M proteins with V8 protease were observed between the human and pig isolates, while more marked differences were noted in the peptide maps of the glycoproteins of the C/Georgia/1/69, C/Yamagata/10/81 and C/Yamagata/ll/81 viruses compared to the other human strains and the pig strains. The overall conclusion is that the proteins of human influenza C viruses isolated over a 35 year period and those of recent pig influenza C virus isolates are highly conserved. INTRODUCTION Influenza C viruses are distinguished from influenza A and B viruses by immunological properties, the nature of the virion glycoprotein and the number of genome segments (Air & Compans, 1983; Palese et al., 1980). Influenza C viruses do not contain neuraminidase (Kendal, 1975; Nerome et al., 1976), which is present in influenza A and B viruses, though the single glycoprotein (gp88) found in influenza C virions has both haemagglutinating and receptor-destroying activities (Meier-Ewert et al., 1978; Herrler et al., 1981). Five further structural proteins have been reported in influenza C virions: a nucleoprotein (NP), a membrane (M) protein (Kendal, 1975; Compans et al., 1977; Herrler et al., 1979; Sugawara et al., 1981), and three minor, high molecular weight proteins thought to be the polymerase (P) proteins (Petri et al, 1980). In infected cells, Yokota et al. (1983) have observed a number of other virus-specific proteins, one of which appears to be equivalent to the NS1 protein encoded by influenza A and B viruses, whereas others are thought to be degradation products of the M protein. Influenza C virus has a worldwide distribution, but the virus is rarely isolated in the diagnostic laboratory because it causes only a mild upper respiratory tract infection in humans (Chakraverty, 1978; O'Callaghan et al., 1980; Homma et al., 1982). It was generally thought that, in contrast to influenza A viruses, there was no natural animal reservoir for influenza C viruses. However, Guo et al. (1983) reported the isolation of influenza C virus from pigs in China in 1981 ; furthermore, pig-to-pig transmission of influenza C virus strains under experimental conditions was demonstrated. It was of interest, therefore, to determine whether there were bio /84/ $ SGM

2 1874 R.M. ELLIOTT, Y. J. GUO AND U. DESSELBERGER chemical differences between the pig isolates and human influenza C virus strains. Accordingly we have analysed the proteins of seven pig influenza C viruses isolated in 1981 and 1982, and of six human influenza C viruses isolated between 1947 and 1981, by SDS-polyacrylamide gel electrophoresis and one-dimensional peptide mapping. Analyses of the genomes of these viruses are presented in the accompanying paper (Guo & Desselberger, 1984). METHODS Cells and viruses. The Madin-Darby canine kidney (MDCK) cell line was maintained in Eagle's MEM supplemented with 10~ foetal bovine serum. The origins of the following influenza C virus strains were as described by Guo et al. (1983): C/Taylor/1233/47, C/Great Lakes/1167/54, C/Georgia/I/69, C/New Jersey/I/76 (C/N J/76), C/pig/Beijing/10/81, C/pig/Beijing/32/81, C/pig/Beijing/107/81, C/pig/Beijing/115/81, C/pig/Beijing/ 123/81, C/pig/Beijing/818/81; in addition, a more recent pig strain, C/pig/Beijing/439/82 (Y. J. Guo, unpublished results) was included. Drs K. Nakamura and K. Sugawara generously provided the C/Yamagata/ 10/81 (C/Ya/10/81 ) and C/Yam agata/11/81 (C/Ya/11 / 81 ) influenza viruses (Sugawara et al., 1983). Virus stoc ks were prepared by growing the viruses in the amniotic cavity of 9- to 10-day-old embryonated hens' eggs (Guo et al., 1983). Virus infectivity was measured by plaque titration in MDCK cells in the presence of 1 lig/ml TPCK-trypsin (Sigma, Type XIII). Purification of influenza C viruses by sucrose gradient centrifugation followed the method of Ritchey et al. (1976). lntracellularprotein labelling. Monolayers of MDCK cells in 30 mm Petri dishes were infected at a multiplicity of 5 to 10 p.f.u./cell and the virus was allowed to adsorb at 34 C for 2 h. The inoculum was then removed, the monolayers washed with medium and incubation continued at 34 C. At 40 h post-infection, the medium was replaced with hypertonic buffer [phosphate-buffered saline (PBS) containing 125 mm excess NaC1 (Nuss et al., 1975)]. After 15 min at 34 C, this was replaced with 1 ml/dish of labelling medium [hypertonic buffer containing either 30 IxCi/ml [35S]methionine (sp. act. 900 Ci/mmol), or 50 I~Ci/ml ps]cysteine (sp. act. 600 Ci/mmol) or 100 p.ci/ml [3H]mannose (sp. act. 16 Ci/mmol) (all from Amersham)]. After 2 h at 34 C, the labelling medium was removed, the monolayers were washed with cold PBS, and the cells scraped into 200 ~tl dissociation buffer (0.125M-Tris-HC1 ph6.8, 4~ SDS, 10~ 2-mercaptoethanol, 20~ glycerol, 0.1~ bromophenol blue). Radiolabelled proteins were separated on 15 ~ polyacrylamide gels (acrylamide : bisacrylamide ratio, 75:1) using the discontinuous buffer system of Laemmli (1970). One-dimensionalpeptide mapping. Partial proteolysis of radiolabelled proteins basically followed the method of Cleveland et al. (1977). Briefly, the radioactive proteins were located in dried gels by autoradiography, appropriate gel slices cut out with a scalpel and rehydrated in TSE buffer (125 mm-tris-hc1 ph 6.8, 0.1 ~ SDS, 1 mm-edta) for 1 h at room temperature. The gel strips were then located in the wells of a 3 ~ stacking gel and overlaid with 20 ~tl TSE buffer containing 20~ glycerol, 0-01 ~ bromophenol blue and either 1 p.g Staphylococcus aureus V8 protease or 5 Ixg ct-chymotrypsin (both from Sigma). Electrophoresis at 40 ma was performed until the bromophenol blue was about 3 mm from the resolving gel, and then the current was switched off for 30 rain. Peptides were separated by subsequent electrophoresis through 20~ resolving gels, and the gels prepared for fluorography with En3Hance (New England Nuclear). RESULTS Identification of influenza C virus polypeptides in infected MDCK MDCK cells infected with the different influenza C virus isolates were labelled with [35 S]methionine in the presence of M excess NaCI after 40 h incubation at 34 C. A number of virus-specific polypeptides were observed (Fig. 1) and have been designated according to the nomenclature of Yokota et al. (1983). These were gp88, NP protein, NS protein [also called C1 by Yokota et al. (1983)], M protein and a small polypeptide, Cs, which was shown by Yokota et al. (1983) to be a breakdown product of the M protein. Proteins equivalent to C2 to C~ (Yokota et al., 1983) could not be identified unambiguously and no high molecular weight proteins equivalent to the P proteins could be detected using these labelling and electrophoresis conditions. The electrophoretic migration of the identified proteins was indistinguishable for the different viruses, with the following exceptions: the C/Georgia/1/69 strain had a faster migrating gp88; the NP proteins of the C/Taylor/1233/47 and C/Great Lakes/1167/54 influenza viruses migrated slightly faster than the NP proteins of the other strains; and there were migration differences in the Cs proteins of C/Taylor/t233/47, C/Great Lakes/l167/54 and cells

3 Influenza C virus polypeptides 1875 q gp88 gp88 NP NP NS M C5 NS M Cs Fig. 1. Identification of influenza C virus-specificproteins in MDCK cells. Infected cells were labelled with [3sS]methionineat 40 h post-infection in the presence of 125 mmexcess NaC1. Radiolabelled proteins in cell lysates were fractionated on a 15~ polyacrylamidegel. Virus proteins are indicated by solid arrows. Open arrows indicate protein bands of different mobilities. All the pig viruses were isolated in Beijing (see Methods). C/Georgia/1/69 isolates. The protein profiles in infected cells of the C/Yamagata/10/81 or C/Yamagata/11/81 isolates were indistinguishable from those of C / N e w Jersey/I/76 virus and the pig influenza C viruses (results not shown). I n order to detect other virus-specified proteins and to identify glycoproteins, infected cells were labelled with [35S]cysteine or [3 H]mannose. [35 S]Cysteine had proved useful for labelling

4 1876 R. M. ELLIOTT, Y. J. GUO AND U. DESSELBERGER r.) ~ r.) ~ r.) ~ ~ r.) (.) r,.) r) Fig. 2. Partial proteolysis maps of the M protein of influenza C viruses generated by digestion with V8 protease. Differences in the maps are indicated by arrows. The inset shows a longer exposure of the area of the maps showing the differences (lanes 1 to 5). the M2 protein in influenza A virus-infected cells (Lamb & Choppin, 1981) but no additional proteins were detected in cells infected with either pig or human influenza C viruses (results not shown). Radiolabelling with [3 H]mannose confirmed that the protein designated gp88 in Fig. 1 was indeed a glycoprotein (results not shown). The proteins of radiolabelled, purified virus were also analysed. MDCK cells infected with either C/pig/Beijing/10/8l or C/New Jersey/I/76 virus were incubated at 34 C in the presence of 1 lig/ml trypsin and labelled with 100 ~tci/ml [35S]methionine from 48 to 72 h post-infection. Virus in clarified cell supernatant fluid was purified and the viral proteins were separated on a polyacrylamide gel. The protein profiles of the two viruses were indistinguishable. In the presence of trypsin, in both cases, the intracellular gp88 was cleaved to give two products of molecular weight and (results not shown), in agreement with observations of Herrler et al. (1979). Comparison of influenza C virus proteins by peptide mapping In order to compare the proteins of the pig and human influenza C viruses by a more sensitive method, the [35S]methionine-labelled peptides produced by partial proteolysis with either V8

5 Influenza C virus polypeptides 1877 o~ o = "~ ~ ~, F~ ~ ~ - Fig. 3. Peptide maps of the C5 polypeptide of influenza C viruses generated by V8 protease digestion. Differences in the maps are indicated by arrows. protease or ct-chymotrypsin were examined. Fig. 2 shows separation of peptides generated from the M protein and minor differences were noted in the patterns ofpeptides produced by V8 protease between the human and the pig viruses. No differences were noted when digestion with ctchymotrypsin was performed (results not shown). Partial proteolysis of the C5 polypeptide, a breakdown product of the M protein (Yokota et al., 1983), displayed in Fig. 3, showed differences between the early human isolates. However, the patterns of generated from the C5 polypeptides of C/New Jersey/I/76 and the pig influenza C virus strains appeared to be identical. Attempts to produce peptide maps of the NS proteins were unsuccessful because of contamination by the M protein. The NP proteins of the pig and human influenza C viruses appeared to be very similar because no differences could be detected in the peptide maps generated by digestion with either V8 protease or 0t-chymotrypsin (results not shown). Peptide maps of the surface glycoprotein, gp88, are displayed in Fig. 4. Differences' were noted between the peptide patterns of the gp88 of three of the human isolates, but the maps of the other human strains and the pig isolates were indistinguishable. C/Georgia/1/69 virus, which showed a faster migrating gp88 (Fig. 1), had one additional and one missing peptide (Fig. 4). Interestingly, the two recent human isolates, C/Yamagata/10/81 and C/Yamagata/11/81 (Fig. 4), had gp88 proteins that generated peptide maps which could be distinguished from both the other, older human influenza C virus isolates and the pig strains which were isolated at around the same time. Digestion with ct-chymotrypsin (results not shown) indicated that the gp88 proteins of the pig influenza C viruses and C/New Jersey/I/76 virus were indistinguishable.

6 1878 R. M. ELLIOTT, Y. J. GUO AND U. DESSELBERGER _ 70 Fig. 4. Peptide maps of the gp88 protein of influenza C viruses produced by digestion with V8 protease. Differences in the maps are indicated by arrows. DISCUSSION Influenza C viruses have maintained a remarkable degree of antigenic stability since the first isolation in 1947 (Air & Compans, 1983), and the apparent lack of non-human hosts of influenza C viruses has been advanced as one possible explanation for this observation (Palese et al., 1981). Hence, it was of interest to compare the proteins of human influenza C viruses with those of influenza C viruses recently isolated from pigs in China (Guo et al., 1983). We found no differences in the electrophoretic mobility of the proteins of the pig influenza C virus isolates when compared to recent human isolates, but differences were noted in the mobilities of the NP proteins of C/Taylor/1233/47 and C/Great Lakes/1167/54 and the gp88 protein of C/Georgia/ 1/69, three human strains, compared to the other influenza C viruses examined. This finding contrasts with observations on influenza A and B viruses, where migrational differences have been demonstrated even between viruses isolated temporally much closer to each other (Oxford et al., 1981 ; Hugentobler et al., 1981 ; Nakamura et al., 1981). Our findings are in general agreement with the recent data of Sugawara et al. (1983) who, however, did not detect differences in the migration of the NP proteins of the C/Taylor/1233/47 virus and the two Yamagata strains; this may be because of differences in the electrophoresis conditions or differences in the passage histories of the viruses used. We have also compared corresponding proteins of the pig and human influenza C viruses by one-dimensional peptide mapping. Only minor differences were noted between the mapping patterns of the pig virus proteins and those of the human virus proteins, although interestingly the maps of the gp88 proteins of the human influenza C virus strains isolated in 1981 in Japan (C/Yamagata/10/81 and C/Yamagata/11/81) differed from maps generated from the gp88 proteins of the pig viruses isolated in the same year in China. It should be noted that Sugawara et al. (1983) failed to detect differences in the peptides generated from the gp88 proteins of C/Taylor/1233/47 virus and the 1981 Yamagata isolates. Again this may be due to different passage histories of the viruses or subtle differences in the limited proteolysis experiments. Notwithstanding this point, our data allow us to extend the observations of Sugawara et al.

7 Influenza C virus polypeptides 1879 (1983) and we can conclude that the proteins of influenza C virus strains isolated at different times, in different geographical areas and from different hosts are highly conserved. This conclusion is supported by the oligonucleotide mapping results presented in the accompanying paper (Guo & Desselberger, 1984), although more differences were detected at the RNA level than at the protein level, possibly reflecting the occurrence of silent mutations in the genomes of these influenza C viruses. We thank Fiona Hundley and Margaret Wilkie for excellent technical assistance. Thanks are due to Professor J. H. Subak-Sharpe for critically reading the manuscript. G.Y.J. was supported by the British Council under the Academic Links with China Scheme and a donation from The Henry Lester Trust Ltd. REFERENCES AIR, G. M. & COMPANS, R. W. (1983). Influenza B and influenza C viruses. In Genetics oflnfluenza Viruses, pp Edited by P. Palese & D. W. Kingsbury. Wien & New York: Springer-Verlag. CI-I~RAVERTY, P. (1978). Antigenic relationship between influenza C viruses. Archives of Virology 58, CLEVELAND, D. W., FISCHER, S. G., KIRSCHER, M. W. & LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. Journal of Biological Chemistry 252, 1 i02-i 106. COMPANS, R. W., BISHOP, D. H. L. & MEIER-EWERT, H. (1977). Structural components of influenza C virions. Journalof Virology 21, GUO, Y. I. & DESSELBERGER, U. (1984). Genome analysis of influenza C viruses isolated in 1981/82 from pigs in China. Journal of General Virology 65, GUO, Y. J., ]'IN, F. G., WANG, P., WANG, M. & ZHU, J. M. (1983). Isolation of influenza C virus from'pigs and experimental infection of pigs with influenza C virus. Journal of General Virology 64, HERRLER, G., COMPAN$, R. W. & MEIER-EWERT, H. (1979). A precursor glycoprotein in influenza C virus. Virology 99, HERRLER, G., NAGELE, A., MEIER-EWERT, H., BHOWN, A. S. & COMPANS, R. W. (1981). Isolation and structural analysis of influenza C virion glycoproteins. Virology 113, 439~451. HOMMA, M., OHYAMA, S. & KATAGIRI, S. (1982). Age distribution of the antibody to type C influenza virus. Microbiology and Immunology 26, HUGENTOBLER, A. L., SCHILD, G. C. & OXFORD, J. S. (1981). Differences in the electrophoretic migration rates of polypeptides and RNAs of recent isolates of influenza B viruses. Archives of Virology 69, KENDAL, A. E. (1975). A comparison of 'influenza C' with prototype myxoviruses: receptor-destroying activity (neuraminidase) and structural polypeptides. Virology 65, LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, LAMB, R. A. & CHOI~PIN, P. W. (1981). Identification of a second protein (M2) encoded by RNA segment 7 of influenza virus. Virology 112, MEIER-EWERT, H., COMPANS, R. W., BISHOP, D. H. t. & HERRLER, G. (1978). Molecular analysis of influenza C virus. In Negative Strand Viruses and the Host Cell, pp Edited by B. W. J. M ahy & R. D. Barry. New York: Academic Press. NMOdaURA, K., KITAME, F. & HOMMA, M. (1981). A comparison of proteins among various influenza B virus strains by one-dimensional peptide mapping. Journal of General Virology 56, NEROME, K., ISHIDA, M. & NAKAYAMA, M. (1976). Absence of neuraminidase from influenza C virus. Archives of Virology.50, NUSS, D. L., OPPERMANN, H. & KOCH, G. (1975). Selective blockage of initiation of host protein synthesis in RNAvirus-infected cells. Proceedings of the National Academy of Sciences, U.S.A. 72, O'CALLAGHAN, R. J., GODH, R. S. & LABAT, D. D. (1980). Human antibody to influenza C virus: its age-related distribution and distinction from receptor analogs. Infection and Immunity 30, OXFORD, J. S., CORCORAN, T. & SCHILD, G. C. (1981). Intratypic electrophorectic variation of structural and nonstructural polypeptides of human influenza A viruses. Journal of General Virology 56, 431~436. PALESE, P., RACANIELLO, V. R., DESSELBERGER, U., YOUNG, I. & BAEZ, M. (1980). Genetic structure and genetic variation of influenza viruses. Philosophical Transactions of the Royal Society' of London, series B 288, PALESE, P., ELLIOTT, R. M., BAEZ, M. I., ZAZRA, J. J. & YOUNG, J. F. (1981). Genome diversity among influenza A, B and C viruses and genetic structure of RNA 7 and RNA 8 of influenza A viruses. In Genetic Variation Among Influenza Viruses, pp Edited by D. P. Nayak. New York: Academic Press. PETRI, T., FIERRLER, G., COMPANS, R. W. & MEIER-EWERT, H. (1980). Gene products of influenza C virus. FEMS Microbiology Letters 9, RITCHEY, M. B., PALESE, P. & KILBOURNE, E. D. (1976). The RNAs of influenza A, B and C viruses. Journal of Virology 18, SUGAWARA, K., OHUCHI, M., NAKAMURA, K. & HOMMA, M. (1981). Effects of various proteases on the glycoprotein composition and the infectivity of influenza C virus. Archives of Virology 68,

8 1880 R. M. ELLIOTT, Y. J. GUO AND U. DESSELBERGER SUGAWARA, K., NAKAMURA, K. & HOMMA, M. (1983). Analyses of structural polypeptides of seven different isolates of influenza C virus. Journal of General Virology 64, YOKOTA, M., N~P.A, K., SUGAWAnA, K. ~ nomma, M. (1983). The synthesis of polypeptides in influenza C virusinfected cells. Virology 130, (Received 30 May 1984)