Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1
Author for correspondence: Kiyoto Nakamura. Fax +81 23 628 5250.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, Marschall et al. (1999) detected a 27 kDa polypeptide in influenza C virus-infected cells that was reactive with antiserum against a glutathione S-transferase (GST) fusion protein constructed to contain NS1. However, influenza C virus NS2 has not yet been identified unequivocally in infected cells, although there are two earlier papers that reported synthesis of a protein of about 15 kDa that might be a counterpart of the NS2s of influenza A and B viruses (Petri et al., 1980
; Nakada et al., 1986
). The second aim of this study was to confirm that the NS2 amino acid sequence is conserved among different influenza C virus strains.
By comparing the haemagglutininesterase (HE) and NS gene sequences among various strains in different parts of the world over a long period of time, Buonagurio et al. (1985) suggested that influenza C virus epidemiology may be characterized by the presence of many co-circulating variants belonging to different lineages. We also compared previously the HE gene sequence among 25 isolates obtained during 19641988 and showed the existence of four discrete lineages, represented by C/Yamagata/26/81, C/Aichi/1/81, C/Aomori/74 and C/Mississippi/80, three of which (C/Yamagata/26/81-, C/Aichi/1/81- and C/Mississippi/80-related lineages) co-circulated in the 1980s in Japan (Muraki et al., 1996
). Therefore, mixed infection with influenza C viruses belonging to different lineages is likely to occur in nature, resulting in the emergence of reassortment viruses. In fact, evidence was obtained that several virus strains (C/Yamagata/64, C/Kanagawa/1/76, C/Miyagi/77, C/England/83, C/Nara/1/85, C/Yamagata/9/88 and C/Yamagata/5/92) are naturally occurring reassortants (Peng et al., 1994
, 1996
; Tada et al., 1997
). However, the significance of genetic reassortment in influenza C virus epidemiology is totally unknown. The third aim of this report was to identify additional reassortants by comparing the phylogenetic positions of the individual isolates between the evolutionary trees for the HE and NS genes and to obtain information about the epidemiological significance of reassortment of influenza C viruses. For these purposes, we compared the nucleotide sequences of the NS gene among 34 influenza C virus isolates obtained between 1947 and 1992. We also present evidence for the synthesis of the NS2 protein with a molecular mass of 22 kDa in influenza C virus-infected cells.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Production of antisera against the C-terminal regions of the NS1 and NS2 proteins.
To create a GST fusion protein containing the C-terminal region (residues 225246) of NS1 (GST/NS1-C), a 69 bp DNA fragment corresponding to positions 700768 of the NS gene was prepared by PCR by using plasmid pCN5-8 (which contains nucleotides 5923 of the YA188 virus NS gene; Hongo et al., 1992 ) as a template and two primers: a plus-sense primer (5' dCGTGGATCCGGAAATGAAACACCAGATATTGACAAG) containing a BamHI site (underlined) followed by sequence corresponding to positions 700726 and a minus-sense primer (5' dCTCGAATTCTTATGGTCCAGCTTCTCTAAGCGAG) containing an EcoRI site (underlined) followed by sequence corresponding to positions 768744. This DNA fragment was digested with BamHI and EcoRI and then cloned into the BamHI and EcoRI sites of pGEX-2T (Pharmacia) to give pGEX/NS1-C. A GST fusion protein containing the C-terminal region (residues 121182) of NS2 (GST/NS2-C) was generated as follows: a 189 bp DNA fragment corresponding to positions 701889 of the NS gene was amplified by PCR with a plus-sense primer composed of a BamHI site followed by sequence corresponding to positions 701728 (5'-dCGTGGATCCGAAATGAAACACCAGATATTGACAAGAC; BamHI site underlined) and a minus-sense primer composed of a SmaI site followed by sequence corresponding to positions 889859 (5' dCTCCCCGGGTTATATAAGTGAATTACACAAAGATTTTATC; SmaI site is underlined). The PCR product, after digestion with BamHI and SmaI, was cloned into the BamHI and SmaI sites of pGEX-2T to give pGEX/NS2-C. pGEX/NS1-C and pGEX/NS2-C were each transformed into E. coli strain DH5
. Cultures of the bacteria were grown to mid-exponential phase and treated with 0·1 M IPTG for 4 h. The cells were then collected by centrifugation, resuspended in Bug Buster reagent (Novagen) and incubated on a rotating mixer for 10 min at room temperature. After centrifugation at 16000 g for 20 min, the GST fusion protein was purified from the supernatant by glutathioneSepharose 4B (Pharmacia) affinity chromatography. Antisera against the GST/NS1-C and GST/NS2-C proteins were raised in New Zealand white rabbits according to procedures described previously (Hongo et al., 1994
).
Radioimmunoprecipitation.
HMV-II cells infected with YA188 virus at an m.o.i. of 10 p.f.u. per cell were labelled for 1 h at 12 h post-infection (p.i.) with 50 µCi/ml [35S]methionine (Amersham) in methionine-free RPMI 1640 medium. Cells were then disrupted in 0·01 M TrisHCl, pH 7·4, containing 1% Triton X-100, 1% sodium deoxycholate, 0·1% SDS, 0·15 M NaCl and a cocktail of protease inhibitors (Hongo et al., 1997 ) and immunoprecipitated as described previously (Sugawara et al., 1986
), utilizing rabbit antiserum against GST/NS1-C or GST/NS2-C. The immunoprecipitates obtained were analysed by SDSPAGE on 17·5% gels containing 4 M urea under reducing conditions and processed for analysis by fluorography (Yokota et al., 1983
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Phylogenetic tree for NS genes
A phylogenetic tree for the NS genes, constructed by using the nucleotide sequences of 27 virus strains determined here as well as the previously determined sequences of seven strains (Nakada et al., 1985 ; Buonagurio et al., 1986
; Hongo et al., 1992
) (nucleotide positions 2441 of these strains, except for CAL78 and YA188, were sequenced in this study and were found to possess the same sequences as those of the other 29 strains, including CAL78 and YA188) is shown in Fig. 2
. The 34 NS genes were split into two distinct groups, designated A and B. Group A contains six strains isolated between 1947 and 1980 in the USA (TAY47, AA50, GL54, CAL78 and MS80) and the Republic of South Africa (JHG67) in addition to 10 strains isolated in Japan between 1964 and 1983. It should be noted that, among the 34 strains analysed, the 12 isolates obtained before 1980 were all within this group. Group B contains 16 virus strains isolated in Japan during 19811992 as well as two foreign isolates from China (PB11581) and the UK (ENG83). Interestingly, the 15 isolates obtained after 1985 were all found to belong to this group. Pairwise comparison of the NS gene sequences among the 34 isolates showed that nucleotide sequence differences between the isolates of the same group were 0·12·5% (group A) and 01·8% (group B), while differences between the isolates belonging to different groups ranged from 1·5 to 4·0%.
|
Deduced amino acid sequences of NS1 and NS2
Deduced amino acid sequences of the NS1 and NS2 proteins were compared among the 34 isolates and the results are summarized in Fig. 3. Amino acid substitutions were observed at 24 positions (9·8%) among the 246 amino acid residues of NS1. The degrees of NS1 protein sequence identity between isolates belonging to the same group were 97·2100% (group A) or 98·4100% (group B), while those between isolates belonging to different groups ranged from 96·3 to 99·6%. It was impressive that the NS1 protein of the prototype strain TAY47 (group A) differed by only one or two amino acids from those of several group A strains (MS80, AI181, NA82, KY4182 and HY183) isolated at least 33 years later and that two strains belonging to different groups (HY183 of group A and MI991 of group B) that were isolated 8 years apart had NS1 proteins that differed by only one amino acid residue, indicating that the amino acid sequence of NS1 is highly conserved.
|
Detection of NS2 protein in influenza C virus-infected cells
In order to identify the NS2 protein unequivocally in influenza C virus-infected cells as well as to obtain direct evidence that NS1 and NS2 are synthesized according to the coding strategy shown in Fig. 1(b), we produced antisera against the GST fusion proteins GST/NS1-C and GST/NS2-C, which contain residues 225246 of the 246 amino acid NS1 protein and residues 121182 of the 182 amino acid NS2 protein, respectively. It should be stressed that the sequence of residues 225246 of the 246 amino acid NS1 is completely different from that of the putative 286 amino acid NS1 and that residues 121182 of the 182 amino acid NS2 are not contained in the putative 121 amino acid NS2 protein. YA188 virus-infected HMV-II cells were labelled with [35S]methionine for 1 h at 12 h p.i. and then subjected to immunoprecipitation with each of these two antisera. As seen clearly in Fig. 4
, immune sera against GST/NS1-C and GST/NS2-C immunoprecipitated polypeptides with apparent molecular masses of 31 and 22 kDa, respectively.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previously, we provided evidence that suggested that reassortment of the genome between different influenza C virus strains occurs frequently in nature (Peng et al., 1994 , 1996
; Kimura et al., 1997
; Tada et al., 1997
). Here, we have obtained data that show that seven 19861991 isolates with HE genes on the AI181 virus lineage (YA186, NA186, YA988, YA589, MI190, MI591 and MI791) are all reassortants that inherited their HE and NS genes from AI181-like and YA2681-like viruses, respectively, and that all five 19811990 strains (YA2681, NA285, YA188, YA788 and YA190) with HE genes on the YA2681 virus lineage are also reassortants, which acquired HE genes from an SA71-like virus and NS genes from an unknown parent. These observations, together with those reported previously (Peng et al., 1996
; Kimura et al., 1997
), suggest that most if not all influenza C viruses currently circulating in Japan arose by reassortment, which presumably occurred either in the 1970s or in the early 1980s. A phylogenetic tree of the NS genes was unique in that 34 influenza C virus strains isolated during 19471992 were split into two different groups (A and B) and that the recent isolates, irrespective of their HE gene lineage, had group B NS genes, whereas the older ones had group A NS genes. In any of the trees for the other six genes, constructed on the basis of total (HE and M genes) or partial nucleotide sequences (PB2, PB1, P3 and NP), three or four distinct lineages were identified, each of which contained older isolates as well as recent ones (Peng et al., 1996
; Muraki et al., 1996
; Kimura et al., 1997
; Tada et al., 1997
). These observations lead us to postulate that influenza C viruses that acquired group B NS genes through the reassortment events described above dominantly replaced the parental viruses with group A genes, forming stable viral lineages.
The roles of NS1 and NS2 in influenza C virus replication are not known. The C-terminal half of influenza A virus NS1 is highly variable (Nakajima et al., 1990 ; Ludwig et al., 1991
; Kawaoka et al., 1998
; Suarez & Perdue, 1998
) and may be dispensable, since its deletion does not lead to loss of virus infectivity (Norton et al., 1987
). In contrast, the C-terminal half of influenza C virus NS1 is highly conserved, suggesting that the NS1 protein of this virus may be structurally and functionally different from that of influenza A virus. Indeed, it has been shown that, while the steady-state levels of M gene-derived spliced mRNAs are only 510% of that of the unspliced mRNA in influenza A virus-infected cells (Lamb et al., 1981
), the predominant M gene-derived mRNA synthesized in influenza C virus-infected cells is a spliced one (Yamashita et al., 1988
; Hongo et al., 1994
), raising the possibility that influenza C virus NS1 may lack the ability to inhibit splicing of pre-mRNAs, one of the important functions identified for influenza A virus NS1 (Fortes et al., 1994
; Lu et al., 1994
). The functions of the influenza C virus NS gene products must be investigated extensively in future studies.
Interestingly, one and three amino acid positions were found in the NS1 and NS2 proteins, respectively, that differentiate between groups A and B. Group A NS1 proteins have Gln at position 212 in common, whereas all group B NS1 proteins have Arg at this position. This was the only amino acid difference detected between the NS1 proteins of strains NA82 (group A) and NA185 (group B) as well as between those of strains HY183 (group A) and MI991 (group B). In the NS2 protein, group-specific amino acid differences were found at positions 76 (Lys in group A; Arg in group B), 108 (Lys or Arg in group A; Glu in group B) and 142 (Leu in group A; His in group B), the former two positions flanking the first heptad repeat motif HR1 (residues 84105) and the last being located within the second motif HR2 (residues 130158). It may be interesting to investigate the possibility that one or more of these amino acid changes might have caused the apparent increase in the epidemiological potential of influenza C viruses with group B NS genes compared with that of viruses with group A NS genes.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Buonagurio, D. A., Nakada, S., Desselberger, U., Krystal, M. & Palese, P. (1985). Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates.Virology146, 221-232.[Medline]
Buonagurio, D. A., Nakada, S., Fitch, W. M. & Palese, P. (1986). Epidemiology of influenza C virus in man: multiple evolutionary lineages and low rate of change.Virology153, 12-21.[Medline]
Desselberger, U., Racaniello, V. R., Zazra, J. J. & Palese, P. (1980). The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity.Gene8, 315-328.[Medline]
Felsenstein, J. (1989). PHYLIP phylogeny inference package.Cladistics5, 164-166.
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology.Systematic Zoology20, 406-416.
Fortes, P., Beloso, A. & Ortin, J. (1994). Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport.EMBO Journal13, 704-712.[Abstract]
Hongo, S., Kitame, F., Sugawara, K., Nishimura, H. & Nakamura, K. (1992). Cloning and sequencing of influenza C/Yamagata/1/88 virus NS gene.Archives of Virology126, 343-349.[Medline]
Hongo, S., Sugawara, K., Nishimura, H., Muraki, Y., Kitame, F. & Nakamura, K. (1994). Identification of a second protein encoded by influenza C virus RNA segment 6.Journal of General Virology75, 3503-3510.[Abstract]
Hongo, S., Sugawara, K., Muraki, Y., Kitame, F. & Nakamura, K. (1997). Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus.Journal of Virology71, 2786-2792.[Abstract]
Kawaoka, Y., Gorman, O. T., Ito, T., Wells, K., Donis, R. O., Castrucci, M. R., Donatelli, I. & Webster, R. G. (1998). Influence of host species on the evolution of the nonstructural (NS) gene of influenza A viruses.Virus Research55, 143-156.[Medline]
Kimura, H., Abiko, C., Peng, G., Muraki, Y., Sugawara, K., Hongo, S., Kitame, F., Mizuta, K., Numazaki, Y., Suzuki, H. & Nakamura, K. (1997). Interspecies transmission of influenza C virus between humans and pigs.Virus Research48, 71-79.[Medline]
Lamb, R. A., Lai, C.-J. & Choppin, P. W. (1981). Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins.Proceedings of the National Academy of Sciences, USA78, 4170-4174.[Abstract]
Lu, Y., Qian, X.-Y. & Krug, R. M. (1994). The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing.Genes & Development8, 1817-1828.[Abstract]
Ludwig, S., Schultz, U., Mandler, J., Fitch, W. M. & Scholtissek, C. (1991). Phylogenetic relationship of the nonstructural (NS) genes of influenza A viruses.Virology183, 566-577.[Medline]
Lupas, A. (1996). Coiled coils: new structures and new functions.Trends in Biochemical Sciences21, 375-382.[Medline]
Marschall, M., Helten, A., Hechtfischer, A., Zach, A., Banaschewski, C., Hell, W. & Meier-Ewert, H. (1999). The ORF, regulated synthesis, and persistence-specific variation of influenza C viral NS1 protein.Virology253, 208-218.[Medline]
Matsuzaki, Y., Muraki, Y., Sugawara, K., Hongo, S., Nishimura, H., Kitame, F., Katsushima, N., Numazaki, Y. & Nakamura, K. (1994). Cocirculation of two distinct groups of influenza C virus in Yamagata City, Japan.Virology202, 796-802.[Medline]
Muraki, Y., Hongo, S., Sugawara, K., Kitame, F. & Nakamura, K. (1996). Evolution of the haemagglutininesterase gene of influenza C virus.Journal of General Virology77, 673-679.[Abstract]
Nakada, S., Graves, P. N., Desselberger, U., Creager, R. S., Krystal, M. & Palese, P. (1985). Influenza C virus RNA 7 codes for a nonstructural protein.Journal of Virology56, 221-226.[Medline]
Nakada, S., Graves, P. N. & Palese, P. (1986). The influenza C virus NS gene: evidence for a spliced mRNA and a second NS gene product (NS2 protein).Virus Research4, 263-273.[Medline]
Nakajima, K., Nobusawa, E. & Nakajima, S. (1990). Evolution of the NS genes of the influenza A viruses. II. Characteristics of the amino acid changes in the NS1 proteins of the influenza A viruses.Virus Genes4, 15-26.[Medline]
Norton, G. P., Tanaka, T., Tobita, K., Nakada, S., Buonagurio, D. A., Greenspan, D., Krystal, M. & Palese, P. (1987). Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides.Virology156, 204-213.[Medline]
Ohyama, S., Adachi, K., Sugawara, K., Hongo, S., Nishimura, H., Kitame, F. & Nakamura, K. (1992). Antigenic and genetic analyses of eight influenza C strains isolated in various areas of Japan during 19859.Epidemiology and Infection108, 353-365.[Medline]
Peng, G., Hongo, S., Muraki, Y., Sugawara, K., Nishimura, H., Kitame, F. & Nakamura, K. (1994). Genetic reassortment of influenza C viruses in man.Journal of General Virology75, 3619-3622.[Abstract]
Peng, G., Hongo, S., Kimura, H., Muraki, Y., Sugawara, K., Kitame, F., Numazaki, Y., Suzuki, H. & Nakamura, K. (1996). Frequent occurrence of genetic reassortment between influenza C virus strains in nature.Journal of General Virology77, 1489-1492.[Abstract]
Petri, T., Herrler, G., Compans, R. W. & Meier-Ewert, H. (1980). Gene products of influenza C virus.FEMS Microbiology Letters9, 43-47.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors.Proceedings of the National Academy of Sciences, USA74, 5463-5467.[Abstract]
Suarez, D. L. & Perdue, M. L. (1998). Multiple alignment comparison of the non-structural genes of influenza A viruses.Virus Research54, 59-69.[Medline]
Sugawara, K., Nishimura, H., Kitame, F. & Nakamura, K. (1986). Antigenic variation among human strains of influenza C virus detected with monoclonal antibodies to gp88 glycoproteins.Virus Research6, 27-32.[Medline]
Tada, Y., Hongo, S., Muraki, Y., Sugawara, K., Kitame, F. & Nakamura, K. (1997). Evolutionary analysis of influenza C virus M genes.Virus Genes15, 53-59.[Medline]
Yamashita, M., Krystal, M. & Palese, P. (1988). Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA.Journal of Virology62, 3348-3355.[Medline]
Yokota, M., Nakamura, K., Sugawara, K. & Homma, M. (1983). The synthesis of polypeptides in influenza C virus-infected cells.Virology130, 105-117.[Medline]
Received 8 February 2000;
accepted 27 March 2000.