熱帯医学 Tropical medicine 33(1,2). p23

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1 NAOSITE: Nagasaki University's Ac Title Author(s) Citation Homology among Eleven Flavivirus by of Geomic RNAs and Deduced Amino Ac Tanaka, Mariko; Haishi, Shozo; Aira Morita, Kouichi; Igarashi, Akira 熱帯医学 Tropical medicine 33(1,2). p23 Issue Date URL Right This document is downloaded

2 Trop. Med., 33 (1,2), 23-33, June, Homology among Eleven Flavivirus by Comparative Nucleotide Sequence of Geomic RNAs and Deduced Amino Acid Sequences of Viral Proteins Mariko TANAKA, Shozo HAISHI1, Yoshikazu AIRA2, Shintaro KURIHARA, Kouichi MORITA and Akira IGARASHI Department of Virology, Institute of Tropical Medicine, Nagasaki University, 12-4 Sakamoto-machi, Nagasaki 852, Japan Abstract: Homology among 11 flaviviruses was compared by nucleotide sequence of genomic RNAs and deduced amino acid sequences of viral proteins. Overall amino acid homology was higher among the viruses in the same (66-90%) than those in different subgroups (39-51%). In a subgroup consisting Japanese encephalitis (JE), West Nile (WN), Kunjin (KUN), Murray Valley encephalitis (MVE), and St. Louis encephalitis (SLE) viruses, homology of the core protein (C) was less than that of the envelope glycoprotein (E). Homology of the nonstructural proteins was also higher among the viruses belonging to the same than those in different subgroups. The results suggested that selection by the neutralizing antibodies was not the determinant factor for the evolution and differentiation of these viruses. High homology of NS3 and particularly NS5 nonstructural proteins than other viral proteins indecated thier vital role in viral replication process. Nucleotide sequence homology in the 3'-and 5'-noncoding region did not reflect serological relationships of flaviviruses, although several conserved sequences were present in these regions. Key words: Flaviviruses, Homology, Nucleotide sequence, Amino acid sequence INTRODUCTION Family Flaviviridae (Westaway et at., 1985) contains more than 60 members of animal viruses, which were classified into 8 serological complexes or subgroups by cross -neutralization using polyclonal antisera (Porterfield, 1980; de Madrid and Porterfield, 1974; Calisher et al., 1989). Most of flaviviruses are transmitted among susceptible virtebrates by hematophagous arthropods in nature, therefore, arthropod-borne vertebrate viruses, or arboviruses (Berge, 1975). Many flaviviruses have been recognized as disease agents of medical Receive for Publication May 22, 1991 Contribution No from the Institute of Tropical Medicine, Nagasaki University. 1 Present address: Department of Neurology, Sanwa Hospital, Nunomaki, Sanwa-cho, Nishisonogi-gun, Nagasaki , Japan. 2 Present address: Department of Parasitology, Aichi Medical College, 21 Karimata, Aza Iwasa, Nagakute-cho, Aichigun, Aichi 480- ll, Japan.

3 24 or veterinary significance (Shope, 1980; Monath, 1986), such as yellow fever (YF), Japanese encephalitis (JE), dengue fever (DEN), Murray Valley encephalitis (MVE), St. Louis encephalitis (SLE) and tickborne encephalitis (TBE). After nucleotide sequence and deduced amino acid sequence of YF (RICE et at., 1985) and West Nile (WN) viruses (Castle et al, 1985, 1986; Wengler et al, 1985) were analyzed, genomic RNA of several other fiaviviruses were completely or partially sequenced. These results showed a commonfeature of flavivirus genomic organization in a linear single-stranded and positivesense RNA of about ll,000 nucleotide residues (Westaway et al, 1985; Rice et al, 1986). The genomic RNA consists of 5'-noncoding region of approximately 100 nucleotides, followed by a single long open reading frame encoding a polyprotein of about 3,500 amino acid residues and 3'-noncoding region of approximately 500 nucleotides without poly(a) tract. The 5' - one-fourth of the open reading frame encodes 3 structural proteins in the order of: core protein (C), precursor (PreM) of membrane protein (M), and envelope glycoprotein (E). While, remaining part of the open reading frame encodes 7 nonstructural proteins: NS1, ns2a, ns2b, NS3, ns4a, ns4b and NS5 according to the nomenclature of Rice et al (1985). Recently, 4 small nonstructural proteins, ns2a, ns2b, ns4a and ns4b were designated as NS2A, NS2B, NS4A, and NS4B, respectively, according to different cleavage sites of Val-X-Ala tripeptide by host signal peptidase (von Heijne, 1985; Speight et al, 1988). Recent genomic analysis of pestiviruses (Collet et al, 1988a, b, c, 1989; Meyers et al, 1989) and hepatitis C virus (Choo et al, 1989; Okamoto et al, 1990) showed that these viruses possess commongenomic organization with that of fiaviviruses; that is a single long open reading frame with structural protein genes in its 5'-side and nonstructural protein genes in its 3'-side, respectively. In this report, homology among ll fiaviviruses were compared by nucleotide sequence of genomic RNAs and deduced amino acid sequence of viral proteins, in order to obtain the idea on the degree of homology in relation to classical serological relatedness of the viruses. Homology was shown by percentage instead of the score which was used in our previous report (Haishi et al, 1990). MATERIALS AND METHODS Sources of the nucleotide and deduced amino acid sequence informations: Flavivirus sequence data were obtained from following publications: YF (Rice et al, 1985); WN (Castle et al, 1985, 1986; Wengler et al, 1985); MVE (Dalgarno et al, 1986); SLE (Trent et al, 1987); JE(Sumiyoshi et al, 1987); Kunjin (KUN: Coia et al, 1988); type 1 DEN (DEN1: Mason et al, 1987); type 2 DEN (DEN2: Hahn et al, 1988; Deubel et al, 1988); type 3 DEN (DENS: Osatomi, 1988); type 4 DEN (DEN4: Mackow et al, 1987; Zhao et al, 1986); TBE (Mandl et al, 1989);. Comparison of nucleotide and amino acid sequences: Virus genomic RNA sequences were divided into each gene according to Rice et al. (1985), excepting ns2a and ns2b

4 25 genes which were divided as NS2A and NS2B accoring to Speight et al. (1988). Since different flaviviruses possessed somewhat different numbers of nucleotides in each gene, their nucleotide and amino acid sequences were compared after alignment to get maximal matching. This procedure was carried out by the Hitachi DNASIS Version 6.0 software system 1989 for all ll flaviviruses, homology was presented by percentage. RESULTS Comparative nucleotide and amino acid sequences of flaviviruses: Results on each flavivirus gene coding region were shown in Fig. 1-6, in which nucleotide sequence homology was shown in left-lower half and amino acid homology in right-upper half, respectively. Nucleotide and amino acid homology of the structural and nonstructural protein gene was shown in Fig. 7, and overall amino acid homology in Fig. 8, respectively. In general, homology of nucleotide sequence paralleled with that of deduced amino acid sequence. Viruses belonging to the same subgroup showed higher homology than those belonging to different subgroups. Homology of the E protein, the major protective antigen of flaviviruses, was 71-93% for JE-WN-KUN-MVE-SLE subgroup and 61-76% for Dl-D2-D3-D4 subgroup. While the figure was 38-51% for those of different subgroups. Similar tendency was observed for the C and PreM or M proteins; the C protein amino acid homology was 57-89% in JE-WN-KUN-MVE-SLE subgroup and 54-77% in D1-D2-D3-D4 subgroup, while the figure was <30%-37% for different subgroups. Thus, homology of the C protein was lower than that of E protein in JE-WN- C protein PrM protein Fig. 1. Nucleotide and ammoacid sequence homology of C and PreM proteins. Figures in the left lower hald show nucleotide and in the right upper half amino acid sequence homology, respectively.

5 26 Mprotein E protein Fig. 2. Nucleotide and ammoacid sequence homology of M and E proteins, Figures in the left lower half show nucleotide and in the right upper half amino acid sequence homology, respectively. NS1 protein NS2A protein Fig. 3. Nucleotide and amino acid sequence homology of NS1 and NS2A proteins. Figures in the left lower half show nucleotide and in the right upper half amino acid sequence homology, respectively.

6 27 NS2B protein NS3 protein Fig. 4. Nucleotide and amino acid sequence homology of NS2B and NS3 proteins. Figures in the left lower half show nucleotide and in the right upper half amino acid sequence homology, respectively. NS4A protein NS4B protein Fig. 5. Nucleotide and amino acid sequence homology of NS4A and NS4B proteins. Figures in the left lower half show nucleotide and in the right upper half amino acid sequence homology, respectively.

7 28 NS5 protein Fig. 6. Nucleotide and amino acid sequence homology of NS5 proteins. Figures in the left lower half show nucleotide and in the right upper half amino acid sequence homology, respectively. Structural protein Non structural protein Fig. 7. Amino acid sequence homology of structural and nonstructural proteins.

8 29 Fig. 8. Amino acid sequence homology of the overall coding region. 5 '-non coding 3 '-non coding Fig. 9. Nucleotide sequence homology of 5'- and 3'-noncoding regions.

9 30 KUN-MVE-SLE subgroup, while, the difference between these figures in Dl-D2 -D3-D4 subgroup was less remarkable. Amirio acid homology of PreM protein was 59-89% in JE-WN-KUN-MVE-SLE subgroup and 61-73% in D1-D2-D3-D4 subgroup, respectively, while the figure was <30%-42% for different subgroups. The results in the M protein was almost the same as PreM protein. Among nonstructural proteins, homology of NS3 and NS5 proteins was significantly higher than other proteins. In JE-WN-KUN subgroup, these two proteins showed 80-92% and 81-94% amino acid homology, respectively. These figures were 76-80% and 71-78% in D2-D3-D4 subgroup, which were lower than the former but still higher than other in different subgroups: 44-64% and 55-66%, respectively. Amino acid homology of other nonstructural proteins less than those for NS3 and NS5, except WN-KUN combination which showed exceptionally high homology of 89-97%, Amino acid homology of NS1 protein was 63-90% for JE-WN-KUN-MVE-SLE subgroup, 71-73% for D2-D3-D4 subgroup, and 40-64% for different subgroups. The amino acid homology of other nonstructural proteins was shown below: NS2A protein 63-98% in JE-WN-KUN-MVE-SLE subgroup, 34-38% in D2-D3-D4 subgroup, and <30% in different subgroups; 34-38% in D2-D3-D4 subgroup, and <30% in different subgroups; NS2B protein 51-97% in- JE-WN-KUN-MVE-SLE subgroup, 55-61% in D2-D3-D4 subgroup, and <30%-35% in different subgroups; NS4A protein 77-94% in JE-WN-KUN subgroup, 59-62% in D2-D3-D4 subgroup, and <30%-43% in different subgroups; NS4B protein 64-89% in JE-WN-KUN subgroup, 75-76% in D2-D3-D4 subgroup, and <30%-40% in different subgroups. Amino acid homology data of the structural and nonstructural protein were summarized in Fig. 7. Homology of the structural proteins was 68-93% in JE-WN-KUN-MVE-SLE,subgroup, 61-75% in D1-D2-D3-D4 subgroup, and 31-46% in different subgroups. The figure of the nonstructural protein was 76-93% in JE-WN-KUN subgroup, 69-73% in D2-D3-D4 subgroup, and 35-54% in different subgroup. Therefore, the structural and nonstructural protein showed almost similar homology percent in the same subgroup. These figures were summarized into overall amino acid homology as shown in Fig. 8: 75-90% in JE-WN-KUN subgroup, 66-69% in D2-D3-D4 subgroup, and 39-50% in different subgroup, respectively. Nucleotide sequence homology in the 5*- and 3'-noncoding regions was shown in Fig. 9. Homology of the 5'-noncoding region in JE-MV-SLE subgroup (<30%-63%) was significantly lower than those in coding regions, while the figure was higher in D2-D3-D4 subgoup (80-85%), and homology in different subgroups was <30%-46%. Homology in the 3'-noncoding region was 71% in JE-WN, 76% in D2-D3, and <30%-57% in different subgroups. Thus, nucleotide sequence homology in noncoding regions did not reflect classical serological relationships although conserved sequences were present.

10 31 DISCUSSION Amino acid homology percent in coding regions generally paralleled to the similarities by classical serology. Higher homology of the E protein than the C protein in JE-WN-KUN-MVE-SLEsubgroup suggests that the selection by neutralizing antibodies was not the determinant factor in the evolution and differentiation of these flaviviruses, suppose they have originated from commonancestor(s). This conjecture was supported by the fact that amino acid homology of the nonstructural proteins was almost the same as that of the structural proteins. Exceptionally high homology around 90% between WN and KUN viruses may suggest that these viruses are more close to the different strains of the same species than different viral species. Several strains of JE virus showed amino acid homology above 93% as shown in accompanying paper (Tanaka et al., 1991). While, amino acid homology percent around 60-70% appear to indicate the membership in the same subgroup, and the percentage below 40% in different subgroups of the Family Flaviviridae, respectively. These figures should be substantiated by further data. ACKNOWLEDGMENTS The study was supported by a Grant-in Aid for Scientific Research No from the Ministry of Education, Science and Culture of Japan, and also by a Grant-in Aid for nona-nonbhepatitis from the Minsitry of Health and Welfare of Japan. Excellent technical help by Ms. C. Uchida and Ms. K. Jodai is appreciated. REFERENCES 1 ) Berge, T. 0. (ed.) (1975): International Catalogue of Arboviruses. DHEW Publication No. (CDC) Government Printing Office, Washington, D. C. 2 ) Calisher, C. H., Karabatsos, N., Dalrymple, J. M., Shope, R. E., Porterfield, J. S., Westaway, E. G. & Brandt, W. (1989): Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J. Gen. ViroL, 70, ) Castle, E., Nowak, T., Leidner, U., Wengler, G. & Wengler, G. (1985): Sequence analysis of the viral core protein and the membrane associated proteins VI and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins. Virology, 145, ) Castle, E., Leidner, U., Nowak, T., Wengler, G. & Wengler, G. (1986): Primary structure of the West Nile flavivirus genome region coding for all nonstructural proteins. Virology, 149, ) Choo, Q-L., Kuo, G., Weiner, A. J., Overy, L. R., Bradley, D. W. & Houghton, M. (1989): Isolation of a CDNA clone derived from a blood-borne nona, nonb viral hepatitis genome. Science, 244, ) Coia, G., Parker, M. D., Speight, G., Byrne, M. E. & Westaway, E. G. (1988): Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virusspecified proteins. J. Gen. ViroL, 69, 1-21.

11 32 7 ) Collet, M. S., Larson, R., Gold, C., Strick, D., Anderson, P. K. & Purchio, A. F. (1988a): Molecular cloning and nucelotide sequence of the pestivirus bovine viral diarrhaea virus. Virology, 165, ) Colet, M. S., Larson, R., Belzer, S. K. & Retzerl, E. (1988b): Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus. Virology, 165, ; 9 ) Collet, M. S., Anderson, D. & Retzel, E. (1988c): Comparisons of the pestivirus bovine viral diarrhea virus with members of the flaviviridae. J. Gen. ViroL, 69, ) Collet, M. S., Moenning, V. & Horzinek, M. C. (1989): Recent advances in pestivirus research. J. Gen Virol., 70, ll) Dalgrno, L., Trent, D. W., Strauss, J. H. & Rice, C. M. (1986): Partial nucleotide sequence of the Murray Valley encephalitis virus genome. J. Mol. Biol., 187, ) Deubel, V., Kinney, R. M. & Trent, D. W. (1986): Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype. Virology, 155, ) Hahn, Y. S., Galler, R., Hunkapiller, T., Dalrymple, J. M., Strauss, J. H. & Strauss, E. G. (1988): Nucleotide sequence of dengue 2 RNA and comparion of the encoded proteins with those of other flaviviruses. Virology, 162, ) Haishi, S. (1990): Comparative nucleotide sequence of flavivirus genomic RNA and deduced amino acid sequences of viral proteins. Trop. Med., 32, ; 15) von Heijne, G. (1985): Signal sequences. The limits of variation. J. Mol. Biol., 184, ) Lipman, D. J. & Pearson, W. R. (1985): Rapid and sensitive protein similarity searches. Science, 227, ) Mackow, E., Makino, Y., Zhao, B., Zhang, Y. M., Markoff, L., Buckler-White, A., Guiler, M., Chanock, R. & Lai, C.: The nucleotide sequence of dengue type 4 virus: analysis of genes coding for nonstructural proteins (1987). Virology, 159, ) de Madrid, A. T. & Porter field, J. S. (1974): The flaviviruses (group B arboviruses): a crossneutralization. J. Gen. Virol., 23, ) Mandl, C., Heinz, F., Stockl, E. & Kunz, C. (1989): Genome sequence of tick-bone encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses. Virology, 173, ) Mason, P. W., McAda, P. C., Mason, T. L. & Fournier, M. J. (1987): Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1. Virology, 161, ) Meyers, G., Rumenapf, T. & Thiel, H-J. (1989): Molecular cloning and nucleotide sequence of the genomre of hog cholera virus. Virology, 171, ) Monath, T. P. (1986): Pathology of flaviviruses. pp In S. Schlesinger & M, J. Schlesinger (ed.). The Togaviridae and Flaviviridae. H. Fraenkel-Conrat & R. R. Wagner (Ser. ed.) The Viruses. Plenum Press, New York. 23) Okamoto, H., Okada, S., Sugiyama, Y., Yotsumoto, S., Tanaka, T., Yoshizawa, H., Tsuda, F., Miyakawa, Y. & Mayumi, M. (1990): The 5'-terminal sequence of the hepatitis C virus genome. Jpn. J. Exp. Med., 60,

12 33 24) Osatomi, K. (1988): Complete nucleotide sequence of type 3 dengue virus genomic RNA and analysis of structural proteins. PhD Thesis for the Faculty of Pharmaceutical Sciences, Nagasaki University. 25) Porter field, J. S. (1980): Antigenic characterization and classification of Togaviridae. pp In R. W. Schlesinger (ed.). The Togaviruses, Biology, Structure and Replication. Academic Press, New York. 26) Rice, C. M., Lenches, E. M., Eddy, S. E., Shin, S. J., Sheets, R. L. & Strauss, J. H. (1985): Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science, 229, ) Rice, C. M., Strauss, E. G. & Strauss, J. H. (1986): Structure of the flavivirus genome, pp In S. Schlesinger & M. J. Schlesinger (ed.). Togaviridae and Flaviviradae. H. Fraenkel-Conrat & R. R. Wagner (Ser. ed.) The Viruses. Plenum Press, New York. 28) Shope, R. E. (1980): Medical significance of togaviruses: an overview of diseases caused by togaviruses in man and domestic and wild vertebrate animals, pp In R. W. Schlesinger (ed.). The Togaviruses, Biology, Structure, Replication. Academic Press, New York. 29) Speight, G., Coia, G., Parker, M. D. & Westaway, E. G. (1988): Gene mapping and positive identification of the nonstructural proteins NS2A, NS2B, NS3, NS4B and NS5 of the flavivirus Kunjin and their cleavage sites. J. Gen. ViroL, 69, ) Sumiyoshi, H. Mori, C., Fuke, L, Morita, K., Kuhara, S., Kondou, J., Kikuchi, Y., Nagamatsu, M. & Igarashi, A. (1987): Complete nucleotide sequence of the Japanese encephalitis virus genome RNA. Virology, 161, ) Tanaka, M., Aira, Y. & Igarashi, A. (1991): Comparative nucleotide and amino acid sequences of five Japanese encephalitis virus strains isolated in Japan and China. Trop. Med., 33 (1, 2), ) Trent, D. W., Kinny, R. M., Johnson, B. J. B., Vorndam, A. M., Grant, J. A., Deubel, V., Rice, C. M. & Hahn, C. (1987): Partial nucleotide sequence of St. Louis encephalitis virus RNA: structural proteins, NS1, ns2a, and ns2b. Virology, 156, ) Wengler, G., Castle, E., Leidner, IL, Nowak, T. & Wengler, G. (1985): Sequence analysis of the membrane protein V3 of the flavivirus West Nile virus and of its gene. Virology, 147, ) Westaway, E. G., Brinton, M. A., Gaidamovich, S. Y., Horzinek, M. C., Igarashi, A., Kaariainen, L., Lvov, D. K., Porterfield, J. S., Russell, P. K. & Trent, D. W. (1985): Flaviviridae. Intervirology, 24, ) Zhao, B., Mackow, E., Buckler-White, A., Markoff, L., Chanock, R. M., Lai, C. J. & Makino, Y. (1986): Cloning full-length dengue type 4 viral DNA sequences: analysis of genes codin'g for structural proteins. Virology 155,