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Complete nucleotide sequence of segment 5 of epizootic haemorrhagic disease virus; the outer capsid protein VP5 is homologous to the VP5 protein of bluetongue virus
Wus Research, 20 (1991) 273-281 0 1991 Elsevier Science Publishers B.V. 016%1702/91/$03.50 ADONIS 0168170291001087
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VIRUS 00691
Complete nucleotide sequence of segment 5 of epizootic haemorrhagic disease virus; the outer capsid protein VP5 is homologous to the VP5 protein of bluetongue virus H. Iwata ‘, T. Hirasawa ’ and P. Roy ‘J 1Department of Environmental Health, University of Alabama at Birmingham, University Station, Birmingham, AL 35294, U.S.A. and 2 Laboratory of Molecular Biophysics, Oxford Universiry, South Parks Road, Oxford, OXI 3QU, U.K.
(Received 1 March 1991; revision received 2 May 1991; accepted 2 May 1991)
Summary The complete nucleotide sequence of a cDNA clone representing the segment 5 RNA of epizootic haemorrhagic disease virus (EHDV) United States serotype 1 was determined. The 5’ and 3’ termini of the RNA are complementary and are capable of forming secondary structures. The comparison of the predicted amino acid sequence of the encoded outer capsid protein (VP5) with the sequences of VP5 from four serotypes of bluetongue virus, the prototype orbivirus, revealed that the protein shares 59% to 62% homologies with various BTV serotypes, including a single conserved glycine residue at the amino terminus. The sequence has been submitted to the Genebank databox (X55782). Epizootic hemorrhagic M5
disease virus; Outer capsid protein VP5; Genome segment
Epizootic haemorrhagic disease virus (EHDV) causes haemorrhagic disease in white-tail deer. The disease is endemic in many parts of the world, including North Correspondence to: P. Roy, Laboratory of Molecular Biophysics, Oxford University, South Parks Road, Oxford, OX1 3QU, U.K.
274
America, Australia, Japan and South Africa. In deer, infection is characterized by mortality and high morbidity. The virus is transmitted by a Cuclicoides vector, as is bluetongue virus the prototype Orbivirus. However, bluetongue virus (BTV) causes disease primarily in sheep. Little information is available on the genetics and molecular properties of EHDV. The virus is similar to BTV, both in its morphological structure and biochemical properties (Gorman et al., 1983; Huismans et al., 1979; Kusarit and Roy, 1986). Like BTV, EHDV virions consist of seven structural proteins and ten double-stranded RNA segments. The outer capsid is composed of two major proteins (VP2 and VP5) while the inner capsid consists of two major (VP3 and VP71 and three minor proteins (VPl, VP4 and VP6). Each of the genome segments codes for the synthesis of one viral protein. The genome segments 2 and 5 encode VP2 and VP5 respectively (Mertens et al., 1984; see review article Roy et al., 1990a). Whilst VP2 has been shown to be the major determinant of serotype specificity and responsible for eliciting neutralizing antibody (in case of BTV), no conclusive function has been assigned yet to VP5 (Huismans and Erasmus, 1981; Kahlon et al., 1983; Huismans et al., 1983; Roy et al., 1990b). However, both proteins (of various BTV serotypes) have been sequenced extensively and VP2 appears to be the most variable protein among the 10 BTV proteins (see review article, Roy, 1989). The second outer capsid protein VP5, on the other hand, is less variable, indicating that only a limited portion of the protein is perhaps exposed on the surface of the virion. In this report we present the complete sequence of genome segment M5 of EHDV-1 from a full length cDNA clone and document its very close homology to the VP5 of various BTV serotypes (Gould and Pritchard, 1988; Wade-Evans et al., 1988; Purdy et al., 1986; Hirasawa and Roy 1990; Oldfield et al., 1991). From predicted amino acid sequence analyses, a potential conserved myristylation site has also been identified for VP5 of EHDV-1 and VP5 of four BTV serotypes. In addition, the 5’ and 3’ terminal regions of the gene have been analysed and their potential secondary structure compared to the M5 gene of four BTV serotypes. The procedure used to obtain the complete nucleotide sequence of M5 RNA species of the EHDV-1 US isolate involved the synthesis of cDNA followed by cloning and sequencing of the derived M5 specific plasmid DNA, as fully described previously (Purdy et al., 1984). In brief, viral M5 RNA species were polyadenylated at their 3’ ends and cDNA copies of both strands were synthesized using reverse transcriptase, deoxyribonucleoside triphosphates and an oligo (dT) primer (Purdy et al., 1984). After removal of the RNA templates by hydrolysis with KOH, cDNA products were self-annealed and, to ensure that the products were full-length, their 3’ ends were repaired with the Klenow fragment of Escherichiu co/i DNA polymerase I. The DNA species were then tailed with dC and cloned into the Pst I site of pBR322. Many clones representing the M5 gene were identified by colony hybridization using a short-copy cDNA probe which was transcribed from polyadenylated viral RNA using an oligo (dT) primer. Subsequently, the length of each clone was estimated by restriction enzyme digestion as described previously
Fig. 1. The nucleotide sequence of the M5 gene of EHDV-1 shown as the cDNA of the message-sense RNA. The open reading frame begins at positions 28-30 and is terminated by the TGA codon at positions 1609-1611. Amino acids are shown above their respective DNA codons. Asterisk indicates termination codon.
et al., 1984). One clone which appeared to be a full-length copy of M5 RNA segment was selected for sequence analyses. In order to confirm that the clone was indeed full length the terminal sequences were determined by the method of Maxam and Gilbert (1980) while the remaining sequences of the clone were determined by the dideoxy method @anger et al., 1977). The 5’ and 3’ termini of the clone were found to contain the characteristic consensus sequences of orbivirus RNA segments (Rao et al., 1983; Mertens and Sanger, 1985; Wilson et al., 1990), namely 5’ GTTAAA.. . and . . . ACITAC 3’ (Fig. 11, indicating that the EHDV-1 M5 clone was full-length. For confirmation, two additional incomplete but overlapping clones were also sequenced. The complete nucleotide sequence of the cDNA of M5 in its coding (mRNA) sense is presented in Fig. 1, with the predicted amino acid sequence of the single long open reading frame shown above the cDNA. The M5 segment is 1640 base pairs long. The M, value of the dsRNA is calculated to be 1.1 X lo6 Da. The coding strand of the EHDV-1 M5 RNA has a calculated base composition of 27.5% U, 26.4% A, 19.7% C, and 26.5% G, i.e., comparable to the base composition of the RNA segment M5 of BTV-10 (Purdy et al., 1986). The M5 strand of (Purdy
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Fig. 2. Predicted secondary structures of the message sense cDNAs of the M5 of EHDV-1, BTV-1 (Aus and SA), BTV-2, BTV-10 and BTV-13.
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EHDV-1 has a short non-coding region (25 nucleotides at the 5’ terminus and 29 at the 3’ terminus, Fig. 2). The protein is negatively charged ( - 5.0) at neutral pH, equivalent to the VP5 of US BTV-10 which also is negatively charged (-4.5). The 5’ and 3’ termini of EHDV-1 are complementary and capable of forming secondary structures as shown in Fig. 2 for the message sense cDNA sequence. Similar complementary sequences were identified from the previously published sequence data of the M5 species of BTV-10, BTV-13, BTV-2 and BTV-1 (Purdy et al., 1985; Oldfield et al., 1991; Hirasawa and Roy 1990; Wade-Evans et al., 1988; Gould and Pritchard, 1989, see Fig. 2). Such secondary structures, albeit of different lengths and composition, are common to all the RNA segments of BTV and other Orbiviruses (e.g., segment 7 of African Horsesickness Virus, AHSV-4, Roy et al., 1991) that have been determined so far. As has been reported previously, all the proposed structures share some common features, for example, there is a looped out sequence (CACA for the M5 cDNAs shown in Fig. 2), proximally to the 3’ terminus, common to all mRNA of orbiviruses so far analysed. All the 3’ and 5’ ends have the potential for two alternative forms, either that shown in Fig. 2, or with a 5’ overhang (G) when the third 5’ nucleotide (T) pairs with the second 3’ nucleotide (A>. These secondary structures probably play a vital role either by masking the mRNA and thereby conferring protection against exonucleases or are responsible for segregation of the 10 mRNA species. It is also possible that these structures may be somehow involved in translation termination signals. In this context it is noteworthy that 5’ and 3’ terminal sequences of wound tumor virus genomic segments consist of segment-specific inverted repeats which are responsible for intramolecular interaction. It has been postulated that these terminal domains play a role in expression, sorting and packaging of the RNA segments (Xu et al., 1989). The single major open reading frame of the M5 RNA of EHDV-1 predicts a polypeptide of 527 amino acids, one amino acid longer than the VP5 of BTV-10. The molecular size CM,) of VP5 is estimated to be 59,119 Da, similar to the VP5 of BTV-10 which is 59,163 Da (Purdy et al., 1986). In order to determine the degree of homology between the VP5 protein of EHDV-1 and that of BTV (Purdy et al., 19861, the predicted amino acid sequence of VP5 was aligned with the VP5 of US serotypes of BTV-10, BTV-2, BTV-13, and BTV-1 of both Australian and South African isolates (shown in Fig. 3) using computer alignment programmes (Lipman and Pearson, 1985). Each VP5 sequence was also compared to that of BTV-10. The close similarities of the VP5 sequence of EHDV with that of BTV are indicated by the fact that only 3 gaps were required for maximum homologous alignment between these two Orbiviruses. The amino acid sequence of EHDV VP5 shares 59% to 62% homologies with various BTV serotypes, the lowest being with BTV-1 @A) and highest the BTV-10. However, the homologies among BTV serotypes are between 75 and 82% (Oldfield et al., 1991). Thus EHDV appears to be only slightly more distant from BTV-10 than BTV-10 is from other BTV serotypes. In general the differences in amino acids among five BTV proteins and the
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BTV-10 BTV-2 BTV-1 a"~ BTV-1 sa BTV-13 mm"-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 ?.a BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 BllS BTV-1 sa BTV-13 EXDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EKDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1
Fig. 3. Alignment of the predicted amino acid sequences of the VP5 of EHDV-1 with VP5 amino acid sequences of five serotypes of BTV. Residues identical to those of BTV-10 are indicated by asterisks.
EHDV proteins are distributed evenly throughout the whole protein, but several highly variable regions can be identified. These regions vary anywhere from between 70 to 95%, and include amino acid residues 141-146, 159-170, 279-298, 279-319,435-445 etc. Conversely, there are a number of highly conserved regions (indicated by asterisks) which are quite clear. A long stretch at the amino terminus (from amino acids residues 1 to 112) is highly conserved, with a few isolated amino acid changes scattered throughout the region. Similarly two middle regions (aa 190-273, and aa 361-420) and the carboxy-terminal region (aa 478-515) are highly conserved. These conserved regions are probably masked by the neutralization
279
protein VP2, the other outer capsid protein, and are probably essential for maintenance of the structural conformation of the VP5 molecule. The variable regions may be exposed on the surface of the particles which are altering constantly due to the immunological pressure imposed by its host. The other striking similarity between the VP5 of these two Orbiviruses is the conserved cysteine residues at position 323 (indicated by the dot). This may indicate that disulfide bridges are important for protein structure. The common phylogenetic origin of these two Orbiviruses was revealed by the diagonal line when VP5 protein of EHDV-1 and BTV-10 were compared although a few small gaps are noticeable
:
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Fig. 4. Diagon analyses. The VP5 proteins of EHDV-1 and BTV-10 were aligned using the Diagon program of Staden (1982). For the protein comparison an 11 amino acid span and a proportional index of 131 was employed.
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(Fig. 41. Also the hydropathic profiles of the two VP5 sequences are similar (data not shown). Perhaps the most significant finding from the sequence data presented in Fig. 3 is the identification of the single glycine (indicated by an arrow) residue at the amino terminus of the proteins. All six VP5 proteins possess this residue following the starting methionine residue. Both the position and conservative nature of this residue imply that the presence of glycine is not coincidental. Similar glycine residues have been implicated to be responsible for myristylation of a number of viral proteins (see review article, Magee and Hanby, 1988). Glycine has been recognised as the donor of the a-amino group to the amide linkage with myristate and other residues. Since the covalent modification of proteins is considered to be the signal for protein trafficking to intracellular sites and metabolic regulation, myristoylation of the proteins could be essential in the assembly and structure of the virus outer capsids. In fact, recently, the amino termini of the outer capsid protein of reovirus has been shown to be modified with an amide-linked myristoyl group (Field et al., personal communication). It is highly likely that the amino terminal glycine of Orbivirus VP5 is similarly modified. However, no experimental data are available to substantiate this speculation. Currently we are investigating this possibility by using baculovirus expressed BTV proteins. If this is the case, it might lead to a better understanding of the morphogenesis of the virus. We thank Miss Stephanie Clarke for typing and Mr Christopher Hatton for photographic work. This work was supported partially by Oxford Virology, Alabama State Grant AE 89-401 and NIH Grant A126879. Gorman, B.M., Taylor, J. and Walker, P.J. (1983) Orbiviruses. In: W.K. Joklik (Ed.), Reoviridiae. pp. 287-357. Plenum, New York. Gould, A.R. and Pritchard, L.I. (1988) The complete nucleotide sequence of the outer coat proteins, VP5, of the Australian bluetongue serotype 1 reveals conserved and non-conserved sequences. Virus Res. 98, 285-292. Hirasawa, T., Fernandez, M. and Roy, P. (1991) Sequence analysis of major capsid protein VP7 of African Horsesickness virus serotype 4 (recently isolated from Spain) reveals an unexpected close relationship with bluetongue virus. (submitted). Huismans, H., Bremer, C.W. and Barber, T.L. (1979) The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoort J. Vet. Res. 46, 95-104. Huismans, H. and Erasmus, B.J. (1981) Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort J. Vet. Res. 48, 51-58. Huismans, Ii., Van Der Walt, N.T., Cloete, M. and Erasmus, B.J. (1983) The biochemial and immunological characterization of bluetongue virus outer capsid polypeptides. In: R.W. Compans and D.H.L. Bishop (Eds.), Double-Stranded RNA Viruses, pp. 165-172. Elsevier, New York. Kahlon, J., Sugiyama. K. and Roy, P. (1983) Molecular basis of bluetongue virus neutralization. J. Virol. 48, 627-632. Knudson, D.L. and Shope, R.E. (1985) Overview of the Orbiviruses. In: T.L. Barker and M.M. Jochim (Eds.), Bluetongue and Related Orbiviruses, pp. 255-256. Alan R. Liss, New York. Kusari, J. and Roy, P. (1986) Molecular and genetic comparisons of two U.S. epizootic haemorrhagic disease viruses. Am. J. Vet. Res. 47, 1-7. Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132.
281 Lipman, D.J. and Pearson, W.R. (1985) Rapid and sensitive protein similarity by searches. Science 227, 1435-1441. Magee, T. and Hanley, M. (1988) Sticky fingers and CAAX boxes. Nature 355, 114-115. Maxam, A.M. and Gilbert, W. (19801 Sequence of end-labelled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499-560. Mecham, J.O., and Dean, V.C. (1988) Protein coding assignment for the genome of epizootic haemorrhagic disease virus. J. Gen. Viral. 69, 1255-1262. Oldfield, S., Hirasawa, T. and Roy, P. (1991) Sequence conservation of the outer capsid protein, VP5, of bluetongue virus, a contrasting feature to the outer capsid protein VP2. J. Gen. Virol. 72, 449-451. Purdy, M.A., Petre, J. and Roy, P. (1984) Cloning of the bluetongue virus L3 gene. Virology 51, 754-759. Purdy, M.A., Ritter, G.D. and Roy, P. (1986) Nucleotide sequence of cDNA clones encoding the outer capsid protein, VP5, of bluetongue virus serotype 10. J. Gen. Virol. 67, 957-962. Roy, P. (1989) Bluetongue virus genetics and genome structure. Virus Res. 13, 179-206. Roy, P., Marshall, J.J.A. and French, T.J. (1990a) Structure of bluetongue virus genome and its encoded proteins. In: P. Roy and B.M. Gorman (Eds.), Current Topics in Microbiology and Immunology: Bluetongue Virus, pp. 43-87. Springer-Verlag, Heidelberg. Roy, P., Urakawa, T., Van Dijk, A.A. and Erasmus, B.J. (1990b) Recombinant virus vaccine for bluetongue disease in sheep. J. Virol. 64, 1998-2003. Sanger, F., Nicklen, S. and Coulson A. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. U.S.A. 74, 5463-5467. Staden, R. (1982) An interactive graphics program for comparing and aligning nucleic acid and amino acid sequences. Nucleic Acids Res. 10, 2951-2961. Tsai, K.S. and Karsted, L. (1973) Ultrastructural characterization of the genome of epizootic hemorrhagic disease virus. Infect. Immun. 8, 463-474. Wade-Evans, A.M., Pan, Z.O. and Mertens, P.P.C. (1988) Sequence analysis and in vitro expression of a cDNA clone of genome segment 5 from bluetongue virus, serotype 1 from South Africa (VRR 00446). Virus Res. 11, 227-240. Xu, Z., Anzola, J.V., Nalin, CM. and Nuss, D.L. (1989) The 3’-terminal sequence of a wound tumor virus transcript can influence conformational and functional properties associated with the 5terminus. Virology 170, 511-522.
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VIRUS 00691
Complete nucleotide sequence of segment 5 of epizootic haemorrhagic disease virus; the outer capsid protein VP5 is homologous to the VP5 protein of bluetongue virus H. Iwata ‘, T. Hirasawa ’ and P. Roy ‘J 1Department of Environmental Health, University of Alabama at Birmingham, University Station, Birmingham, AL 35294, U.S.A. and 2 Laboratory of Molecular Biophysics, Oxford Universiry, South Parks Road, Oxford, OXI 3QU, U.K.
(Received 1 March 1991; revision received 2 May 1991; accepted 2 May 1991)
Summary The complete nucleotide sequence of a cDNA clone representing the segment 5 RNA of epizootic haemorrhagic disease virus (EHDV) United States serotype 1 was determined. The 5’ and 3’ termini of the RNA are complementary and are capable of forming secondary structures. The comparison of the predicted amino acid sequence of the encoded outer capsid protein (VP5) with the sequences of VP5 from four serotypes of bluetongue virus, the prototype orbivirus, revealed that the protein shares 59% to 62% homologies with various BTV serotypes, including a single conserved glycine residue at the amino terminus. The sequence has been submitted to the Genebank databox (X55782). Epizootic hemorrhagic M5
disease virus; Outer capsid protein VP5; Genome segment
Epizootic haemorrhagic disease virus (EHDV) causes haemorrhagic disease in white-tail deer. The disease is endemic in many parts of the world, including North Correspondence to: P. Roy, Laboratory of Molecular Biophysics, Oxford University, South Parks Road, Oxford, OX1 3QU, U.K.
274
America, Australia, Japan and South Africa. In deer, infection is characterized by mortality and high morbidity. The virus is transmitted by a Cuclicoides vector, as is bluetongue virus the prototype Orbivirus. However, bluetongue virus (BTV) causes disease primarily in sheep. Little information is available on the genetics and molecular properties of EHDV. The virus is similar to BTV, both in its morphological structure and biochemical properties (Gorman et al., 1983; Huismans et al., 1979; Kusarit and Roy, 1986). Like BTV, EHDV virions consist of seven structural proteins and ten double-stranded RNA segments. The outer capsid is composed of two major proteins (VP2 and VP5) while the inner capsid consists of two major (VP3 and VP71 and three minor proteins (VPl, VP4 and VP6). Each of the genome segments codes for the synthesis of one viral protein. The genome segments 2 and 5 encode VP2 and VP5 respectively (Mertens et al., 1984; see review article Roy et al., 1990a). Whilst VP2 has been shown to be the major determinant of serotype specificity and responsible for eliciting neutralizing antibody (in case of BTV), no conclusive function has been assigned yet to VP5 (Huismans and Erasmus, 1981; Kahlon et al., 1983; Huismans et al., 1983; Roy et al., 1990b). However, both proteins (of various BTV serotypes) have been sequenced extensively and VP2 appears to be the most variable protein among the 10 BTV proteins (see review article, Roy, 1989). The second outer capsid protein VP5, on the other hand, is less variable, indicating that only a limited portion of the protein is perhaps exposed on the surface of the virion. In this report we present the complete sequence of genome segment M5 of EHDV-1 from a full length cDNA clone and document its very close homology to the VP5 of various BTV serotypes (Gould and Pritchard, 1988; Wade-Evans et al., 1988; Purdy et al., 1986; Hirasawa and Roy 1990; Oldfield et al., 1991). From predicted amino acid sequence analyses, a potential conserved myristylation site has also been identified for VP5 of EHDV-1 and VP5 of four BTV serotypes. In addition, the 5’ and 3’ terminal regions of the gene have been analysed and their potential secondary structure compared to the M5 gene of four BTV serotypes. The procedure used to obtain the complete nucleotide sequence of M5 RNA species of the EHDV-1 US isolate involved the synthesis of cDNA followed by cloning and sequencing of the derived M5 specific plasmid DNA, as fully described previously (Purdy et al., 1984). In brief, viral M5 RNA species were polyadenylated at their 3’ ends and cDNA copies of both strands were synthesized using reverse transcriptase, deoxyribonucleoside triphosphates and an oligo (dT) primer (Purdy et al., 1984). After removal of the RNA templates by hydrolysis with KOH, cDNA products were self-annealed and, to ensure that the products were full-length, their 3’ ends were repaired with the Klenow fragment of Escherichiu co/i DNA polymerase I. The DNA species were then tailed with dC and cloned into the Pst I site of pBR322. Many clones representing the M5 gene were identified by colony hybridization using a short-copy cDNA probe which was transcribed from polyadenylated viral RNA using an oligo (dT) primer. Subsequently, the length of each clone was estimated by restriction enzyme digestion as described previously
Fig. 1. The nucleotide sequence of the M5 gene of EHDV-1 shown as the cDNA of the message-sense RNA. The open reading frame begins at positions 28-30 and is terminated by the TGA codon at positions 1609-1611. Amino acids are shown above their respective DNA codons. Asterisk indicates termination codon.
et al., 1984). One clone which appeared to be a full-length copy of M5 RNA segment was selected for sequence analyses. In order to confirm that the clone was indeed full length the terminal sequences were determined by the method of Maxam and Gilbert (1980) while the remaining sequences of the clone were determined by the dideoxy method @anger et al., 1977). The 5’ and 3’ termini of the clone were found to contain the characteristic consensus sequences of orbivirus RNA segments (Rao et al., 1983; Mertens and Sanger, 1985; Wilson et al., 1990), namely 5’ GTTAAA.. . and . . . ACITAC 3’ (Fig. 11, indicating that the EHDV-1 M5 clone was full-length. For confirmation, two additional incomplete but overlapping clones were also sequenced. The complete nucleotide sequence of the cDNA of M5 in its coding (mRNA) sense is presented in Fig. 1, with the predicted amino acid sequence of the single long open reading frame shown above the cDNA. The M5 segment is 1640 base pairs long. The M, value of the dsRNA is calculated to be 1.1 X lo6 Da. The coding strand of the EHDV-1 M5 RNA has a calculated base composition of 27.5% U, 26.4% A, 19.7% C, and 26.5% G, i.e., comparable to the base composition of the RNA segment M5 of BTV-10 (Purdy et al., 1986). The M5 strand of (Purdy
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Fig. 2. Predicted secondary structures of the message sense cDNAs of the M5 of EHDV-1, BTV-1 (Aus and SA), BTV-2, BTV-10 and BTV-13.
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EHDV-1 has a short non-coding region (25 nucleotides at the 5’ terminus and 29 at the 3’ terminus, Fig. 2). The protein is negatively charged ( - 5.0) at neutral pH, equivalent to the VP5 of US BTV-10 which also is negatively charged (-4.5). The 5’ and 3’ termini of EHDV-1 are complementary and capable of forming secondary structures as shown in Fig. 2 for the message sense cDNA sequence. Similar complementary sequences were identified from the previously published sequence data of the M5 species of BTV-10, BTV-13, BTV-2 and BTV-1 (Purdy et al., 1985; Oldfield et al., 1991; Hirasawa and Roy 1990; Wade-Evans et al., 1988; Gould and Pritchard, 1989, see Fig. 2). Such secondary structures, albeit of different lengths and composition, are common to all the RNA segments of BTV and other Orbiviruses (e.g., segment 7 of African Horsesickness Virus, AHSV-4, Roy et al., 1991) that have been determined so far. As has been reported previously, all the proposed structures share some common features, for example, there is a looped out sequence (CACA for the M5 cDNAs shown in Fig. 2), proximally to the 3’ terminus, common to all mRNA of orbiviruses so far analysed. All the 3’ and 5’ ends have the potential for two alternative forms, either that shown in Fig. 2, or with a 5’ overhang (G) when the third 5’ nucleotide (T) pairs with the second 3’ nucleotide (A>. These secondary structures probably play a vital role either by masking the mRNA and thereby conferring protection against exonucleases or are responsible for segregation of the 10 mRNA species. It is also possible that these structures may be somehow involved in translation termination signals. In this context it is noteworthy that 5’ and 3’ terminal sequences of wound tumor virus genomic segments consist of segment-specific inverted repeats which are responsible for intramolecular interaction. It has been postulated that these terminal domains play a role in expression, sorting and packaging of the RNA segments (Xu et al., 1989). The single major open reading frame of the M5 RNA of EHDV-1 predicts a polypeptide of 527 amino acids, one amino acid longer than the VP5 of BTV-10. The molecular size CM,) of VP5 is estimated to be 59,119 Da, similar to the VP5 of BTV-10 which is 59,163 Da (Purdy et al., 1986). In order to determine the degree of homology between the VP5 protein of EHDV-1 and that of BTV (Purdy et al., 19861, the predicted amino acid sequence of VP5 was aligned with the VP5 of US serotypes of BTV-10, BTV-2, BTV-13, and BTV-1 of both Australian and South African isolates (shown in Fig. 3) using computer alignment programmes (Lipman and Pearson, 1985). Each VP5 sequence was also compared to that of BTV-10. The close similarities of the VP5 sequence of EHDV with that of BTV are indicated by the fact that only 3 gaps were required for maximum homologous alignment between these two Orbiviruses. The amino acid sequence of EHDV VP5 shares 59% to 62% homologies with various BTV serotypes, the lowest being with BTV-1 @A) and highest the BTV-10. However, the homologies among BTV serotypes are between 75 and 82% (Oldfield et al., 1991). Thus EHDV appears to be only slightly more distant from BTV-10 than BTV-10 is from other BTV serotypes. In general the differences in amino acids among five BTV proteins and the
278
BTV-10 BTV-2 BTV-1 a"~ BTV-1 sa BTV-13 mm"-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 ?.a BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 BllS BTV-1 sa BTV-13 EXDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EKDV-1 BTV-10 BTV-2 BTV-1 aus BTV-1 sa BTV-13 EHDV-1
Fig. 3. Alignment of the predicted amino acid sequences of the VP5 of EHDV-1 with VP5 amino acid sequences of five serotypes of BTV. Residues identical to those of BTV-10 are indicated by asterisks.
EHDV proteins are distributed evenly throughout the whole protein, but several highly variable regions can be identified. These regions vary anywhere from between 70 to 95%, and include amino acid residues 141-146, 159-170, 279-298, 279-319,435-445 etc. Conversely, there are a number of highly conserved regions (indicated by asterisks) which are quite clear. A long stretch at the amino terminus (from amino acids residues 1 to 112) is highly conserved, with a few isolated amino acid changes scattered throughout the region. Similarly two middle regions (aa 190-273, and aa 361-420) and the carboxy-terminal region (aa 478-515) are highly conserved. These conserved regions are probably masked by the neutralization
279
protein VP2, the other outer capsid protein, and are probably essential for maintenance of the structural conformation of the VP5 molecule. The variable regions may be exposed on the surface of the particles which are altering constantly due to the immunological pressure imposed by its host. The other striking similarity between the VP5 of these two Orbiviruses is the conserved cysteine residues at position 323 (indicated by the dot). This may indicate that disulfide bridges are important for protein structure. The common phylogenetic origin of these two Orbiviruses was revealed by the diagonal line when VP5 protein of EHDV-1 and BTV-10 were compared although a few small gaps are noticeable
:
.,; .fl. .: il.
..
;.
Fig. 4. Diagon analyses. The VP5 proteins of EHDV-1 and BTV-10 were aligned using the Diagon program of Staden (1982). For the protein comparison an 11 amino acid span and a proportional index of 131 was employed.
280
(Fig. 41. Also the hydropathic profiles of the two VP5 sequences are similar (data not shown). Perhaps the most significant finding from the sequence data presented in Fig. 3 is the identification of the single glycine (indicated by an arrow) residue at the amino terminus of the proteins. All six VP5 proteins possess this residue following the starting methionine residue. Both the position and conservative nature of this residue imply that the presence of glycine is not coincidental. Similar glycine residues have been implicated to be responsible for myristylation of a number of viral proteins (see review article, Magee and Hanby, 1988). Glycine has been recognised as the donor of the a-amino group to the amide linkage with myristate and other residues. Since the covalent modification of proteins is considered to be the signal for protein trafficking to intracellular sites and metabolic regulation, myristoylation of the proteins could be essential in the assembly and structure of the virus outer capsids. In fact, recently, the amino termini of the outer capsid protein of reovirus has been shown to be modified with an amide-linked myristoyl group (Field et al., personal communication). It is highly likely that the amino terminal glycine of Orbivirus VP5 is similarly modified. However, no experimental data are available to substantiate this speculation. Currently we are investigating this possibility by using baculovirus expressed BTV proteins. If this is the case, it might lead to a better understanding of the morphogenesis of the virus. We thank Miss Stephanie Clarke for typing and Mr Christopher Hatton for photographic work. This work was supported partially by Oxford Virology, Alabama State Grant AE 89-401 and NIH Grant A126879. Gorman, B.M., Taylor, J. and Walker, P.J. (1983) Orbiviruses. In: W.K. Joklik (Ed.), Reoviridiae. pp. 287-357. Plenum, New York. Gould, A.R. and Pritchard, L.I. (1988) The complete nucleotide sequence of the outer coat proteins, VP5, of the Australian bluetongue serotype 1 reveals conserved and non-conserved sequences. Virus Res. 98, 285-292. Hirasawa, T., Fernandez, M. and Roy, P. (1991) Sequence analysis of major capsid protein VP7 of African Horsesickness virus serotype 4 (recently isolated from Spain) reveals an unexpected close relationship with bluetongue virus. (submitted). Huismans, H., Bremer, C.W. and Barber, T.L. (1979) The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoort J. Vet. Res. 46, 95-104. Huismans, H. and Erasmus, B.J. (1981) Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort J. Vet. Res. 48, 51-58. Huismans, Ii., Van Der Walt, N.T., Cloete, M. and Erasmus, B.J. (1983) The biochemial and immunological characterization of bluetongue virus outer capsid polypeptides. In: R.W. Compans and D.H.L. Bishop (Eds.), Double-Stranded RNA Viruses, pp. 165-172. Elsevier, New York. Kahlon, J., Sugiyama. K. and Roy, P. (1983) Molecular basis of bluetongue virus neutralization. J. Virol. 48, 627-632. Knudson, D.L. and Shope, R.E. (1985) Overview of the Orbiviruses. In: T.L. Barker and M.M. Jochim (Eds.), Bluetongue and Related Orbiviruses, pp. 255-256. Alan R. Liss, New York. Kusari, J. and Roy, P. (1986) Molecular and genetic comparisons of two U.S. epizootic haemorrhagic disease viruses. Am. J. Vet. Res. 47, 1-7. Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132.
281 Lipman, D.J. and Pearson, W.R. (1985) Rapid and sensitive protein similarity by searches. Science 227, 1435-1441. Magee, T. and Hanley, M. (1988) Sticky fingers and CAAX boxes. Nature 355, 114-115. Maxam, A.M. and Gilbert, W. (19801 Sequence of end-labelled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499-560. Mecham, J.O., and Dean, V.C. (1988) Protein coding assignment for the genome of epizootic haemorrhagic disease virus. J. Gen. Viral. 69, 1255-1262. Oldfield, S., Hirasawa, T. and Roy, P. (1991) Sequence conservation of the outer capsid protein, VP5, of bluetongue virus, a contrasting feature to the outer capsid protein VP2. J. Gen. Virol. 72, 449-451. Purdy, M.A., Petre, J. and Roy, P. (1984) Cloning of the bluetongue virus L3 gene. Virology 51, 754-759. Purdy, M.A., Ritter, G.D. and Roy, P. (1986) Nucleotide sequence of cDNA clones encoding the outer capsid protein, VP5, of bluetongue virus serotype 10. J. Gen. Virol. 67, 957-962. Roy, P. (1989) Bluetongue virus genetics and genome structure. Virus Res. 13, 179-206. Roy, P., Marshall, J.J.A. and French, T.J. (1990a) Structure of bluetongue virus genome and its encoded proteins. In: P. Roy and B.M. Gorman (Eds.), Current Topics in Microbiology and Immunology: Bluetongue Virus, pp. 43-87. Springer-Verlag, Heidelberg. Roy, P., Urakawa, T., Van Dijk, A.A. and Erasmus, B.J. (1990b) Recombinant virus vaccine for bluetongue disease in sheep. J. Virol. 64, 1998-2003. Sanger, F., Nicklen, S. and Coulson A. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. U.S.A. 74, 5463-5467. Staden, R. (1982) An interactive graphics program for comparing and aligning nucleic acid and amino acid sequences. Nucleic Acids Res. 10, 2951-2961. Tsai, K.S. and Karsted, L. (1973) Ultrastructural characterization of the genome of epizootic hemorrhagic disease virus. Infect. Immun. 8, 463-474. Wade-Evans, A.M., Pan, Z.O. and Mertens, P.P.C. (1988) Sequence analysis and in vitro expression of a cDNA clone of genome segment 5 from bluetongue virus, serotype 1 from South Africa (VRR 00446). Virus Res. 11, 227-240. Xu, Z., Anzola, J.V., Nalin, CM. and Nuss, D.L. (1989) The 3’-terminal sequence of a wound tumor virus transcript can influence conformational and functional properties associated with the 5terminus. Virology 170, 511-522.