Nucleotide Sequence of a eDNA Clone of RNA Segment ... - CiteSeerX

J. gen. Virol. (1986), 67,2833-2837. Printed in Great Britain

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Key words: BTV/genome segment lO/nucleotide sequence

Nucleotide Sequence of a eDNA Clone of RNA Segment 10 of Bluetongue Virus (Serotype 10) By J. W. L E E AND P O L L Y R O Y * Department o f Environmental Health Sciences, School o f Public Health, University of Alabama at Birmingham, Birmingham, Alabama 35294, U.S.A. (Accepted 9 September 1986)

SUMMARY

The complete sequence of the double-stranded RNA segment that codes for a nonstructural protein (P8) of bluetongue virus serotype 10 has been determined from a eDNA clone inserted into the plasmid pBR322. The segment 10 RNA of the virus (S10 RNA) is deduced to be 822 base pairs long (0.5 x 106 daltons) and has an open reading frame in one strand capable of coding for a protein with a calculated size of 25 572 daltons (229 amino acids) and a net charge of +5.5 at neutral pH. Bluetongue virus (BTV) is the prototype virus of the Orbivirus genus in the Reoviridae family. The ten segment, double-stranded RNA genome of BTV (dsRNA segments LI to L3, M4 to M6 and $7 to S10) is located in a core particle which exhibits icosahedral symmetry (Els & Verwoerd, 1969). The core is composed of two major (VP3, VP7) and three minor protein components (VP1, VP4, VP6) and is surrounded by an outer capsid protein layer consisting of two major species, VP2 and VP5 (Verwoerd et al., 1972). The sequences of representative BTV serotype 10 (BTV-10) (or BTV-17) L2, L3 and M5 RNA species have been reported previously (Purdy et al., 1984, 1985, 1986; Ghiasi et al., 1985). The L2 RNA species codes for the outer typespecific VP2 protein (Kahlon et al., 1983; Sangar & Mertens, 1983). VP2 has been shown to be the antigen recognized by neutralizing antibodies; it is the main determinant of serotype specificity (Huismans & Erasmus, 1981; Kahlon et al., 1983; Huismans et al., 1983). The L3 RNA codes for the internal group-specific VP3 protein which has a highly conserved sequence (Roy et al., 1985). The M5 RNA codes for the VP5 gene, representing the other outer protein of the virus. Other than a structural role, functions of the VP5 protein have not been elucidated. In addition to structural proteins, BTV codes for a variety of non-structural proteins. These include proteins variously designated as P5A (NS1), P6A (NS2) and the smallest, P8 or NS3 (Gorman et al., 1981 ; Sangar & Mertens, 1983). In vitro translation studies have indicated that RNA segment S10 codes for the P8 protein, as well as a product designated P8A, which by tryptic peptide analyses appears to be related to P8 (Sangar & Mertens, 1983). The sizes of these two proteins have been estimated to be in the range of 1.5 x 104 to 2.0 x 104 daltons. As a first step to investigating the identity and function of the S10 gene product(s), we have cloned and sequenced DNA copies of the S10 gene of BTV-10 (BTV CA 8; California, 1953, sheep; Sugiyama et al., 1981). The results are reported. The procedure used to obtain the complete sequences of the BTV-10 S10 RNA species involved the synthesis o f c D N A by reverse transcriptase followed by cloning and sequencing the derived clones. To this end, the denatured viral S 10 RNA species were polyadenylated at their 3' ends, and cDNA copies synthesized with reverse transcriptase, deoxyribonucleoside triphosphates, and an oligo(dT)l 2-18 primer as described previously (Purdy et al., 1984). The RNA template strands were removed by RNase H treatment (Purdy et al., 1985; Maniatis et al., 1982), the cDNA products self-annealed and, after tailing with dC, the duplexes were cloned into the PstI site of pBR322. Approximately 30 clones representing the S10 gene were identified by colony hybridization. The HinfI restriction patterns of the clones were compared as described 0000-7308 © 1986 SGM

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Fig. 1. Hybridization of 3zp-labelled DNA of clones no. 90 and no. 95 to BTV-10 genomic RNA. Nicktranslated plasmid DNA of two clones were hybridized to blots of BTV-10 RNA and autoradiographed. The numbers at the sides of the figure represent the numbering system for BTV genomic RNAs in agarose gel.

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Fig. 2. Sequence strategy used to analyse the cDNA clones of BTV-10 segment 10. The restriction fragments of one clone, clone no. 90, used to obtain the cDNA sequence are shown by the arrows representing the individual strands that were sequenced and the direction in which they were sequenced (Maxam & Gilbert, 1980). Restriction site symbols: D, DdeI; H, HinfI; N, N¢il.

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M L S G L I O R F 5- E E I' M ~. H 1+q ~ D ~ V E E • S L V R V D D • I S GTTAAAAAGTGTCGCqfGCCAT BCTA TCCB~JGCT3 ATCCAAAI~Gr '~CBAAGAAGA~ AAGATBAAACACAA TCAABATAGAGTTGAAGAACTG~~ r T TAGTGC~CB TGGATGATACCAT TT C 10 20 3O ~o 50 too 70 6,3 9O I O0 110 120

g P P R Y A P S A P M P S S M P ]" v A • E I L D K A Fl S N T T G A T ~.~ • 0 ~-- A E TCAACCSCCAAGSTATGCTCCGABTGCACCGA TGCCATCA]"CCAl GCCAACI3GT3"BUCF.TTGAAA FAI2I BBACAAAGCGATGTCAAACACAACTGGTGCAACGCAAACACAAAABGCGBA 130 1~0 150 I bO 170 180 190 200 210 22O 23O 240

K A ~ F ~ S Y A E A F R D D v R L R O I t. R H V N E O ! L P ~ L K 5 D L S E L K c~A~T~ATT~CATCGT~CGCAGAAG~:GTT~C~T~ATGAC~TAAGACTAA~CAGATTA~CGC~AT~TAAATGAACA~ATTTTAC~TAAATTGAAAAGTGATCTAA~T~AATTGAA 250 2~0 270 280 290 300 310 320 33O 340 350 3bO

K K R A I l H T T L L V A A V V A L L T S V C • L S S O M S V A F K I N G T K ¥ ~ f l @ I a C ~ , I ~ T C , ~ T , ~ , C ~ A C TACTCTACTAGTBGCTGCTGTGGTTGI:GCTSCTGACA TCAGTTTGCACCUTT TCAAG•3AI ATGAG113TSGUCTTUAAAATAAA TGGGACCA.AAAC 370 380 390 400 4 ~0 42O 430 440 450 460 470 4eO

E V P S W F K S L N P M L G V v N L G A T F • M M V C A ,k" 5 E R A L N Q Q I D M AGAAGTGCCTTCATGGTTTAAAAGCC TTAATCCAATGCTTGGCGTTG TCAACT TGGGAGCAAC t TTTTTGATC-ATGGT TTBCGCAAAGAG•GAAAGAGCCTTBAATCAACAGAT AGATAT 490 5O0 5 t0 520 530 540 550 560 57O 580 590 600

I K K E V M K K Q S Y N 0 A V R M 5 F T E F S S I P L O G F E M P L T * fid~T#~G~AAGAAGTBATGA~A4W~ACAATCATATAA r GACGCAGTGAGGATGAGr T T TACAGAGT r c TCGTCGATCCCGCTGGAXBGTTT•GAAATGCCATTAACCTGAGGACAGTA6GT 610 620 b30 640 bSO b60 b70 680 690 700 710 720

AC~,ABTBBCBCCCCAABGTT TACGTCGTGCI~(3r,?~(3TGGTTGACC TCGCGGCG TAAA TTCCCACTGCTGTATAACGGGGGAGGB [ GCGCGATACT A C A C ~ T I~C 730 740 750 760 770 780 790 800 BIO 820

Fig. 3. DNA sequence of the positive sense strand of BTV-10 segment 10. The open reading frame begins with the ATG codonat residues 20 to 22 and terminates at the TGA stop codonat residues 707 to 709. The largest predicted gene product is shown by the single-letter amino acid convention. Amino acid residues are centred over the corresponding codons. Nucleotides are numbered below their respective residues.

previously (Purdy et al., 1984) and confirmed to represent BTV-10 S10 R N A by Northern hybridization as shown in Fig. 1. The position of each viral R N A segment in the agarose gel used for the Northern analyses was determined by ethidium bromide staining as described previously (Purdy et al., 1984). The sequence of the S10 gene was determined on strand-separated, endqabelled, restriction D N A samples using the restriction endonuclease fragments shown for clone no. 90 in Fig. 2. The extent to which each D N A strand was sequenced is indicated by arrows. The complete nucleotide sequence of the S10 eDNA is given in Fig. 3. Excluding the homopolymeric tails, it is deduced to be 822 nucleotides in length. The comparable R N A sequence contains the BTV 5' and 3' conserved terminal sequences identified previously (mRNA sense strand, i.e. G U U A A A . . . and ... CACUUAC, respectively) (Rao et al., 1983; Mertens & Sangar, 1985) which indicates that the clone is a full-length eDNA copy of the R N A segment 10. Based on these data the double-stranded S!0 RNA is estimated to have a mass of 0.5 x 106 daltons, somewhat higher than the estimates made for the S 10 RN A of another BTV strain by Verwoerd et al. (1970). A single open reading frame exists in one strand of the viral RNA, presumably equivalent to the S10 m R N A species. Beginning with the A U G triplet at residues 20 to 22, the RNA codes for a primary gene product composed of 229 amino acids. A second A U G codon at positions 60 to 62 is in the same open reading frame. Which methionine is used to initiate translation of the gene product is not known. The first translation initiation codon is present at residues 20 to 22, i.e. comparable in position to those of the L2 and L3 R N A species of some BTV-10 isolates (Purdy et al., 1984, 1985). The open reading frame terminates with a U G A translation termination codon at residues 709 to 711. It is followed by a 3' non-coding region of 113 nucleotides, substantially longer than that identified for the L2 RNA (36 nucleotides; Purdy et al., 1984), L3 RNA (49 nucleotides; Purdy et al., 1985) or M6 R N A (28 nucleotides; Purdy et al., 1986). The indicated primary gene product of the S10 R N A codes for a protein (P8) that is estimated to have a size of 25 572 daltons (Table 1) and an overall net positive charge of + 5.5 at neutral pH. The protein has a somewhat higher relative abundance of serine, threonine and methionine residues and relative paucity of histidine, tyrosine and glycine residues by comparison with the other BTV proteins that have been characterized (Table 1). However, the significance of these observations is not known.

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Fig. 4. Hydropathic plot of the BTV-10 segment 10 gene product. The regions of the predicted protein with a net hydrophobicity (areas above the centre line) or hydrophilicity (areas below the centre line) are displayed according to Kyte & Doolittle (1982). The distribution of the charged residues (D, E, K and R) are displayed below the hydropathic plot. Cysteine positions are indicated by the bars above the graph.

T a b l e 1. Amino acid (symbol) Alanine (A) Arginine (R) Aspartate (D) Asparagine (N) Cysteine (C) Glutamate (E) Glutamine (Q) Glycine(G) Histidine (H) Isoleucine (I) Leucine (L) Lysine (K) Methionine (M) Phenylalanine (F) Proline (P) Serine (S) Threonine (T) Tryptophan (W) Tyrosine (Y) Valine (V) Total amino acids New charge Size (daltons)

Amino acid compositions of four proteins of BTV-IO

r VP2 53 65 69 41 16 60 37 49 29 70 90 61 20 43 36 47 48 13 47 62 956 +11.5 111112

No. of residues ~ VP3 VP5 63 49 69 28 59 26 41 16 5 3 50 55 42 19 43 33 16 17 65 48 82 43 27 40 36 17 39 20 47 15 42 31 53 22 10 2 39 14 73 28 901 526 -5.0 -4.5 103344 59163

Mol ~ P8 20 11 11 8 2 15 10 6 3 11 23 19 13 9 11 21 15 1 3 17 229 +5.5 25572

~ VP2 6 7 7 4 2 6 4 5 3 7 9 6 2 4 4 5 5 1 5 6

VP3 7 8 7 5 1 6 5 5 2 7 9 3 4 4 5 5 6 1 4 8

VP5 9 5 5 3 1 11 4 6 3 9 8 8 3 4 3 6 4 0 3 5

P8 9 5 5 3 1 7 4 3 1 5 10 8 6 4 5 9 7 0 1 7

T h e h y d r o p a t h i c profile and c h a r g e d a m i n o acids distribution (D, E, K, L) of the protein (Fig. 4) indicate that the protein c o n t a i n s two regions o f h y d r o p h o b i c i t y (Fig. 4, regions a b o v e the central line, i.e. a m i n o acid residues 115 to 145 and 160 to 185), and an a m i n o t e r m i n a l d o m a i n o f hydrophilic a m i n o acids (Fig. 4, r e g i o n below the central line). T h e r e are only two cysteine residues, b o t h located in the h y d r o p h o b i c domains. O f the four B T V proteins t h a t h a v e b e e n c h a r a c t e r i z e d so far (VP2, VP3, VP5 and PS) the S10 gene p r o d u c t is the only protein w h i c h contains any m a j o r h y d r o p h o b i c region.

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C o m p a r i s o n o f t h e d e d u c e d a m i n o a c i d s e q u e n c e o f t h e S 1 0 - c o d e d P8 p r o t e i n o f B T V - 1 0 a n d t h e 41 164 d a l t o n $4 g e n e p r o d u c t o f r e o v i r u s t y p e 3 (the s m a l l e s t r e o v i r u s g e n e ; G i a n t i n i et al., 1984) d i d n o t i n d i c a t e a n y d i r e c t a m i n o acid s e q u e n c e h o m o l o g y b e t w e e n t h e t w o p r o t e i n s . H o w e v e r , b o t h p r o t e i n s are r i c h in m e t h i o n i n e ( 6 ~ ) a n d h a v e h y d r o p h o b i c d o m a i n s . W h e t h e r t h e y serve t h e s a m e f u n c t i o n s is n o t k n o w n . N o s e q u e n c e h o m o l o g i e s h a v e b e e n d e t e c t e d b e t w e e n t h e $2 or $3 g e n e p r o d u c t s o f r e o v i r u s a n d t h a t o f t h e S10 g e n e o f B T V - 1 0 virus. This work was supported by U.S. Department of Agriculture grant 84CRSR-2-2453. REFERENCES ELS, n. L • VERWOERD, D. W. (1969). Morphology of bluetongue virus. Virology 38, 213-219. GHIASI,~., PURDY,M. A. & ROY, P. (1985). The complete sequence of bluetongue virus serotype 10 segment 3 and its predicted VP3 polypeptide compared with those of BTV serotype 17. Virus Research 3, 181-190. GIANTINI, M., SELIGER, L. S., FURUICHI, Y. & SHATKIN, A. J. (1984). Reovirus type 3 genome segment $4: nucleotide sequence of the gene encoding a major virion surface protein. Journal of Virology 52, 984-987. GORMAN, B. M., TAYLOR, J., WALKER, P. J., DAVIDSON, W. L. & BROWN, F. (1981). Comparison of bluetongue type 20 with certain viruses of the bluetongue and Eubenangee serological groups of orbiviruses. Journal of General Virology 57, 251-261. ~IS~t~NS, H. • ERASMUS, B. J. (1981). Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort Journal of Veterinary Research 48, 51-58. HtJIS~NS, H., VAN DER WALT, N. T., eLOETE, M. & ERASMUS,B. J. (1983). The biochemical and immunological characterization of bluetongue virus outer capsid polypeptides. In Double-stranded RNA Viruses,pp. 165-172. Edited by R. W. Compans & D. H. L. Bishop. New York: Elsevier. ~ L O N , L, SUGIY~, K. & ROY, P. (1983). Molecular basis of bluetongue virus neutralization. Journal of Virology 48, 627-632. KVTE, J. & r~OOLITrLE,R. r. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105-132. MANIATIS,T., FRITSCH,E. F. & SAMBROOK,J. (1982). Molecular Cloning: A Laboratory Manual, pp. 168-169, 230-234, 239-242. New York: Cold Spring Harbor Laboratory. M~, A. M. & GILBERT,W. (1980). Sequencing end-labeled DNA with basespecific chemical cleavages. Methods in Enzymology 65, 499-560. MERTENS, P. P. C. & SANGm~,D. V. (1985). Analysis of the terminal sequences of the genome sequences of four orbiviruses. Virology 140, 55-67. PURDY, M. A., PETRE, L & ROY, P. (1984). Cloning of the bluetongue virus L3 gene. Journal of Virology 51,754-749. PURDY, M. A., GHI~I, H., ~ O , C. D. & ROY, P. (1985). The complete sequence of bluetongue virus L2 RNA that codes for the antigen recognized by neutralizing antibodies. Journal of Virology 55, 826-830. PURDY, M. A., RI~ER, G. D. & ROY, P. (1986). Nucleotide sequence of cDNA clones encoding the outer capsid protein, VP5, of bluetongue virus serotype 10. Journal of General Virology 67, 957-962. ~ o , c. D., KIUeHI, A. & ROY, P. (1983). Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus. Journal of Virology 46, 378 383. ROY, P., RIYrER, G. D., JR, A~SHI, H., COLLISSOY,E. & INABA,V. (1985). A genetic probe for identifying bluetongue virus infections in vivo and in vitro. Journal of General Virology 66, 1613 1619. SANG~, D. V. & MERTENS,P. P. ¢. (1983). Comparison of type 1 bluetongue virus protein synthesis in vivo and in vitro.In Double-stranded RNA Viruses,pp. 183-191. Edited by R. W. Compans & D. H. L. Bishop. New York: Elsevier. SUGIYAMA,K., BISHOP,D. H. L. & ROY, P. (1981). Analyses of the genomes of bluetongue viruses recovered in the United States. I. Oligonucleotide fingerprint studies that indicate the existence of naturally occurring reassortant BTV isolates. Virology 114, 210-217. WRWOERD, D. W., LOtJW, a. & OELLERMA~q, g. A. (1970). Characterization of bluetongue virus ribonucleic acid. Journal of Virology 5, 1-7. VERWOERD, D. W., ELS, H. J., DE VILLIERS, E. M. & HUISMANS,H. (1972). Structure of the bluetongue virus capsid. Journal of Virology 10, 783-794.

(Received 16 June 1986)