VIRUS GENES 6:4, 387-392, 1992 © Kluwer Academic Publishers, Manufactured in The Netherlands

Nucleotide Sequence of the Genome Segment Encoding Nonstructural Protein NS1 of Bluetongue Virus Serotype 20 from Australia JEFF A. COWLEY

Queensland Department of Primary Industries, Animal Research Institute. Yeerongpilly, Australia Received November 1, 1991 Accepted December 24, 1991 Requests for reprints should be addressed to Jeff A. Cowley, CSIRO Division of Tropical Animal Production, Private Bag No. 3, Indooroopilly, 4068, Australia.

Key words: bluetongue virus type 20, nonstructural protein NSI, nucleotide sequence

Abstract

The nucleotide sequence of the genome segment ($6) encoding the nonstructural protein NS1 of an Australian isolate of bluetongue virus serotype 20 (BTV 20) has been determined from a series of overlapping cDNA clones synthesized using two terminal 15-mer otigonucleotides as primers. The gene consists of 1769 nucleotides with an open reading frame between nucleotides 35 and 1690 encoding a protein of 552 amino acids (molecular weight 64,506 Da; net charge - 2 at pH 7). Comparison of the nucleotide and deduced amino acid sequence of this genome segment with cognate segments of isolates of BTV 1 from Australia and South Africa, and BTV 10 and BTV 17 from the United States, revealed homologies of 98%, 80%, 79%, and 79%, respectively, at the nucleotide level and 98%, 90%, 89%, and 90% identity, respectively, at the amino acid level. The data indicate that the evolutionary divergence between NSI genes of two different Australian BTV serotypes (BTV 20 and BTV 1) is less than that between isolates of the same (BTV 1) or different serotypes from different geographical locations. Bluetongue virus (BTV) is the prototype member of the orbivirus genus of the Reoviridae family. Of the 24 serotypes of BTV currently recognized worldwide, eight s e r o t y p e s - - l , 3, 9, 15, 16, 20, 21, and 23--have been isolated in Australia. The EMBL Data Library sequence accession Code is X56735 BLUETONGUE VIRUS RNA SEGMENT 6.

388

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The BTV particle contains a genome comprising 10 segments of double-stranded RNA (1) encapsidated in a double-layered shell comprising seven proteins (2,3). In addition to the seven structural proteins, two major (NS 1, NS2) and one minor (NS3) nonstructural protein are synthesized in BTV-infected cells (4,5). The largest and most abundant nonstructural protein, NSI (encoded by genome segment 6), is responsible for the formation of cytoplasmic tubular structures (6) of as yet undefined function. Northern hybridizations with dsRNA from isolates of the 24 BTV serotypes and DNA probes to genome segments 1-8 of a BTV 4 isolate from South Africa have been used to determine the levels of sequence homology among these BTV isolates (7). Hybridization data indicated that the genome segment ($6) encoding NS1 was the most highly conserved of those segments analyzed. In addition, hybridization signals with probes to this segment, and some of the more conserved segments, namely, 1, 3, 4, and 8, distinguished the South African viruses from viruses isolated in Australia, Pakistan, and the United States. Sequence conservation within distinct geographical groupings of BTV isolates has been found by comparing sequences of genome segment 8 (NS2) (8) and complete and partial sequences of genome segment 3 (VP3) (9). The complete nucleotide sequences of cognate segments 6 of isolates of BTV 1 from Australia (Aus) and South Africa (SA), and BTV 10 and BTV 17 from the United States (USA) have revealed that they are between 1769 and 1773 nucleotides in length, with each encoding a protein of 552 amino acids (10-13). Although the sequence of this segment was highly conserved among these viruses, homology levels suggested that the Australian isolate of BTV I had evolved in geographical isolation (I0). The nucleotide sequence of genome segment 6 of an Australian isolate of BTV 20 presented here reveals high levels of homology (i.e., 98%) with BTV 1 (Aus). This level of homology is similar to that observed between NS1 genes of the BTV 10 and BTV 17 (USA), suggesting that NS1 could be used to identify geographically and evolutionarily divergent BTV isolates. A large plaque type of BTV 20 (strain CSIRO 19) was grown in PS-EK cells, total nucleic acid was extracted with 3% diethylpyrocarbonate in I% sodium dodecyl sulphate, and dsRNA was purified on cellulose (Whatman CF1 I) chromotography columns (4). Double-stranded cDNA was prepared as described by Wade-Evans et al. (14) using synthetic I5-mer oligonucleotide primers (A) 5'..GTTAAAAAAGTTCTC..3' and (B) 5'..GTAAGTTGTAAAGTT..3' complementary to the 3'-terminal ends of each RNA strand of genome segment 6 (15). Whole genomic RNA (5~g) rather than purified segment 6 was used as the template for cDNA synthesis. This appeared feasible as BTV 20 dsRNA separated on a 0.8% agarose gel, Northern blotted onto Hybond N (Amersham), and hybridized with both oligonucleotides 5'-end labeled with [32p]-ATP and T4 polynucleotide kinase (16) indicated that both oligonucleotides hybridized specifically with segment 5, equivalent to segment 6 in polyacrylamide gels (17; data not shown). The cDNA was collected by extraction with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and ligated into the dephosphorylated

NUCLEOTIDE SEQUENCE OF BTV 20 NSI GENE

389

~AA~TTCTCT~TGGC~CCACC~CAT~A~GCTTTTTGAC~TACAACATTAGTGGAGATTAT~T~TGCCACGAG M E R F L R K Y N I S G D Y A N A T R ~CTTTTTTGGCTATATC~C~TGGACTTC~ACTCACTTAAAAAG~TT~TTATCC~T~ATGTGT~GAAACA~TTTTGA T F L A I S P Q W T C S H L K R N C L S N G M C A K Q N F E CAGA~TGAT~CAGC~CGCA~CCGA~CC~T~GCATTCC~T~TAG~GCCAI~CA~GATGTATGATCGAGA R A M I A A T D A E E P I K A F R L I E L A K E A M Y D R E GACCGTT~CC~AGTGTTTT/u%AAGCT~TCTCA~CGTA~AGGA~TATC~GGC~GATA~CGA~CCGT~ACAGCTACT T V W L Q C F K S F S Q P Y E E D I E G K I K R C G A Q L L I 0 9 TG~GACTACCGCAAAAGTGG~T~TGGA~CTATT~TCT~TTTGATT~CTCGGAG~GGTTAGATTAGACGATTCGCT E D Y R K S G M M E E A I K Q S A L I N S E R V R L D D S L I 3 9 TTCGGCCATTCCCTATATTTATGTTCCCATA~GGTC~TTGT~CCCAACGTTCATCTCCAGGTATCGCCAGAT~GTACTA $ A I P Y I Y V P l K E G Q I V N P T F I S R Y R Q I A Y Y I 6 9 TTTCTAC~TCCAGATGCAGCTGACGATTGGATAGAT~C~TTTATTT~GGTT~A~ACAGCATCATC~ATT~GCGAGAAGTTGA F Y N P D A A D D W I D P N L F G V R C Q H H Q I K R E V E I 9 9 GAGGCAGATC~CACTTGCCCTTATACTGGATACAAAGGAGG~TTTTC~GTGATGTATTTACC~TTC~CT~T~CTTCCT~G R Q I N T C P Y T G Y K G G I F Q V M Y L P I Q L I N F L R 2 2 9 GATGGATGATTTTGCA~CATTTC~TAGGTATGCTTCGAT~CCATA~GC~TATCT~GAGTT~ATATTTAGAAGAGATTAGGTA M D D F A K H F N R Y A S M A I Q Q Y L R V G Y L E E I B Y 2 5 9 TGTGC~TTATTCGGG~GGTTCCATCTGGC~TTCCCACTGCATCAGATGAT~TT~TGAGA~GTGATTT~C~CGCGC~CCG V Q Q L F G K V P S G E F P L H Q M M L M R R D F P T R D R 2 8 9 T~CATCGTGG~GCGCGTGTGAAAAGGTC~GTGACGAAAATTGGC~GCTGGTTATTACC~TGGTC~TGGTGCGAG~GGGCTAGA N I V E A R V K R S G D E N W Q S W L L P M V L V R E G L D 3 1 9 TCAGCA~AG~GTGGG~TGGCTTCTAGAGTATAT~ATAGAAAACACATATGCCAGCTCTG~TATTTG~CATTC~GCAGATACA Q Q E K W E W L L E Y M D R K H I C Q L C Y L K H S K Q I Q 3 4 9 GACGTGTAGCGT~TAGATGTTCGCGCTTCAGAGTTGATCGGTTG~TCACCCTTCAG~CTGT~GATCGAGGAGCACGTAGGT~TGA T C S V I D V R A S E L I G C S P F R T V K I E E H V G N E 3 7 9 ACCAGTCTTTAAAACT~T~TTCGTGATCAGC~TCGGCAG~TT~GGATCATTATTACAC~CGAGCTGCTATACCGGAGCTGA P V F K T K L I R D Q Q I G R I G D H Y Y T T S C Y T G A E 4 0 9 AGCGTT~TTAC~CTGCTATCCATATACACCGATG~TCAGA~GTGT~TATCTGGAATGATG~ATGGCAGGAGGGAGTGTTTAT A L V T T A I H I H R W I R G C G I W N D E G W Q E G V F M 4 3 9 GCTGGGACGTGT~TATT~GGTG~AGCTTACG~GCAGCGCAGC~ACTGCTTAGGCT~TTcTGTTTTGT~TGTTATGGATATGC L G R V L L R W E L T K A Q R S A L L R L F C F V C Y G Y A 4 6 9 T~C~CGCGCGGATGG~C~TACCTGATTGG~C~TTTAGGC~TTTCTTAGATATCATCCTG~GGACCG~GCTCA~G~GATGA P R A D G T V P D W N N L G S F L D I I L K G P E L S E D E 4 9 9 GGATG~GAGCTTATGCTAc~TGTTTG~T~TTCGGTGTATTATCACCTTGTGTTAT~TGAG~GTT~AcTTTGCTGGAT~A~ D E R A Y A T M F E M V R C I I T L C Y A E K V H F A G F T 5 2 9

90 19 180 49 270 79 360 450 540 630 720 810 900 990 1080 1170 1260 1350 1440 1530 1620

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Fig. I. The nucleotide sequence of cDNA to BTV 20 segment 6 (NS1) and its predicted 552 amino acid sequence for the long open reading frame from nucleotide 35 to 1690. The nucleotide sequence in bold at the 5'- and 3'-terminals was not determined from sequence data but lies within the conserved terminal hexanucleotide sequences (15). Underlined nucleotides represent the inverted terminal repeat sequences predicted by the computer program FOLD for determining secondary structures in RNA (2 I).

3 m a l site o f pUC18. R e c o m b i n a n t plasmids were transformed into E. coli X L I Blue host cells (Stratagene) and white c o l o n i e s were identified on LB/Amp/X-gat plates. Inserts in recombinant plasmids were sequenced in both orientations with M13 universal forward- and reverse-sequencing primers (Beohringer Mannheim) and T7 D N A potymerase sequencing reagents (Pharmacia). The sequence o f B T V 20 segment 6 from nucleotide 4 to 1765 w a s generated from five overlapping c D N A inserts and several subclones that were generated from these plasmids. The nucleotide s e q u e n c e o f c D N A to B T V 20 segment 6 (NS1) and its predicted 552 amino acid s e q u e n c e (molecular weight 64,507 Da; estimated pI 6.7; net charge - 2 at pH7) for the long open reading frame from nucleotide 35 to 1690 are s h o w n in Fig. 1. The 5'-terminal three nucleotides and the 3'-terminal four nucleotides, which reside within the c o n s e r v e d terminal hexanucleotide sequences of B T V g e n o m e segments (15) and represent the ends of the BTV20specific primers, have been included to complete the sequence. Thus, the c o m plete segment 6 o f B T V 20 p o s s e s s e s 1769 nucleotides with 5'- and 3'-noncoding regions o f 34 and 79 nucleotides, respectively. The sequence flanking the A U G

390

COWLEY

initiation codon (AACAUGG) indicates strong translation initiation based on the hypothesis of Kozak (18) that predicts purine residues at positions - 3 and +4 in relation to the A of the AUG triplet. Comparison of the NS1 nucleotide sequence of BTV 20 (Aus) with cognate genome segments of BTV 1 (Aus, SA) (10) and BTV 10 (USA) (12) and BTV 17 (USA) (11,13) revealed homologies of 98%, 80%, 79%, and 79%, respectively (data not shown). The homology between BTV 20 and BTV 1 (Aus) (98%) was similar to that between BTV 10 and BTV 17 (USA) but greater than that (80%) reported between the BTV 1 (Aus and SA) isolates from different geographical locations (I0). In addition, both the 5'- and 3'-near terminal sequences were highly conserved among the five BTV isolates. The 56 nucleotides from the 5'-terminus were conserved, except for two nucleotide mismatches with BTV 1 (SA), as were the 33 nucleotides from the 3'-terminus, except for one nucleotide mismatch with the BTV 20 sequence. Similar to segment 8 (NS2) genes (8) but contrary to data on other BTV genome segments (19), the 5'-noncoding region of the NS1 genes appears to be more highly conserved than the longer 3'-noncoding region. Comparison of the amino acid sequence of the NS1 protein of BTV 20 (Aus) with those of isolates of BTV 1 (Aus, SA) (10), BTV 10 (USA) (12), and BTV 17 (USA) (11,13) revealed homologies of 98%, 90%, 89%, and 90% identity, respectively (Fig. 2). These homology levels further increased to 99.6%, 96.6%, 96.6%, and 96.9%, respectively, when functional similarities defined by charge and polarity conservation and matching of aromatic residues (P,G; S,T; A) (F,Y,W; I,L,M,V) (D,E; N,Q) (K,R,H) (C) were considered (20). Secondary structure analysis of BTV 20 NS1 mRNA using the computer algorithm FOLD (21) predicted base pairing of 10 nucleotides in an imperfect inverted terminal repeat adjacent to the conserved terminal hexanucleotide sequences (Fig. 3). In addition, base pairing was predicted between the 5'-terminal GTT and the CAA in the 3'-terminal region, similar to genome segment 4 of BTV 10 (USA) (22), and a small stem-loop structure occurred in the 5'-noncoding sequence. Near identical terminal secondary structures were predicted for NS1 mRNAs of the other BTV isolates (data not shown), reflecting the high level of sequence conservation in their terminal noncoding regions. Secondary structure data on reovirus and phytoreovirus mRNA has indicated that terminal stem loops and the availability of 5'-terminal unpaired nucleotides can regulate the association of mRNA with initiation factors and ribosomal subunits, thus influencing translation efficiency (23-25). Furthermore, the conserved nature of terminal stem-loop structures in mRNAs of cognate genes of segmented dsRNA viruses, more commonly observed in their longer 3'-noncoding regions, suggests that they may constitute selection and sorting motifs for gene packaging during virus morphogenesis (22,23,25). Determination of the nucleotide and amino acid sequence of the NSI gene of BTV 20 (Aus) has confirmed the extensive conservation observed amongst cognate genes of other BTV isolates. Sequence comparisons with NS 1 genes of other BTV isolates from Australia, South Africa, and the United States indicated a

391

NUCLEOTIDE SEQUENCE OF BTV 20 NS1 GENE

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Fig. 2. Comparison of the amino acid sequence of the NS1 protein (encoded by genome segment 6) of BTV 20 (Aus) with cognate proteins of isolates of BTV 1 (Aus, SA), BTV 10 (USA), and BTV 17 (USA). Only those amino acids that differ from those of BTV 20 (Aus) are indicated.

closer evolutionary relationship between BTV 20 and BTV 1 (Aus), and between BTV 10 and BTV 17 (USA) than between isolates from distinct geographical locations, irrespective of whether they were the same serotype (BTV 1). This suggests that the NS1 gene represents a suitable marker for determining the geographical origin of BTV isolates. In addition, corroborative data from comparisons of NSI (10) and other more highly conserved BTV genome segments encoding VP3 (9) and NS2 (8) lend support to the hypothesis that geographical clusters of BTV strains have evolved in isolation for considerable time periods. A

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392

COWLEY

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. t3. t4. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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