Nucleotide Sequence of the Australian Bluetongue Virus Serotype 1 RNA Segment 10

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1 J. gen. Virol. (1988), 69, Printed in Great Britain 945 Key words: BTV/genome segment lo/nucleotide sequence Nucleotide Sequence of the Australian Bluetongue Virus Serotype 1 RNA Segment 10 By ALLAN R. GOULD CSIRO, Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia (Accepted 14 January 1988) SUMMARY The complete nucleotide sequence of the RNA segment l0 of Australian BTV serotype 1 has been deduced from a combination of sequencing cdna inserts cloned into pbr322 and synthetic deoxynucleotide priming on purified double-stranded RNA molecules. The gene segment was 822 nucleotides in length and capable of coding for two proteins on either 229 or 216 amino acids, having net charges of either +4.5 or respectively at neutral ph. Comparison with the RNA segment 10 from BTV serotype 10 revealed a high degree of amino acid sequence conservation as well as regions of nucleic acid sequence conservation. Bluetongue virus (BTV) is the type member of the orbivirus genus, which is one of six genera in the family Reoviridae. A total of 24 serotypes of BTV have been identified, on the basis of serum neutralization tests, and at present eight of these have been identified in Australia (Gould, 1988). The genome of this arthropod-borne virus consists of 10 double-stranded RNA (dsrna) molecules which are enclosed by a double-shelled capsid. The inner core of the virus is composed of two major (VP3 and VP7) and three minor (VP1, VP4 and VP6) proteins which in turn are surrounded by an outer coat of two proteins, VP2 and VP5 (Verwoerd et al., 1972). Apart from the seven structural viral proteins, three non-structural proteins are also synthesized from individually purified segments of the BTV genome. In vitro translation studies revealed that NS1, NS2 and NS3 were coded for by RNA segments 5, 8 and 10, respectively (Sangar & Mertens, 1983; Eaton & Gould, 1987) (Fig. 1). In particular, RNA segment 10 was capable of coding for two proteins (termed VP8 and VP8a or NS3 and NS3a) either in vitro or in vivo in approximately equimolar proportions. Tryptic peptide analyses (Sangar & Mertens, 1983; B. T. Eaton, unpublished observations) have shown NS3 and NS3a to be related. Comparative sequence analyses of genome segments from North American BTV, South African BTV and Australian BTV isolates have demonstrated that different BTV serotypes have variable nucleic acid sequences for each segment, although the proteins that they encode remain highly conserved. The exception to this appeared to be VP2, the protein responsible for the elicitation of virus-neutralizing antibody (Huismans & Erasmus, 1981; Inumaru & Roy, 1987). In VP2, only certain well defined regions were conserved while the other regions appeared to be variable (Gould, 1987b). To date no comparative sequence data have been available for the non-structural proteins of BTV. Hybridization analyses (Huismans & Cloete, 1987) have suggested that the most highly conserved RNA segment for BTV serotypes from all geographical regions was that coding for NS1. Hybridization responses for RNA segment 5, which codes for NS1, appeared to be similar to that for VP3; the latter is one of the major core antigens and recombinant DNA probes from its gene were capable of differentiating BTV on a geographical basis (Gould, 1987a, 1988), as it also preferentially hybridized to BTV isolates from the same geographical region. Kowalik & Li (1987) showed that NSI, NS2 and NS3 were all highly conserved within the North American BTV isolates. However no data were available (either by hybridization or sequence analysis) for the relatedness of nucleotide sequences of BTV NS3 (RNA segment 10) across geographical SGM

946 Short communication VP1-- VP2-- VP3 VP4~ 1 2 3 4 VP5 NSI~ NS2 VP6 VP7~ NS3~ NS3a- Fig. l. In vitro translation products of total BTV genome dsrna or individually purified RNA segments.

2 946 Short communication VP1-- VP2-- VP3 VP4~ VP5 NSI~ NS2 VP6 VP7~ NS3~ NS3a- Fig. l. In vitro translation products of total BTV genome dsrna or individually purified RNA segments. Lane 1, total genomic dsrna; lane 2, RNA segment 8; lane 3, RNA segment 9; lane 4, RNA segment 10 from BTV serotype 1 (CSIRO 156). Translations were carried out using rabbit reticulocyte lysate as described in Eaton & Gould (1987) and electrophoresed on a 10~ SDSpolyacrylamide gel. boundaries; this is necessary for the development of a recombinant DNA probe capable of distinguishing BTV from the other orbiviruses. The procedures used to clone the RNA segment 10.of Australian BTV serotype 1 and to deduce the complete nucleotide sequence have been described fully (Gould, 1987 a). Essentially, cdna was transcribed from heat-denatured dsrna by the use of synthetic random hexamer deoxynucleotide primers and reverse transcriptase. After removal of RNA by alkali digestion, ds cdna was formed by self-annealing, C-tailed and cloned into the PstI site of G-tailed pbr322. One clone (SG93) containing an insert of 693 nucleotides (nucleotides 32 to 724) was subcloned and sequenced using the dideoxynucleotide methods of Sanger & Coulson (1978) or Chen & Seeburg (1985) while purified dsrna of segment 10 was fully sequenced using synthetic deoxynucleotide primers (Bassel-Duby et al., 1986). Fig. 1 illustrates that purified RNA segment 10 from BTV1 coded for two proteins of approximate Mr 2-5 x l04 and 2.4 x 10 ~ (termed NS3 and NS3a, respectively). This was almost double the expected coding capacity for that RNA species. Fig. 2 shows the complete nucleotide sequence of the sense strand of RNA segment 10 and, for comparative purposes, the nucleotide sequence of the North American BTV10 RNA segment 10 as well as any deduced amino acid differences are also given. The complete sequence of RNA segment 10 was 822 nucleotides long and contained one open reading frame which began at the presumed initiator codon (nucleotides

Short communication 947 I 19 GTTAAAAAGTGTCGCTGCC ATG CTA TCC GGG CTG Met Leu Set Gly Leu 37 55 73 90 A G C T A T ATC CAA AGG TTC GAG GAA GAA AAA ATG AAA CAT AAC CAG GAG AGA GTT GAA GAA Ile Gln Arg

3 Short communication 947 I 19 GTTAAAAAGTGTCGCTGCC ATG CTA TCC GGG CTG Met Leu Set Gly Leu A G C T A T ATC CAA AGG TTC GAG GAA GAA AAA ATG AAA CAT AAC CAG GAG AGA GTT GAA GAA Ile Gln Arg Phe Glu GlU Glu Lys Met Lys His ASh Gln Glu Arg Val Glu GIu Asp G T G C T G A A A C A G CTA AGT CTA GTA CGC GTG GAT GAT ACA ATT TCA CAA CCA CCG AGG TAT GCT CCG AGT GCC CCG ATG CCG TCA TCA ATG CCG ACC GTT GCC Leu Set Leu Val Arg Val Asp Asp Tbr Ile Set Gln Pro Pro Arg Tyr Ala Pro Ser Ala Pro Met Pro Set Ser Met Pro Thr Val Ala C C A A G G CTT G~A ATA TTG GAT AAA GCG ATG TCA AAC ACT ACT GGT GCT ACG CAA ACA CAA AAG GCG GAA AAA GCT GCA TTC GCA TCG TAC GCA GAA Leu GIU Ile Leu Asp Lys Ala Met Set ASh Thr Thr Gly Ala Thr GID Thr Gln Lys Ala GIU Lys Ala Ala Phe Ala Ser Tyr Ala GIu C A A A A T A C A A T A G A C AT AA G GCG TTT CGT GAT GAT GTG AGG CTG AGG CAG ATC AAC CGT CAT GTT AAT GAA CAG ATT TTC CCA AAG TTA AAG AGT GAT TTA GGC GGT TTA Ala Phe Arg Asp Asp Val Arg Leu Arg Gln Iie ASh Arg His Val ASh GIu Gln Ile Phe Pro Lys Leu Lys Set Asp Leu Gly Gly Leu Lys Set Glu G C A CT T C A C A G T G T C C A T C AAG AAA AAG AGA GCT ATC ATA CAC ATG ACG TTG TTG GTC GCC GCT GTC GTA GCG TTG TTG ACA TCG GTA TGC ACA CTT TCA AGT GAT ATG Lys Lys Lys Arg Ala Ile Ile His Met Thr Leu Leu Val Ala Ala Val Val Ala Leu Leu Thr Set Val Cys Tbr Leu Ser Set Asp Met Thr ~ 5&O C C A A A G C AA A A G G T TCA A C T A C T C C T A AGT GTG GCA TTT AAG CTT AAT GGT ACA TCA GCG GAA ATA CCA CAG TGG TTT AAG AGT CTA AAT CCC ATG CTT GGA GTA GTG AAT CTG GGT Ser Val Ala Phe Lys Leu Ash Gly Thr set Ala Glu Ile Pro Gln Trp Phe Lys Ser Leu ASh Pro Met Leu Gly Val Val ASh Leu Gly Ile Lys Thr Val Ser A T T G C A T CC G A A T A A G GCG ACT TTC ATT ATG ATG GTT TGT GCG A~G AGC GAA AGA GGG TTA /~AT CAG CAG ATT GAC ATG ATT AAG AAG GAA GTT ATG AAA AAA CAA Ala Tnr Phe Ile Met Met Val Cys Ala Lys Set Giu Arg Giy Leu ASh Gln Oln Ile Asp Met Ile Lys Lys GIu ~al Met Lys Lys Gln Leu Ala T G A G T A GA TCA TAT AAT GAC GCA GTG AGG ATG AGT TTC ACA GAG TTC TCG TCA GTC CCG CTA GAT GGT TTC GA.A CTG CCA TTA ACC TGA GATCAGTAGGTA Set Tyr Ash Asp Ala Val Arg Met Ser Phe Thr Glu Phe Ser Ser Val Pro Leu Asp Gly Phe GIU Leu Pro Leu Thr *** rle Met G A GT TGCA G C TC G GC T C C ATA C GAGTTGcGccccAAGGTTTGcAccGcATGGAGT GTTGATccAGAGATGcAAATTccTAcTGcTGTATAAcGGGGGAGGGTGTGcGGcGcTATAcAcTTAc Fig. 2. The complete nucleotide sequence of RNA segment 10 from the Australian BTV serotype 1 and its deduced amino acid sequence. Nucleotide position numbers and the differences with respect to the BTV 10 RNA segment 10 are given above the nucleotide sequence while amino acid changes reflected by sequence differences are given below the amino acid sequence. 20 to 22) and terminated at a TGA codon (nucleotides 707 to 709). This enabled a protein of 229 amino acids or Mr to be translated. The presence of a second AUG codon at nucleotide positions 59 to 61, previously reported by Lee & Roy (1986), was also conserved and raised the question as to which methionine was actually used to initiate synthesis of NS3. Unlike the other BTV gene segments previously sequenced (Purdy et al., 1985, 1986; Ghiasi et al., 1985 ; Gould, 1987a, b; Gould & Pritchard, 1987), the RNA segment coding for NS3 was the only one in which the presumed initiator codon did not conform to the Kozak consensus sequence (Kozak, 1981, 1984). NS3 is poorly translated in vivo, which may be a consequence of the pyrimidine present at position +4. In v#ro, two proteins of approximate Mr 2.5 x 104 and 2.4 x 104 were translated from RNA segment 10 (Fig. 1, lane 4). This may have arisen from inefficient recognition of the initial AUG codon by ribosomes scanning the 5' terminus of the sense strand and initiation of synthesis at the second in-phase AUG codon, which has a purine at position +4 (Fig. 2; Kozak, 1981). This explanation appears to be reasonable as the apparent difference in M r between NS3 and NS3a (Fig. 1, lanes 1 and 4) approximated to the calculated difference of deduced from their sequence differences (Fig. 2). The 5' terminus of RNA segment 10 showed another feature unusual for BTV RNA segments in that only two nucleotide changes were observed in the initial 66 nucleotides. The remainder of the gene was 82~ homologous at the nucleotide level, which was similar to comparisons between BTV1 (Australia) and BTV10 (North America) for VP3 genes (80~; Gould, 1987a) but

4 948 Short communication higher than that for VP5 (68~; Gould & Pritchard, 1987) or VP2 (52~; Gould, 1987b). The 3' terminus of RNA 10 also differed from that of other RNA segments in that it was unusually long, having 113 nucleotides, as well as being G + C-rich (nucleotides 727 to 757 and 790 to 822 were 63 ~ and 67 ~ G + C, respectively). Other BTV gene segments examined so far have been slightly A + T-rich (56~ to 58~). It is not clear why a 3' terminus that was three to four times longer than any previously sequenced BTV RNA segment should be conserved. Presumably some sequences were conserved as either RNA replication signals and/or for encapsidation, whilst other areas may have been involved in the formation of'head to tail' concatemers of this RNA segment (Eaton & Gould, 1987). If this were the case then it would be expected that RNA segment 9, which can also form concatemers, should show similar features at its 3' terminus. The deduced amino acid sequences for BTV1 RNA segment 10 codes for an NS3 protein very similar in composition to that of BTV10 from North America (Lee & Roy, 1986). Of the 14 deduced amino acid differences, exactly half were conservative changes and thus the distribution of cysteine residues, charged amino acids and the hydropathy profiles were almost identical to those of BTV10 NS3 (Lee & Roy, 1986). To date no function has been assigned to NS3. However, as this protein is so highly conserved, it may well play an essential role in either BTV replication or morphogenesis. The author wishes to acknowledge the excellent technical assistance of L. I. Pritchard and to thank Dr B. T. Eaton for translation of RNA segments. REFERENCES BASSEL-DUBY, R., SPRIGGS, D. R., TYLER, K. L. & FIELDS, B. N. (1986). Identification of attenuating mutations on the reovirus type 3 S 1 double-stranded RNA segment with a rapid sequencing technique. Journal of Virology 60, CHEN, E. V. & S~EBURG, V. R. (1985). Supercoil sequencing" a fast and simple method for sequencing plasmid DNA. DNA 4, EATON, B. T. & GOULD, A. R. (1987). Isolation and characterization of orbivirus genotypic variants. Virus Research 6, GHIASI, H., 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, GOULD, A. R. (1987a). The complete nucleotide sequence of bluetongue virus serotype 1RNA3 and a comparison with other geographic serotypes from Australia, South Africa and the United States of America, and with other orbivirus isolates. Virus Research 7, GOULD, A. R. (1987b). Conserved and non-conserved regions of the outer coat protein, VP2, of the Australian bluetongue serotype 1 virus revealed by sequence comparison to the VP2 of North American BTV serotype 10. Virus Research (in press). GOULD, A. R. (1988). The use of recombinant DNA probes to group and type orbiviruses. A comparison of Australian and South African isolates. Archives of Virology (in press). GOULD, A. R. & PRITCHARD, L. I. (1987). The complete nucleotide sequence of the outer coat protein, VP5, of the Australian bluetongue serotype 1 reveals conserved and non-conserved sequences. Virus Research (in press). HUISMANS, H. & CLOETE, M. (1987). A comparison of different cloned bluetongue virus genome segments as probes for the detection of virus-specified RNA. Virology 158, HUISMANS, H. & ERASMUS, B. L (1981). Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort Journal of Veterinary Research 48, INUMARU, S. & ROY, P. (1987). Production and characterization of the neutralization antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector. Virology 157, KOWALIK, T. F. & LI, J. K.-K. (1987). The genetic relatedness of United States prototype bluetongue virus by RNA/RNA hybridization. Virology 158, KOZAK, M. (1981). Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Research 9, KOZAK, M. (1984). Selection of initiation sites by eukaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence of preproinsulin. Nucleic Acids Research 12, L~E, J. W. & ROY, P. (1986). Nucleotide sequence of a cd N A clone of R NA segment 10 of bluetongue virus (serotype 10). Journal of General Virology 67, PURDY, M. A., GHIASI, H., RAt), C. D. & ROY, P. (1985). Complete sequence of bluetongue virus L2 RNA that codes for the antigen recognized by neutralizing antibodies. Journal of Virology 55, PURDY, M. A., RITTER, 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,

5 Short communication 949 SANGAR, D. V. & MERTENS, P. P. C. (1983). Comparison of type 1 bluetongue virus protein synthesis in vivo and in vitro. In Double-stranded RNA Viruses, pp Edited by R. W. Compans and D. H. L. Bishop. New York: Elsevier. SANGER, F. & COULSON, A. R. (1978), The use of thin acrylamide gels for DNA sequencing. FEBS Letters 87, VERWOERD, D. W., ELS, H. J., DE V[LLIERS, E. M. & HUISMANS, n. (1972). Structure of the bluetongue virus capsid. Journal of Virology 10, (Received 23 Ocwber 1987)

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative