Virus Research, 18 (1990) 21-28 Else-her

21

VIRUS 00615

Expression of bluetongue virus serotype 17 NSl protein from a cloned gene Marvin J. Grubman United States Deportment okAgri~~~~re, ARS, NAA, Plum Island Animal Disease Center, Greenport, New York, U. S.A. (Accepted 14 August 1990)

Summary

A full-length copy of the coding region of segment 6 from bluetongue virus (BTV) serotype 17 was constructed from five overlapping cDNA clones. The gene coding for the NSl protein was cloned into an expression plasmid under the control of a bacteriophage T7 promoter and expressed both in vitro and in ~sc~eric~i~ co& BL21(DE3) cells which contain a T7 RNA polymerase gene in their chromosome. Expression in both systems resulted in the synthesis of a protein comigrating with NSl and a minor polypeptide comigrating with another viral-induced protein, NSla, sometimes seen in BTV-infected cells. The proteins induced in E. coli were synthesized to high levels as insoluble products, Bluetongue virus; Segment 6; cDNA clone: NSl protein; Expression

Bluetongue virus (BTV) induces the synthesis of at least ten proteins, three of which are nonstruct~~ (Roy, 1989). Nonst~~tur~ protein NSl, which is coded for by genome segment 6 in BTV serotype 17 (Grubman et al., 1983), forms tubular structures in infected cells (Huismans and Els, 1979) and has also been located at the periphery of virus inclusion bodies perhaps associated with loose fibrillar material (Eaton et al., 1988). The genome segment coding for NSl from serotypes 17 and 10 North America, 1 South Africa and 1 Australia has been sequenced and the degree of homology at both the nucleic acid and amino acid levels is high Correspondence fo: M.J. Grubmau, U.S. Dept. of Agriculture, ARS, NAA, Plum Island Animal Disease Center, P.O. Box 848, Greenport, NY 11944, U.S.A.

22

(Grubman and Samal, 1989; Wang et al., 1989b; Lee and Roy, 1987; Gould et al., 1988). Because of the conservation of this genome segment within the various BTV serotypes and its selective hybridization with BTV and not other closely related orbiviruses (Huismans and Cloete, 1987; Unger et al., 1988) the protein product coded for by segment 6 may be a useful diagnostic reagent. Five overlapping clones, from a BTV-17 cDNA library, hybridized to BTV-17 segment 6 and were shown by nucleic acid sequence analysis to represent 97% of the gene, i.e., bases l-1712 out of 1769 (Grubman and Samal, 1989; Grubman et al., 1990; Wang et al., 1989b). The open reading frame begins with the AUG codon at bases 35-37 and terminates with a stop codon at bases 1691-1693. These clones were therefore used to construct a copy of the complete protein coding region of the NSl gene by standard recombinant DNA techniques (Ausubel et al., 1987; Maniatis et al., 1989; Guo and Wu, 1983). This construct, however, contained both a poly A tail (added to the RNA template) and a GC tail (the cDNA was ligated into the Pst I site of pUC18) at the 5’ end of the gene. The homopolymer tails were removed by utilizing an Mae I site upstream of the initiation codon and cloning the purified gene into the transcription and bacterial expression vector pT7-7 (Tabor and Richardson, 1985; Studier and Moffat, 1986). This vector contains a bacteriophage T7 promoter followed by a Shine-Dalgarno sequence and a polylinker region. The NSl gene was cloned into the NdeI site of the polylinker region of pT7-7 resulting in the gene containing its own translation initiation and termination codons (plasmid pT7-NSl-45.1). Thus upon induction a full-length, nonfusion, NSl protein would be synthesized. Transformants from the various constructs were analyzed by restriction enzyme analysis and sequenced at the junctions by the dideoxy chain termination method (Sanger et al., 1977) prior to the subsequent step in construction of the full-length gene. Run-off transcripts of the plasmid pT7-NSl-45.1, linearized with EcoRI, which cuts immediately downstream of base 1712 and the GC tail of the NSl gene, were synthesized in vitro with T7 RNA polymerase and translated in a rabbit reticulocyte cell-free system (Grubman and Baxt, 1982). The major translation product derived from this clone was a protein which co-migrated with NSl from BTV-infected cells (Fig. 1A). In addition, a minor protein was also synthesized which co-migrated with a polypeptide, NSla, sometimes found in BTV-infected cells. Furthermore, these products as well as infected cell lysates were immunoprecipitated by both BTV-17 polyclonal antiserum and a monoclonal antibody against NSl, but not by normal serum (Fig. 1B) (Appleton and Letchworth, 1983). Expression of plasmid pT7-NSl-45.1 was also followed in the E. cofi strain BL21(DE3) which contains the T7 RNA polymerase gene, inducible with isopropylP-D-thiogalactopyranoside (IPTG), in the chromosome. Addition of IPTG therefore results in the expression of the NSl gene which is under the control of a T7 promoter. Cell lysates were prepared prior to and at various times after induction and analyzed by SDS-PAGE and Coomassie blue staining (Fig. 2). TWO new proteins of approximately 54 kDa and 50.5 kDa (identified by arrowheads in Fig. 2) are present by one hour after induction. These proteins are not visible prior to induction nor are they present in induced BL21(DE3) cells containing the vector

23

A12

B

34

12

3

456

VP1

VP2 VP3

NSI NS2

I NSl NSla

4 NSI 4 NSla

VP6 VP7

Fig. 1. In vitro translation of BTV-17 NSl gene transcripts. RNA generated by in vitro transcription of EcoRI linearized pT7-NSl-45.1 was translated in a rabbit reticulocyte cell-free system and the products analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% slab gel (Laemmli, 1970) either prior to (Panel A) or after (Panel B) immunoprecipitation with various antisera. Panel A: Lane 1, mock-infected baby hamster kidney (BHK-21) cell lysate; 2, BTV-17 infected BHK-21 cell lysate; 3, in vitro translation in the absence of exogenous RNA; 4, in vitro translation in the presence of pT7-NSl-45.1 transcripts; Panel B: Lanes 1-3, immunoprecipitation of BTV-17 infected BHK-21 cell lysate and Lanes 4-6, immunoprecipitation of pT7-NSl-45.1 translation products with normal mouse serum, BTV-17 polyclonal mouse antiserum and monoclonal antibody 8BSB.1, respectively.

pT7-7 lacking the NSl gene (lanes 1 and 2). The amount of these proteins increases with time of induction (lanes 3-5). Both proteins are greatly enriched in the pellet fraction of lysed cells (compare lanes 7-11 and 13-17) and by 4 h after induction they represent two of the major products of the cell. Treatment of the pellet fraction with the nonionic detergents Nonidet P-40 and sodium desoxycholate or with high salt did not solubilize the proteins (data not shown). Expression of NSl was also monitored by pulse labeling cells with [ 35S]methionine prior to and at various times after induction. The results shown in Fig. 3 reveal that by 1 h after addition of IPTG one major (54 kDa) and one other (50.5 kDa) new

24 Total hrs IPTG

Pellet hrs IPTG

Super hrs IPTG

-iI 4

0

t

24

M

40

124

M

4

2104

kDa 11692.5-

66”

NSl NSla 45-

31-

1 2

3

4 5

6

7 6 9 10 11 12

1314

1516

17

Fig. 2. PAGE analysis of BL21(DE3) cells carrying the NSl gene prior to and after induction with IPTG. Aliquots of BL21(DE3) cells containing the expression plasmid pT7-NSl-45.1 were removed prior to and at 1, 2 and 4 h after induction with IPTG. Also BL21(DE3) cells containing the vector pT7-7 were induced with IPTG for 4 h. Cells were lysed either in 0.063 M Tris-HCl, pH 6.8, 2% SDS, 0.68 M 2-mercaptoethanol, 10% glycerol and 0.05% bromophenol blue, boiled and pelleted (total, lanes l-5) or lysed with lysozyme, frozen and thawed, digested with DNase (Rosenberg and Studier, 1987) and separated into pellet (lanes 7-11) and supernatant (super, lanes 13-17) fractions and examined by PAGE on a 10% slab gel. The gel was stained with Coomassie brilliant blue. Lanes 1, 7 and 17 refer to pT7-7 total, pellet and super-fractions, respectively, while lanes designated M refer to molecular weight markers.

polypeptide are synthesized as compared to uninduced cells (Fig. 3B). These polypeptides are found solely in the pellet fraction of cells and they comigrate with NSl and NSla (Fig. 3A-C). Both the 54 and 50.5 kDa polypeptides reacted with BTV-17 polyclonal antiserum as shown by immunoblot analysis (Fig. 3D). The NSl protein from serotype 10 has also been expressed to high levels by recombinant baculoviruses (Urakawa and Roy, 1988) while a truncated NSl fusion protein from serotype 17 was expressed at relatively low levels in E. co& (Wang et al., 1989a). To determine if isolated NSl or NSla could react with BTV specific serum in a solid phase assay the pellet fraction from induced BL21(DE3) cells containing the NSl gene was fractionated by preparative SDS-PAGE. Proteins were visualized after soaking in 0.25 M KC1 and NSl and NSla separately eluted (Hager and

25 IPTG 1 +-

Pellet

qNNS1 (I NSla

1234 Fig. 3. PAGE analysis of pulp-labeled BL21(DE3) cells carrying the NSl gene prior to and after induction with IPTG. Aliquots of BL21(DE3) cells containing the expression plasmid pT7-NSl-45.1 were radiolabeled with [ “S]methionine for 30 min prior to and at various times after induction with IPTG. Cells were treated with lysozyme, frozen and thawed, lysates separated into pellet and supernatant (super) fractions and examined on 10% slab gels (panels A-C). Nonradioactive pellet fractions were prepared and examined immunologically for NSl and NSla production by Western bIot analysis (panel D). Panel A: Lanes 1, supematant fraction prior to induction; 2, supematant fraction 1 h after induction; panel B: Lanes 1, pellet fraction 1 h after induction; 2, pellet fraction prior to induction; panel C: Lanes 1, [35S]met~o~ne-labeled mock-infected BHK cell lysate; 2, peliet fraction prior to induction; 3, pellet fraction 4 h after induction; 4, [35S]methionine-labeled BTV-infected BEK cell lysate. Panel D: Pellet fractions were prepared from BL21(DE3) cells containing the vector pT7-7 induced for 4 h with WIG (lane l), uninduced BL21(DE3) cells containing pT7-NSl-45.1 (lane 2) or 4 h induced cells containing pT7-NSl-45.1 (lane 3). Equivalent A,, amounts of the pellet fractions of each sample were separated on 10% slab gels and i~unoblott~ to nitrocellulose (Devaney et al., 1988). Proteins were detected with a 1: 50 dilution of BTV-17 polyclonal mouse as&tic fluid followed by 4~ 10’ cpm of ‘251-labeled

staphylococcal protein A per ml,

Burgess, 1980). Each protein remained soluble after elution and was approximately 90-95% pure as shown by SDS-PAGE and Coomassie blue staining. Varying concentrations of the two proteins were used in a solid phase assay and maximal reactivity, with a 1: 10 dilution of BTV serotype 17 polyclonal serum, occurred with

2h

between SO-150 ng of either protein. Preliminary results suggest that a 1 : 10 dilution of polyclonal serum against the orbivirus African horsesickness virus serotype 3 does not react with either protein. It has previously been demonstrated by in vitro translation of BTV genome segments that segment 6 from serotypes 17 and 1 codes for two polypeptides, a major protein termed NSl and a minor, lower molecular weight, polypeptide, NSla (Mertens et al., 1984; Grubman et al., 1983). Based on immunoprecipitation of both polypeptides with polyclonal antisera and comparison by partial protease digestion NSl and NSla appeared to be similar to each other and to two proteins synthesized in infected cells (Grubman et al., 1983; Mertens et al., 1984). Expression of the NSl gene in both the in vitro transcription-translation system and in transformed E. co& described here also resulted in synthesis of the major, 54 kDa, NSl protein and lesser amounts of NSla (50.5 kDa) (Figs. l-3). However, it should be noted that the level of synthesis of NSla relative to NSl in E. co/i is considerably greater than in infected BHK cells or in vitro. Immunoprecipitation of NSla by a monoclonal antibody against NSl suggests that this protein is translated from gene 6 in the same reading frame as NSl. It has been suggested that synthesis of NSla probably results from either late initiation or early termination of translation of segment 6 (Mertens et al., 1984). Of these possibilities it would appear that synthesis of NSla is a result of alternate initiation at a downstream site rather than early termination since there is no early in-frame stop codon in gene 6 and NSla appears as a discrete rather than a broad band on PAGE. A second in-frame AUG codon occurs 120 bases downstream from the major initiation codon and initiation at this site would result in the synthesis of a protein of approximately the calculated molecular weight of NSla. This codon, however, is not in a favorable Kozak environment (Kozak, 1981, 1983). Lacking N-terminal amino acid sequence information on NSla it is not possible, at this time, to determine if this second AUG codon is utilized. Nucleic acid sequence information, Northern blot hybridization, as well as immunological evidence indicate that gene 6 and its protein product are highly conserved among different BTV serotypes, but not among other orbivirus serogroups (Grubman and Samal, 1989; Wang et al., 1989b; Lee and Roy, 1987; Gould et al., 1988; Huismans and Cloete, 1987; Unger et al., 1988; Appleton and Letchworth, 1983). Considering these results our preliminary solid phase radioimmune assays with gel purified NSl or NSla from induced E. cob as antigen suggest that both of these proteins may be useful in a diagnostic test for identifying serum from BTV-infected animals. Similar results have been reported by Urakawa and Roy (1988) utilizing NSl expressed in insect cells infected with a recombinant baculovirus.

Acknowledgement I thank Marla Zellner the manuscript.

for technical

assistance

and Adriene

Ciupryk

for preparing

27

References Appleton, J.A. and Letchworth, G.J. (1983) Monoclonal antibody analysis of serotype-restricted and unrestricted bluetongue viral antigenic deter~n~ts. Virology 124: 286-299. Ausubel, FM., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1987) Current protocols in molecular biology. Greene Publishing Associates and Wiley-Interscience, New York. Devaney, M.A., Vakharia, V.N., Lloyd, R.E., Ehenfeld, E. and Grubman, M.J. (1988) Leader protein of foot-and-mouth disease virus is required for cleavage of the ~220 component of the cap-binding protein complex. J. Viral. 62, 4407-4409. Eaton, B.T., Hyatt, A.D. and White, J.R. (1988) UltrastNctural location of NSl in bluetongue virus infected cells and its presence in ViNS particles. Virology 163, 527-537. Gould, A.R., Pritchard, L.I. and Tavaria, M.D. (1988) Nucleotide and deduced amino acid sequences of the non-structural protein, NSl, of Australian and South African bluetongue virus serotype 1. Virus Res. 11, 97-107. Grubman, M.J., Appleton, J.A. and Letchworth, G.J. (1983) Identification of bluetongue virus type 17 genome segments coding for polypeptides associated with virus neutralization and intergroup reactivity. Virology 131, 355-366. Grubman, M.J. and Baxt, B. (1982) Translation of foot-and-mouth disease virion RNA and processing of the primary cleavage products in a rabbit reticulocyte lysate. Virology 116, 19-30. Grubman, M.J. and Samal, S. (1989) Nucleotide and deduced amino acid sequence of the nonstructural protein, NSl, of the U.S. bluetongue virus serotype 17. Nucleic Acids Res. 17, 10498. Grubman, M.J., Zellner, M. and Samal, S. (1990) Nucleotide and deduced amino acid sequence of the nonst~ctural phosphoprotein, NS2, of bluetongue virus serotype 17: Comparison to two isolates of serotype 10. Virus Res. 15, 243-250. Guo, L. and Wu, R. (1983). Exonuclease III: Use for DNA sequence analysis and in specific deletions of nucleotides. In: K. Moldave and L. Grossman (Eds), Methods in Enzymology, Vol. 100, pp. 60-96. Academic Press, New York. Hager, D.A. and Burgess, R.R. (1980) Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with the sigma subunit of E~cherichiu coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal. B&hem. 109,76-86. Huismans, H. and Cloete, M. (1987) A comparison of different cloned bluetongue virus genome segments as probes for the detection of virus-specific RNA. Virology 158, 373-380. Huismans, H. and Els, H.J. (1979) Characterization of the tubules associated with the replication of three different orbiviruses. Virology 92, 397-406. Kozak, M. (1981) Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res. 9, 5233-5252. Kozak, M. (1983) Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbial. Rev. 47, I-5. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. Lee, J. and Roy, P. (1987) Complete sequence of the NSl gene (M6 RNA) of U.S. bluetongue virus serotype 10. Nucleic Acids Res. 15, 7207. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Mertens, P.D.C., Brown, F. and Sangar, D.V. (1984) Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology 135, 207-217. Rosenberg, A.H. and Studier, W.F. (1987) T7 RNA polymerase can direct expression of influenza virus cap-binding protein (PB2) in Escherichia coli. Gene 59, 191-200. Roy, P. (1989) Bluetongue virus genetics and genome structure. Virus Res. 13, 179-206. Sanger, F., Nicklen, S. and Coulsen, A.R. (1977) DNA sequencing with khan-ter~nating inhibitors. Proc. Nati. Acad. Sci. USA 74, 5463-5467.

28 Studier, F.W. and Moffat, B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130. Tabor, S. and Richardson, C.C. (1985) A bacteriophage 77 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82, 1074-1078. Unger, R.E., Chuang, R.Y., Chuang, L.F., Osbum, B.I. and Doi, R.H. (1988) The cloning of full-length genome segments 2, 5, 6 and 8 of bluetongue virus (BTV) serotype 17 and studies of their genetic relatedness to United States BTV serotypes. Viroiogy 167, 296-298. Urakawa, T. and Roy, P. (1988) Bluetongue virus tubules made in insect cells by recombinant baculovirus: expression of NSI gene of bluetongue virus serotype 10. J. Viral. 62, 3919-3927. Wang, L-F, Doi, R.H., Chuarg, L.F., Osbum, B.I., Maisonnave, J., Benjamini, E. and Chuang, R.Y. (1989a) Bluetongue virus-17 fusion protein NSl expressed in Escherichia coli by pUC vectors. Biochem. Biophys. Res. Comm. 162, 892-899. Wang, L-F, Doi, R.H., Osburn, B.I. and Chuang, R.Y. (1989b) Complete sequence of the NSl gene (S6 RNA) of U.S. bluetongue virus serotype 17. Nucleic Acids Res. 17, 8002. (Received

12 June 1990; revision

received

14 August

1990)