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Journal of General Virology (1998), 79, 47–50. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION An RNA-binding domain in the viral haemorrhagic septicaemia virus nucleoprotein Thania Said, Henriette Bruley, Annie Lamoureux and Michel Bre! mont Unite! de Virologie et Immunologie Mole! culaires, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France The gene encoding the nucleoprotein (N) and PCRderived subfragments from viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were overexpressed in Escherichia coli BL21(DE3) transformed by recombinant expression vector pET-14b containing N and PCR-generated subfragment cDNAs under the control of the T7 RNA polymerase promoter. Following induction with IPTG, recombinant His-tagged proteins were expressed, purified by affinity metal chelation chromatography under denaturing conditions and renatured. Protein blots were hybridized with various radiolabelled nucleic acid probes. Results obtained using genomic or messenger virus RNA as a probe indicated that the middle part of N was possibly an RNA-binding domain. To confirm this observation, two more accurate approaches were undertaken : (i) a gel retardation assay of RNA and purified protein complexes was done ; and (ii) RNA–protein complexes were cross-linked by UV light and analysed on a denaturing polyacrylamide gel. All these experiments led us to conclude that the middle part of N is the domain which interacts with RNA, despite the absence of homology with known consensus amino acid sequences of other RNA-binding proteins. Viruses of the family Rhabdoviridae, identified as bulletshaped, enveloped viruses with a single-stranded RNA genome of negative polarity, are frequently isolated from a wide range of host fish (Wolf, 1988). Until recently, based on the electrophoretic pattern of the structural polypeptides, they were classified as members of either the genus Vesiculovirus, e.g. spring viraemia of carp virus, or the genus Lyssavirus, e.g. viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). VHSV and IHNV have been the most extensively studied, due to their economic impact. VHSV is one of the causative agents of an acute disease Author for correspondence : Michel Bre! mont. Fax 33 1 34 65 26 21. e-mail bremont!biotec.jouy.inra.fr in salmon and trout. The genomes of VHSV and IHNV encode six proteins in the order 3« N-M1-M2-G-Nv-L 5« (Kurath et al., 1985 ; Kurath & Leong, 1985 ; Benmansour et al., 1994). The virion is composed of five structural proteins : a glycoprotein (G) which is embedded in the virus membrane ; a matrix protein (M2) ; a polymerase-associated protein (M1) ; a minor component, the polymerase (L) ; and the nucleoprotein (N). N, which is tightly bound to the genomic negative-strand RNA, is 404 amino acids long and represents approximately 800 molecules per virion (Leong et al., 1983). The gene encoding N has been sequenced for the 07-71 (French ; pathogenic) (Bernard et al., 1990) and Makah (Washington state, USA ; asymptomatic) (Bernard et al., 1992) isolates of VHSV. Comparison of the deduced amino acid sequences shows 94±8 % similarity and 91±1 % identity between the two VHSV strains, and 62±1 % and 37±1 %, respectively, between VHSV and IHNV. The central portion of this protein is the most highly conserved, suggesting that this region has functional importance. To map the RNA-binding domain on the VHSV N, the corresponding gene and subfragments were amplified by PCR from a cDNA clone (Bernard et al., 1992) using specific primers. Primers were chosen in order to generate N subfragments dividing the N gene into three overlapping fragments : A, the 5« end (1–471 nt) ; B, the middle part (442–771 nt) ; and C, the 3« end (673–1224 nt). The resulting DNA fragments were subcloned into the blunt-ended NdeI site of the pET-14b expression vector (Novagen). Following Escherichia coli BL21(DE ) transformation (Studier & Moffatt, 1986), recom$ binant clones were screened by hybridization using a radiolabelled cDNA N probe and analysed for insert orientation by restriction enzyme digestion of mini lysates. Using the pET14b expression vector, six His residues were fused to the amino-terminal end of the recombinant proteins, enabling purification by metal chelation affinity chromatography. Expression of recombinant proteins was induced by the addition of IPTG to exponential phase cultures and aliquots were analysed on a 12 % SDS–PAGE gel and visualized after Coomassie-blue staining. As shown in Fig. 1 (a), proteins of the expected molecular mass were synthesized at a very high level. The yield was estimated at between 80 and 120 mg}l induced bacteria. To confirm the authenticity of the expressed recombinant proteins, aliquots of induced recombinant clones separated on 0001-5070 # 1998 SGM EH Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:42:06 T. Said and others (a) N (b) A B C N (c) A B C C B A N Fig. 1. Expression of N and A, B and C proteins in E. coli BL21(DE3) transformed by the respective recombinant pET constructs and their reactivity and purification. (a) Following IPTG induction, aliquots were boiled in buffer described by Laemmli (1970) and loaded on a 12 % SDS–PAGE gel, which was then stained with Coomassie blue. (b) North-Western blot analysis. Recombinant E. coli cell lysates separated on a 12 % SDS–PAGE gel were transferred onto PVDF membranes and saturated as described in the text. The blot was probed with [α-32P]UTP radiolabelled M1 RNA. (c) Coomassie blue-stained gel analysis of the recombinant proteins purified by metal chelation chromatography. a 12 % SDS–PAGE gel were electrotransferred onto ProBlott membranes (Applied Biosystems) in 3-(cyclohexylamino)-1propanesulfonic acid buffer containing 10 % methanol, saturated for 2 h at room temperature in 3 % BSA in PBS (PBSA) and then incubated with anti-VHSV polyclonal or anti-N monoclonal antibodies diluted in PBSA containing 0±05 % Tween 20. After washing, the bound antibodies were detected using alkaline phosphatase-conjugated anti-mouse or antirabbit immunoglobulin (BYOSIS). 4-Nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (GibcoBRL) were used as substrates for the alkaline phosphatase. N and the A, B and C proteins were recognized by the 616 antiVHSV polyclonal antiserum, confirming the authenticity of the recombinant proteins. When blots were incubated with anti-N monoclonal antibodies, all eight monoclonal antibodies reacted with N and either the A or C proteins, but none reacted with the B part of N. It should be noted that this lack of reactivity of specific monoclonal antibodies against the middle part of a nucleoprotein has been described previously (Chen et al., 1991) for IHNV, another fish rhabdovirus. It could be hypothesized that this domain in the virus particle is masked and poorly accessible to the immune system. The first approach developed to detect binding between the recombinant N-derived proteins and nucleic acids was North-Western blotting, as first described by Boyle & Holmes (1986). Recombinant E. coli lysates were separated on a 12 % SDS–PAGE gel, transferred onto PVDF membranes and blocked in PBSA for 1 h at room temperature. The incubation medium was replaced by fresh PBSA containing 250 µg}ml yeast tRNA and incubated for 30 min. Blots were then incubated for 1 h in PBSA containing DNA or RNA probes at 10& c.p.m.}ml, obtained, respectively, by either in vitro transcription or random priming radiolabelling of cloned M1 and EI M2 VHSV genes. For the in vitro transcription reaction, a pBluescript-M2 plasmid was either linearized by BsmI and used to produce a 150 nt genomic sense RNA probe using the T3 RNA polymerase, or linearized by BsaBI and transcribed with the T7 RNA polymerase to produce a 250 nt messenger sense RNA probe. In both reaction mixtures, [α-$#P]UTP was included at a final concentration of 30 µCi. The specific activity of the probes was 7¬10( c.p.m.}µg. After four washes in PBSA, blots were dried and autoradiographed. A typical result obtained using RNA probes is shown in Fig. 1 (b), where hybridization was conducted with the M2 RNA probe (the same result was obtained with an M1 RNA probe). This result indicates that only the B protein seems able to interact with RNA. When DNA probes were employed, no proteins were detected (data not shown). It should be noted that the B protein interacts with the RNA in either the plus or minus sense. A longer time of exposure was required to visualize the RNA–N interaction. This is probably due to the less efficient blotting of N versus the B protein and thus less N molecules are available to bind the probe. To ascertain that binding of the B protein and RNA observed in the North-Western blotting experiment was not an artifact, binding was assayed by electrophoretic gel retardation in non-denaturing polyacrylamide gels. Recombinant proteins were first purified using His-bind resin columns (Novagen). Recombinant clones were inoculated into 3 ml LB medium containing 50 µg}ml carbenicillin, grown to OD 0±6, then stored overnight at 4 °C. Cells were pelleted, '!! then resuspended in 50 ml fresh LB medium and agitated at 37 °C to OD 0±8. Expression of recombinant proteins was '!! induced with 0±4 mM IPTG after 3 h incubation at 37 °C. Cells were pelleted at 5000 g for 5 min, resuspended in 12±5 ml cold buffer A (50 mM Tris–HCl, pH 8 ; 2 mM EDTA), centrifuged Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:42:06 VHSV nucleoprotein RNA-binding domain (a) (a) BSA N added N A C B Complex RNase T1 Free (b) BSA A B C A N N A B C BS C on tro l (b) Monomer Complex Free Fig. 2. Gel retardation analysis of VHSV RNA. (a) RNA binding to purified N ; 20 fmol of 150 nt 32P-radiolabelled RNA was added to increasing amounts of N and analysed on a non-denaturing polyacrylamide gel. (b) Same as (a) using N and A, B and C proteins (6 pmol each). BSA was also included as a negative control. The control lane contained RNA probe incubated with binding buffer only. and the final pellets were stored at ®70 °C. For purification, cell pellets were resuspended in 20 ml buffer B (5 mM imidazole ; 500 mM NaCl ; 20 mM Tris–HCl, pH 7±9) containing 200 units benzonase (Merck). After sonication, lysates were centrifuged and the resulting pellets were resuspended with 5 ml buffer B containing 6 M urea. Solubilization was achieved by gentle agitation overnight at 4 °C. Solubilized proteins were clarified by ultracentrifugation (100 000 g for 30 min) and supernatants were loaded onto His-bind resin columns pre-equilibrated in buffer B containing 6 M urea. Resins were washed and recombinant proteins were eluted with 5 ml buffer C (400 mM imidazole ; 500 mM NaCl ; 20 mM Tris–HCl, pH 7±9). Purified proteins were refolded by gradual removal of the 6 M urea by a step-by-step dialysis (0±5 M urea step). After the final dialysis against PBS, purified recombinant Fig. 3. Analysis of RNA–protein complexes following UV light cross-linking. Mixtures of RNA probe and proteins were irradiated with UV light for various lengths of time, digested with RNase T1 and loaded on a 12 % SDS–PAGE gel. The gel was then stained with Coomassie blue (a) and autoradiographed (b). proteins were stored at ®70 °C until use. Purity was estimated on Coomassie blue-stained SDS–PAGE gels (Fig. 1 c). For the gel retardation assay, purified proteins were mixed with riboprobes in binding buffer (10 mM Tris–HCl, pH 8 ; 100 mM NaCl ; 1 mM EDTA) to give a total volume of 20 µl. The mixtures were incubated at 4 °C for 20 min, after which 5 µl loading buffer (10 mM Tris–HCl, pH 8 ; 5 % glycerol ; 1 mM EDTA ; 0±05 % xylene cyanol ; 0±05 % bromophenol blue) was added to each. The mixtures were then separated by electrophoresis on 5 % non-denaturing polyacrylamide gels (60 : 1 acrylamide : bisacrylamide) in 0±5¬ TBE (45 mM Tris– borate ; 45 mM boric acid ; 1 mM EDTA) for 5 h at 50 V and 4 °C. Gels were then dried and exposed to X-ray films. Conditions for binding were first optimized using N and the genomic sense M2 riboprobe (20 fmol). Using low protein concentrations (0±6–3 pmol corresponding to 28–140 ng), the radioactive probe migrated as the protein-free RNA, but with 6 pmol (280 ng) of added N, the probe was retarded, indicating the formation of a complex between the purified N and the riboprobe (Fig. 2 a). The same experiment with N and the A, B Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:42:06 EJ T. Said and others and C proteins is presented in Fig. 2 (b) and it shows that N and the B protein interact with the RNA in a similar manner. The same experiment using the messenger sense M2 riboprobe gave similar results (data not shown), except that part of the probe migrated faster in lanes N and B. This increased mobility is probably due to the large size (250 nt) of the riboprobe used (Lane et al., 1992). Thus, these results are in agreement with those obtained with the North-Western blot, confirming that the B protein, the middle part of N, is able to complex the RNA, whereas A and C proteins are not. This result also indicates that the observed binding for N and the B protein was not due to the six His residues present at the amino terminus of each recombinant protein, and therefore that the B protein could represent an RNA-binding domain. To exclude the possibility that the interaction observed between RNA and N or the B protein was due to contaminant E. coli proteins, we developed a UV cross-linking assay in which RNA–protein complexes were analysed on a denaturing polyacrylamide gel. Mixtures of riboprobes and proteins as described above were UV-irradiated for 5 or 10 min using a UV General Electric USB (1±125 mW}cm#) ; complexes were then digested with 50 U RNase T1 for 15 min at 37 °C. RNA–protein complexes were run on 15 % SDS–PAGE gels, which were then stained with Coomassie blue, dried and autoradiographed. Superimposing the stained gel (Fig. 3 a) and resulting autoradiograph (Fig. 3 b) clearly indicated that the B protein is cross-linked to the RNA probe. Moreover, the additional bands visualized on the autoradiograph could correspond, by size estimation, to dimer and trimer forms of the B protein and aggregates which are not resolved on that gel. A longer period of UV irradiation of the samples resulted in the appearance of N, but it was not visible for 5 min irradiation (data not shown). In the past, many studies on vesicular stomatitis virus or rabies virus, the two rhabdovirus prototypes, have focused on the interaction between the virus RNA and the nucleoprotein. The specificity of this interaction requires the presence of the phosphoprotein. In the absence of this protein, the nucleoprotein is able to bind any RNA (Masters & Banerjee, 1988). In this study, we showed that a short region on VHSV N is implicated in RNA binding, even in the absence of the M1 phosphoprotein. A comparison of N from two fish rhabdoviruses, VHSV and IHNV, showed 37 % identity at the amino acid level, with the strongest identity between amino acids localized in the B fragment and more precisely in the carboxyterminal half. This observation reinforces the importance of this conserved central region in interaction with the virus genomic RNA ; nevertheless, a search for homology with other known RNA-binding proteins failed to show any significant data. Detailed analysis of the B fragment sequence did not reveal any special features such as stretches of basic amino acids. Nevertheless, the presence of a repeat of ELAD motifs has been detected, as well as the presence of 12 arginine and lysine residues, but these observations cannot be related to any FA previously described signature for an RNA-binding domain. Taking account of the fact that the three recombinant proteins A, B and C produced in E. coli overlap, we may assume that the RNA-binding domain is restricted to 68 amino acid residues and could represent a new RNA-binding motif. The authors are grateful to Dr J. M. Coll (CISA, Spain) for supplying some of the anti-N monoclonal antibodies. Drs C. Aponte and D. Poncet (INRA, France) are fully acknowledged for their valuable suggestions for this study. References Benmansour, A., Paubert, G., Bernard, J. & de Kinkelin, P. (1994). The polymerase-associated protein (P) and the matrix protein (M2) from a virulent and an avirulent strain of viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. Virology 198, 602–612. Bernard, J., Lecocq-Xhonneux, F., Rossius, M., Thiry, M. E. & de Kinkelin, P. (1990). Cloning and sequencing the messenger RNA of the N gene of viral haemorrhagic septicaemia virus. Journal of General Virology 71, 1669–1674. Bernard, J., Bremont, M. & Winton, J. (1992). Nucleocapsid gene sequence of a North American isolate of viral haemorrhagic septicaemia virus, a fish rhabdovirus. Journal of General Virology 73, 1011–1014. Boyle, J. F. & Holmes, K. V. (1986). RNA-binding proteins of bovine rotavirus. Journal of Virology 58, 561–568. Chen, L., Ristow, S. & Leong, J. C. (1991). Epitope mapping and characterization of the infectious hematopoietic necrosis virus nucleoprotein. In Proceedings of the Second International Symposium on Viruses of Lower Vertebrates, pp. 65–72. Corvallis, Oreg. : Oregon State University Press. Kurath, G. & Leong, J. A. C. (1985). Characterization of infectious hematopoietic necrosis virus mRNA species reveals a non virion rhabdovirus protein. Journal of Virology 53, 462–468. Kurath, G., Ahern, K. G., Pearson, G. D. & Leong, J. A. C. (1985). Molecular cloning of the six mRNA species of infectious hematopoietic necrosis virus, a fish rhabdovirus, and gene order determination by Rloop mapping. Journal of Virology 53, 469–476. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lane, D., Prentki, P. & Chandler, M. (1992). Use of gel retardation to analyze protein–nucleic acid interactions. Microbiological Reviews 56, 509–528. Leong, J. C., Hsu, Y. L. & Engelking, H. M. (1983). Synthesis of the structural proteins of infectious hematopoietic necrosis virus. In Bacterial and Viral Diseases of Fish : Molecular Studies, pp. 61–71. Edited by J. H. Crosa. Seattle : Washington Sea Grant Program. Masters, P. S. & Banerjee, A. K. (1988). Complex formation with vesicular stomatitis virus phosphoprotein NS prevents binding of nucleocapsid protein N to nonspecific RNA. Journal of Virology 62, 2658–2664. Studier, F. W. & Moffatt, B. A. (1986). Use of T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology 189, 113–130. Wolf, K. (1988). Fish Viruses and Fish Viral Diseases. New York : Cornell University Press. Received 7 July 1997 ; Accepted 9 September 1997 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:42:06