Homotypic Interaction of Bunyamwera Virus Nucleoca
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病菌学杂志 2005年第20期
Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 5JR, Scotland, United Kingdom
ABSTRACT
The bunyavirus nucleocapsid protein, N, plays a central role in viral replication in encapsidating the three genomic RNA segments to form functional templates for transcription and replication by the viral RNA-dependent RNA polymerase. Here we report functional mapping of interacting domains of the Bunyamwera orthobunyavirus N protein by yeast and mammalian two-hybrid systems, immunoprecipitation experiments, and chemical cross-linking studies. N forms a range of multimers from dimers to high-molecular-weight structures, independently of the presence of RNA. Deletion of the N- or C-terminal domains resulted in loss of activity in a minireplicon assay and a decreased capacity for N to form higher multimers. Our data suggest a head-to-head and tail-to-tail multimerization model for the orthobunyavirus N protein.
TEXT
Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae, a family of mainly arthropod-borne viruses. The family is divided into five genera, Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus; the latter genus comprises plant-infecting viruses. The Orthobunyavirus genus includes important human pathogens, such as La Crosse virus, a leading cause of pediatric encephalitis in North America; Oropouche virus, which causes a debilitating febrile illness in South America; and Tahyna virus, which causes an influenza-like syndrome in central Europe (8).
The bunyavirus genome consists of three segments of single-stranded RNA of negative, or ambisense, polarity. The largest segment (L) codes for an RNA-dependent RNA polymerase (L protein), the medium segment (M) codes for the two glycoproteins (Gn and Gc), and the smallest segment (S) encodes the nucleoprotein N. Viruses of some genera also encode nonstructural proteins either on the M (called NSm) or the S segment (called NSs). Viral replication takes place in the cytoplasm, while budding generally occurs at the Golgi apparatus (3, 26). As in other negative-stranded viruses, the RNA genome serves as the template for the synthesis of mRNAs and full-length, positive-sense antigenomic RNAs that in turn are templates for synthesis of progeny negative-stranded genomes. Reverse genetic systems developed for BUNV (7), La Crosse (4), Rift Valley fever (16, 22), Toscana (1), Uukuniemi (12), Crimean-Congo hemorrhagic fever (11), and Hantaan (10) viruses have shown that transcription and replication of artificial minigenomes require only two viral proteins, the polymerase L and N proteins.
The primary function of the N protein is the encapsidation of genome and antigenome RNAs to form a biologically active structure, the viral ribonucleoprotein or RNP. The RNPs have a "panhandle" structure in which the 3' and 5' genome termini are able to base pair (21). For BUNV, N was shown to bind specifically to the 5' terminus of the S genome segment, suggesting cotranscriptional encapsidation (24) of nascent viral RNA.
Bunyavirus N proteins vary in size from 25 to 30 kDa (orthobunya-, phlebo-, and tospoviruses) to 50 kDa (hanta- and nairoviruses), with little homology detected between the N proteins of viruses in different genera. A homotypic N-protein interaction implicated in the nucleocapsid structure has been extensively studied for the viruses of the Hantavirus genus; the N protein forms stable trimers (2, 18) that seem to bind specifically the panhandle structure of the RNA (23). The interaction sites on N proteins of several hantaviruses have been mapped and were shown to be principally in the N-terminal and C-terminal domains (see reference 20). Here we analyzed the homotypic interaction of the 26-kDa N protein of BUNV, the prototype virus of the Orthobunyavirus genus, using yeast and mammalian two-hybrid systems, coimmunoprecipitation, and chemical cross-linking experiments.
BUNV N-protein homotypic interaction detected in yeast and mammalian two-hybrid systems. BUNV N-N interaction was tested using a commercial yeast two-hybrid system, Matchmaker 3, obtained from Clontech (Palo Alto, CA). The full-length N open reading frame (ORF) was amplified by PCR using pTM1-BUNN (9) as the template and was cloned into EcoRI/PstI-digested binding domain (BD)-containing plasmid pGBKT7 (pBD) or EcoRI/XhoI-digested activation domain (AD)-containing plasmid pGADT7 (pAD) to produce pBD-BUNN or pAD-BUNN, respectively. Saccharomyces cerevisiae AH109 strain (17) was cotransformed using the lithium acetate method (14). Yeast cotransformed with the plasmid of interest and the other respective empty vector (pBD or pAD) was used as a negative control. Further controls for protein-protein interaction provided in the Matchmaker 3 system included pGADT7-T and pGBKT7-53 (positive controls) and pGADT7-T and pGBKT7-Lam (negative controls).
Yeast growth was observed only in the case of pAD-BUNN and pBD-BUNN cotransformants and the pGADT7-T plus pGBKT7-53 positive control (Fig. 1A, sectors a and c). No growth was observed when one N construct was cotransformed with the other empty vector or with the pGADT7-T plus pGBKT7-Lam negative control (Fig. 1A, sectors b, d, and e). These results show that N can homodimerize in the yeast two-hybrid system.
To confirm the data obtained with the yeast two-hybrid system, we investigated interaction of N using a modified two-hybrid assay adapted for mammalian cells (28, 30). The N ORF was cloned into the BD-containing plasmid pSG424 (41) to give pSGN, and two fusions of N with AD-containing plasmids were also constructed, one using pVP16 (Clontech) to give pVPBUNN (N-terminal AD fusion tag) and another using pVP16AASV19N (41) to give pAASN (C-terminal AD fusion tag). HeLa cells were transfected with 1 μg each of the N-expressing plasmids together with the reporter plasmid pG5CAT (which contains five GAL4 binding sites upstream of the adenovirus E1b promoter [28]) using liposomes as previously described (31). As a positive control, plasmid pM3VP16 (containing both AD and BD; Clontech) and plasmids pGalMxA and pVPMxA (a kind gift of F. Weber and previously shown to interact in this assay) were used. Strong chloramphenicol acetyltransferase (CAT) activity could be observed with the positive controls, pM3VP16 or the cotransfected plasmids pVPMxA and pGalMxA (Fig. 1B, lanes 1 and 2). The combination of pSGN and pVPBUNN gave weak CAT activity (lane 3), whereas a stronger interaction was achieved when the C-terminal AD-fusion pAASN was used in combination with pSGN (lane 4). No CAT signal was observed when pSGN, pVPBUNN, or pAASN was cotransfected with the reporter plasmid pG5CAT alone (Fig. 1B, lanes 5 to 7). These results show that CAT activity was the outcome of coexpression and interaction of N proteins and not due to a nonspecific interaction. Thus, an interaction between BUN N-protein molecules can be observed in both yeast and mammalian cells.
Transiently expressed N protein multimerizes. To extend the above results, we studied N-N interaction during transient expression in mammalian cells. The plasmid pBD-BUNN contains a T7 RNA polymerase promoter sequence downstream of the BD, allowing expression of a mRNA encoding a c-Myc-tagged version of the N protein. To assess the capacity of the c-Myc-tagged N protein to form nucleocapsid structures, we used the previously described BUNV minireplicon system (31). BSR-T7/5 cells, which stably express T7 RNA polymerase (5), were cotransfected with pTM1-BUNL, pTM1-FF-Luc, the minigenome pT7riboBUNMRen(–), and increasing amounts of the N-encoding plasmid pBD-BUNN or pT7riboBUNN. The pT7riboBUNN plasmid was chosen as the positive control for this test, as it produces mRNA encoding native N protein under control of the T7 RNA polymerase promoter. The c-Myc-tagged N protein was functional in the minireplicon system (Fig. 2A) though it was less active than the native N protein: when 1 μg of each N-expressing plasmid was transfected, the tagged N resulted in 14-fold-less luciferase signal. This result indicated that the tagged N protein was able to encapsidate minireplicon RNA and multimerize to form a functional RNP; therefore, we used it in coimmunoprecipitation experiments.
CV-1 cells were infected with the recombinant vaccinia virus vTF7-3, which synthesizes bacteriophage T7 RNA polymerase (13), and transfected with pTM1-BUNN and pBD-BUNN, and following radioactive labeling with [35S]methionine, cell lysates were immunoprecipitated with an anti-c-Myc antibody. As shown in Fig. 2B, lane 1, two bands were revealed, one corresponding to the immunoprecipitated c-Myc N and a second signal corresponding to the native N. The signal obtained for the native N was much stronger than that of the tagged N, suggesting that multimeric N complexes were formed containing more native than tagged N molecules. These results are consistent with the facts that the native protein was expressed from a plasmid containing an internal ribosome entry site (pTM1 [13]), allowing more efficient translation than for c-Myc N expressed on its own (lane 2), and that the tagged N protein showed decreased biological activity, suggesting the native form is preferably incorporated into N multimers. The c-Myc antibody did not recognize native N (lane 3) nor any cellular proteins in the empty-plasmid-transfected cells (lane 4). Previously we showed that N protein strongly bound RNA and formed ribonucleoprotein-like structures (24). To investigate whether the observed N-N interaction shown here involved RNA, a similar coimmunoprecipitation experiment was carried out in the presence of 5 μg/ml RNase A, a concentration shown to digest RNA when in RNA/N complexes (24), and also sufficient to digest the cellular RNA in the extract (data not shown). However, RNase A treatment failed to interfere with the formation of N multimers (Fig. 2C), indicating that the N-N interaction was direct and not mediated by RNA.
To study the possibility of a higher degree of N multimerization, suggested by the coimmunoprecipitation experiment, transiently expressed wild-type N protein was cross-linked with 1 mM dithiobis(succinimidylpropionate) (DSP) (Pierce), a homobifunctional and reduction-sensitive cross-linking agent. Transfected cells were treated with DSP as recommended by the manufacturer, and cell extracts were separated on a denaturing polyacrylamide gel followed by Western blotting using an anti-N polyclonal antibody (Fig. 3A). A ladder of proteins with molecular masses corresponding to those of N multimers (50 kDa, 75 kDa, 100 kDa, etc.) (Fig. 3A, lane 1) was detected in the absence of reducing agent. When ?-mercaptoethanol was added to the loading buffer, the cross-linking agent was reduced, and a single band corresponding to N monomers was observed (Fig. 3A, lane 3). This experiment was repeated with RNase A treatment of the extract with a concentration of 10 μg/ml prior to cross-linking, but no difference was observed (Fig. 3B). The authenticity of N-protein multimerization was confirmed by the appearance of a similar pattern of N-protein bands following cross-linking of BUNV-infected cell extract (Fig. 3C).
Involvement of the N and C termini of BUNV N in homotypic interaction. Previous studies on oligomerization of hantavirus and tospovirus N proteins identified the N and C termini in homotypic interactions (summarized in reference 20). Therefore, we focused our study primarily on these regions of the BUNV N protein. Alignment of a number of orthobunyavirus N-protein sequences (8) shows that the N-terminal 10 amino acids contain a cluster of residues conserved throughout the genus, while there is a highly conserved motif from residues 17 to 20 (FDP[D/E]). Two N-terminal deletion mutants of N protein were generated by PCR (Fig. 4A). The sequence encoding amino acids 11 to 233 was cloned between BsmBI/PstI restriction sites of pTM1 plasmid, producing pTM1-BUNN11-233, and the sequence encoding amino acids 11 to 20 was deleted by ExSite mutagenesis (Stratagene) to produce pTM1-BUNNdel11-20. The mutants were tested for activity in the minireplicon system (Fig. 4B) and showed no activity compared to the native N protein (note that the light units are presented on a logarithmic scale). Expression of the proteins in the minireplicon samples was tested by Western blotting using an anti-N polyclonal antibody. The level of expression of the N11-233 protein was comparable to that of the native N; however, Ndel11-20 could not be detected. Similar results were obtained with a deletion mutant lacking only the conserved FDPE motif (data not shown). These results suggest that the FDPE motif might be structurally essential for protein folding and/or stability.
To investigate the reason why the N11-233 mutant was inactive in the minireplicon system, a cross-linking experiment was performed on transiently expressed protein (Fig. 4C). While the native N protein produced the typical ladder-like pattern, the mutant N protein did not efficiently multimerize into oligomers higher than dimers; quantification of the signal on the blot indicated that trimers and higher multimers were decreased by at least sixfold compared to those obtained with native N. This result suggests that the N-terminal 10 amino acids of the protein are important for efficient multimerization. The presence of a significant level of dimers in the N11-233 sample is consistent with there being two interaction domains involved in the multimerization process.
The C-terminal 17 amino acids of the orthobunyavirus N proteins also show a high degree of conservation (8). We investigated the importance of this region in N multimerization by generating a C-terminally truncated construct of the N protein lacking amino acids 217 to 233. A double mutant, lacking amino acids 1 to 10 and 217 to 233 was also generated by PCR. Both products were cloned into the restriction sites BsmBI/PstI of pTM1, producing pTM1-BUNN1-216 and pTM1-BUN11-216, respectively (Fig. 5A). Both mutants were tested for activity in the minireplicon system (Fig. 5B), and neither showed activity compared to the native N protein. Both mutant proteins had slightly decreased stability, and the amount of mutant N-encoding plasmid was adjusted empirically to ensure a level of expression similar to that of the native N-protein positive control. To investigate the loss of activity, the multimerization capacity of both mutants was assessed by cross-linking (Fig. 5C). The N1-216 mutant was able to form dimers and small amounts of trimers but was severely impaired in its ability to form higher multimers. In contrast, the double mutant was not able to form any oligomers, suggesting that the deleted sequences are essential for N multimerization. Thus, we have identified sequences involved in both binding sites of the interaction. The homodimerization observed for both the N-terminal and C-terminal single deletion mutants suggests that BUNV N can form head-to-head and tail-to-tail types of interaction.
The low levels of multimers produced by the N-terminal and C-terminal single deletion mutants might be due to the presence of weak interaction sites involved in the interaction between successive helical turns. These interactions could be stabilized by the formation of dimers. This would explain the absence of such multimers in the double mutant N11-216. The presence of one binding site between successive N proteins is consistent with the necessity for the RNP to form a flexible structure. As discussed by Schoehn et al. (27) the formation of a nucleocapsid using only one point of contact between adjacent monomers increases flexibility, allowing formation of a panhandle structure for influenza virus RNP. The panhandle structure observed for bunyavirus RNPs suggests a similar necessity for flexibility.
The presence of substantial amounts of tetramers in the cross-linking experiment (Fig. 3) indicates that the oligomerization strategy of BUNV N protein differs from the model proposed for hantavirus N multimerization. The BUNV nucleocapsid protein seems to form multimers by addition of one N molecule at a time and not by association of preformed trimers (19). Our results are thus in agreement with those obtained with tomato spotted wilt tospovirus N protein (29), where the interaction of N in solution was shown to be a continuous process, leading to the formation of multimers, including tetramers; interaction was shown to involve amino acids 1 to 39 and 233 to 248 of the 258-residue-long tospovirus N protein. The apparent conservation of the multimerization mechanism of the N proteins of orthobunyaviruses and tospoviruses, coupled with previous sequence comparisons of the viral polymerase ORFs that demonstrated the orthobunyavirus L protein was more homologous to the tospovirus L than to the hantavirus or phlebovirus polymerase proteins (25), indicate closer evolutionary similarity of these two groups of animal- and plant-infecting bunyaviruses.
ACKNOWLEDGMENTS
We thank Anne Bridgen and Friedemann Weber for helpful discussions during the course of this work and Friedemann Weber and Bernard Moss for supplying reagents.
J.C.O. received a CASE studentship from BBSRC and Roche Products Ltd. Research in R.M.E.'s laboratory is funded by the Wellcome Trust.
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ABSTRACT
The bunyavirus nucleocapsid protein, N, plays a central role in viral replication in encapsidating the three genomic RNA segments to form functional templates for transcription and replication by the viral RNA-dependent RNA polymerase. Here we report functional mapping of interacting domains of the Bunyamwera orthobunyavirus N protein by yeast and mammalian two-hybrid systems, immunoprecipitation experiments, and chemical cross-linking studies. N forms a range of multimers from dimers to high-molecular-weight structures, independently of the presence of RNA. Deletion of the N- or C-terminal domains resulted in loss of activity in a minireplicon assay and a decreased capacity for N to form higher multimers. Our data suggest a head-to-head and tail-to-tail multimerization model for the orthobunyavirus N protein.
TEXT
Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae, a family of mainly arthropod-borne viruses. The family is divided into five genera, Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus; the latter genus comprises plant-infecting viruses. The Orthobunyavirus genus includes important human pathogens, such as La Crosse virus, a leading cause of pediatric encephalitis in North America; Oropouche virus, which causes a debilitating febrile illness in South America; and Tahyna virus, which causes an influenza-like syndrome in central Europe (8).
The bunyavirus genome consists of three segments of single-stranded RNA of negative, or ambisense, polarity. The largest segment (L) codes for an RNA-dependent RNA polymerase (L protein), the medium segment (M) codes for the two glycoproteins (Gn and Gc), and the smallest segment (S) encodes the nucleoprotein N. Viruses of some genera also encode nonstructural proteins either on the M (called NSm) or the S segment (called NSs). Viral replication takes place in the cytoplasm, while budding generally occurs at the Golgi apparatus (3, 26). As in other negative-stranded viruses, the RNA genome serves as the template for the synthesis of mRNAs and full-length, positive-sense antigenomic RNAs that in turn are templates for synthesis of progeny negative-stranded genomes. Reverse genetic systems developed for BUNV (7), La Crosse (4), Rift Valley fever (16, 22), Toscana (1), Uukuniemi (12), Crimean-Congo hemorrhagic fever (11), and Hantaan (10) viruses have shown that transcription and replication of artificial minigenomes require only two viral proteins, the polymerase L and N proteins.
The primary function of the N protein is the encapsidation of genome and antigenome RNAs to form a biologically active structure, the viral ribonucleoprotein or RNP. The RNPs have a "panhandle" structure in which the 3' and 5' genome termini are able to base pair (21). For BUNV, N was shown to bind specifically to the 5' terminus of the S genome segment, suggesting cotranscriptional encapsidation (24) of nascent viral RNA.
Bunyavirus N proteins vary in size from 25 to 30 kDa (orthobunya-, phlebo-, and tospoviruses) to 50 kDa (hanta- and nairoviruses), with little homology detected between the N proteins of viruses in different genera. A homotypic N-protein interaction implicated in the nucleocapsid structure has been extensively studied for the viruses of the Hantavirus genus; the N protein forms stable trimers (2, 18) that seem to bind specifically the panhandle structure of the RNA (23). The interaction sites on N proteins of several hantaviruses have been mapped and were shown to be principally in the N-terminal and C-terminal domains (see reference 20). Here we analyzed the homotypic interaction of the 26-kDa N protein of BUNV, the prototype virus of the Orthobunyavirus genus, using yeast and mammalian two-hybrid systems, coimmunoprecipitation, and chemical cross-linking experiments.
BUNV N-protein homotypic interaction detected in yeast and mammalian two-hybrid systems. BUNV N-N interaction was tested using a commercial yeast two-hybrid system, Matchmaker 3, obtained from Clontech (Palo Alto, CA). The full-length N open reading frame (ORF) was amplified by PCR using pTM1-BUNN (9) as the template and was cloned into EcoRI/PstI-digested binding domain (BD)-containing plasmid pGBKT7 (pBD) or EcoRI/XhoI-digested activation domain (AD)-containing plasmid pGADT7 (pAD) to produce pBD-BUNN or pAD-BUNN, respectively. Saccharomyces cerevisiae AH109 strain (17) was cotransformed using the lithium acetate method (14). Yeast cotransformed with the plasmid of interest and the other respective empty vector (pBD or pAD) was used as a negative control. Further controls for protein-protein interaction provided in the Matchmaker 3 system included pGADT7-T and pGBKT7-53 (positive controls) and pGADT7-T and pGBKT7-Lam (negative controls).
Yeast growth was observed only in the case of pAD-BUNN and pBD-BUNN cotransformants and the pGADT7-T plus pGBKT7-53 positive control (Fig. 1A, sectors a and c). No growth was observed when one N construct was cotransformed with the other empty vector or with the pGADT7-T plus pGBKT7-Lam negative control (Fig. 1A, sectors b, d, and e). These results show that N can homodimerize in the yeast two-hybrid system.
To confirm the data obtained with the yeast two-hybrid system, we investigated interaction of N using a modified two-hybrid assay adapted for mammalian cells (28, 30). The N ORF was cloned into the BD-containing plasmid pSG424 (41) to give pSGN, and two fusions of N with AD-containing plasmids were also constructed, one using pVP16 (Clontech) to give pVPBUNN (N-terminal AD fusion tag) and another using pVP16AASV19N (41) to give pAASN (C-terminal AD fusion tag). HeLa cells were transfected with 1 μg each of the N-expressing plasmids together with the reporter plasmid pG5CAT (which contains five GAL4 binding sites upstream of the adenovirus E1b promoter [28]) using liposomes as previously described (31). As a positive control, plasmid pM3VP16 (containing both AD and BD; Clontech) and plasmids pGalMxA and pVPMxA (a kind gift of F. Weber and previously shown to interact in this assay) were used. Strong chloramphenicol acetyltransferase (CAT) activity could be observed with the positive controls, pM3VP16 or the cotransfected plasmids pVPMxA and pGalMxA (Fig. 1B, lanes 1 and 2). The combination of pSGN and pVPBUNN gave weak CAT activity (lane 3), whereas a stronger interaction was achieved when the C-terminal AD-fusion pAASN was used in combination with pSGN (lane 4). No CAT signal was observed when pSGN, pVPBUNN, or pAASN was cotransfected with the reporter plasmid pG5CAT alone (Fig. 1B, lanes 5 to 7). These results show that CAT activity was the outcome of coexpression and interaction of N proteins and not due to a nonspecific interaction. Thus, an interaction between BUN N-protein molecules can be observed in both yeast and mammalian cells.
Transiently expressed N protein multimerizes. To extend the above results, we studied N-N interaction during transient expression in mammalian cells. The plasmid pBD-BUNN contains a T7 RNA polymerase promoter sequence downstream of the BD, allowing expression of a mRNA encoding a c-Myc-tagged version of the N protein. To assess the capacity of the c-Myc-tagged N protein to form nucleocapsid structures, we used the previously described BUNV minireplicon system (31). BSR-T7/5 cells, which stably express T7 RNA polymerase (5), were cotransfected with pTM1-BUNL, pTM1-FF-Luc, the minigenome pT7riboBUNMRen(–), and increasing amounts of the N-encoding plasmid pBD-BUNN or pT7riboBUNN. The pT7riboBUNN plasmid was chosen as the positive control for this test, as it produces mRNA encoding native N protein under control of the T7 RNA polymerase promoter. The c-Myc-tagged N protein was functional in the minireplicon system (Fig. 2A) though it was less active than the native N protein: when 1 μg of each N-expressing plasmid was transfected, the tagged N resulted in 14-fold-less luciferase signal. This result indicated that the tagged N protein was able to encapsidate minireplicon RNA and multimerize to form a functional RNP; therefore, we used it in coimmunoprecipitation experiments.
CV-1 cells were infected with the recombinant vaccinia virus vTF7-3, which synthesizes bacteriophage T7 RNA polymerase (13), and transfected with pTM1-BUNN and pBD-BUNN, and following radioactive labeling with [35S]methionine, cell lysates were immunoprecipitated with an anti-c-Myc antibody. As shown in Fig. 2B, lane 1, two bands were revealed, one corresponding to the immunoprecipitated c-Myc N and a second signal corresponding to the native N. The signal obtained for the native N was much stronger than that of the tagged N, suggesting that multimeric N complexes were formed containing more native than tagged N molecules. These results are consistent with the facts that the native protein was expressed from a plasmid containing an internal ribosome entry site (pTM1 [13]), allowing more efficient translation than for c-Myc N expressed on its own (lane 2), and that the tagged N protein showed decreased biological activity, suggesting the native form is preferably incorporated into N multimers. The c-Myc antibody did not recognize native N (lane 3) nor any cellular proteins in the empty-plasmid-transfected cells (lane 4). Previously we showed that N protein strongly bound RNA and formed ribonucleoprotein-like structures (24). To investigate whether the observed N-N interaction shown here involved RNA, a similar coimmunoprecipitation experiment was carried out in the presence of 5 μg/ml RNase A, a concentration shown to digest RNA when in RNA/N complexes (24), and also sufficient to digest the cellular RNA in the extract (data not shown). However, RNase A treatment failed to interfere with the formation of N multimers (Fig. 2C), indicating that the N-N interaction was direct and not mediated by RNA.
To study the possibility of a higher degree of N multimerization, suggested by the coimmunoprecipitation experiment, transiently expressed wild-type N protein was cross-linked with 1 mM dithiobis(succinimidylpropionate) (DSP) (Pierce), a homobifunctional and reduction-sensitive cross-linking agent. Transfected cells were treated with DSP as recommended by the manufacturer, and cell extracts were separated on a denaturing polyacrylamide gel followed by Western blotting using an anti-N polyclonal antibody (Fig. 3A). A ladder of proteins with molecular masses corresponding to those of N multimers (50 kDa, 75 kDa, 100 kDa, etc.) (Fig. 3A, lane 1) was detected in the absence of reducing agent. When ?-mercaptoethanol was added to the loading buffer, the cross-linking agent was reduced, and a single band corresponding to N monomers was observed (Fig. 3A, lane 3). This experiment was repeated with RNase A treatment of the extract with a concentration of 10 μg/ml prior to cross-linking, but no difference was observed (Fig. 3B). The authenticity of N-protein multimerization was confirmed by the appearance of a similar pattern of N-protein bands following cross-linking of BUNV-infected cell extract (Fig. 3C).
Involvement of the N and C termini of BUNV N in homotypic interaction. Previous studies on oligomerization of hantavirus and tospovirus N proteins identified the N and C termini in homotypic interactions (summarized in reference 20). Therefore, we focused our study primarily on these regions of the BUNV N protein. Alignment of a number of orthobunyavirus N-protein sequences (8) shows that the N-terminal 10 amino acids contain a cluster of residues conserved throughout the genus, while there is a highly conserved motif from residues 17 to 20 (FDP[D/E]). Two N-terminal deletion mutants of N protein were generated by PCR (Fig. 4A). The sequence encoding amino acids 11 to 233 was cloned between BsmBI/PstI restriction sites of pTM1 plasmid, producing pTM1-BUNN11-233, and the sequence encoding amino acids 11 to 20 was deleted by ExSite mutagenesis (Stratagene) to produce pTM1-BUNNdel11-20. The mutants were tested for activity in the minireplicon system (Fig. 4B) and showed no activity compared to the native N protein (note that the light units are presented on a logarithmic scale). Expression of the proteins in the minireplicon samples was tested by Western blotting using an anti-N polyclonal antibody. The level of expression of the N11-233 protein was comparable to that of the native N; however, Ndel11-20 could not be detected. Similar results were obtained with a deletion mutant lacking only the conserved FDPE motif (data not shown). These results suggest that the FDPE motif might be structurally essential for protein folding and/or stability.
To investigate the reason why the N11-233 mutant was inactive in the minireplicon system, a cross-linking experiment was performed on transiently expressed protein (Fig. 4C). While the native N protein produced the typical ladder-like pattern, the mutant N protein did not efficiently multimerize into oligomers higher than dimers; quantification of the signal on the blot indicated that trimers and higher multimers were decreased by at least sixfold compared to those obtained with native N. This result suggests that the N-terminal 10 amino acids of the protein are important for efficient multimerization. The presence of a significant level of dimers in the N11-233 sample is consistent with there being two interaction domains involved in the multimerization process.
The C-terminal 17 amino acids of the orthobunyavirus N proteins also show a high degree of conservation (8). We investigated the importance of this region in N multimerization by generating a C-terminally truncated construct of the N protein lacking amino acids 217 to 233. A double mutant, lacking amino acids 1 to 10 and 217 to 233 was also generated by PCR. Both products were cloned into the restriction sites BsmBI/PstI of pTM1, producing pTM1-BUNN1-216 and pTM1-BUN11-216, respectively (Fig. 5A). Both mutants were tested for activity in the minireplicon system (Fig. 5B), and neither showed activity compared to the native N protein. Both mutant proteins had slightly decreased stability, and the amount of mutant N-encoding plasmid was adjusted empirically to ensure a level of expression similar to that of the native N-protein positive control. To investigate the loss of activity, the multimerization capacity of both mutants was assessed by cross-linking (Fig. 5C). The N1-216 mutant was able to form dimers and small amounts of trimers but was severely impaired in its ability to form higher multimers. In contrast, the double mutant was not able to form any oligomers, suggesting that the deleted sequences are essential for N multimerization. Thus, we have identified sequences involved in both binding sites of the interaction. The homodimerization observed for both the N-terminal and C-terminal single deletion mutants suggests that BUNV N can form head-to-head and tail-to-tail types of interaction.
The low levels of multimers produced by the N-terminal and C-terminal single deletion mutants might be due to the presence of weak interaction sites involved in the interaction between successive helical turns. These interactions could be stabilized by the formation of dimers. This would explain the absence of such multimers in the double mutant N11-216. The presence of one binding site between successive N proteins is consistent with the necessity for the RNP to form a flexible structure. As discussed by Schoehn et al. (27) the formation of a nucleocapsid using only one point of contact between adjacent monomers increases flexibility, allowing formation of a panhandle structure for influenza virus RNP. The panhandle structure observed for bunyavirus RNPs suggests a similar necessity for flexibility.
The presence of substantial amounts of tetramers in the cross-linking experiment (Fig. 3) indicates that the oligomerization strategy of BUNV N protein differs from the model proposed for hantavirus N multimerization. The BUNV nucleocapsid protein seems to form multimers by addition of one N molecule at a time and not by association of preformed trimers (19). Our results are thus in agreement with those obtained with tomato spotted wilt tospovirus N protein (29), where the interaction of N in solution was shown to be a continuous process, leading to the formation of multimers, including tetramers; interaction was shown to involve amino acids 1 to 39 and 233 to 248 of the 258-residue-long tospovirus N protein. The apparent conservation of the multimerization mechanism of the N proteins of orthobunyaviruses and tospoviruses, coupled with previous sequence comparisons of the viral polymerase ORFs that demonstrated the orthobunyavirus L protein was more homologous to the tospovirus L than to the hantavirus or phlebovirus polymerase proteins (25), indicate closer evolutionary similarity of these two groups of animal- and plant-infecting bunyaviruses.
ACKNOWLEDGMENTS
We thank Anne Bridgen and Friedemann Weber for helpful discussions during the course of this work and Friedemann Weber and Bernard Moss for supplying reagents.
J.C.O. received a CASE studentship from BBSRC and Roche Products Ltd. Research in R.M.E.'s laboratory is funded by the Wellcome Trust.
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