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Mutational analysis of positively charged amino acid residues of Uukuniemi phlebovirus nucleocapsid protein
Virus Research 167 (2012) 118–123
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Short communication
Mutational analysis of positively charged amino acid residues of Uukuniemi phlebovirus nucleocapsid protein Anna Katz a,∗ , Alexander N. Freiberg b , Vera Backström a,1 , Liisa Holm c , Antti Vaheri a , Ramon Flick b,2 , Alexander Plyusnin a a
Research Programs Unit, Infection Biology Research Program, Department of Virology, Haartman Institute, P.O. Box 21, University of Helsinki, 00014 Helsinki, Finland Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA c Structural Genomics Group, Institute of Biotechnology, P.O. Box 56, University of Helsinki, 00014 Helsinki, Finland b
a r t i c l e
i n f o
Article history: Received 17 February 2012 Received in revised form 16 March 2012 Accepted 4 April 2012 Available online 17 April 2012 Keywords: Negative-strand RNA virus Phlebovirus Uukuniemi virus Nucleocapsid protein Mutational analysis RNA binding Oligomerization
a b s t r a c t The aim of this study was to evaluate the contribution of positively charged amino acid residues for the Uukuniemi virus (UUKV) N protein functionality. Based on phlebovirus nucleocapsid (N) protein alignments and 3D-structure predictions of UUKV N protein, 14 positively charged residues were chosen as targets for the mutagenesis. The impact of mutations to the N protein functionality was analyzed using minigenome-, virus-like particle-, and mammalian two-hybrid-assays. Seven of the mutations affected the functional competence in all three assays, while others had milder impact or no impact at all. In the 3D-model of UUKV N protein, five of the affected residues, R61, R64, R73, R98 and R115, were located either within or in close proximity to the central cavity that could potentially bind RNA. © 2012 Elsevier B.V. All rights reserved.
Uukuniemi virus (UUKV) belongs to the family Bunyaviridae, and there to the genus Phlebovirus, which contains important pathogens, such as Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Punta Toro virus (PTV) (Bouloy, 2011). The virus was originally isolated from Ixodes ricinus ticks in Finland (Oker-Blom et al., 1964) serving ever since as a convenient model for studying the bunyavirus structure and function (Flick and Pettersson, 2001; Flick et al., 2004; Katz et al., 2010; Pettersson and von Bonsdorff, 1975; Överby et al., 2008). The viral genome is composed of three single-stranded RNA segments: negative-sense L (large) and M (medium) segments, whereas the S (small) segment encodes the nucleocapsid (N) and non-structural (NSs) proteins using an ambisense strategy. The RNA segments are encapsidated by the N protein to form ribonucleoprotein complexes (RNPs), the functional templates for viral RNA synthesis (Bouloy, 2011). This mechanism
∗ Corresponding author. Tel.: +358 9 19126707; fax: +358 9 19126491. E-mail addresses: anna.katz@helsinki.fi (A. Katz), [email protected] (A.N. Freiberg), [email protected] (V. Backström), liisa.holm@helsinki.fi (L. Holm), antti.vaheri@helsinki.fi (A. Vaheri), rfl[email protected] (R. Flick), alexander.plyusnin@helsinki.fi (A. Plyusnin). 1 Present address: Software Point, Valkjärventie 1, 02130 Espoo, Finland. 2 Present address: BioProtection Systems, A Subsidiary of Newlink Genetics Corporation, 2901 South Loop Drive, Suite 3360, Ames, IA 50010, USA. 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.04.003
is shared by all negative-strand RNA viruses (NSRV) (Ruigrok et al., 2011). The details of the RNP-formation remained unsolved for many NSRV until very recently. Progress in cryo-electron tomography and crystallization techniques has led to solving various N protein and RNP structures for several NSRV (Ruigrok et al., 2011). The structures of RNPs vary from more relaxed in segmented bunya-, arena-, and influenza viruses (Ferron et al., 2011; Hastie et al., 2011a, 2011b; Raymond et al., 2010; Ye et al., 2006) to more helical in nonsegmented rhabdo- and filoviruses, giving the characteristic shape for the virion (Bharat et al., 2011; Ge et al., 2010). The ring-shaped RNPs of respiratory syncytial, rabies, and vesicular stomatitis viruses contain 10–11 N protein molecules (Albertini et al., 2006; Green et al., 2006; Tawar et al., 2009), whereas the N protein of Borna disease virus was crystallized as a tetramer (Rudolph et al., 2003). In most viruses, RNA was observed inside the N ring-structures. The nucleoprotein of influenza virus forms trimers (Ng et al., 2008; Ye et al., 2006), suggesting a ring of nine molecules as an RNA-binding unit with a positively charged cleft that probably binds the RNA (Ng et al., 2008). The first N protein structures of RVFV (Raymond et al., 2010) and Lassa virus (LASV, genus Arenavirus) (Qi et al., 2010) were recently defined, followed by more detailed structures revealing the mechanism for the N protein oligomerization and RNA-binding (Ferron et al., 2011; Hastie et al., 2011b). Interestingly, in both viruses the shift
A. Katz et al. / Virus Research 167 (2012) 118–123
of the N-terminal arm of N protein was crucial for RNA-binding. For LASV N protein, a specific gating mechanism was a key in the presented model (Hastie et al., 2011b), showing that RNA-free N protein trimer was unable to bind RNA. After a conformational change the shift of the N-terminal arm revealed the RNA-binding cavity. Ferron et al. (2011) presented a hexameric ring structure for RVFV N protein with proposed sites for RNA-binding and oligomerization. In contrast to the first structure (Raymond et al., 2010), here the N-terminal arm was extended from the molecule core, exposing the RNA-binding cavity in the central part of the protein. Even though the N protein of RVFV can oligomerize without RNA, Ferron et al. (2011) suggested that stabilization of the N protein oligomers might require an association with RNA. The aim of this study was to evaluate the contribution of positively charged aa residues to the functionality of UUKV N protein. Toward this aim we analyzed sequence alignments of the Phlebovirus N proteins and found 11 positively charged residues, conserved throughout the genus (Fig. 1). To gain more information of the fold of UUKV N protein, the 2D-structure was predicted using Jpred-, Psipred- and PredictProtein-servers (Cole et al., 2008; Buchan et al., 2010; Rost et al., 2003). The analyses suggested N protein to be a compact, globular protein formed of 11–13 ␣-helices. Since at the starting point of this study there were no N protein 3D-structures solved for any bunyavirus, the ab initio protein structure prediction server, Robetta (Bonneau et al., 2002) was chosen to guide our mutational analysis. Ten models were created for UUKV N protein and examined to identify surfaces suitable for putative RNA-binding. Since UUKV N was predicted to be a globular protein, we concentrated mostly on Robetta models fulfilling these criteria. In most of the models, the cluster of positively charged residues was observed in the central part of the molecule. When choosing the aa residues for mutation, models with a cavity in the central part of the N protein were favored, since in other NSRV, this kind of structures are known to bind RNA (Ruigrok et al., 2011). The conserved aa residues were chosen for the mutational analysis, some of them located in the potential RNA-binding cavity. The resulting set contained 13 single and two double mutants: R44A, KK50-51AA, R61A, R64A, R73A, K76A, KR82-83AA, R98A, R115A, H178A, R187A, R194A, K223A, R224A and K238A. All mutants, except the double mutant KR82-83AA were generated and tested. To investigate the N–RNA interactions directly, we attempted to establish a gel-shift assay using N protein mutants and in vitro-transcribed RNAs. Unfortunately our efforts were not successful, therefore the N protein mutants were analyzed using the minigenome- and virus-like particle (VLP)-systems, where the RNA-binding ability of the N protein is crucial (Flick and Pettersson, 2001; Överby et al., 2006). The N protein functionality was also tested in the mammalian two-hybrid (M2H) system, which has been shown to be efficient in studying bunyavirus N protein oligomerization (N–N-interactions) (Kaukinen et al., 2003; Alminaite et al., 2008; Katz et al., 2010). First, the functional competence of the mutants was investigated using the RNA Pol I-driven minigenome system (Flick and Pettersson, 2001), which requires that the N protein mutants are capable to bind to the viral RNA and support its transcription (and replication) by the viral polymerase. The plasmid pcDNAUUKV-N encoding the wild type (wt) UUKV N protein was used to generate point mutations using a site-directed mutagenesis kit (Stratagene) as described previously (Katz et al., 2010). Integrity of all constructs was verified by both restriction analysis and sequencing, and expression of the N protein mutants was confirmed by immunoblotting (Fig. S1) as described previously (Katz et al., 2010). BHK-21 cells were transfected with the UUKV N-, UUKV L- and UUKV GN /GC -protein expressing plasmids and the UUKV M-CAT minigenome, an UUKV M segment-based minigenome containing
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the reporter gene chloramphenicol acetyltransferase (CAT). The functionality of the N protein mutants was monitored by CAT reporter gene expression. Five mutations (R61A, R64A, R73A, R115A and R223A) severely affected the N protein functionality and three mutations (R98A, H178A and R224A) had a more moderate impact on the functionality (Fig. 2A and Table 1). The remaining six mutations did not show any altering effect (Fig. 2A and Table 1). Next, the mutants were tested in the previously established Uukuniemi VLP-system (Överby et al., 2006). This system allows to investigate if the N protein mutants, as an integral part of RNPs, can get incorporated into VLPs and drive minigenome transcription in VLP-infected cells. Even if the N mutant would be fully functional in the minigenome system and capable to support RNA transcription/replication, the VLP system should reveal the defects in the VLP assembly, e.g. packaging of vRNA into the VLPs. The method for VLP generation and infection was described previously (Katz et al., 2010). Briefly, minigenome and expression plasmids were transfected into BHK-21 cells and supernatant harvested 24 h post-transfection. VLP/minigenome-containing supernatants were then passaged to fresh cells, pre-transfected with the wt UUKV N- and L-expressing plasmids to support minigenome expression. When all components were functional, UUKV minigenomecontaining VLPs were generated, which was monitored by the expression of CAT-reporter gene in the VLP-infected cells. As expected, in the negative control for VLP-infected cells (transfection without GN /GC ) (Fig. 2B, lane 2), no CAT activity was detected. The positive control containing the wt UUKV N and GN /GC showed CAT activity (Fig. 2B, lane 3), demonstrating that VLPs were generated and minigenomic RNA was successfully transferred into VLP-infected cells. The results observed with the VLP system were in agreement with those of the minigenome system. The overall CAT activities were lower than in minigenome system, since VLP-infected cells receive only a few copies of minigenomic RNA through VLP infection. The N protein functionality was also tested in the M2H assay, which is primarily designed to test the protein–protein interactions. Whether the N protein oligomerization and interaction with RNA are coupled in all bunyaviruses remains to be seen. For Tula hantavirus N protein, it was shown that RNase treatment weakens the N–N-interaction in M2H system (Alminaite, 2010). In contrast, Leonard et al. (2005) showed that the N protein of Bunyamwera virus can oligomerize in the presence of high RNase concentrations, and it was suggested also for the N protein of Lassa virus that the RNA-binding might not be required for the N protein oligomerization (Hastie et al., 2011b). For RVFV, it was suggested that the N protein can oligomerize without RNA, but the stabilization of the oligomers might need an RNA bound to the structure (Ferron et al., 2011). Next, the M2H assay was employed to evaluate the overall impact of the mutations to the UUKV N protein functionality. Altogether 11 out of 14 mutations were introduced into plasmids pM-UUKV-N (DNA-BD) and pVP-UUKV-N (DNA-AD) as described earlier (Katz et al., 2010). In this study, the N protein mutations were introduced to both DNA-BD- and DNA-AD-counteracting plasmids. The effect of having the mutation in only one of the partners was tested with the mutation R73A. When the mutation was introduced into both partners (e.g. pM-R73A vs. pVP-R73A), the interaction, in three separate assays, was decreased to 12% (Table 1). However, when the mutation was tested against wt N protein, e.g. pM-wt-N vs. pVP-R73A, the interaction was decreased to 38–46%, while in the assay with pM-R73A vs. pVP-N-wt these values were 73–89%. These data suggested, that the wt N protein can compensate the harmful affect of the mutation in the other counterpart in the M2H assay. Therefore each mutation was tested against the same mutation in the other counterpart, e.g. pM-R73A vs. pVP-R73A. The expression
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Fig. 1. Multiple alignment on phlebovirus N proteins shows the positively charged residues throughout the genus. Conserved aa residues are shown in white on red background, invariant residues in red on white background and the variable residues in black on white background. The UUKV N protein aa residues which were mutated are marked with triangles (). This ClustalW alignment shows also RVFV N protein (PDB: 3OV9) aa residues R64, K67 and K74, marked with stars (), which were suggested to bind RNA. The viruses and GenBank accession numbers for the N protein sequences are: Uukuniemi virus [GI 38371708], Toscana virus [GI 52627074], Corfou virus [GI 146336856], Punta Toro virus [GI 127918], Candiru virus [GI 328545956], Aguacate virus [GI 330850814], and Rift Valley fever virus [GI 87622293]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of the N fusion protein mutants was confirmed using immunoblotting as described previously (Katz et al., 2010). Three mutations, R187A, R194A and K238A were excluded from the M2H assay since these were fully competent in the minigenome system, while two mutations, R44A and KK50-51AA, were tested to have representatives of fully competent mutations also in the M2H system. The M2H assay was performed as described previously (Katz et al., 2010). Briefly, HeLa cells were co-transfected with plasmids expressing the N protein mutants and reporter plasmids expressing the firefly (FL) luciferase and renilla (RL) luciferase
(Promega). Reporter gene activities were determined in triplicates with the Dual-Luciferase Reporter Assay System (Promega). Each experiment was performed three times and the FL-values were normalized using the RL-values. The M2H results correlated with those in the minigenome system: seven mutations (R64A, R73A, R98A, R115A, H178A, K223A and R224A) appeared detrimental for the N protein functionality while the impact of mutations R61A and K76A was minor (Fig. 2C and Table 1). The mutants R44A and KK50-51AA, which were fully competent in the minigenome system, were found functional in the M2H assay as well. The mutation
Table 1 Functionality data of UUKV N protein point mutants in the minigenome, VLP and mammalian two-hybrid (M2H) assays. UUKV N protein point mutations
Minigenomea
VLPa
M2H % of interactionb
wt N protein R44A KK50-51AA R61A R64A R73A K76A R98A R115A H178A R187A R194A K223A R224A K238A
+++ +++ +++ − − − ++ + − + +++ +++ − + +++
++ ++ ++ − − − + − − − ++ ++ − − ++
100 89 ± 11 >100 82 ± 25 7±4 12 ± 6 78 ± 18 32 ± 19 18 ± 10 18 ± 6 ND ND 3±1 35 ± 12 ND
a
In minigenome (and VLP systems) the level of CAT expression ranged between non-affected (+++), reduced (++), substantially reduced (+), and completely abolished (−). Full-length N–N protein interaction (100%, +++) was compared to the N–N interaction of point mutants. The mutations were introduced to both interacting plasmid partners in the M2H system. The interaction ranged from not affected (+++) to reduced (++) and to substantially reduced interaction (+). Each test was performed three times in triplicates. b
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Fig. 2. (A) Reporter gene activity based on UUKV N protein functionality in minigenome transcription: expression of N protein mutants was determined in a minigenome system using CAT reporter gene expression as described earlier (Katz et al., 2010). BHK-21 cells were transfected with UUKV M segment-based minigenome plasmid (UUKV M-CAT) and expression plasmids coding for the viral polymerase (pCMV-UUKV-L), glycoproteins (pCMV-GN /GC ), and N protein mutants. Lane 1: negative control (without UUKV L), lane 2: VLP control (without UUKV GN /GC ) lane 3: positive control (with wt UUKV N, UUKV L and UUKV GN /GC ). Single and double aa point mutations within the N protein are listed above the individual CAT signals. (B) Reporter gene activity based on UUKV N protein functionality in VLP assembly, minigenome packaging and transcription: BHK-21 cells were first transfected with UUKV L and wt UUKV N expression plasmids before VLPs with minigenome-containing supernatant aliquots from the minigenome-expressing cells (Fig. 1a) were transferred. VLP infection incorporates the minigenome encapsidated by the different UUKV N mutants and transcription levels are monitored by CAT assay. (C) Reporter gene expression based on UUKV N protein functionality in the M2H assay: the impact of the N protein mutations to the N–N interactions was monitored as firefly luciferase expression. The values were normalized with the renilla luciferase and compared to the wt N expression (set as 100%, lane 3). Negative control (vectors pM1 and pVP16 without N protein) is shown in lane 2 and positive control (wt N protein in both vectors, pM1 and pVP16) in lane 3 (ND = not determined).
R61A appeared to be peculiar: in the M2H assay the impact of this mutation was minor, but in the minigenome system it damaged the protein functionality severely. This could imply that this residue is directly involved in the RNA-binding process but not in the N protein oligomerization. Two recent publications allowed for gaining more insights into the RNA-binding process in phleboviruses. Raymond et al. (2010) presented RVFV N protein structure, the first among the Bunyaviridae. It was soon followed by another RVFV N protein structure (Ferron et al., 2011), which also revealed the mechanism of N protein oligomerization. In this structure, the patch of positively
charged residues in the inner cleft of the N protein probably accommodates vRNA. We utilized the latter structure (RVFV N protein, PDB code 3OV9) to create new 3D-models for the UUKV N protein, using three established servers: I-Tasser (Roy et al., 2010), Phyre2 (Kelley and Sternberg, 2009), and Swissmodel (Arnold et al., 2006), all highly ranked in the CASP modeling contests (Raman et al., 2009). Three resulting models resembled each other and adopted the folds very similar to that of RVFV N protein. The model created using I-Tasser is shown in Fig. 3. The majority of our experimental data with the N protein mutants (Table 1 and Fig. 2) was in good agreement with the presented 3D-fold of the UUKV N protein.
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Fig. 3. The I-Tasser server 3D-model for UUKV N protein. The model was built based on the RVFV N protein structure (PDB code: 3OV9) (Ferron et al., 2011), showing that most of the affected aa residues (shown in red) are located within the central cavity of the molecule or in the close proximity of it, in the proposed RNA-binding surface of UUKV N protein. The aa residues which were not affected by the mutations are shown in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Mutation of six aa residues, R44, KK50-51, R187, R194 and K238A had no affect in the N protein functionality in the minigenome and M2H assays (Table 1). These residues are located on the outer surfaces of the molecule, distant from the RNA-binding cleft. Some of these residues are seen in Fig. 3. In contrast, mutation of residues R61, R64, R73, K76, R98 and R115, located either within or next to the central RNA-binding cavity of the molecule, affected the N protein functionality severely (Fig. 3 and Table 1). Ferron et al. (2011) proposed that three RVFV N protein residues, R64, K67 and K74, are most directly involved in the RNA-binding based on the finding that the triple mutant R64D/K67D/K74D lost its ability to bind RNA. These residues correspond to R73, K76 and K82 in the UUKV N protein. In our experiments, the mutation R73A strongly affected the UUKV N protein function in minigenome/VLP and M2H systems, and the functionality of K76A was reduced. The UUKV N protein functionality was affected also by mutation of residues H178, K223 and R224, of which residues K223 and R224 correspond to residues R214 and R215 in the N protein of RVFV. According to Ferron et al. (2011), these residues are located in the proposed oligomerization groove. Involvement in the oligomerization could explain the affect we observed in our assays, although the role of the residue H178 remained unclear. The RVFV N protein model (Ferron et al., 2011) shows also that the N-terminal arm of the protein is involved in the oligomerization through interaction(s) with the C-terminal part of the adjacent N protein molecule. This is in good agreement with the results of our previous work on UUKV N protein (Katz et al., 2010), where we observed that the N-terminus of the protein is crucial for oligomerization. To summarize, minigenome-, VLP- and M2H-data support the 3D-models of UUKV N protein based on the solved structure of RVFV N protein. Residues R61, R64, R73, R98 and R115, located in the central cavity of the N protein molecule, might contribute to the RNA-binding, while some other positively charged residues, such as K223 and R224, might be important for the oligomerization.
Acknowledgments This work was supported by the Helsinki Biomedical Graduate School, Sigrid Jusélius Foundation, and Finnish Cultural Foundation. We are grateful to late Ralf Pettersson for valuable comments and friendly support.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.virusres.2012.04.003. References Albertini, A.A., Wernimont, A.K., Muziol, T., Ravelli, R.B., Clapier, C.R., Schoehn, G., Weissenhorn, W., Ruigrok, R.W., 2006. Crystal structure of the rabies virus nucleoprotein–RNA complex. Science 313 (5785), 360–363. Alminaite, A., Backström, V., Vaheri, A., Plyusnin, A., 2008. Oligomerization of hantaviral nucleocapsid protein: charged residues in the N-terminal coiled-coil domain contribute to intermolecular interactions. Journal of General Virology 89, 2167–2174. Alminaite, A., 2010. Structure and functions of hantavirus nucleocapsid protein. Doctoral Dissertation. Helsinki University Press, University of Helsinki. http://ethesis.helsinki.fi. Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201. Bharat, T.A., Riches, J.D., Kolesnikova, L., Welsch, S., Krähling, V., Davey, N., Parsy, M.L., Becker, S., Briggs, J.A., 2011. Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells. PLoS Biology 9 (11), e1001196. Bonneau, R., Strauss, C.E., Rohl, C.A., Chivian, D., Bradley, P., Malmström, L., Robertson, T., Baker, D., 2002. De novo prediction of three-dimensional structures for major protein families. Journal of Molecular Biology 322 (1), 65–78. Bouloy, M., 2011. Molecular biology of Phleboviruses. In: Plyusnin, A., Elliott, R.M. (Eds.), Bunyaviridae: Molecular and Cellular Biology. Caister Academic Press, Norfolk, UK, pp. 95–128. Buchan, D.W., Ward, S.M., Lobley, A.E., Nugent, T.C., Bryson, K., Jones, D.T., 2010. Protein annotation and modelling servers at University College London. Nucleic Acids Research 38 (Suppl.), W563–W568. Cole, C., Barber, J.D., Barton, G.J., 2008. The Jpred secondary structure prediction server. Nucleic Acids Research 36 (Suppl.), W197–W201. Ferron, F., Li, Z., Danek, E.I., Luo, D., Wong, Y., Coutard, B., Lantez, V., Charrel, R., Canard, B., Walz, T., Lescar, J., 2011. The hexamer structure of Rift Valley fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes. PLoS Pathogens 7 (5), e1002030. Flick, R., Pettersson, R.F., 2001. Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs. Journal of Virology 75 (4), 1643–1655. Flick, K., Katz, A., Överby, A., Feldmann, H., Pettersson, R.F., Flick, R., 2004. Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments. Journal of Virology 78 (21), 11726–11738. Ge, P., Tsao, J., Schein, S., Green, T.J., Luo, M., Zhou, Z.H., 2010. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science 327 (5966), 689–693. Green, T.J., Zhang, X., Wertz, G.W., Luo, M., 2006. Structure of the vesicular stomatitis virus nucleoprotein–RNA complex. Science 313 (5785), 357–360. Hastie, K.M., Kimberlin, C.R., Zandonatti, M.A., MacRae, I.J., Saphire, E.O., 2011a. Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3 to 5 exonuclease activity essential for immune suppression. Proceedings of the National Academy of Sciences of the United States of America 108 (6), 2396–2401.
A. Katz et al. / Virus Research 167 (2012) 118–123 Hastie, K.M., Liu, T., Li, S., King, L.B., Ngo, N., Zandonatti, M.A., Woods Jr., V.L., de la Torre, J.C., Saphire, E.O., 2011b. Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding. Proceedings of the National Academy of Sciences of the United States of America 108 (48), 19365–19370. Katz, A., Freiberg, A.N., Backström, V., Schulz, A.R., Mateos, A., Holm, L., Pettersson, R.F., Vaheri, A., Flick, R., Plyusnin, A., 2010. Oligomerization of Uukuniemi virus nucleocapsid protein. Virology Journal 7, 187. Kaukinen, P., Vaheri, A., Plyusnin, A., 2003. Mapping of the regions involved in homotypic interactions of Tula hantavirus N protein. Journal of Virology 77 (20), 10910–10916. Kelley, L.A., Sternberg, M.J.E., 2009. Protein structure prediction on the web: a case study using the Phyre server. Nature Protocols 4 (3), 363–371. Leonard, V.H., Kohl, A., Osborne, J.C., McLees, A., Elliott, R.M., 2005. Homotypic interaction of Bunyamwera virus nucleocapsid protein. Journal of Virology 79 (20), 13166–13172. Ng, A.K., Zhang, H., Tan, K., Li, Z., Liu, J.H., Chan, P.K., Li, S.M., Chan, W.Y., Au, S.W., Joachimiak, A., Walz, T., Wang, J.H., Shaw, P.C., 2008. Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. FASEB Journal 22 (10), 3638–3647. Oker-Blom, N., Salminen, A., Brummer-Korvenkontio, M., Kääriäinen, L., Weckström, P., 1964. Isolation of some viruses other than typical tick-borne encephalitis viruses from Ixodes ricinus ticks in Finland. Annales medicinae experimentalis et biologiae Fenniae 42, 109–112. Överby, A.K., Popov, V., Neve, E.P., Pettersson, R.F., 2006. Generation and analysis of infectious virus-like particles of Uukuniemi virus (Bunyaviridae): a useful system for studying bunyaviral packaging and budding. Journal of Virology 80 (20), 10428–10435. Överby, A.K., Pettersson, R.F., Grünewald, K., Huiskonen, J.T., 2008. Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus.
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Proceedings of the National Academy of Sciences of the United States of America 105 (7), 2375–2379. Pettersson, R.F., von Bonsdorff, C.H., 1975. Ribonucleoproteins of Uukuniemi virus are circular. Journal of Virology 15 (2), 386–392. Qi, X., Lan, S., Wang, W., Schelde, L.M., Dong, H., Wallat, G.D., Ly, H., Liang, Y., Dong, C., 2010. Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature 468 (7325), 779–783. Raman, S., Vernon, R., Thompson, J., Tyka, M., Sadreyev, R., Pei, J., Kim, D., Kellogg, E., DiMaio, F., Lange, O., Kinch, L., Sheffler, W., Kim, B.H., Das, R., Grishin, N.V., Baker, D., 2009. Structure prediction for CASP8 with all-atom refinement using Rosetta. Proteins 77 (Suppl. 9), 89–99. Raymond, D.D., Piper, M.E., Gerrard, S.R., Smith, J.L., 2010. Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation. Proceedings of the National Academy of Sciences of the United States of America 107 (26), 11769–11774. Rost, B., Yachdav, G., Liu, J., 2003. The PredictProtein Server. Nucleic Acids Research 32 (Suppl.), W321–W326. Roy, A., Kucukural, A., Zhang, Y., 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols 5 (4), 725–738. Rudolph, M.G., Kraus, I., Dickmanns, A., Eickmann, M., Garten, W., Ficner, R., 2003. Crystal structure of the Borna disease virus nucleoprotein. Structure 11 (10), 1219–1226. Ruigrok, R.W., Crepin, T., Kolakofsky, D., 2011. Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Current Opinion in Microbiology 14 (4), 504–510. Tawar, R.G., Duquerroy, S., Vonrhein, C., Varela, P.F., Damier-Piolle, L., Castagné, N., MacLellan, K., Bedouelle, H., Bricogne, G., Bhella, D., Eléouët, J.F., Rey, F.A., 2009. Crystal structure of a nucleocapsid-like nucleoprotein–RNA complex of respiratory syncytial virus. Science 326 (5957), 1279–1283. Ye, Q., Krug, R.M., Tao, Y.J., 2006. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444 (7122), 1078–1082.
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Short communication
Mutational analysis of positively charged amino acid residues of Uukuniemi phlebovirus nucleocapsid protein Anna Katz a,∗ , Alexander N. Freiberg b , Vera Backström a,1 , Liisa Holm c , Antti Vaheri a , Ramon Flick b,2 , Alexander Plyusnin a a
Research Programs Unit, Infection Biology Research Program, Department of Virology, Haartman Institute, P.O. Box 21, University of Helsinki, 00014 Helsinki, Finland Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA c Structural Genomics Group, Institute of Biotechnology, P.O. Box 56, University of Helsinki, 00014 Helsinki, Finland b
a r t i c l e
i n f o
Article history: Received 17 February 2012 Received in revised form 16 March 2012 Accepted 4 April 2012 Available online 17 April 2012 Keywords: Negative-strand RNA virus Phlebovirus Uukuniemi virus Nucleocapsid protein Mutational analysis RNA binding Oligomerization
a b s t r a c t The aim of this study was to evaluate the contribution of positively charged amino acid residues for the Uukuniemi virus (UUKV) N protein functionality. Based on phlebovirus nucleocapsid (N) protein alignments and 3D-structure predictions of UUKV N protein, 14 positively charged residues were chosen as targets for the mutagenesis. The impact of mutations to the N protein functionality was analyzed using minigenome-, virus-like particle-, and mammalian two-hybrid-assays. Seven of the mutations affected the functional competence in all three assays, while others had milder impact or no impact at all. In the 3D-model of UUKV N protein, five of the affected residues, R61, R64, R73, R98 and R115, were located either within or in close proximity to the central cavity that could potentially bind RNA. © 2012 Elsevier B.V. All rights reserved.
Uukuniemi virus (UUKV) belongs to the family Bunyaviridae, and there to the genus Phlebovirus, which contains important pathogens, such as Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Punta Toro virus (PTV) (Bouloy, 2011). The virus was originally isolated from Ixodes ricinus ticks in Finland (Oker-Blom et al., 1964) serving ever since as a convenient model for studying the bunyavirus structure and function (Flick and Pettersson, 2001; Flick et al., 2004; Katz et al., 2010; Pettersson and von Bonsdorff, 1975; Överby et al., 2008). The viral genome is composed of three single-stranded RNA segments: negative-sense L (large) and M (medium) segments, whereas the S (small) segment encodes the nucleocapsid (N) and non-structural (NSs) proteins using an ambisense strategy. The RNA segments are encapsidated by the N protein to form ribonucleoprotein complexes (RNPs), the functional templates for viral RNA synthesis (Bouloy, 2011). This mechanism
∗ Corresponding author. Tel.: +358 9 19126707; fax: +358 9 19126491. E-mail addresses: anna.katz@helsinki.fi (A. Katz), [email protected] (A.N. Freiberg), [email protected] (V. Backström), liisa.holm@helsinki.fi (L. Holm), antti.vaheri@helsinki.fi (A. Vaheri), rfl[email protected] (R. Flick), alexander.plyusnin@helsinki.fi (A. Plyusnin). 1 Present address: Software Point, Valkjärventie 1, 02130 Espoo, Finland. 2 Present address: BioProtection Systems, A Subsidiary of Newlink Genetics Corporation, 2901 South Loop Drive, Suite 3360, Ames, IA 50010, USA. 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.04.003
is shared by all negative-strand RNA viruses (NSRV) (Ruigrok et al., 2011). The details of the RNP-formation remained unsolved for many NSRV until very recently. Progress in cryo-electron tomography and crystallization techniques has led to solving various N protein and RNP structures for several NSRV (Ruigrok et al., 2011). The structures of RNPs vary from more relaxed in segmented bunya-, arena-, and influenza viruses (Ferron et al., 2011; Hastie et al., 2011a, 2011b; Raymond et al., 2010; Ye et al., 2006) to more helical in nonsegmented rhabdo- and filoviruses, giving the characteristic shape for the virion (Bharat et al., 2011; Ge et al., 2010). The ring-shaped RNPs of respiratory syncytial, rabies, and vesicular stomatitis viruses contain 10–11 N protein molecules (Albertini et al., 2006; Green et al., 2006; Tawar et al., 2009), whereas the N protein of Borna disease virus was crystallized as a tetramer (Rudolph et al., 2003). In most viruses, RNA was observed inside the N ring-structures. The nucleoprotein of influenza virus forms trimers (Ng et al., 2008; Ye et al., 2006), suggesting a ring of nine molecules as an RNA-binding unit with a positively charged cleft that probably binds the RNA (Ng et al., 2008). The first N protein structures of RVFV (Raymond et al., 2010) and Lassa virus (LASV, genus Arenavirus) (Qi et al., 2010) were recently defined, followed by more detailed structures revealing the mechanism for the N protein oligomerization and RNA-binding (Ferron et al., 2011; Hastie et al., 2011b). Interestingly, in both viruses the shift
A. Katz et al. / Virus Research 167 (2012) 118–123
of the N-terminal arm of N protein was crucial for RNA-binding. For LASV N protein, a specific gating mechanism was a key in the presented model (Hastie et al., 2011b), showing that RNA-free N protein trimer was unable to bind RNA. After a conformational change the shift of the N-terminal arm revealed the RNA-binding cavity. Ferron et al. (2011) presented a hexameric ring structure for RVFV N protein with proposed sites for RNA-binding and oligomerization. In contrast to the first structure (Raymond et al., 2010), here the N-terminal arm was extended from the molecule core, exposing the RNA-binding cavity in the central part of the protein. Even though the N protein of RVFV can oligomerize without RNA, Ferron et al. (2011) suggested that stabilization of the N protein oligomers might require an association with RNA. The aim of this study was to evaluate the contribution of positively charged aa residues to the functionality of UUKV N protein. Toward this aim we analyzed sequence alignments of the Phlebovirus N proteins and found 11 positively charged residues, conserved throughout the genus (Fig. 1). To gain more information of the fold of UUKV N protein, the 2D-structure was predicted using Jpred-, Psipred- and PredictProtein-servers (Cole et al., 2008; Buchan et al., 2010; Rost et al., 2003). The analyses suggested N protein to be a compact, globular protein formed of 11–13 ␣-helices. Since at the starting point of this study there were no N protein 3D-structures solved for any bunyavirus, the ab initio protein structure prediction server, Robetta (Bonneau et al., 2002) was chosen to guide our mutational analysis. Ten models were created for UUKV N protein and examined to identify surfaces suitable for putative RNA-binding. Since UUKV N was predicted to be a globular protein, we concentrated mostly on Robetta models fulfilling these criteria. In most of the models, the cluster of positively charged residues was observed in the central part of the molecule. When choosing the aa residues for mutation, models with a cavity in the central part of the N protein were favored, since in other NSRV, this kind of structures are known to bind RNA (Ruigrok et al., 2011). The conserved aa residues were chosen for the mutational analysis, some of them located in the potential RNA-binding cavity. The resulting set contained 13 single and two double mutants: R44A, KK50-51AA, R61A, R64A, R73A, K76A, KR82-83AA, R98A, R115A, H178A, R187A, R194A, K223A, R224A and K238A. All mutants, except the double mutant KR82-83AA were generated and tested. To investigate the N–RNA interactions directly, we attempted to establish a gel-shift assay using N protein mutants and in vitro-transcribed RNAs. Unfortunately our efforts were not successful, therefore the N protein mutants were analyzed using the minigenome- and virus-like particle (VLP)-systems, where the RNA-binding ability of the N protein is crucial (Flick and Pettersson, 2001; Överby et al., 2006). The N protein functionality was also tested in the mammalian two-hybrid (M2H) system, which has been shown to be efficient in studying bunyavirus N protein oligomerization (N–N-interactions) (Kaukinen et al., 2003; Alminaite et al., 2008; Katz et al., 2010). First, the functional competence of the mutants was investigated using the RNA Pol I-driven minigenome system (Flick and Pettersson, 2001), which requires that the N protein mutants are capable to bind to the viral RNA and support its transcription (and replication) by the viral polymerase. The plasmid pcDNAUUKV-N encoding the wild type (wt) UUKV N protein was used to generate point mutations using a site-directed mutagenesis kit (Stratagene) as described previously (Katz et al., 2010). Integrity of all constructs was verified by both restriction analysis and sequencing, and expression of the N protein mutants was confirmed by immunoblotting (Fig. S1) as described previously (Katz et al., 2010). BHK-21 cells were transfected with the UUKV N-, UUKV L- and UUKV GN /GC -protein expressing plasmids and the UUKV M-CAT minigenome, an UUKV M segment-based minigenome containing
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the reporter gene chloramphenicol acetyltransferase (CAT). The functionality of the N protein mutants was monitored by CAT reporter gene expression. Five mutations (R61A, R64A, R73A, R115A and R223A) severely affected the N protein functionality and three mutations (R98A, H178A and R224A) had a more moderate impact on the functionality (Fig. 2A and Table 1). The remaining six mutations did not show any altering effect (Fig. 2A and Table 1). Next, the mutants were tested in the previously established Uukuniemi VLP-system (Överby et al., 2006). This system allows to investigate if the N protein mutants, as an integral part of RNPs, can get incorporated into VLPs and drive minigenome transcription in VLP-infected cells. Even if the N mutant would be fully functional in the minigenome system and capable to support RNA transcription/replication, the VLP system should reveal the defects in the VLP assembly, e.g. packaging of vRNA into the VLPs. The method for VLP generation and infection was described previously (Katz et al., 2010). Briefly, minigenome and expression plasmids were transfected into BHK-21 cells and supernatant harvested 24 h post-transfection. VLP/minigenome-containing supernatants were then passaged to fresh cells, pre-transfected with the wt UUKV N- and L-expressing plasmids to support minigenome expression. When all components were functional, UUKV minigenomecontaining VLPs were generated, which was monitored by the expression of CAT-reporter gene in the VLP-infected cells. As expected, in the negative control for VLP-infected cells (transfection without GN /GC ) (Fig. 2B, lane 2), no CAT activity was detected. The positive control containing the wt UUKV N and GN /GC showed CAT activity (Fig. 2B, lane 3), demonstrating that VLPs were generated and minigenomic RNA was successfully transferred into VLP-infected cells. The results observed with the VLP system were in agreement with those of the minigenome system. The overall CAT activities were lower than in minigenome system, since VLP-infected cells receive only a few copies of minigenomic RNA through VLP infection. The N protein functionality was also tested in the M2H assay, which is primarily designed to test the protein–protein interactions. Whether the N protein oligomerization and interaction with RNA are coupled in all bunyaviruses remains to be seen. For Tula hantavirus N protein, it was shown that RNase treatment weakens the N–N-interaction in M2H system (Alminaite, 2010). In contrast, Leonard et al. (2005) showed that the N protein of Bunyamwera virus can oligomerize in the presence of high RNase concentrations, and it was suggested also for the N protein of Lassa virus that the RNA-binding might not be required for the N protein oligomerization (Hastie et al., 2011b). For RVFV, it was suggested that the N protein can oligomerize without RNA, but the stabilization of the oligomers might need an RNA bound to the structure (Ferron et al., 2011). Next, the M2H assay was employed to evaluate the overall impact of the mutations to the UUKV N protein functionality. Altogether 11 out of 14 mutations were introduced into plasmids pM-UUKV-N (DNA-BD) and pVP-UUKV-N (DNA-AD) as described earlier (Katz et al., 2010). In this study, the N protein mutations were introduced to both DNA-BD- and DNA-AD-counteracting plasmids. The effect of having the mutation in only one of the partners was tested with the mutation R73A. When the mutation was introduced into both partners (e.g. pM-R73A vs. pVP-R73A), the interaction, in three separate assays, was decreased to 12% (Table 1). However, when the mutation was tested against wt N protein, e.g. pM-wt-N vs. pVP-R73A, the interaction was decreased to 38–46%, while in the assay with pM-R73A vs. pVP-N-wt these values were 73–89%. These data suggested, that the wt N protein can compensate the harmful affect of the mutation in the other counterpart in the M2H assay. Therefore each mutation was tested against the same mutation in the other counterpart, e.g. pM-R73A vs. pVP-R73A. The expression
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Fig. 1. Multiple alignment on phlebovirus N proteins shows the positively charged residues throughout the genus. Conserved aa residues are shown in white on red background, invariant residues in red on white background and the variable residues in black on white background. The UUKV N protein aa residues which were mutated are marked with triangles (). This ClustalW alignment shows also RVFV N protein (PDB: 3OV9) aa residues R64, K67 and K74, marked with stars (), which were suggested to bind RNA. The viruses and GenBank accession numbers for the N protein sequences are: Uukuniemi virus [GI 38371708], Toscana virus [GI 52627074], Corfou virus [GI 146336856], Punta Toro virus [GI 127918], Candiru virus [GI 328545956], Aguacate virus [GI 330850814], and Rift Valley fever virus [GI 87622293]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of the N fusion protein mutants was confirmed using immunoblotting as described previously (Katz et al., 2010). Three mutations, R187A, R194A and K238A were excluded from the M2H assay since these were fully competent in the minigenome system, while two mutations, R44A and KK50-51AA, were tested to have representatives of fully competent mutations also in the M2H system. The M2H assay was performed as described previously (Katz et al., 2010). Briefly, HeLa cells were co-transfected with plasmids expressing the N protein mutants and reporter plasmids expressing the firefly (FL) luciferase and renilla (RL) luciferase
(Promega). Reporter gene activities were determined in triplicates with the Dual-Luciferase Reporter Assay System (Promega). Each experiment was performed three times and the FL-values were normalized using the RL-values. The M2H results correlated with those in the minigenome system: seven mutations (R64A, R73A, R98A, R115A, H178A, K223A and R224A) appeared detrimental for the N protein functionality while the impact of mutations R61A and K76A was minor (Fig. 2C and Table 1). The mutants R44A and KK50-51AA, which were fully competent in the minigenome system, were found functional in the M2H assay as well. The mutation
Table 1 Functionality data of UUKV N protein point mutants in the minigenome, VLP and mammalian two-hybrid (M2H) assays. UUKV N protein point mutations
Minigenomea
VLPa
M2H % of interactionb
wt N protein R44A KK50-51AA R61A R64A R73A K76A R98A R115A H178A R187A R194A K223A R224A K238A
+++ +++ +++ − − − ++ + − + +++ +++ − + +++
++ ++ ++ − − − + − − − ++ ++ − − ++
100 89 ± 11 >100 82 ± 25 7±4 12 ± 6 78 ± 18 32 ± 19 18 ± 10 18 ± 6 ND ND 3±1 35 ± 12 ND
a
In minigenome (and VLP systems) the level of CAT expression ranged between non-affected (+++), reduced (++), substantially reduced (+), and completely abolished (−). Full-length N–N protein interaction (100%, +++) was compared to the N–N interaction of point mutants. The mutations were introduced to both interacting plasmid partners in the M2H system. The interaction ranged from not affected (+++) to reduced (++) and to substantially reduced interaction (+). Each test was performed three times in triplicates. b
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Fig. 2. (A) Reporter gene activity based on UUKV N protein functionality in minigenome transcription: expression of N protein mutants was determined in a minigenome system using CAT reporter gene expression as described earlier (Katz et al., 2010). BHK-21 cells were transfected with UUKV M segment-based minigenome plasmid (UUKV M-CAT) and expression plasmids coding for the viral polymerase (pCMV-UUKV-L), glycoproteins (pCMV-GN /GC ), and N protein mutants. Lane 1: negative control (without UUKV L), lane 2: VLP control (without UUKV GN /GC ) lane 3: positive control (with wt UUKV N, UUKV L and UUKV GN /GC ). Single and double aa point mutations within the N protein are listed above the individual CAT signals. (B) Reporter gene activity based on UUKV N protein functionality in VLP assembly, minigenome packaging and transcription: BHK-21 cells were first transfected with UUKV L and wt UUKV N expression plasmids before VLPs with minigenome-containing supernatant aliquots from the minigenome-expressing cells (Fig. 1a) were transferred. VLP infection incorporates the minigenome encapsidated by the different UUKV N mutants and transcription levels are monitored by CAT assay. (C) Reporter gene expression based on UUKV N protein functionality in the M2H assay: the impact of the N protein mutations to the N–N interactions was monitored as firefly luciferase expression. The values were normalized with the renilla luciferase and compared to the wt N expression (set as 100%, lane 3). Negative control (vectors pM1 and pVP16 without N protein) is shown in lane 2 and positive control (wt N protein in both vectors, pM1 and pVP16) in lane 3 (ND = not determined).
R61A appeared to be peculiar: in the M2H assay the impact of this mutation was minor, but in the minigenome system it damaged the protein functionality severely. This could imply that this residue is directly involved in the RNA-binding process but not in the N protein oligomerization. Two recent publications allowed for gaining more insights into the RNA-binding process in phleboviruses. Raymond et al. (2010) presented RVFV N protein structure, the first among the Bunyaviridae. It was soon followed by another RVFV N protein structure (Ferron et al., 2011), which also revealed the mechanism of N protein oligomerization. In this structure, the patch of positively
charged residues in the inner cleft of the N protein probably accommodates vRNA. We utilized the latter structure (RVFV N protein, PDB code 3OV9) to create new 3D-models for the UUKV N protein, using three established servers: I-Tasser (Roy et al., 2010), Phyre2 (Kelley and Sternberg, 2009), and Swissmodel (Arnold et al., 2006), all highly ranked in the CASP modeling contests (Raman et al., 2009). Three resulting models resembled each other and adopted the folds very similar to that of RVFV N protein. The model created using I-Tasser is shown in Fig. 3. The majority of our experimental data with the N protein mutants (Table 1 and Fig. 2) was in good agreement with the presented 3D-fold of the UUKV N protein.
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Fig. 3. The I-Tasser server 3D-model for UUKV N protein. The model was built based on the RVFV N protein structure (PDB code: 3OV9) (Ferron et al., 2011), showing that most of the affected aa residues (shown in red) are located within the central cavity of the molecule or in the close proximity of it, in the proposed RNA-binding surface of UUKV N protein. The aa residues which were not affected by the mutations are shown in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Mutation of six aa residues, R44, KK50-51, R187, R194 and K238A had no affect in the N protein functionality in the minigenome and M2H assays (Table 1). These residues are located on the outer surfaces of the molecule, distant from the RNA-binding cleft. Some of these residues are seen in Fig. 3. In contrast, mutation of residues R61, R64, R73, K76, R98 and R115, located either within or next to the central RNA-binding cavity of the molecule, affected the N protein functionality severely (Fig. 3 and Table 1). Ferron et al. (2011) proposed that three RVFV N protein residues, R64, K67 and K74, are most directly involved in the RNA-binding based on the finding that the triple mutant R64D/K67D/K74D lost its ability to bind RNA. These residues correspond to R73, K76 and K82 in the UUKV N protein. In our experiments, the mutation R73A strongly affected the UUKV N protein function in minigenome/VLP and M2H systems, and the functionality of K76A was reduced. The UUKV N protein functionality was affected also by mutation of residues H178, K223 and R224, of which residues K223 and R224 correspond to residues R214 and R215 in the N protein of RVFV. According to Ferron et al. (2011), these residues are located in the proposed oligomerization groove. Involvement in the oligomerization could explain the affect we observed in our assays, although the role of the residue H178 remained unclear. The RVFV N protein model (Ferron et al., 2011) shows also that the N-terminal arm of the protein is involved in the oligomerization through interaction(s) with the C-terminal part of the adjacent N protein molecule. This is in good agreement with the results of our previous work on UUKV N protein (Katz et al., 2010), where we observed that the N-terminus of the protein is crucial for oligomerization. To summarize, minigenome-, VLP- and M2H-data support the 3D-models of UUKV N protein based on the solved structure of RVFV N protein. Residues R61, R64, R73, R98 and R115, located in the central cavity of the N protein molecule, might contribute to the RNA-binding, while some other positively charged residues, such as K223 and R224, might be important for the oligomerization.
Acknowledgments This work was supported by the Helsinki Biomedical Graduate School, Sigrid Jusélius Foundation, and Finnish Cultural Foundation. We are grateful to late Ralf Pettersson for valuable comments and friendly support.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.virusres.2012.04.003. References Albertini, A.A., Wernimont, A.K., Muziol, T., Ravelli, R.B., Clapier, C.R., Schoehn, G., Weissenhorn, W., Ruigrok, R.W., 2006. Crystal structure of the rabies virus nucleoprotein–RNA complex. Science 313 (5785), 360–363. Alminaite, A., Backström, V., Vaheri, A., Plyusnin, A., 2008. Oligomerization of hantaviral nucleocapsid protein: charged residues in the N-terminal coiled-coil domain contribute to intermolecular interactions. Journal of General Virology 89, 2167–2174. Alminaite, A., 2010. Structure and functions of hantavirus nucleocapsid protein. Doctoral Dissertation. Helsinki University Press, University of Helsinki. http://ethesis.helsinki.fi. Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201. Bharat, T.A., Riches, J.D., Kolesnikova, L., Welsch, S., Krähling, V., Davey, N., Parsy, M.L., Becker, S., Briggs, J.A., 2011. Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells. PLoS Biology 9 (11), e1001196. Bonneau, R., Strauss, C.E., Rohl, C.A., Chivian, D., Bradley, P., Malmström, L., Robertson, T., Baker, D., 2002. De novo prediction of three-dimensional structures for major protein families. Journal of Molecular Biology 322 (1), 65–78. Bouloy, M., 2011. Molecular biology of Phleboviruses. In: Plyusnin, A., Elliott, R.M. (Eds.), Bunyaviridae: Molecular and Cellular Biology. Caister Academic Press, Norfolk, UK, pp. 95–128. Buchan, D.W., Ward, S.M., Lobley, A.E., Nugent, T.C., Bryson, K., Jones, D.T., 2010. Protein annotation and modelling servers at University College London. Nucleic Acids Research 38 (Suppl.), W563–W568. Cole, C., Barber, J.D., Barton, G.J., 2008. The Jpred secondary structure prediction server. Nucleic Acids Research 36 (Suppl.), W197–W201. Ferron, F., Li, Z., Danek, E.I., Luo, D., Wong, Y., Coutard, B., Lantez, V., Charrel, R., Canard, B., Walz, T., Lescar, J., 2011. The hexamer structure of Rift Valley fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes. PLoS Pathogens 7 (5), e1002030. Flick, R., Pettersson, R.F., 2001. Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs. Journal of Virology 75 (4), 1643–1655. Flick, K., Katz, A., Överby, A., Feldmann, H., Pettersson, R.F., Flick, R., 2004. Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments. Journal of Virology 78 (21), 11726–11738. Ge, P., Tsao, J., Schein, S., Green, T.J., Luo, M., Zhou, Z.H., 2010. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science 327 (5966), 689–693. Green, T.J., Zhang, X., Wertz, G.W., Luo, M., 2006. Structure of the vesicular stomatitis virus nucleoprotein–RNA complex. Science 313 (5785), 357–360. Hastie, K.M., Kimberlin, C.R., Zandonatti, M.A., MacRae, I.J., Saphire, E.O., 2011a. Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3 to 5 exonuclease activity essential for immune suppression. Proceedings of the National Academy of Sciences of the United States of America 108 (6), 2396–2401.
A. Katz et al. / Virus Research 167 (2012) 118–123 Hastie, K.M., Liu, T., Li, S., King, L.B., Ngo, N., Zandonatti, M.A., Woods Jr., V.L., de la Torre, J.C., Saphire, E.O., 2011b. Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding. Proceedings of the National Academy of Sciences of the United States of America 108 (48), 19365–19370. Katz, A., Freiberg, A.N., Backström, V., Schulz, A.R., Mateos, A., Holm, L., Pettersson, R.F., Vaheri, A., Flick, R., Plyusnin, A., 2010. Oligomerization of Uukuniemi virus nucleocapsid protein. Virology Journal 7, 187. Kaukinen, P., Vaheri, A., Plyusnin, A., 2003. Mapping of the regions involved in homotypic interactions of Tula hantavirus N protein. Journal of Virology 77 (20), 10910–10916. Kelley, L.A., Sternberg, M.J.E., 2009. Protein structure prediction on the web: a case study using the Phyre server. Nature Protocols 4 (3), 363–371. Leonard, V.H., Kohl, A., Osborne, J.C., McLees, A., Elliott, R.M., 2005. Homotypic interaction of Bunyamwera virus nucleocapsid protein. Journal of Virology 79 (20), 13166–13172. Ng, A.K., Zhang, H., Tan, K., Li, Z., Liu, J.H., Chan, P.K., Li, S.M., Chan, W.Y., Au, S.W., Joachimiak, A., Walz, T., Wang, J.H., Shaw, P.C., 2008. Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. FASEB Journal 22 (10), 3638–3647. Oker-Blom, N., Salminen, A., Brummer-Korvenkontio, M., Kääriäinen, L., Weckström, P., 1964. Isolation of some viruses other than typical tick-borne encephalitis viruses from Ixodes ricinus ticks in Finland. Annales medicinae experimentalis et biologiae Fenniae 42, 109–112. Överby, A.K., Popov, V., Neve, E.P., Pettersson, R.F., 2006. Generation and analysis of infectious virus-like particles of Uukuniemi virus (Bunyaviridae): a useful system for studying bunyaviral packaging and budding. Journal of Virology 80 (20), 10428–10435. Överby, A.K., Pettersson, R.F., Grünewald, K., Huiskonen, J.T., 2008. Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus.
123
Proceedings of the National Academy of Sciences of the United States of America 105 (7), 2375–2379. Pettersson, R.F., von Bonsdorff, C.H., 1975. Ribonucleoproteins of Uukuniemi virus are circular. Journal of Virology 15 (2), 386–392. Qi, X., Lan, S., Wang, W., Schelde, L.M., Dong, H., Wallat, G.D., Ly, H., Liang, Y., Dong, C., 2010. Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature 468 (7325), 779–783. Raman, S., Vernon, R., Thompson, J., Tyka, M., Sadreyev, R., Pei, J., Kim, D., Kellogg, E., DiMaio, F., Lange, O., Kinch, L., Sheffler, W., Kim, B.H., Das, R., Grishin, N.V., Baker, D., 2009. Structure prediction for CASP8 with all-atom refinement using Rosetta. Proteins 77 (Suppl. 9), 89–99. Raymond, D.D., Piper, M.E., Gerrard, S.R., Smith, J.L., 2010. Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation. Proceedings of the National Academy of Sciences of the United States of America 107 (26), 11769–11774. Rost, B., Yachdav, G., Liu, J., 2003. The PredictProtein Server. Nucleic Acids Research 32 (Suppl.), W321–W326. Roy, A., Kucukural, A., Zhang, Y., 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols 5 (4), 725–738. Rudolph, M.G., Kraus, I., Dickmanns, A., Eickmann, M., Garten, W., Ficner, R., 2003. Crystal structure of the Borna disease virus nucleoprotein. Structure 11 (10), 1219–1226. Ruigrok, R.W., Crepin, T., Kolakofsky, D., 2011. Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Current Opinion in Microbiology 14 (4), 504–510. Tawar, R.G., Duquerroy, S., Vonrhein, C., Varela, P.F., Damier-Piolle, L., Castagné, N., MacLellan, K., Bedouelle, H., Bricogne, G., Bhella, D., Eléouët, J.F., Rey, F.A., 2009. Crystal structure of a nucleocapsid-like nucleoprotein–RNA complex of respiratory syncytial virus. Science 326 (5957), 1279–1283. Ye, Q., Krug, R.M., Tao, Y.J., 2006. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444 (7122), 1078–1082.