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1 Department of Virology, Haartman Institute, PO Box 21, FIN-00014 University of Helsinki, Finland
2 Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland
Correspondence
Agne Alminaite
agne.alminaite{at}helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-helix are especially important. Here, the importance of charged amino acid residues located within the coiled-coil for trimer formation and oligomerization was analysed. To predict the interacting surfaces of the monomers, the previous in silico model of TULV coiled-coils was first upgraded, taking advantage of the recently published crystal structure of the N-terminal coiled-coil of the Sin Nombre virus N protein. The results obtained using a mammalian two-hybrid assay suggested that conserved, charged amino acid residues within the coiled-coil make a substantial contribution to N protein oligomerization. This contribution probably involves (i) the formation of interacting surfaces of the N monomers (residues D35 and D38, located at the tip of the coiled-coil loop, and R63 appear particularly important) and (ii) stabilization of the coiled-coil via intramolecular ionic bridging (with E55 as a key player). It is hypothesized that the tips of the coiled-coils are the first to come into direct contact and thus to initiate tight packing of the three structures.
These authors contributed equally to this paper. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The S RNA genome segment of hantaviruses carried by Arvicolinae, Sigmodontinae and Neotominae rodents, in addition to the N protein, encodes a non-structural (NSs) protein in an overlapping (+1) open reading frame (Jääskeläinen et al., 2007). Currently, 22 hantavirus species are known and the list is increasing. Hantaviruses are carried by rodents and insectivores, and some cause haemorrhagic fever with renal syndrome or hantavirus (cardio)pulmonary syndrome when transmitted to humans (Vapalahti et al., 2003).
The hantaviral N protein is the most abundant viral component in both virions and infected cells. It is a multifunctional molecule involved in several important steps of virus replication and assembly, initially, in RNA encapsidation (Kaukinen et al., 2005). In the course of infection, the N protein interacts with viral RNAs to form ribonucleoprotein (RNP), with the L protein and probably also with the cytoplasmic tail of Gn, as well as with different cellular proteins (Ravkov & Compans, 2001; Li et al., 2002; Xu et al., 2002; Kaukinen et al., 2003a; Maeda et al., 2003; Kukkonen et al., 2004; Severson et al., 2005). The N protein is synthesized in the cytoplasm and is later transported to the perinuclear region, where assembly of new virions begins (Ravkov & Compans, 2001; Kaukinen et al., 2004; Kukkonen et al., 2004).
The molecular mass of the hantaviral N protein is approximately 50 kDa and its length varies from 429 to 433 aa residues in different hantaviruses. Sequence analysis suggests three conserved domains separated by two more-variable regions. The RNA-binding domain, which includes aa 175–217, has been located in the central part of the molecule (Xu et al., 2002; Severson et al., 2005). Regions involved in interactions with viral or cellular proteins remain to be mapped.
Similar to the nucleocapsid proteins of other negative-strand RNA viruses, hantaviral N protein can oligomerize. It forms some dimers but mostly trimers, which are thought to serve as intermediates in the process of oligomerization and RNP formation (Alfadhli et al., 2001; Kaukinen et al., 2001, 2003b, 2004; Mir & Panganiban, 2004). Both C- and N-terminal domains of the N protein contribute to the oligomerization. The N-terminal domain folds into a coiled-coil (Alfadhli et al., 2001; Alminaite et al., 2006; Boudko et al., 2007) and the helix–loop–helix structure has been found to be essential for the oligomerization capacity of the C-terminal domain (Kaukinen et al., 2004). The current data are consistent with the ‘head-to-head, tail-to-tail’ model for hantavirus N protein interaction (Kaukinen et al., 2003b, 2004; Alminaite et al., 2006). This model suggests a two-step process that involves an initial interaction between the N-terminal coiled-coils followed by a consolidating interaction between the C-terminal domains. According to our model of the N protein trimer, anti-parallel coiled-coils of the monomers do not undergo intensive conformational changes and stay folded; thus, the interacting forces are generated mainly by charged or polar residues, which are located on opposite sides of the helix relative to the hydrophobic residues (Alminaite et al., 2006). This model is supported by earlier data (Alfadhli et al., 2002) and also by our observation that increased ionic strength inhibits interaction(s) between N protein molecules (Kaukinen et al., 2003b). Notably, some charged residues located at ‘e’ positions of heptad repeats contribute towards stabilizing the coiled-coil structure by forming intramolecular ionic bridges (Boudko et al., 2007). Further details of the interaction between the N-terminal coiled-coils remain unknown.
The aim of this study was to evaluate the contribution of charged residues within the N-terminal coiled-coil to N protein oligomerization. Towards this aim, we improved our previous model of the Tula virus (TULV) N-terminal coiled-coil monomer, which was based on a DNA topoisomerase dispensable domain (Alminaite et al., 2006), taking into account the recently published crystal structure of the N-terminal coiled-coil of Sin Nombre virus (SNV) N protein (Boudko et al., 2007). Targets for point mutagenesis were selected based on this improved model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Alignment of the TULV N protein (GenBank accession no. Z69991
[GenBank]
) and the SNV N protein (Uniprot Q89462) sequences was used to construct a model of the TULV N protein coiled-coil domain using the MODELLER program, version 8.1 (Sali & Blundell, 1993). Validation of the model was carried out by calculating solvation profiles with the SolvX Server (Holm & Sander, 1992) and evaluating the Ramachandran plot (Ramachandran et al., 1963) provided by the WhatIf webserver (Vriend, 1990).
In silico docking of three N protein monomers.
A combination of manual and automatic docking using the ClusPro server (Comeau et al., 2004) was applied to bring together three monomers in a putative oligomerization mode. The two highest ranked models of trimers were chosen and verified by visual inspection.
Point mutagenesis.
The plasmids pM1-TULVN and pVP16-TULVN encoding the N protein of TULV, strain Moravia, fused with either the GAL4 DNA-binding domain (DBD) or VP16 activation domain (AD) have been generated previously (Kaukinen et al., 2003b). Point mutations were introduced into these plasmids using a site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. New constructs were characterized by sequencing using an ABI Prism Dye Terminator sequencing kit (Perkin-Elmer). A mammalian two-hybrid (M2H) assay was performed essentially as described previously (Kaukinen et al., 2004). In brief, HeLa cells were cultivated in Eagle's minimal essential medium supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, and penicillin and streptomycin on 24-well culture plates to approximately 70 % confluency and transfected with 0.3 µg plasmid construct based on pM1-TULVN or pVP16-TULVN, 0.5 µg reporter plasmid pGluc (expressing firefly luciferase) and 0.01 µg control plasmid pRL-SV40 (expressing Renilla luciferase; Promega). Except for controls, in which plasmids pM1-TULVN and pVP16-TULVN encoding the wild-type N protein of TULV were used, both interacting partners contained point mutations. Transfections were performed in triplicate with 3 µl FuGENE 6 reagent for each transfection according to the manufacturer's instructions (Roche Diagnostics) and, after 24 h, the luciferase activities were determined using a Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase values were normalized to the Renilla luciferase values and the interaction was calculated as described previously (Kaukinen et al., 2003b). P values were calculated using Student's t-test and an f-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), we built a model of the N-terminal domain of TULV N protein (aa 3–73) based on the crystal structure of the DNA topoisomerase dispensable domain (PDB 1K4T, chain A; Staker et al., 2002). This model, based on a modest sequence identity (26 %), nevertheless proved useful for prediction of amino acid residues that stabilize the coiled-coil (Fig. 1a). Following the recent release of the SNV N protein coiled-coil domain crystal structure (PDB 2IC6, chain B; Boudko et al., 2007), we constructed a new, hopefully more accurate, molecular model for TULV N protein (Fig. 1b). The sequence identity between the N-terminal regions of TULV and SNV N proteins (aa 1–76) is 71 % (Fig. 2). It is widely accepted (see, for example, Sander & Schneider, 1991 and Rost, 1997) that such a value is high enough to imply identical c- traces (positions of atoms in the main chain) of TULV and SNV N-terminal domains.
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). The only visible difference was that the new model had a slightly stronger curvature than the old one. The solvation profile (a measure of solvent accessibility for each residue within a protein; a well-packed structure should have an overall solvation preference below zero) calculated for assessing the quality of the new model using the SolvX server corresponded well to that of the crystal structure. The overall residue-by-residue solvation preference for the TULV N protein model and for the original crystal structure of SNV N protein were in the same range (–51.4 and –40.1, respectively), indicating that both structures are well-refined. Also, the Ramachandran Z scores (which express how well the backbone conformations of all residues correspond to the known allowed areas in the Ramachandran plot) for the two structures were within the range expected for well-refined structures: 5.143 and 4.423, respectively.
Prediction of interacting surfaces
In order to picture possible N protein trimerization modes, we docked three monomers, both manually and automatically. The results of the automatic docking were inspected visually and two trimer models were chosen. These models were used to predict the interacting surfaces of the monomers.
Our previous results showing that an increase in ionic strength abolished the interaction between N protein molecules (Kaukinen et al., 2003b) suggested that at least some charged amino acid residues belonged to the interacting surface. We examined the conservation of charged residues among different hantaviruses and, considering their spatial position (relative to the leucine zipper) in the 3D TULV N models, selected the most probable candidates to form interacting surfaces involved in the oligomerization. The following charged residues were selected for point mutagenesis: R22, K24, K26, E29, K30, E33, D35, D37, D38, K41, R47, R48, R63, D67 and K73. We also selected residue E76, which is located just outside the crystallized part of the coiled-coil, but which, according to our docking results, also belonged to the interacting surface.
According to the crystal structure of the SNV N protein N-terminal domain, three of these residues, R22, E29 and R48, are involved in the formation of intramolecular ionic bridges: R22–E55 and E29–R48 (Boudko et al., 2007). Based on these data, E55 was added to the mutagenesis list.
Point mutagenesis of charged amino acid residues
The TULV N-terminal coiled-coil region (aa 1–73) contains 29 conserved charged amino acid residues (Fig. 2). Based on the predictions of interacting surfaces, we mutated 16 selected charged residues to alanine and evaluated the interacting capacity of the resultant mutants using a M2H assay. It was expected that the removal of a relevant charge would have an inhibitory effect in the N–N interaction. The results of point mutagenesis showed that the majority of charged amino acid residues did indeed contribute to the interaction. The data presented in Fig. 3 show the N protein mutants ascribed to three groups according to their interacting capacities. Group 1 comprised four mutants that showed a non-inhibited interacting capacity of the N protein molecules: K24A, K41A, R47A and K73A (interacting activity 
100 %). Group 2 comprised nine mutants with moderately affected oligomerization capacity (remaining interacting activity 80 %): R22A, K26A, E29A, K30A, E33A, D37A, R48A, D67A and E76A. Group 3 comprised four mutants showing somewhat more strongly affected oligomerization (remaining interacting activity 44–80 %): D35A, D38A, E55A and R63. Taking into account the fact that the values shown in Fig. 3 are for single mutants, such effects of single-point mutations should be considered substantial. To confirm that the reduced oligomerization capacity of the group 3 mutants was not due to lower protein expression levels, we tested the capacity of the mutants to interact with the wild-type N protein. With one exception (mutant D35A; see Discussion), no reduced interaction capacity of the mutants was seen (data not shown), thus confirming that they were expressed properly.
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-helices, the key players in the oligomerization process. This was in agreement with our previous observations (Kaukinen et al., 2003b; Alminaite et al., 2006).
The amino acid residues from mutant groups 2 and 3 were not scattered randomly throughout the coiled-coil 3D structure (Fig. 4). Some formed clusters (e.g. R22, E29 and E33 in the first helix and D35, D37 and D38 in the tip), whilst the side chains of others were similarly oriented and also came into close proximity to each other (e.g. R48 and E55 in the helix). Residues K26 and K30 in the first helix and residues R63 and D67 in the second helix pointed slightly outwards, but could also form an interacting surface together with the R22/E29/E33 cluster (note that the interacting surface is probably not flat). Interestingly, when subjected to a M2H assay, mutations D35A, D37A and D38A had different impacts on the oligomerization capacity and the double mutation D37A/D38A showed a statistically significant cumulative effect (Fig. 3). This implied that D37, together with D35 and D38, belongs to the interacting surface.
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). It cannot be excluded that, due to their flexibility, they could also be involved in intermolecular interactions between the N protein monomers. The mutants E29A and R48A showed a reduced interacting capacity and, most importantly, the cumulative effect of the two mutations was apparent: the remaining interaction capacities of the corresponding constructs were 57 % compared with 88 and 90 %, respectively (Fig. 5). Unexpectedly, the double mutant R22A/E55A did not show a substantially reduced oligomerization capacity, although mutation of one of the residues, E55, had a strong effect. The reason(s) for this remains unknown.
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). The double mutation E33A/R48A showed a cumulative effect when compared with the single mutations E33A and R48A: the remaining interaction capacities of the corresponding constructs were 67 % compared with 83 and 90 %, respectively. Furthermore, the single mutation R48E had a similar (also statistically significant) effect on oligomerization (64 %), suggesting an important role for the R48 residue in the interacting surface. Interestingly, the oligomerization capacity was not restored in the mutant E33R/R48E (Fig. 5), suggesting that not only charges but also the nature of the two amino acid residues is important. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), we demonstrated that an intact coiled-coil structure of the N terminus is crucial for the oligomerization capacity of the N protein and that the hydrophobic ‘a’ residues from the second -helix are especially important. Here, we demonstrated that conserved charged amino acid residues within the coiled-coil also make a critical contribution to the oligomerization. This contribution probably involves (i) formation of interacting surfaces of the N monomers and (ii) stabilization of the coiled-coil via intramolecular ionic bridging. Our main instrument, the M2H assay, of course, has its limitations, such as modest sensitivity. Nevertheless, the efficiency of mutant evaluation using the M2H assay was reasonably high and our data were in agreement with the results of cross-linking experiments using synthetic peptides described by Alfadhli et al. (2002).
To predict the interacting surfaces of the monomeric coiled-coils, we first tried to upgrade our in silico model of the N-terminal coiled-coil. Our previous model was based on the crystal structure of DNA topoisomerase and 26 % sequence identity between its dispensable domain and TULV N protein N-terminal domain. We took advantage of the recently published crystal structure of the N-terminal coiled-coil of SNV N protein (Boudko et al., 2007), which has 71 % of its sequence identical to the TULV N coiled-coil, allowing us to imply identical c- traces. The resulting model showed excellent Ramachandran Z scores and a solvation preference value, and provided important new knowledge, for example about intramolecular ionic bridges. It also allowed stronger confidence in prediction of the interacting surfaces of monomers (Fig. 4) and higher precision of in silico docking of the monomers into a trimer (Fig. 6). Finally, the new model confirmed that our selection of the first set of amino acid residues, which supposedly stabilize the coiled-coil structure (Alminaite et al., 2006), was well justified.
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and 5). Notably, the amino acid residues supposedly contributing to interaction(s) between the coiled-coils appeared clustered within the 3D structure (Fig. 4). Two of the four most crucial residues, D35 and D38, are located at the tip, and the double mutation D37A/D38A had a cumulative effect, suggesting that the tips of the coiled-coils are highly reactive and are especially important for the oligomerization. Interestingly, the mutant D35A showed a reduced interaction capacity (remaining activity of approximately 70 %, for both DBD and AD domains), even in combination with the wild-type N protein. This result could reflect unusually low expression levels for both mutant constructs. Alternatively, it could suggest that residue D35 is an essential player in the interacting surface of the coiled-coil. One would imagine that the tips are the first to come into direct contact and thus to initiate tight packing of the three coiled-coils (Fig. 6).
The results of the M2H assay demonstrated an important contribution of charges, which are involved in the formation of ionic bridges, both intra- and intermolecular, in the oligomerization process. The capacity of the ‘e’ residues R22, E29, E55 and especially R48 both to stabilize the coiled-coil and to form an interacting surface raises the intriguing possibility of conformational change(s) of the N protein molecule, which might follow trimerization or even be a prerequisite for the formation of longer oligomers. For instance, ‘switching’ of R48 from intra- to intermolecular ionic bridging when the other N protein molecule comes into close proximity could cause a conformational change and imply a certain degree of asymmetry to the N-trimer structure, which is probably needed for oligomerization to continue along the viral RNA molecule. Naturally, the binding of the N protein to the viral RNA is another likely candidate for the driving force behind the conformation change(s), in a way similar to other RNA viruses, including negative-strand RNA viruses (van Marle et al., 1995; Spencer & Hiscox, 2006; Albertini et al., 2008).
One cannot exclude the possibility of indirect N–N protein interaction(s) via cellular proteins and/or RNA. However, the results of our in silico docking (Fig. 6) suggest that the contribution of direct interaction(s) is substantial.
Coiled-coils are well-known protein-interacting modules (for a review, see Lupas, 1996; Burkhard et al., 2001). There are many examples of coiled-coils mediating interactions between viral proteins and also between viral and cellular proteins (Bullough et al., 1994; Chen et al., 1999; Möller et al., 2005; DiCarlo et al., 2007). Characteristically, such famous interactors as sarc homology 2 domains (which bind to phosphorylated tyrosine residues and are intensively involved in signalling) often include a coiled-coil as their structural element (see, for example, Hale et al., 2008). In addition to hantaviruses, the involvement of coiled-coils in nucleocapsid protein oligomerization was recently reported for Marburg virus (family Filoviridae) (DiCarlo et al., 2007), suggesting that this might be a general mechanism shared by several negative-strand RNA viruses. This would make these structures a promising target for antiviral therapies.
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ACKNOWLEDGEMENTS |
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Received 8 May 2008;
accepted 29 May 2008.
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80 %) as open bars. The interaction value of the wild-type N protein is shown as a hatched bar. Numbers above the columns show the means±SD of normalized luciferase activity values (%). The mean values were calculated from at least three independent experiments and each mutant was tested in triplicate. *, P<0.05; **, P<0.1.
