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1 Stockholm Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
2 Institut für Virologie, The Calicilab, Medizinisch-Theoretisches Zentrum, Dresden, Germany
3 Institute of Cell and Molecular Biology, Uppsala University, Sweden
Correspondence
Jacques Rohayem
Jacques.Rohayem{at}mailbox.tu-dresden.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
Published online ahead of print on 7 November 2008 as DOI 10.1099/vir.0.005629-0
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2008). NV belongs to the family Caliciviridae, a virus family including human and non-human pathogenic strains. The genus Norovirus is divided into five genogroups. Strains belonging to genogroups 1, 2 and 4 infect humans, with the genogroup 2 strains being predominantly detected over the years. Animal NV strains, i.e. murine or bovine NV strains cluster mainly within genogroup 5 or 3, respectively (Zheng et al., 2006). The murine NV so far remains the only strain of the NV species that can be propagated in cultured cells.
NV is a non-enveloped RNA virus with a single-stranded positive-oriented genome ranging from 7.3 to 8.5 kb (Green, 2007). The viral genome is organized into three open reading frames (ORF): ORF-1 encodes the non-structural proteins, ORF-2 encodes the capsid protein or virion protein 1 (VP1) and ORF-3 encodes the virion protein 2 (VP2) (Fig. 1a). Downstream from ORF-2, an untranslated region (UTR) is present (Green, 2007), which is followed by a poly(A) tail of variable length. ORF-1 is predicted to encode a single polyprotein that is co-translationally processed, resulting in the generation of the viral enzymes of replication. Among those, the RNA-dependent RNA polymerase (RdRp) non-structural protein 7 (NS7) plays a key role in the synthesis and amplification of genomic RNA.
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have described the interaction between the NV RdRp and its RNA template. However, it remains unclear as to whether RNA synthesis by the NV RdRp is regulated by the interaction of two monomeric forms, resulting in positive cooperativity. In this study, the structural and functional features of NV NS7 homodimers have been investigated. The structure of the protein in a new crystal form has been resolved by X-ray crystallography to 2.3 Å (0.23 nm) resolution, displaying a dimer-of-dimers arrangement in the crystal lattice. The possible three-dimensional structural arrangement of NV NS7 homodimers was analysed using the five available crystal forms of the protein. Furthermore, we present the first experimental evidence for positive cooperativity of NV NS7 monomers, showing that RNA synthesis by NV NS7 is increased by dimerization of the monomers.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, b). Protein concentration was determined with the BCA Protein assay kit (Pierce) based on the Biuret reaction. The fraction containing the recombinant protein was diluted in glycerol (50 % v/v) and stored at –20 °C.
Crystallization of NV NS7 protein.
The purified NV NS7 was concentrated to 9 mg ml–1. The concentrated sample was centrifuged at 10 000 g for 5 min to remove aggregates arising from the concentration procedure. Subsequent crystallization trials were carried out at room temperature and 4 °C leading to optimized conditions of 1 M tri-sodium citrate, 125 mM NaCl and 0.1 M sodium cacodylate, pH 6.5 at 20 °C.
The diffraction data (Table 1) were collected by using the beamline ID23-1 (European Synchotron Radiation Facility). The diffraction data were measured to 2.3 Å resolution and the data were processed and scaled using the programs XDS and XSCALE. The phases were obtained by molecular replacement using the program MOLREP of the CCP4 program package and NV RdRp (PDB ID 1SH0
[PDB]
) as the model (Ng et al., 2004). The crystals were primitive orthorhombic in space group P212121 with unit cell dimensions of a=90.5 (9.05 nm), b=125.6 (12.56 nm) and c=218.1 Å (21.81 nm), and contain four molecules in the asymmetric unit. The model was built using consecutive rounds of ARP/wARP (Lamzin et al., 1999), manual building in O (Jones et al., 1991) and refinement with REFMAC5 (Murshudov et al., 1997). The coordinates and structure factors have been deposited in the PDB with ID code 2B43. A summary of the model quality is given in Table 1.
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). Briefly, the purified proteins were mixed with 2x native PAGE sample buffer (100 mM Tris/HCl, pH 9.0, 40 % glycerol (v/v), 0.5 % Coomassie brilliant blue G) and incubated for 10 min at room temperature. The samples were loaded onto a 6–12 % gradient gel. Cathode (100 mM Tris-histidine, pH 8.0, 0.002 % Coomassie brilliant blue G) and anode buffer (100 mM Tris/HCl, pH 8.8) were applied to a vertical electrophoresis chamber (Hoefer) and Coomassie brilliant blue G (Serva) was added to a final concentration of 0.002 % (w/v) to the cathode buffer. Electrophoresis was carried out at 15 V cm–1 for 14–16 h at 4 °C. The gel was then washed with stripping solution (25 % methanol, 10 % acetic acid). For verification of the correct size of the proteins, the protein band was excised from the native PAGE and incubated for 20 min in denaturing solution (350 mM Tris/HCl, pH 6.8, 10.0 % SDS, 36.0 % glycerol, 5.0 % β-mercaptoethanol, 0.012 % bromphenol blue). The gel stripes were then embedded in the stacking gel of a vertical electrophoresis chamber (Hoefer). Electrophoresis for separation of the proteins was carried out at 80 V for the stacking gel (5 %) and at 120 V for the separation gel (15 %). Proteins were subsequently visualized by Western blot using an anti-NS7 polyclonal antibody as described previously (Rohayem et al., 2006b).
Investigation of the equilibrium conditions of RNA synthesis by NV NS7.
The equilibrium conditions were characterized by measuring the amount of RNA synthesized through de novo initiation by NV NS7 in vitro using homopolymeric poly(C)20-RNA templates (GE Healthcare) in the presence of GTP as described previously with slight modifications (Rohayem et al., 2006a, b). Briefly, the reaction mixture consisted of 5 µg synthetic poly(C)20-RNA template (20 nt in length, yielding a final concentration of 14.7 µM), 50 mM HEPES (pH 7.0), 3 mM magnesium acetate, 4 mM DTT, 6 µM ZnCl2, 50 U RNase inhibitor (RNAsin; Promega), 0.5 mM rGTP and RNase-DNase-free water to a final volume of 50 µl. In each reaction, 1.25 µM purified NV NS7 was added and the reaction carried out at 37 °C. It was stopped by adding 50 µl stop solution (4 M ammonium acetate, 100 mM EDTA) and purified with the Microcon Ultracel JM-10 columns (Millipore) according to manufacturer's instructions. The amount of RNA synthesized was measured in a time-course experiment at 5, 10, 15, 20 and 25 min after initiation of the reaction. For this purpose, 3 µl of the reaction was used in the Quant-iT RiboGreen RNA assay kit (Invitrogen) according to manufacturer's instructions. Fluorescence was measured on an Infinite F200 reader (Tecan).
Characterization of the Kd of homodimerization of NS7 monomers.
To determine the affinity constants for NS7 dimerization, the RdRp enzymic reaction was used as a tool to measure an apparent Kd for the interaction of the NS7 monomers. Therefore, the enzymic activity of NS7 was measured in the presence of increasing concentrations of the active-site mutant µNS7. The Kd measured here depends on the values Pi and Pmax, where Pi is the amount of RNA synthesized in a set period of time, here 15 min, and Pmax is the maximal amount of RNA synthesized in a set time period in the presence of saturating concentrations of NS7 or µNS7. The Kd as well as the Pi and Pmax were determined using a non-linear regression curve-fitting program with the Prism 4 software (GraphPad software).
Assessment of the cooperativity between NS7 monomers.
The cooperativity between two NS7 monomers was investigated using the Hill plots as described by others (Goodrich & Kugel, 2007). Therefore, NS7 was titrated and the amount of synthesized RNA was determined at each concentration of NS7. Next, the ratio P=Pi/Pmax was calculated. Pi relates to the amount of RNA synthesized for a given concentration, and Pmax to the maximal amount of RNA synthesized. To determine the Hill coefficient, the value log (P/1–P) was plotted against the logarithmic value of the NS7 concentration. Importantly, only log (P/1–P) values ranging between –0.9 and 0.9 were used, resulting in a linear regression. The correlation coefficient as well as the slope of the line were measured, giving the Hill coefficient nH. An nH>1 indicates positive cooperativity between NS7 monomers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Analogously, the active-site mutant of NV NS7 (µNS7) was expressed and purified. The active-site mutant displayed the same characteristics in terms of solubility and migration in SDS-PAGE (Fig. 1b).
Structure of NV NS7 protein
The structure of the NS7 protein from NV strain Hu/NLV/Dresden174/1997/GE has been determined to 2.3 Å resolution (PDB ID 2B43). The model was refined to an R-factor of 19.2 % and free R-factor of 25.7 % and contains 501 aa (aa 6–506) and 1122 water molecules. The overall structure (Fig. 1c) is, as expected, very similar to the previously determined structure of NV NS7 (Ng et al., 2004). A comparison of the structural similarities of different RdRps of the families Caliciviridae and Pircornaviridae is shown in Table 2.
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) and the recent structure of NV polymerase in complex with primer–template RNA (Zamyatkin et al., 2008) is most likely due to structural flexibility, since the sequence identity is very high 98–99 % in the structured regions. The main differences are observed in the loop region made up of residues 369–380 (Fig. 1d) and in the C-terminal region. In the uncomplexed structures, this region is located in the active-site cleft (Fig. 1e). In the previously published NV structure (PDB ID 1SH0
[PDB]
), the C-terminal residues interact mainly with residues 218–220 preceding the 
7 helix, while in the structure published in this study (PDB ID 2B43), the C terminus interacts mainly with residues 441–443 of the 13 helix in the active-site cleft. These interactions also lead to slight changes in the backbone structure of the 13 helix and the N-terminal part of the 7 helix. In the RNA complex structure (PDB ID 3BSO), RNA binding displaces the C-terminal segment from the active-site cleft and it is not observable in the electron density map (Zamyatkin et al., 2008).
NV NS7 forms homodimers in a concentration-dependent manner
In order to investigate the interaction of NV NS7 monomers, native PAGE was run using increasing concentrations of the purified protein. As shown in Fig. 2(a), a protein of a molecular mass of about 120 kDa was observed starting at a concentration of about 0.500 µM. The intensity of the protein stained on the native gel increased with increased concentration of NS7. For further characterization, the protein at a concentration of 2.000 µM was excised from the native gel, denatured and analysed by SDS-PAGE. Western blot analysis revealed that the protein migrated at about 57 kDa, indicating that the excised protein of about 120 kDa consisted of two monomers of 57 kDa each (Fig. 2b).
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, the amount of RNA synthesized until 25 min displayed a linear correlation (r2=0.98) with the time of reaction, indicating that the active enzyme complex synthesizes RNA at a constant rate in the presence of saturating amounts of RNA substrate (14.7 µM). Based on this data, the time point chosen where the reaction is at equilibrium was 15 min.
In a further step, the Kd of homodimerization was determined in a series of reactions in which the concentration of NS7 is kept constant (1.25 µM), and the concentration of µNS7 is varied over a range extending from 10-fold below the putative Kd to 10-fold above the putative Kd. By measuring the amount of RNA synthesized at 15 min, the Kd as well as the Pi and Pmax were determined using a non-linear regression curve-fitting program (Fig. 2d). The apparent calculated Kd±SEM was 0.649±0.109 µM.
Multimeric arrangement of NV NS7 RdRp
In the present structure, the NV NS7 crystallizes in space group P212121 with four monomers in the asymmetric unit. The protein arranges as a dimer-of-dimers tetramer (Fig. 3a). Given the biochemical data supporting higher activity of the dimeric protein, an interesting question is whether the active dimer is represented in the crystal packing of this crystal, or the other crystal forms available for the protein. To investigate this issue, we analysed the crystal packing of the five available crystal forms for the NV NS7 protein represented by structures NV NS7 PDB ID 1SH0
[PDB]
, 1SH2, 1SH3, 2B43 and 3BSO (Table 3). The crystal packing was examined to define plausible C2-symmetrical dimeric arrangements. The resulting dimers were analysed for interacting surface area and surface shape complementarity (SC) as defined by others (Lawrence & Colman, 1993). SC values and interaction surface areas were calculated using the CCP4 programs: SC and Areaimol, respectively (Lawrence & Colman, 1993; Lee & Richards, 1971).
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). The interface areas (sum of the interacting area of both subunits) in the different dimeric arrangements ranges from 1528 to 2271 Å2. The buried surfaces are thus in all cases of average size for protein–protein interactions, or even somewhat larger (Lo Conte et al., 1999). The SC statistic on the other hand clearly puts the dimeric arrangements into two groups. The NV NS7 PDB ID 1SH2
[PDB]
and 1SH3 structures produce, as mentioned before, very similar dimers that also have very poor SC values, well below what is to be expected for a protein–protein complex. In contrast, the NV NS7 PDB ID 1SH0
[PDB]
and 2B43 crystal lattices contain dimeric arrangements with very good SC statistics. It should be mentioned that these dimeric interactions, unlike NV NS7 PDB ID 1SH2
[PDB]
and 1SH3, are fundamentally different.
NV NS7 monomers display positive cooperativity
The interdependence between RNA synthesis by NV NS7 and dimerization was investigated using the enzymic assay described previously. When increasing the concentration of NS7 in the reaction, the amount of RNA synthesized increases until a plateau phase (Fig. 4a), indicating an equilibrium shift from the monomeric form of the enzyme into the dimeric form. In order to determine whether a positive cooperativity between the monomers occurs, the Hill plots were used. As shown in Fig. 4(b), the amount of RNA synthesized correlated linearly with the NS7 concentration (r2=0.99). The Hill coefficient nH±SEM was 1.86±0.13, indicating positive cooperativity between the NS7 monomers.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ng et al., 2004) were determined. The NV NS7 RdRp resembles the right-hand configuration of many DNA and RNA polymerases (Biswal et al., 2005; Choi et al., 2004; Lesburg et al., 1999; Ng et al., 2002, 2004; Thompson & Peersen, 2004). Moreover, the functional features of the NV NS7 have been dissected in vitro (Rohayem et al., 2006a, b). However, it remains unclear whether the active form of the NV NS7 RdRp is a homodimer displaying cooperative activity. This question is of relevance, in particular in the light of innovative therapeutic strategies aiming at disassembling the replication complex of NV through inhibition of viral enzymes interaction. In this study, the structure and functional properties of NV NS7 homodimers have been determined for the first time. According to our structural data, NV NS7 monomers interact to form homodimers. Our results also show that homodimerization of NV NS7 monomers is concentration-dependent, and that the apparent Kd of homodimerization lies within the nM range. Moreover, the monomers display a positive cooperativity in vitro.
In order to investigate the structural and functional features of the NV NS7 RdRp, a domain encompassing the predicted NV RNA polymerase motifs A–E was designed. This domain displayed homologies to other RdRps of calicivirus, i.e. the lagovirus, sapovirus and vesivirus. After amplification of the encoding DNA and subsequent cloning in a prokaryotic expression vector, expression and purification yielded the soluble recombinant protein. In addition, an active-site mutant of NV NS7 was generated.
The structure of NV NS7 RdRp from strain Hu/NLV/Dresden174/1997/GE was resolved to 2.3 Å resolution. Although the sequence is highly similar to the previously determined structures of NV NS7 RdRp (PDB ID 1SH0
[PDB]
), some differences in the structure are observed (Fig. 1d and e). Because of the high sequence identity, the observed differences are most likely indicative of structural flexibility in these parts. It is interesting to note that the C terminus in the present structure (PDB ID 2B43) is located in the active-site cleft as in the previous structure (PDB ID 1SH0
[PDB]
), although in a different conformation and making different interactions in the cavity. This is most likely due to a combination of flexibility and the fact that the present structure (PDB ID 2B43) contains a C-terminal His-tag. Although missing from the electron density map, it is interesting to note that this drastic change in physicochemical properties of the C terminus does not prevent binding to the active-site cleft.
In the next step, we investigated whether the NV NS7 RdRp is active in a monomeric or dimeric form. Therefore, dimerization of NS7 was examined on native gels with increasing concentrations of the protein. Discontinuous native gel electrophoresis that is suitable for analysis of oligomeric proteins under native conditions was used (Niepmann & Zheng, 2006). The difference between the discontinuous native gel electrophoresis and the classical native Laemmli Tris-glycine buffer system is the use of Tris-histidine gel buffer and a polyacrylamide gradient, allowing a reliable characterization of the physical properties of a protein consistent with its size, oligomeric state and shape (Niepmann & Zheng, 2006). As observed in Fig. 2(a), NV NS7 dimerizes in a concentration-dependent manner. This observation is in accordance with previous studies in other viral RdRps such as in vesivirus, hepatitis C virus and influenza virus (Jorba et al., 2008; Kaiser et al., 2006; Wang et al., 2002). For those proteins, dimerization but also oligomerization of monomeric subunits of the polymerase complex have been evidenced.
In order to investigate further the dimerization of NV NS7 in solution, size exclusion chromatography (SEC) was performed. With this assay, the NS7 protein eluted as a single peak corresponding in size to the monomer (data not shown). The reasons for such a discrepancy are various. One possible explanation could lie in the lower resolution of SEC in comparison to discontinuous native gel electrophoresis (Niepmann & Zheng, 2006), the dimers not being eluted, as such, out of the column in SEC. Furthermore, discrepancies in the systems used for characterization of the oligomerization status of a protein may be related to the physical separation principles of the systems, such as gravity flow in the case of SEC versus voltage in the case of gel electrophoresis. This is the case for the polypyrimidine tract-binding protein that binds to the picornavirus RNA, where discontinuous native protein gel electrophoresis yields different products than those observed by SEC (Perez et al., 1997; Song et al., 2005), but also for hepatitis C virus NS3 protein (Dumont et al., 2006; Levin & Patel, 1999; Sikora et al., 2008). Moreover, and as observed in other viral RdRps (Wang et al., 2002), the NS7 monomers may not obligatorily interact in a stable complex during RNA synthesis, but rather enhance a certain point of the reaction, such as initiation or release of the product. This may explain the lack of dimerization under SEC conditions, in contrast to RNA synthesis conditions where positive cooperativity was evidenced.
The apparent Kd of the NV NS7–NS7 homodimer was measured using an enzymic assay as described by others (Goodrich & Kugel, 2007). The enzymic assay was designed in such a way that the concentration of the substrate is sufficiently high and the reaction time limited enough to ensure that the amount of RNA synthesized increases linearly over time. This was done by assembling a reaction in the presence of saturating amounts of substrate, and stopping the reaction at 5, 10, 15, 20 and 25 min, measuring the amount of RNA synthesized and plotting it versus time. As observed in Fig. 2(c), the plot is linear through the time points used until 25 min, indicating a non-limiting rate of the reaction. Based on those parameters, a series of reactions was performed in which the concentration of NS7 is kept constant (1.25 µM) and the concentration of µNS7 is varied over a range extending from 10-fold below the putative Kd to 10-fold above the putative Kd. In the case of the addition of µNS7, the µNS7 competes with the NS7 monomer for formation of the NS7–µNS7 heterodimer. If a decrease is observed in the presence of increased concentrations of the µNS7, this will indicate that only active NS7–NS7 homodimers can convert substrate to product, whereas NS7–µNS7 heterodimers will not. Previous reports on the Kd of the RdRp of hepatitis C virus have reported a Kd for homodimerization in the nanomolar range (22 nM; Wang et al., 2002). The concentrations of µNS7 chosen were therefore chosen to range from 0 to 2 µM. The amount of RNA synthesized at 15 min was measured and the Kd as well as the Pi and Pmax determined using a non-linear regression curve-fitting program (Fig. 2d). Here, Pi is the amount of RNA synthesized in a set time period by the NS7 RdRp in the absence of µNS7, whereas Pmax is the amount of RNA synthesized in a set period of time when NS7 is saturated with µNS7. The apparent calculated Kd±SEM was 0.649±0.109 µM.
To investigate further the structural features of NS7 RdRp dimers, crystals of the NS7 protein were grown and the structure resolved at 2.3 Å resolution. The NS7 protein arranges in a dimer-of-dimers tetramer, displaying very different crystal packing than the previous structures of this protein. Biologically relevant multimeric states are commonly represented in the crystal packing even if they are of rather low affinity, although this is not necessarily the case especially for transient ones that may only contribute to some part of the reaction cycle. Counting the present structure, there are now five different crystal forms available for this protein, still, predictions by the PISA-server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) suggest that none of the lattice interactions is expected to form a stable dimer in solution. Since the catalytically relevant multimeric state is a key factor in understanding the protein function, an in-depth investigation of the structural features of multimerization was performed. In order to adopt defined dimeric structures, the protein dimers need to be C2 symmetrical, or at least possess a pseudo C2 symmetry. Indeed, dimers can only form if the same interacting surface is employed in both monomers, otherwise a polymerization or higher oligomerization would occur. We have analysed the molecular packing in the five different crystal forms to evaluate the plausibility of observed dimerization. Four of the five crystal packages produce C2-symmetrical dimers (Table 3 and Fig. 3b). Interestingly, all four dimeric arrangements are different, although the dimers observed in PDB ID 1SH2
[PDB]
and 1SH3 are essentially the same in terms of surface and geometry of interaction. In all cases, the size of the interacting surfaces are as expected for a protein–protein complex of this size, although two of the dimers display a very poor surface SC. The other two dimers, on the other hand, have a very good SC and are thus more probable candidates for a biologically relevant dimer. In order to assess the plausibility of the dimeric arrangements during the different steps of the catalytic cycle, models of the RNA-bound forms of the dimers were constructed based on RNA binding observed in the 3BSO structure. From these models, it is obvious that three of the dimers are not compatible with the full polymerization reaction of the enzyme. The dimers of the 1SH2 or 1SH3 structures would prevent template entrance. In contrast, the NV 2B43 dimer would prevent exit of the product RNA. However, the dimer observed in the 1SH0 crystal lattice seems compatible with the full reaction (Fig. 5).
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), the NS7 monomers may not obligatorily interact in a stable complex during RNA synthesis, but rather enhance a certain point of the reaction, such as initiation or release of product. If this is the case, the NV NS7 PDB ID 1SH0
[PDB]
dimer may not be the only possible form of dimerization. If the dimeric interaction enhances the initiation step, the packing observed in the present (2B43) structure may also represent a plausible arrangement considering its high SC. Based on the interaction and modelling studied performed here we are unable to tell whether the biologically relevant dimerization is represented in the available crystal packing or not; however, the analysis has picked out the most probable candidates and may serve as a good foundation to investigate this further, e.g. by mutational studies. Importantly, the NS7 proteins displayed a cooperative activity (nH±SEM=1.86±0.13), indicating that the interaction of the monomers increases the activity of the protein. Based on the structural analysis it seems most likely that the dimers observed in vitro may reflect a transient state within the replication complex of NV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Choi, K. H., Groarke, J. M., Young, D. C., Kuhn, R. J., Smith, J. L., Pevear, D. C. & Rossmann, M. G. (2004). The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation. Proc Natl Acad Sci U S A 101, 4425–4430.
Dumont, S., Cheng, W., Serebrov, V., Beran, R. K., Tinoco, I., Jr, Pyle, A. M. & Bustamante, C. (2006). RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108.[CrossRef][Medline]
Fullerton, S. W., Blaschke, M., Coutard, B., Gebhardt, J., Gorbalenya, A., Canard, B., Tucker, P. A. & Rohayem, J. (2007). Structural and functional characterization of sapovirus RNA-dependent RNA polymerase. J Virol 81, 1858–1871.
Goodrich, J. A. & Kugel, J. A. ((2007). ). Binding and Kinetics for Molecular Biologists, p. 182. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Green, K. Y. ((2007). ). Caliciviridae: The Noroviruses. In Fields Virology, 5th edn. Edited by D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman & S. E. Straus. Lippincott Williams and Wilkins.
Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47, 110–119.[CrossRef]
Jorba, N., Area, E. & Ortin, J. (2008). Oligomerization of the influenza virus polymerase complex in vivo. J Gen Virol 89, 520–524.
Kaiser, W. J., Chaudhry, Y., Sosnovtsev, S. V. & Goodfellow, I. G. (2006). Analysis of protein–protein interactions in the feline calicivirus replication complex. J Gen Virol 87, 363–368.
Lamzin, V. S., Morris, R. J., Dauter, Z., Wilson, K. S. & Teeter, M. M. (1999). Experimental observation of bonding electrons in proteins. J Biol Chem 274, 20753–20755.
Lawrence, M. C. & Colman, P. M. (1993). Shape complementarity at protein/protein interfaces. J Mol Biol 234, 946–950.[CrossRef][Medline]
Lee, B. & Richards, F. M. (1971). The interpretation of protein structures: estimation of static accessibility. J Mol Biol 55, 379–400.[CrossRef][Medline]
Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F. & Weber, P. C. (1999). Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 6, 937–943.[CrossRef][Medline]
Levin, M. K. & Patel, S. S. (1999). The helicase from hepatitis C virus is active as an oligomer. J Biol Chem 274, 31839–31846.
Lo Conte, L., Chothia, C. & Janin, J. (1999). The atomic structure of protein-protein recognition sites. J Mol Biol 285, 2177–2198.[CrossRef][Medline]
Lopman, B. A., Reacher, M. H., Van Duijnhoven, Y., Hanon, F. X., Brown, D. & Koopmans, M. (2003). Viral gastroenteritis outbreaks in Europe, 1995–2000. Emerg Infect Dis 9, 90–96.[Medline]
Lopman, B., Zambon, M. & Brown, D. W. (2008). The evolution of norovirus, the ‘gastric flu’. PLoS Med 5, e42[CrossRef][Medline]
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240–255.[CrossRef][Medline]
Ng, K. K., Cherney, M. M., Vazquez, A. L., Machin, A., Alonso, J. M., Parra, F. & James, M. N. (2002). Crystal structures of active and inactive conformations of a caliciviral RNA-dependent RNA polymerase. J Biol Chem 277, 1381–1387.
Ng, K. K., Pendas-Franco, N., Rojo, J., Boga, J. A., Machin, A., Alonso, J. M. & Parra, F. (2004). Crystal structure of Norwalk virus polymerase reveals the carboxyl terminus in the active site cleft. J Biol Chem 279, 16638–16645.
Niepmann, M. & Zheng, J. (2006). Discontinuous native protein gel electrophoresis. Electrophoresis 27, 3949–3951.[CrossRef][Medline]
Perez, I., McAfee, J. G. & Patton, J. G. (1997). Multiple RRMs contribute to RNA binding specificity and affinity for polypyrimidine tract binding protein. Biochemistry 36, 11881–11890.[CrossRef][Medline]
Rohayem, J., Jager, K., Robel, I., Scheffler, U., Temme, A. & Rudolph, W. (2006a). Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis. J Gen Virol 87, 2621–2630.
Rohayem, J., Robel, I., Jager, K., Scheffler, U. & Rudolph, W. (2006b). Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol. J Virol 80, 7060–7069.
Sikora, B., Chen, Y., Lichti, C. F., Harrison, M. K., Jennings, T. A., Tang, Y., Tackett, A. J., Jordan, J. B., Sakon, J. & other authors (2008). Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro. J Biol Chem 283, 11516–11525.
Song, Y., Tzima, E., Ochs, K., Bassili, G., Trusheim, H., Linder, M., Preissner, K. T. & Niepmann, M. (2005). Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation. RNA 11, 1809–1824.
Thompson, A. A. & Peersen, O. B. (2004). Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase. EMBO J 23, 3462–3471.[CrossRef][Medline]
Wang, Q. M., Hockman, M. A., Staschke, K., Johnson, R. B., Case, K. A., Lu, J., Parsons, S., Zhang, F., Rathnachalam, R. & other authors (2002). Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase. J Virol 76, 3865–3872.
Zamyatkin, D. F., Parra, F., Alonso, J. M., Harki, D. A., Peterson, B. R., Grochulski, P. & Ng, K. K. (2008). Structural insights into mechanisms of catalysis and inhibition in Norwalk virus polymerase. J Biol Chem 283, 7705–7712.
Zheng, D. P., Ando, T., Fankhauser, R. L., Beard, R. S., Glass, R. I. & Monroe, S. S. (2006). Norovirus classification and proposed strain nomenclature. Virology 346, 312–323.[CrossRef][Medline]
Received 16 July 2008;
accepted 31 October 2008.
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