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1 Institut für Virologie, The Calicilab, Medizinisch-Theoretisches Zentrum, Fiedlerstraße 42, D-01307 Dresden, Germany
2 Institut für Immunologie, Medizinisch-Theoretisches Zentrum, Fiedlerstraße 42, D-01307 Dresden, Germany
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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Patent pending DE no. 10 2005 036 085.8.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Infection with NV leads to vomiting and diarrhoea, which lasts for 24–48 h, generally resolving without vital complications. Despite this self-limiting character, NV is an important human pathogen, being the major cause of food-borne viral gastroenteritis, and a major public health concern (Parashar & Monroe, 2001). So far, studying NV replication has been hampered by the lack of a cell culture system for isolation and propagation of the virus. Therefore, our understanding of NV replication was limited to in vitro studies in cell-free systems. Recently, replication in cell culture of an animal NV strain, the murine NV, was reported (Wobus et al., 2004). In addition, a major breakthrough was achieved by Asanaka et al. (2005) who established a mammalian cell-based system that may allow extensive investigation of NV replication.
NV is a non-enveloped RNA virus with a single-stranded positive-sense genome of approximately 7.5 kb (7338–7708 nt) (Green et al., 2001). The viral genome consists of three open reading frames (ORF) encoding the non-structural proteins (encoded in ORF1), the capsid protein or virion protein 1 (VP1) encoded in ORF2 and a protein termed virion protein 2 (VP2), which is encoded in ORF3 that is located at the 3'-end of the genome (Green et al., 2001). ORF1 is predicted to encode a single polyprotein that is co-translationally processed by the viral protease, resulting in the appearance of non-structural proteins required for replication of the viral genome. Among those, the RNA-dependent RNA polymerase (RdRp) (3Dpol) is predicted to play an important role in the replication of the genome and the synthesis and amplification of an additional subgenomic RNA. The subgenomic RNA corresponds to the region of the genome that contains ORF2 and ORF3, and the 3'-end. Downstream from ORF3, a 42–78 nt untranslated region (UTR) is present (Green et al., 2001), which is followed by a poly(A) tail of variable length.
So far, the biochemical characterization of recombinant NV proteins has remained incomplete, in particular for the NV non-structural proteins. Recently, different authors have shown that NV 3Dpol displays in vitro an RdRp activity (Belliot et al., 2005; Fukushi et al., 2004). In those studies, however, discordant findings were reported with respect to the initiation of RNA synthesis by NV 3Dpol, and terminal transferase activity was not reported. In our study, in an attempt to characterize RNA synthesis by NV 3Dpol in more detail, concentration, temperature and metal-ion dependence of NV 3Dpol activity were examined. Interestingly, in our hands, NV 3Dpol did exhibit terminal transferase activity. Furthermore, we concluded that initiation of RNA synthesis by NV 3Dpol on heteromeric RNA template occurs de novo.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), with slight modifications. Briefly, 100 ng plasmid cDNA was added to the reaction mix (50 µl) consisting of 5 µl 10x Herculase polymerase reaction buffer (Stratagene), 0.2 mM of each of dATP, dCTP, dGTP and dTTP, 5 % DMSO, 1 mM of each primer and 5 U Herculase hotstart polymerase (Stratagene). Cycling conditions were an initial denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation for 15 s at 94 °C, annealing for 30 s at 56 °C and extension for 3 min at 72 °C. A final extension step at 72 °C was carried out for 10 min. PCR products were separated by 1 % agarose gel electrophoresis in the presence of ethidium bromide and the gel was visualized under UV light. The PCR products were purified with a QIAquick PCR purification kit (Qiagen) and sequenced. For in vitro transcription, a Megascript T7 kit (Ambion) was used. The reaction consisted of 1 µg purified cDNA, 2 µl 10x T7 reaction buffer, 20 U RNase inhibitor (40 U RNAsin µl–1; Promega), 7.5 mM of each ATP, CTP, GTP and UTP, 10 U T7-polymerase and RNase-DNase-free water to a final volume of 20 µl. The reaction was run for 2 h at 37 °C, then 20 U DNase I (Ambion) was added and the reaction incubated at 37 °C for 30 min. The reaction products were then purified with the MEGAclear kit (Ambion) according to manufacturer's instructions. RNA concentration in the sample was determined by measuring OD260.
Expression and purification of NV 3Dpol.
The DNA corresponding to the NV 3Dpol coding sequence was generated by PCR from NV full-length cDNA clone pUS-NorII (GenBank accession no. AY741811
[GenBank]
) as described above. The PCR product was molecularly cloned into the pET-28b(+) vector (Novagen) according to the manufacturer's instructions using the restriction sites NcoI and XhoI within the multiple cloning site, yielding clone pWR-Nor-3D. The expression vector was sequenced and used to transform Escherichia coli BL21(DE3)pLysS cells. The 3Dpol active-site mutant was generated from pWR-Nor-3D clone by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene) and primers 149-Nor-µpol-for (5'-CTCTTCTCCTTTTATGGTGGTGGTGAAATTGTTAGTACAG-3') and 150-Nor-µpol-rev (5'-CTGTACTAACAATTTCACCACCACCATAAAAGGAGAAGAG-3') according to the manufacturer's instructions, yielding clone pIR-Nor-3D-D343GD344G. The 3Dpol active-site mutant bears substitutions in the GDD motif of both aspartates with glycine (GD343GD344G). NV 3Dpol and 3Dpol active-site mutants were expressed in E. coli BL21(DE3)pLysS cells. For this purpose, E. coli BL21(DE3)pLysS cells were transformed with the pWR-Nor-3D and pIR-Nor-3D-D343GD344G clones. Cells were grown at 37 °C in Luria–Bertani medium with kanamycin (50 mg l–1). At an optical density of 0.6 (OD600), protein expression was induced by adding IPTG to a final concentration of 1 mM. Cultures were then incubated at 25 °C overnight. Cell pellets obtained from 250 ml cultures were washed once in 4 ml PBS and 1 % Triton X-100 (Merck). Cells were treated with DNase (10 U ml–1) for 15 min at 37 °C, then sonicated on ice and resuspended in 40 ml binding buffer [20 mM Tris/HCl (pH 7.9), 500 mM NaCl, 5 mM imidazole]. After centrifugation at 4300 r.p.m. (Multifuge 4 KS-R; Kendro) at 4 °C for 40 min, cleared lysate was obtained. The (His)6-tagged protein was bound onto a Ni–nitrilotriacetic acid (NTA)–Sepharose resin (Novagen), pre-equilibrated with binding buffer. The bound protein was washed with the binding buffer containing 60 mM imidazole and eluted with binding buffer containing 100 mM imidazole. The eluted protein was then dialysed against buffer A [25 mM Tris/HCl (pH 8.0), 1 mM 
-mercaptoethanol, 100 mM NaCl, 5 mM MgCl2, 10 % glycerol (v/v) and 0.1 % Triton X-100]. Protein concentration was determined with a BCA protein assay kit (Pierce) based on the biuret reaction. The fraction containing the recombinant protein was diluted in glycerol to a final volume of 50 % and stored at –20 °C.
Western blot analysis of the His-tagged protein was performed as described previously (Rohayem et al., 2000).
Micrococcal nuclease treatment of purified NV 3Dpol.
Purified NV 3Dpol was treated with micrococcal nuclease to remove RNA or DNA fragments that might act as primers in the 3Dpol assay. The reaction mixture consisted of purified NV 3Dpol, 1 U micrococcal nuclease (Amersham Biosciences) µl–1, 2.5 mM calcium acetate and RNase–DNase-free water to a final volume of 20 µl. The reaction was incubated at 30 °C for 30 min and stopped by adding EGTA to a final concentration of 5 mM. The activity of the polymerase was subsequently assessed in the RdRp assay.
RdRp assay.
The RdRp activity of NV 3Dpol was assessed in vitro. The reaction mix consisted of 1 µg synthetic RNA template (2473 nt in length, yielding a final concentration of 0.024 µM), 50 mM HEPES (pH 7.0), 3 mM magnesium acetate, 4 mM DTT, 6 µM ZnCl2, 50 U RNase inhibitor (RNAsin; Promega), 0.4 mM each ATP, CTP, GTP and UTP, as well as 0.07 µM [
-32P]UTP (3000 Ci mmol–1; Hartmann Analytic) when [-32P]UMP incorporation was assessed, and RNase–DNase-free water to a final volume of 50 µl. In all reactions, 3 µM of the purified NV 3Dpol was added, and the reaction was carried out at 30 °C for 2 h. It was stopped by adding 50 µl stop solution (4 M ammonium acetate, 100 mM EDTA), and purified with a MEGAclear kit (Ambion) according to the manufacturer's instructions.
Reaction products were separated on agarose gels under non-denaturing or denaturing conditions. They were visualized using either UV transillumination or autoradiography. For non-denaturing conditions, samples were resuspended in RNA-loading buffer [0.2 M formaldehyde, 1 mM EDTA, 0.1 % SDS, 4 % glycerol (v/v), 6 % formamide, 0.5 % bromophenol blue and 0.8 vol formaldehyde gel buffer]. The formaldehyde gel buffer consisted of 20 mM MOPS, 5 mM sodium acetate and 1 mM EDTA (pH 7.0). Samples were separated on 1 % agarose gels buffered with 1x formaldehyde gel buffer, 0.5 M formaldehyde, containing 0.25 µg ethidium bromide ml–1 when visualization was done using UV transillumination. Alternatively, for electrophoresis under denaturing conditions, a buffer containing 1.25 M formaldehyde was used. After heating at 65 °C for 5 min, the samples were incubated on ice for 5 min. For autoradiography, agarose gels were dried and exposed to BAS-SR 2040 film for 1 h. Autoradiographic signals were visualized on the FLA-2000 Phosphorimager (Fuji).
Incorporation of [-32P]UMP was determined by precipitation of the NV 3Dpol products with 10 % trichloroacetic acid (TCA). Therefore, 1 µl of the reaction was diluted into 150 µl sheared salmon sperm DNA (1 mg ml–1; Ambion). Fifty microlitres of the RNA–carrier DNA mixture was counted with a liquid scintillation counter, whereas another 50 µl was resuspended in 2 ml cold 10 % TCA, mixed thoroughly and incubated on ice for 10 min. The precipitate was then applied to a GF/C glass fibre filter (Whatman). The unincorporated nucleotides were removed by washing the filters with 5 ml ice-cold 10 % TCA. Finally, filters were washed with 5 ml 95 % ethanol and dried. The amount of radioactivity present in each filter was determined with a liquid scintillation counter (Winspectral 1414; Wallac). The amount of incorporated [-32P]UMP was calculated from the ratio of radioactivity in the reaction products before and after TCA precipitation.
Nuclease treatment of NV 3Dpol reaction product.
The 3Dpol reaction products were incubated with S1 nuclease (Promega), 1 µg RNA (final concentration of 0.024 µM), 1 µl 10x S1 nuclease buffer [500 mM sodium acetate (pH 4.5), 45 mM ZnSO4] with low (50 mM) or high (250 mM) NaCl concentrations and RNase–DNase-free water to a final volume of 10 µl. The reaction was incubated at 37 °C for 60 min, then stopped by the addition of 1 µl 1 M EDTA. The reaction was resuspended in denaturing loading buffer, heated to 65 °C (or boiled when appropriate) for 5 min then quickly chilled on ice, loaded onto agarose gels, then autoradiographed and/or visualized under UV-light after ethidium bromide staining.
Northern blot analysis of NV 3Dpol reaction products.
Northern blot analysis was performed as described previously (Temme et al., 1998). Briefly, 3Dpol RNA product was resolved on formaldehyde–agarose gel (1x running buffer containing 0.75 M formaldehyde) and transferred onto Hybond-N membranes (Amersham Biosciences). Blots were hybridized with [-32P]UMP radiolabelled RNA probes generated in vitro by T7-polymerase transcription as described above. For generation of sense cDNA probe, primers 67-Nor-Cap-for, consisting of the T7 promoter sequence fused at its 3'-end to a gene-specific sequence (5'-CAGAGATGCATAATACGACTCACTATAGGGAGAGATGTTAGGCAACTGGAACC-3'), and 68-Nor-Cap-rev (5'-AGCACTGCTGGGACCCGTGTA-3') were used. The RNA probe displayed a length of 330 nt and located at position 5521–5851 of the NV genome (pUS-NorII clone, GenBank accession no. AY741811
[GenBank]
). Reaction conditions, amplification cycles and identification of the products were identical to the details described above, except that the PCR extension was for 1 min. Blotted nitrocellulose membranes were subsequently washed with 1x SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0) at room temperature, then with 2x SSC/0.1 % SDS at 68 °C, followed by 0.5x SSC/0.5 % SDS at 68 °C. Membranes were autoradiographed as described.
Assessing terminal transferase activity of NV 3Dpol.
The same conditions described for assessment of NV 3Dpol activity were used with in vitro transcribed subgenomic RNA as template, except that instead of an NTP mixture, only [-32P]ATP, [-32P]GTP, [-32P]CTP or [-32P]UTP were added to the reaction. Incorporation of [-32P]AMP, [-32P]GMP, [-32P]CMP or [-32P]UMP radioactivity as well as visualization of the products were performed as described previously.
Generation of 3'-blocked RNA templates.
Synthetic RNA corresponding to the full-length sequence of NV subgenomic RNA was incubated with cordycepin triphosphate (Sigma) and yeast poly(A) polymerase (Sigma). The reaction mix consisted of 10 µg RNA (final concentration of 0.24 µM), 5 mM cordycepin triphosphate, 1x poly(A) buffer [20 mM Tris/HCl (pH 7.0), 1 mM MgCl2, 25 mM KCl, 200 µM EDTA], 600 U yeast poly(A) polymerase, 50 µg BSA ml–1 and RNase–DNase-free water to an end volume of 50 µl. After 3 h incubation at 37 °C, the reaction was purified with the MEGAclear kit (Ambion) according to the manufacturer's instructions. Products were resolved on denaturing formaldehyde–agarose gels and visualized by autoradiography.
Labelling of RNA synthesis products with [
-32P]GTP.
To test whether NV 3Dpol is able to initiate template elongation de novo, [-32P]GTP was used. The reaction was performed under the same conditions used to assess NV 3Dpol activity, except that [-32P]GTP replaced [-32P]UTP. Products were visualized by electrophoresis on an agarose gel and autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Analogously, an active-site mutant of NV 3Dpol (m3Dpol) was expressed and purified. It displayed the same characteristics in terms of solubility and migration during SDS-PAGE analysis (Fig. 1a). Western blot analysis of the elution fraction with anti-His antibodies showed a specific reactivity for both wild-type and mutated NV 3Dpol (Fig. 1b).
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). Staining of the gel with ethidium bromide revealed a product of the 3Dpol that was about twice the size of the template (Fig. 2b). In contrast, no product was generated by mutated 3Dpol (Fig. 2a and b). Upon denaturation of products from the 3Dpol reaction on a 1.25 M formaldehyde–agarose gel, the reaction product was resolved, indicating that the product resulting from NV 3Dpol RNA synthesis was not covalently linked to template RNA (Fig. 2c).
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). However, decreasing NaCl concentrations (50 mM) in the S1 nuclease reaction allowed partial digestion of the synthesized RNA, as expected in the case of low ionic strength buffer that promotes electrostatic repulsion and reduces association of complementary strands (Fig. 3b). Denaturation of NV 3Dpol products by heating at 95 °C followed by incubation with S1 nuclease allowed their complete digestion (Fig. 3b). Northern blot analysis using a minus-sense RNA probe detected the template strand RNA in the reaction product (Fig. 4a and b), indicating that NV 3Dpol synthesis product consists of two complementary RNA strands, namely the template RNA and its complementary strand.
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-32P]UMP and enzyme concentration in the reaction. As shown in Fig. 5(a), the amount of UMP incorporated increased with the concentration of NV 3Dpol up to 0.9 µM, where the reaction reached its saturation phase. The temperature dependence of 3Dpol activity was also determined. NV 3Dpol was found to display a higher activity at 30 °C compared with 37 °C (Fig. 5b). Based on those results, a time-course experiment was performed at 30 °C with an NV 3Dpol concentration of 3 µM. Accumulation of RNA products was shown to be linear up to 90 min, followed by a saturation phase that remained constant until 180 min (Fig. 5c). In addition, the effect of both actinomycin D and rifampicin on NV 3Dpol activity was examined. Actinomycin D inhibits DNA-dependent synthesis of RNA by binding to double-stranded template DNA (Sambrook & Russel, 2001), whereas rifampicin strongly inhibits prokaryotic DNA-dependent RNA polymerase (Sambrook & Russel, 2001). Neither actinomycin D nor rifampicin were able to inhibit RNA synthesis by NV 3Dpol RNA-polymerase (data not shown), indicating no cross-contamination of the purified protein with bacterial DNA or DNA-dependent RNA polymerase that is responsible for background RNA synthesis that is not specific for NV 3Dpol.
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). In addition, NV activity was up to 30-fold higher in the presence of Mg2+ or Mn2+, in comparison to Fe2+. These results indicate that NV 3Dpol displays flexibility with respect to the use of Mg2+ or Mn2+ as a cofactor.
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) and Poliovirus (Arnold et al., 1999), adding nucleotides to the 3'-ends of newly synthesized RNAs. This activity could potentially be used by RNA viruses as a mechanism to restore the 3'-initiation site of RNA synthesis (Ranjith-Kumar et al., 2001), and is one of the prerequisites for initiation of RNA synthesis by back-priming. Indeed, initiation of RNA synthesis by back-priming relies upon terminal transferase activity of the RNA polymerase, extending at the 3' terminus a heteromeric or homopolymeric tail that primes RNA synthesis. To characterize the terminal transferase activity of NV 3Dpol, the enzyme was incubated with subgenomic RNA template in a reaction mixture containing the [-32P] labelled version of only one of the NTPs. NV 3Dpol displayed terminal transferase activity that was higher in the presence of UTP and CTP than GTP or ATP (Fig. 7a). To investigate the impact of terminal transferase activity in RNA synthesis by NV 3Dpol, a time-course experiment was performed. Terminal transferase activity and RNA synthesis were examined under the same conditions as described above, except that the reaction was stopped at the indicated time points and products were analysed on agarose gel under non-denaturing conditions. As shown in Fig. 7(b), terminal transferase activity could already be detected after 20 s incubation, as evidenced by radiolabelling of the RNA substrate, which steadily increased up to 10 min. In contrast, the double-stranded product of NV 3Dpol-driven RNA synthesis was observed only after 15 min (900 s) (Fig. 7c). This discrepancy in the time of appearance of these products suggests that RNA synthesis by NV 3Dpol does not depend upon terminal transferase activity. As a correlate, RNA synthesis by NV 3Dpol did not lead to an increase in length of the radiolabelled product over time, as would have been expected if it were driven by terminal transferase activity (Fig. 7c). Accordingly, these results suggest that NV 3Dpol does not initiate RNA synthesis by back-priming.
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). As shown in Fig. 8(a), NV 3Dpol terminal transferase activity was inhibited in the presence of template concentrations of up to 1000 ng µl–1. In contrast, under the same reaction conditions and using the same concentrations of cordycepin-blocked templates, synthesis of double-stranded product was not affected (Fig. 8b).
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-32P]GTP to label the nascent RNA. [-32P]GTP was chosen, because in prototypic NV strains, a guanidine 5'-monophosphate is the ultimate nucleotide at the 5' terminus of the genomic and subgenomic RNA (Green et al., 2001). [-32P]GTP has also been used to address the question of initiation of RNA synthesis by the hepatitis C virus RdRp (Zhong et al., 2000). This approach relies upon the observation that [-32P]GTP only labels RNA synthesized through de novo initiation. Upon initiation through back-priming, the label will be lost due to hydrolysis and release of -32Pi. As shown by autoradiography in Fig. 8(c)
, synthesis of RNA by NV 3Dpol led to labelling by [-32P]GTP, strongly suggesting that NV 3Dpol initiates RNA synthesis on heteromeric RNA de novo. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In the first step and in order to investigate the activity of NV 3Dpol in vitro, the recombinant protein was expressed in E. coli and purified by Ni–NTA affinity chromatography. To assess the specificity of recombinant NV polymerase activity, NV 3Dpol mutated in its active site was generated. In the next step, activity of NV 3Dpol on synthetic RNA templates was characterized in vitro. On those templates, NV 3Dpol was able to initiate RNA synthesis in a strictly RNA template-dependent manner. NV 3Dpol activity was found to depend on the presence of Mg2+ or Mn2+, increasing its activity up to 30-fold in comparison to Fe2+. In previous studies, Mn2+ has been shown to preferentially enable RNA synthesis by NV 3Dpol in vitro (Belliot et al., 2005; Fukushi et al., 2004). However, in our study, flexibility with respect to the type of metal ion was observed, with no strong preference for Mg2+ versus Mn2+ usage. This flexibility has already been described for various viral RdRps (Crotty et al., 2003; Vazquez et al., 2000). In Poliovirus, it has also been suggested that Mn2+ may influence 3Dpol activity through an indirect effect on folding of the enzyme (Crotty et al., 2003). In addition, Mn2+ has been reported to reduce the processivity of poliovirus 3Dpol by reducing the rate of nucleotide incorporation (Crotty et al., 2003). This effect, however, cannot be generalized to RNA polymerases of single-stranded RNA viruses, as Mn2+ has been reported to enhance replication initiation by brome mosaic virus RNA polymerase (Kao & Sun, 1996).
Our data also indicate that NV 3Dpol is able to replicate heteromeric subgenomic RNA by a de novo initiation mechanism. In NV, the mechanism of initiation of RNA synthesis remains, so far, unclear. Previous studies have suggested initiation through a template-primed (Fukushi et al., 2004) or back-priming mechanism (Belliot et al., 2005). In the latter study, however, no terminal transferase activity was reported that could sustain this hypothesis. Interestingly, NV 3Dpol has been reported to display a template-switching activity (Belliot et al., 2005), strongly supporting de novo initiation of RNA synthesis. In our study, several lines of evidence indicated that the double-sized product of NV 3Dpol synthesis results from de novo synthesis from the heteromeric subgenomic RNA template rather than from back-priming. First, strand-separation analysis showed that the replication product of NV 3Dpol could be completely denatured, yielding two strands that consisted of the template RNA and its complementary synthesized RNA strand. In the case of back-primed initiation, the two strands are expected to be covalently linked, and no separation should occur. In addition, the double-sized product was not digested by S1 nuclease, suggesting that it does not consist of a hairpin-dimer. Indeed, in the case of a hairpin-dimer, digestion would have generated two template-sized strands that would have been visualized on formaldehyde–agarose gels, as observed in other studies on RdRps in vitro (Behrens et al., 1996; Luo et al., 2000). Second, time-course experiments showed dissociation between terminal transferase and RNA synthesis activities, suggesting that elongation of the template by back-priming did not occur. Third, replication was possible on cordycepin-blocked RNA templates, indicating de novo initiation on those templates. In contrast and as a control, terminal transferase activity was abolished on the same templates. Finally, incorporation of [-32P]GMP in RNA synthesis product was possible, indicating an incorporation of [-32P] in the nascent strand that can only take place by de novo initiation. All in all, these results suggest that initiation of RNA synthesis of non-polyadenylated subgenomic RNA occurs de novo. De novo initiation on single-stranded templates is common in RNA viruses and has been reported in various RdRps like hepatitis C NS5B (Luo et al., 2000), bovine viral diarrhoea virus NS5B (Kao et al., 1999) and brome mosaic virus replicase (Kao & Sun, 1996; Sun et al., 1996). In Brome mosaic virus, de novo initiation on single-stranded templates occurs within a replication complex, allowing discrimination of homologous and heterologous templates. In the case of NV, de novo initiation was driven in vitro by NV 3Dpol alone, without additional factors. In our study, non-polyadenylated subgenomic RNA was used as the template. This template reflects the nature of the antigenomic RNA, that is, in contrast to genomic RNA, not polyadenylated at its 3'-end. On NV polyadenylated genomic RNA, initiation of RNA synthesis by NV 3Dpol is primer-dependent (Fukushi et al., 2004). Furthermore, NV 3Dpol displayed in our hands an activity that is not NV-template-specific, being able to synthesize RNA using a single-stranded RNA template of eukaryotic origin. In this context, further characterization of additional viral proteins allowing a specific recognition of viral genomic template in vivo may help in understanding the replication strategy of NV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Asanaka, M., Atmar, R. L., Ruvolo, V., Crawford, S. E., Neill, F. H. & Estes, M. K. (2005). Replication and packaging of Norwalk virus RNA in cultured mammalian cells. Proc Natl Acad Sci U S A 102, 10327–10332.
Atmar, R. L. & Estes, M. K. (2001). Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin Microbiol Rev 14, 15–37.
Behrens, S. E., Tomei, L. & De Francesco, R. (1996). Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 15, 12–22.[Medline]
Belliot, G., Sosnovtsev, S. V., Chang, K. O., Babu, V., Uche, U., Arnold, J. J., Cameron, C. E. & Green, K. Y. (2005). Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase. J Virol 79, 2393–2403.
Crotty, S., Gohara, D., Gilligan, D. K., Karelsky, S., Cameron, C. E. & Andino, R. (2003). Manganese-dependent polioviruses caused by mutations within the viral polymerase. J Virol 77, 5378–5388.
Fukushi, S., Kojima, S., Takai, R., Hoshino, F. B., Oka, T., Takeda, N., Katayama, K. & Kageyama, T. (2004). Poly(A)- and primer-independent RNA polymerase of norovirus. J Virol 78, 3889–3896.
Green, K. Y., Chanock, R. M. & Kapikian, A. Z. (2001). Human caliciviruses. In Fields Virology, 4th 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 & Wilkins.
Kao, C. C. & Sun, J. H. (1996). Initiation of minus-strand RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase: use of oligoribonucleotide primers. J Virol 70, 6826–6830.
Kao, C. C., Del Vecchio, A. M. & Zhong, W. (1999). De novo initiation of RNA synthesis by a recombinant flaviviridae RNA-dependent RNA polymerase. Virology 253, 1–7.[CrossRef][Medline]
Luo, G., Hamatake, R. K., Mathis, D. M., Racela, J., Rigat, K. L., Lemm, J. & Colonno, R. J. (2000). De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. J Virol 74, 851–863.
Parashar, U. D. & Monroe, S. S. (2001). ‘Norwalk-like viruses' as a cause of foodborne disease outbreaks. Rev Med Virol 11, 243–252.[CrossRef][Medline]
Ranjith-Kumar, C. T., Gajewski, J., Gutshall, L., Maley, D., Sarisky, R. T. & Kao, C. C. (2001). Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis. J Virol 75, 8615–8623.
Rohayem, J., Diestelkoetter, P., Weigle, B., Oehmichen, A., Schmitz, M., Mehlhorn, J., Conrad, K. & Rieber, E. P. (2000). Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients. Cancer Res 60, 1815–1817.
Rohayem, J., Munch, J. & Rethwilm, A. (2005). Evidence of recombination in the norovirus capsid gene. J Virol 79, 4977–4990.
Sambrook, J. & Russel, D. W. (2001). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sun, J. H., Adkins, S., Faurote, G. & Kao, C. C. (1996). Initiation of (–)-strand RNA synthesis catalyzed by the BMV RNA-dependent RNA polymerase: synthesis of oligonucleotides. Virology 226, 1–12.[CrossRef][Medline]
Temme, A., Traub, O. & Willecke, K. (1998). Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes. Cell Tissue Res 294, 345–350.[CrossRef][Medline]
Vazquez, A. L., Alonso, J. M. & Parra, F. (2000). Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase. J Virol 74, 3888–3891.
Wobus, C. E., Karst, S. M., Thackray, L. B. & 7 other authors (2004). Replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol 2, e432.[CrossRef][Medline]
Zhong, W., Uss, A. S., Ferrari, E., Lau, J. Y. & Hong, Z. (2000). De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J Virol 74, 2017–2022.
Received 30 December 2005;
accepted 9 May 2006.
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I. Robel, J. Gebhardt, J. R. Mesters, A. Gorbalenya, B. Coutard, B. Canard, R. Hilgenfeld, and J. Rohayem Functional Characterization of the Cleavage Specificity of the Sapovirus Chymotrypsin-Like Protease J. Virol., August 15, 2008; 82(16): 8085 - 8093. [Abstract] [Full Text] [PDF]
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 U. Scheffler, W. Rudolph, J. Gebhardt, and J. Rohayem Differential cleavage of the norovirus polyprotein precursor by two active forms of the viral protease J. Gen. Virol., July 1, 2007; 88(7): 2013 - 2018. [Abstract] [Full Text] [PDF]
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S. W. B. Fullerton, M. Blaschke, B. Coutard, J. Gebhardt, A. Gorbalenya, B. Canard, P. A. Tucker, and J. Rohayem Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase J. Virol., February 15, 2007; 81(4): 1858 - 1871. [Abstract] [Full Text] [PDF]
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