Institut für Virologie (FB Veterin ärmedizin), Justus-Liebig-Universit ät Giessen, Frankfurter Str. 107, D- 35392 Giessen, Germany1
Author for correspondence: Sven-Erik Behrens.Fax +49 641 99 38359. e- mail Sven-Erik.Behrens{at}vetmed.uni-giessen.de
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Abstract |
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Introduction |
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Studies in vitro based on heterologously expressed proteins have yielded much data concerning the functions of certain NS proteins. The viral protease complex responsible for most of the cleavages that generate the mature NS proteins (Fig. 1) was shown to consist of NS3 and NS2B or of NS3 and NS4A for flaviviruses and pestiviruses/HCV, respectively (reviewed in Rice, 1996
; N. Tautz, personal communication). Moreover, for several members of the Flaviviridae, NS3 was found to exhibit RNA-stimulated NTPase and/or RNA helicase activity (Jin & Peterson, 1995
; Warrener & Collett, 1995
; Wengler & Wengler, 1991
). The NS5B proteins of HCV and bovine viral diarrhoea virus (BVDV) and the NS5 protein of dengue flavivirus type I were demonstrated to function as RNA-dependent RNA polymerases (RdRp) (Behrens et al., 1996
; Tan et al., 1996
; Zhong et al., 1998
). Since these viral polypeptides are suspected to play a key role during catalysis of both steps of the viral RNA replication process, it was considered worthwhile to compare the functional properties of the RdRps of members of each of the different genera of the Flaviviridae.
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Methods |
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DNA fragments were obtained by PCR from cDNA constructs of BVDV, CSFV and WNV that were kindly provided by N. Tautz (Giessen), G. Meyers (Tübingen) and G. Wengler (Giessen), respectively. To allow initiation of transcription, additional ATG codons were engineered upstream of the coding sequences of each DNA fragment by using oligonucleotide primers. Translation termination occurred at the termination codons of the virus polyproteins (Fig. 1). Virus genes were cloned initially into pGEM-T (Promega) and, after verifying the nucleotide sequences, they were subsequently cloned into pBlueBac 4.5 (Invitrogen). The restriction sites used are indicated in Fig. 1
.
Generation and selection of the recombinant baculoviruses BacB5B (expressing BVDV NS5B), BacC5B (CSFV NS5B) and BacW5 (WNV NS5) were performed in Sf21 cells by using the Invitrogen recombination kit as recommended by the manufacturer.
Authenticity of expressed polypeptides.
The authenticity of the polypeptides was verified by SDSPAGE and Western blotting of total protein from cytoplasmic extracts as described by Mondelli et al. (1994) . Antibodies used to detect the various polypeptides were an anti-WNV NS5 antibody (kindly provided by G. Wengler, Giessen), a rabbit antiserum directed against the C terminus of the CSFV polyprotein (aa 35203761) (kindly provided by R. Stark, Giessen) and an anti-HCV NS5B antiserum (Tomei et al., 1993
).
Templates for RdRp assay.
Templates for assay of RdRp activity (as outlined in Fig. 3) were generated as follows. D-RNA represents the mRNA of the liver-specific transcription factor DCoH and was obtained by transcription from BglII-linearized plasmid pT7DCoH (Behrens et al., 1996
). The IRES RNA was obtained by in vitro transcription of EcoRI-linearized plasmid pCITE2A (Novagen). The BVDV 3'-end RNA was a transcript comprising the C terminus of the BVDV CP7 ORF and the authentic 3' UTR (Yu et al., 1999
). The BVDV replicon RNA was obtained from plasmid pA/BVDV/D9 as described previously (Behrens et al., 1998
). The BVDV minigenome RNA was transcribed from a pA/BVDV/D9 derivative, in which the BstEII site at position 421 had been fused to the EcoRV site at position 11888.
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Micrococcal nuclease (MN) treatment of cytoplasmic extracts.
Potential nucleic acid primers were removed from cytoplasmic extracts of baculovirus-infected Sf21 cells by treatment with 10 U MN per µl reaction volume for 1 h at 20 °C in the presence of 2 mM CaCl2. After inactivation of the MN by addition of 2 mM EGTA, the RdRp assay was carried out as described above either in the absence or in the presence of 200 ng oligo(dG) 18 primer.
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Results |
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To allow a comparative analysis of the RdRps encoded by representatives of each of the three genera of Flaviviridae, recombinant baculoviruses were constructed that should be capable of producing enzymatically active NS5B polypeptides from BVDV strain CP7 and CSFV strain Alfort and NS5 from WNV in insect cells.
DNA fragments were obtained by PCR. Additional ATG codons were engineered upstream of the respective coding sequences of each DNA fragment; translation termination occurred at the termination codons of the viral polyproteins (Fig. 1). A certain quantity of the recombinant proteins was expected to differ from the authentic viral polypeptides because of the presence of an additional methionine residue at the N terminus. However, as a result of the activity of cellular methionine aminopeptidases, which remove N-terminal methionine residues co-translationally (Tsunasawa et al., 1985
) when they are adjacent to small amino acids such as glycine (WNV) or serine (BVDV, CSFV and HCV), a considerable amount of heterologously expressed protein was expected to contain the authentic N terminus.
As shown in Fig. 2, infection of Sf21 cells with the different recombinant baculoviruses led to the expression of each of the viral polypeptides. The authenticity of the expressed proteins was verified by SDSPAGE and Western blotting. As expected, bands corresponding to proteins of 65 kDa (HCV NS5B), 77 kDa (BVDV NS5B, CSFV NS5B) and 100 kDa (WN NS5) were detected specifically by antisera that had previously been raised against the respective proteins.
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Initially, we employed a non-viral 399 nucleotide mRNA (D-RNA) as the template in the reaction (Fig. 3) because this RNA was observed previously to be used efficiently as a substrate by HCV RdRp (Behrens et al., 1996
). As shown in Fig. 4
, all experiments including the negative control, the latter carried out with cytoplasmic extract of either uninfected or wild-type baculovirus- infected Sf21 cells, revealed labelling of the input template RNA (as determined by silver staining side by side with the input RNA transcript; data not shown). This observation is in accordance with previous data (Behrens et al., 1996
) and was explained by the activity of cellular terminal nucleotidyl transferases present in the cytoplasmic extracts of Sf21 cells. Accordingly, the addition of a small quantity of a single-strand-specific ribonuclease such as RNase T1 (5 U in 40 µl reaction volume) to the assay mixture in the presence of 500 mM NaCl (high ionic strength) led to the disappearance of this band (Fig. 4
). As with RdRp reactions performed on extracts containing the HCV NS5B, a novel product could be detected in experiments employing extracts that included either the BVDV or CSFV NS5B or WNV NS5 proteins. This product migrated significantly faster on normal denaturing gels (5% acrylamide, 7 M urea, 20 °C) and significantly slower on highly denaturing gels (10% acrylamide, 8 M urea, 60 °C) (not shown) than the template RNA (as also shown in Fig. 4
). Treatment of the products with RNase A at low ionic strength (50 mM NaCl) resulted in the complete disappearance of all radioactively labelled bands (not shown), confirming the nature of this latter reaction product as RNA. In contrast, as outlined above, high-salt conditions (500 mM NaCl) and small quantities of RNase left the migration behaviour of this band largely unaltered (Fig. 4a
), whereas upon addition of large amounts of RNase (3·5 µg RNase A and 25 U RNase T1 per 40 µl reaction volume) it was converted into a new product that co-migrated with the original template (Fig. 4b
).
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A D-RNA hairpin product was obtained with cytoplasmic extracts containing NS5B from BVDV, CSFV and HCV and NS5 from WNV, and RNA synthesis was found to be dependent on the addition of an external RNA substrate, ribonucleotides and magnesium ions (not shown). We therefore concluded that NS5B from BVDV and CSFV and NS5 from WNV exhibited essentially the same activity as HCV NS5B; in other words, they functioned as RdRps.
Generation of dimer-sized RNA hairpin molecules via initiation of RNA polymerization at the 3'-hydroxyl group of the template implies intramolecular priming, which is probably enabled by RNA structures near the 3' terminus of the RNA. This indicates that initiation of RNA polymerization, as catalysed by each of the different Flaviviridae RdRps, represents a primer-dependent mechanism. To address this in more detail, we next tested the different polymerases on a poly(C) template, i.e. a homopolymeric RNA that should be incapable of forming internal structures. In order to deplete the assay mixture of nucleic acid molecules that might conceivably serve as unspecific primers, the cytoplasmic extracts of baculovirus-infected Sf21 cells were treated with MN before determining RdRp activity. After inactivation of the MN, the RdRp assay was carried out either in the absence or presence of an oligo(dG)18 primer. Incorporation of [32P]GTP was monitored by filter-binding. As shown in Fig. 5, experiments with the different RdRps revealed that, in all cases, RNA polymerization occurred exclusively in the presence of oligodeoxynucleotide primer molecules. These data thus show that the RNA-dependent RNA polymerization process of the different Flaviviridae RdRps is strictly primer-dependent. Comparable results were obtained with heteropolymeric substrates such as D-RNA that had previously been 3'-oxidized (oxidation was carried out with sodium metaperiodate, as described by Behrens et al., 1996
). In this case, RNA polymerization was detectable only in the presence of the respective antisense oligodeoxynucleotide primers (data not shown).
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Taken together, we conclude that intramolecular priming represents the preferred mechanism for initiation of RNA polymerization by the different Flaviviridae polymerases on heteropolymeric RNA templates in vitro. `Template priming and copy- back' apparently takes place irrespective of whether or not the templates contain cis-encoded signals that should be essential for specific initiation of the virus replication process. Like IRES- encoding transcripts, the viral RNAs, in particular the UTRs, contain numerous pronounced RNA structural elements (Blight & Rice, 1997 ; Brinton et al., 1986
; Wengler & Castle, 1986
; Yu et al., 1999
). We therefore reasoned that the large number of different RNase-resistant products that were detected during experiments employing the viral RNAs as templates were caused by a significantly lower ability of the enzyme to perform a continuous (processive) polymerization reaction like that carried out on other, less structured, heteropolymeric templates such as the D-RNA. Most likely, RNA polymerization on highly structured templates initially yields RNA hairpin molecules containing antisense strands of variable lengths. These may then be recognized again by the RdRp, which may perform further polymerization cycles, thus leading to a broad spectrum of different RNase-resistant products.
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Discussion |
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As shown in Figs 4 and 5
, RNA synthesis as catalysed by each of the different viral proteins expressed was easily detectable by employing an assay system that had been established initially for the HCV RdRp (Behrens et al., 1996
; De Francesco et al., 1996
). Interestingly, the optimal conditions determined previously for the in vitro reaction (e.g. concentration of magnesium ions, reaction temperature etc.) were found to be perfectly suitable to assay the pestivirus and flavivirus RdRps. Differences in RdRp activity, as observed for example between the BVDV NS5B and WNV NS5 proteins, correlated directly with the level of expression of the viral proteins in insect cells. Hence, in agreement with recent data (M. Collett & J. B. Flanegan, personal communication; Zhong et al. , 1998
), these experiments demonstrate clearly that the product of the NS5B gene of the pestiviruses BVDV and CSFV represents an RdRp. Furthermore, our data on the WNV NS5 confirm and extend previous studies in vitro on the dengue virus RdRp (Tan et al. , 1996
).
As with other virus RdRps studied in vitro (Neufeld et al., 1991 ; Sankar & Porter, 1991
), the activities of the RdRps of the members of the Flaviviridae examined were found not to be restricted to the authentic viral template and, furthermore, were shown to be primer-dependent. In the case of heteropolymeric templates, the formation of stable structures by annealing nucleotides at or near the 3' end of the RNA apparently allowed intramolecular priming at the 3'-hydroxyl group. Priming was then followed by `copy-back' transcription of a complementary RNA strand, thus giving rise to dimer- sized, hairpin-like RNA molecules (Behrens et al., 1996
; Fig. 4
). On homopolymeric templates and 3'-terminally blocked heteropolymeric templates, RNA synthesis was consistently found to be dependent on the presence of RNA (not shown) or DNA primer molecules complementary to the template (Fig. 5
). In contrast to previous experiments with unfractionated cytoplasmic extracts of baculovirus-infected Sf9 cells (Behrens et al., 1996
), annealed primertemplate hybrids apparently remained rather stable within the MN-treated cytoplasmic extracts of Sf21 cells. This may be explained by a significantly lower activity of cellular RNA helicases under the chosen experimental conditions, and turned out to be advantageous, since it enabled the measurement of primer-dependent RNA synthesis without prior purification of the viral protein. Obviously, the experimental test system chosen did not permit the additional testing of the flavivirus and pestivirus RdRps for a co-fractionating terminal nucleotidyl transferase activity, as described for the purified HCV NS5B (Behrens et al., 1996
) and BVDV NS5B (Zhong et al., 1998
).
Priming at the 3'-hydroxyl group of RNA templates appears to be a common property of RNA-polymerizing enzymes in vitro (Cazenave & Uhlenbeck, 1994 ; Konarska & Sharp, 1989
). However, hairpin-like molecules are also formed in vivo during virus infection, as observed in the cases of poliovirus and encephalomyocarditis virus (Senkevich et al., 1980
; Young et al., 1985
). In view of these observations and the fact that data from cell culture-based RdRp assays are either not available or are difficult to compare with the conditions in vitro (Chu & Westaway, 1985
; Grun & Brinton, 1986
), it is not currently possible to decide whether intramolecular priming reflects the situation in vivo . If it does, one would have to postulate a ribonuclease that cleaves specifically RNA hairpins derived from the homologous viral genome. Those cases of positive-strand RNA viruses where specific initiation of RNA synthesis has been detected in vitro have all involved the presence of other virus- and/or host cell-encoded proteins besides the viral RdRp (Ball, 1995
; Blumenthal & Carmichael, 1979
; Hayes & Buck, 1990
; Lemm et al., 1998
; Wu et al., 1992
). Thus, it is tempting to assume that the RdRps of members of the Flaviviridae are necessary but not sufficient to catalyse the initial step of the replication pathway. It will be important in the future to search for factors that either mediate the specificity of the virus RNA polymerization pathway as such or modulate the RdRp in a way to generate specificity for the viral template. The observation that the polymerases are significantly less processive on highly structured templates such as the natural viral RNAs may point in this direction, suggesting, for instance, an important functional role for the viral helicase, which, at least in the case of flaviviruses, was found to be associated with the NS5 RdRp (Chen et al., 1997
; Kapoor et al., 1995
). Future experiments will focus on such virus proteinprotein interactions for pestiviruses and HCV and on the reconstitution of a specific in vitro priming system, similar to that recently developed for the RdRp of poliovirus (Paul et al., 1998
).
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Acknowledgments |
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Footnotes |
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References |
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Received 29 April 1999;
accepted 18 June 1999.