1 Institute of Molecular Biology, Federal Research Centre for Viral Diseases of Animals, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany
2 Institute for Diagnostic Virology, Federal Research Centre for Viral Diseases of Animals, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany
3 Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands
4 Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA
Correspondence
Egbert Mundt
Egbert.Mundt{at}rie.bfav.de
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ABSTRACT |
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INTRODUCTION |
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The coding region of IBDV VP1 (Morgan et al., 1988) was shown to contain motifs I (now termed motif A), II (motif B), III (motif C) and IV (motif E) that are common to RNA-dependent RNA polymerases (RdRp) of positive-strand RNA (ssRNA+) viruses (Gorbalenya & Koonin, 1988
). Subsequent analyses revealed that the originally described motif C is not conserved in other birnaviruses (Shwed et al., 2002
) and may be fortuitous. Its genuine counterpart, which includes the AlaAspAsn rather than the common GlyAspAsp tripeptide, was identified upstream of motif A using a sophisticated computer-assisted analysis that involved newly determined sequences of ssRNA+ Thosea asigna and Euprosterna eleasa viruses (Gorbalenya et al., 2002
). This unique structural organization of VP1 may be compatible with RdRp (Gorbalenya et al., 2002
) as well as other enzymic activities suggested in early studies (Spies et al., 1987
; Spies & Müller, 1990
), although neither of them has been demonstrated yet.
To verify RdRp activity of the IBDV VP1, we expressed this protein in the baculovirus system and analysed its properties in vitro.
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METHODS |
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Lysates were tested for the presence of VP1 and NS5B by discontinuous SDS-10 % PAGE (Sambrook et al., 1989) followed by blotting onto a nitrocellulose membrane (Schleicher and Schüll, Germany). VP1 was detected with rabbit anti-VP1 serum (Birghan et al., 2000
) and NS5B with rabbit anti-NS5B serum (Behrens et al., 1996
). Antigenantibody complex was visualized by incubation with diluted horseradish peroxidase-conjugated goat anti-rabbit antibodies (Sigma) followed by an enhanced chemoluminescence system (Pierce) as described by the manufacturer.
Substrate for RNA polymerase assay.
To assay for polymerase activity, cRNA transcripts were originated from plasmid pP2A (Birghan et al., 2000) and pP2B (Mundt & Vakharia, 1996
). To this end, pP2A was cleaved with RsrII/BglII to remove nearly the complete coding region of segment A (nucleotides 2013020). After fill-in using Klenow enzyme and ligation, a plasmid was obtained containing the 5' and 3' non-coding regions (NCRs) of segment A and a small part of the coding region of the polyprotein gene, nt 131201 (p
P2A, Fig. 1
). pP2B was cleaved with Bsu36I/BsrGI to delete a large part of the coding region of VP1 (nt 2312538). The remaining plasmid was treated as described above to obtain a plasmid which contained the 5' and 3' NCRs and a small part of the coding region of segment B (p
P2B, Fig. 1
). p
P2A was linearized with BsrGI or BglII and p
P2B was cleaved with SgrAI or PstI. The samples were digested with proteinase K (0·5 mg ml1)/SDS (0·5 %) and purified by phenol/chloroform extraction. T7 polymerase reaction was carried out using the following conditions in a 25 µl sample: 2 µg linearized DNA, 1x transcription buffer (Promega), 10 mM DTT, 0·5 mM of each nucleotide, 1 µl RNasin (Promega), 1 µl T7 RNA polymerase. After incubation at 37 °C for 60 min, 1 µl DNase I (Roche) was added for degradation of plasmid DNA. The plus-stranded cRNAs obtained were separated from nucleotides using Probe Quant G50-Micro Columns (Amersham Pharmacia) following the manufacturer's instructions, ethanol precipitated, and subsequently used for further experiments.
cRNA transcribed from pP2A was 32P-labelled during the transcription reaction and eluted from a 1 % agarose gel. To this end, approximately 1 µg linearized p
P2A (5 µl) was added to a mixture consisting of 5 µl 5x transcription buffer (Promega), 2·5 µl 100 mM DTT (Promega), 3 µl of an rNTP mixture (ATP, CTP, GTP, 10 mM each; Roche), 1 µl of 1 mM UTP (Roche), 5 µl [
-32P]UTP [10 µCi, 3000 Ci mmol1 (111 TBq mmol1); ICN], 1·5 µl nuclease-free water, 1 µl RNasin (Promega) and 1 µl T7 RNA polymerase (Promega). After 1 h incubation at 37 °C, reaction products were separated in a 1·5 % low-melting-point agarose gel (Seakem Agarose; Biozym) containing 0·5 µg ethidium bromide. The appropriate RNA was excised and 400 µl of a buffer containing 5 % lithium dodecyl sulphate, 100 mM Tris/HCl pH 8·0, 0·5 M LiCl, 10 mM EDTA and 5 mM DTT (Sawicki & Sawicki, 1990
) was added. The mixture was then incubated at 70 °C for 10 min, followed by phenol/chloroform extraction and ethanol precipitation.
In vitro RdRp assays using Sf9 cell extracts.
The assay was performed using cytoplasmic extracts and in vitro transcribed cRNA derived from pP2A or p
P2B. For the assay, to 10 µl of the cytoplasmic extract were added 8 µl 100 mM Tris/HCl pH 7·5, 2 µl 100 mM MgCl2, 4 µl 10 mM DTT, 1 µl 1 M KCl, 0·8 µl 50 mM EDTA, 0·5 µl RNasin (20 units, Promega), 2 µl actinomycin D (1 mg ml1), 2 µl of ATP, CTP and GTP (each 10 mM), 5 µl cRNA dissolved in RNase-free water (approx. 1 µg) and 1 µl [
-32P]UTP (10 µCi). After incubation of the reaction mixture at 22 °C for 2 h, reaction products were subjected to proteinase K (0·5 mg ml1) digestion in 1 vol. 2x proteinase K buffer (300 mM NaCl, 10 mM Tris/HCl pH 7·5, 1 % SDS) at 37 °C for 30 min, and then isolated. After phenol/chloroform extraction, glycogen (Roche) was added and RNA was ethanol-precipitated.
In a modified polymerase assay [-32P]UTP-labelled cRNA (see above) was used. To this end, radioactively labelled [
-32P]cRNA was ethanol-precipitated and sedimented by centrifugation. After resuspension in RNase-free water, 5 µl was added to the reaction mixture instead of non-labelled cRNA, and 2 µl 10 mM UTP was added. Purification of reaction products was performed as described above.
RNase digestion.
RNase digestion was performed using RNase If (New England Biolabs) for specific digestion of single-stranded RNA, or using RNase III (New England Biolabs) which specifically cleaves double-stranded RNA into fragments of 1215 bp with two-base 3' overhangs. For RNase If assay, reaction products from the polymerase assay were ethanol-precipitated, sedimented by centrifugation, resuspended in 20 µl buffer (50 mM Tris/HCl pH 8·0, 10 mM MgCl2, 1 mM DTT, 100 mM NaCl) and 20 U enzyme was added. To assay for double-stranded RNA, reaction products were resuspended in an appropriate buffer (10 mM Tris/HCl pH 8·0, 10 mM MgCl2, 1 mM DTT, 100 mM NaCl) and 0·2 U RNase III was added. For incubation with RNase T1 (Roche) enzyme was added directly to the RdRp reaction to a final concentration of 1 U µl1. Reaction mixtures were incubated at 37 °C (RNase III, RNase If, RNase T1) for 30 min, digested by incubation with proteinase K/SDS, and purified as described above.
Analysis of RNA.
Reaction products were sedimented, washed with 70 % ethanol and resuspended in loading buffer (95 % formamide, 20 mM EDTA pH 8·0, 0·05 % bromophenol blue, 0·05 % xylene cyanol). Resuspended RNA was separated in a 5 % acrylamide gel containing 8 M urea in TBE using a 60 cm sequencing apparatus (Gibco-BRL) at 70 W for 2 h. Gels were fixed, incubated with water to remove urea, and dried before exposure at 70 °C for at least 1 week to an X-OMAT AR film (Eastman Kodak) using an intensifying screen.
Preparation of RNA markers.
To obtain [-32P]UTP-labelled RNA markers, pP2A was cleaved with RsrII, NdeI and SacI at positions 201, 647 and 868, respectively, of the full-length cDNA sequence of segment A of IBDV strain P2 (Mundt & Müller, 1995
). The labelling reaction for cRNA using T7 polymerase, and purification of the three radioactively labelled cRNAs of lengths 201, 647 and 848 nt, were performed as described above.
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RESULTS |
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Analysis of lysates of Sf9 cells infected with the two recombinant baculoviruses (VP1-Bac and VP1D416A-Bac) showed after probing with the anti-VP1 antiserum that IBDV VP1 protein was expressed (Fig. 2a). The specificity of the detected signal was proved using lysates of mock-infected and IBDV (strain P2)-infected CECs, prepared as described previously by Mundt et al. (1995)
. In the P2-infected CECs we detected the full-sized protein, while in the lysates of VP1-Bac- and VP1D416A-infected Sf9 cells we detected VP1 as well as some degradation products which were not detectable in the control lysates. In a parallel experiment we expressed the HCV NS5B RdRp using an analogous baculovirus approach. The expression of the protein was also confirmed by Western blots applying an anti-HCV NS5B antiserum.
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In the next experiment, we tested cytoplasmic extracts of VP1-Bac-infected and AcNPV-infected Sf9 cells in a parallel approach, using either the above-described assay with unlabelled RNA-A444 substrate and the labelled nucleotide or, alternatively, with a mixture that contained unlabelled nucleotides and a 32P-labelled RNA-A444 substrate. As shown in Fig. 3(a), we detected the aforementioned product with a faster electrophoretic mobility independently of the nature of the reaction mixture in the assays that contained VP1. Thus, in addition to confirming that VP1 has polymerase activity, these experiments demonstrated that the enzyme used the input RNA-A444 transcript as a substrate. The labelling of the previously unlabelled input RNA in the absence of VP1 is likely to be explained by the activity of terminal nucleotidyl transferases (TNTs) that are present in the cytoplasmic extracts of Sf9 cells (Behrens et al., 1996
).
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The IBDV RdRp operates by substrate priming
In order to characterize the product of VP1-mediated reaction further, we analysed it under highly denaturing conditions and tested its accessibility to the ss-specific RNase T1 (Fig. 4). We performed the RdRp assay with cytoplasmic extracts of VP1-Bac-infected and AcNPV-infected Sf9 cells using either 32P-labelled RNA-A444 (lanes 14) or unlabelled RNA-A444 (lanes 6 and 7) templates. Half of each of the assay mixtures was exposed to RNase T1 at 37 °C; the reaction products in each case were resuspended in loading buffer, heated to 95 °C and separated on a pre-heated (approx. 80 °C) denaturing (8 M urea) polyacrylamide gel. In the non-T1-treated samples containing VP1 and a hot or cold template, we observed the dsRNA product (see above) that migrated at a high electrophoretic mobility in the gel (lanes 3 and 6). In addition, a band that migrated significantly slower than the input 32P-labelled RNA-A444 was detected in lane 3 (dHP band). We interpreted the slower-migrating product as corresponding to the denatured ss form of the reaction product. We believe that weak labelling and only partial denaturation of the reaction product were the reasons for the lack of a slower-migrating product in lane 6. This partial denaturation was also evident in lane 3. As the ds (lane 3) and denatured products (lanes 4 and 7) after T1 digestion were found to be labelled in reactions that involved either a labelled or an unlabelled template, we concluded that the original template RNA was part of the reaction product (see above). Thus, in line with previous observations obtained with poliovirus and HCV RdRp, it became apparent that the fast-migrating reaction product probably corresponded to ds hairpin that was generated by the copy-back RNA synthesis mechanism involving template priming. This notion was clearly confirmed in the course of the experiments that applied RNase T1: in the reactions utilizing VP1-Bac lysate, unlabelled RNA template and a labelled nucleotide, we detected a product that was sensitive to the T1 digestion and had the same electrophoretic mobility as the input RNA-A444 in the denatured gel. These properties indicate that the primary product of VP1-mediated reaction corresponded to a hairpin made up from the original template and a complementary negative strand synthesized de novo. T1 converted this hairpin into the dsRNA molecule that was denatured under the conditions of the urea gel into two ss molecules, one of which labelled negative single-strand remained detectable.
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Template specificity of VP1
The next experiment was aimed at testing the substrate specificity of VP1 RNA polymerase (Fig. 5). For this purpose, two different IBDV segment A-specific ssRNAs were tested in the polymerase assay described above, utilizing either IBDV VP1 or HCV NS5B. The first substrate corresponded to RNA-A444, which was transcribed in vitro from p
P2A after linearization with BsrGI, and which contained the 5' as well as the 3' NCR of segment A (Fig. 5a
, lanes 57). The second substrate tested was a 201 nt RNA (RNA-A201) transcribed from p
P2A after linearization with BglII; it contained only the 5' NCR of segment A (Fig. 5a
, lanes 24). After incubation of the RNA-A444 template with lysates containing VP1 or NS5B, in each case we observed the known fast-migrating hairpin product (lanes 6 and 7). However, while the HCV NS5B polymerase was found to be active on both RNA substrates (lanes 4 and 7), which was explained by its unspecific polymerase activity described earlier (Behrens et al., 1996
), this was not the case with VP1. VP1 was evidently inactive on the RNA-A201 substrate that lacked the 3' NCR of segment A (lane 3). To support this finding, we used two additional substrates in addition to RNA-A444 and RNA-A201 for RdRp polymerase assay using lysates containing VP1 (Fig. 5b
). To this end, p
P2B was linearized with SgrAI and PstI to obtain cRNA transcripts lacking the 3' NCR of segment B (RNA-B317) and containing both 5' and 3' NCRs of segment B (RNA-B520), respectively. Here, we observed that VP1 was inactive with RNA-B317 (lane 3). In contrast, a reaction product was observed when RNA-B520 was used in the assay. Hence, we concluded that the 3' NCR of segments A and B was critical for VP1-directed RNA polymerization in vitro.
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DISCUSSION |
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Although E. coli-expressed VP1 failed to show polymerase activity (data not shown), we were able to demonstrate RdRp activity in lysates of insect cells infected with a recombinant baculovirus expressing VP1. This report provides the first direct evidence that VP1 of IBDV is indeed the RdRp of the virus. Related to this finding, we ruled out the possibility that RdRp activity requires an interaction between the two IBDV proteins VP1 and VP3 (Lombardo et al., 1999; Tacken et al., 2000
) as well as binding of double-stranded IBDV RNA by VP3 (Tacken et al., 2002
). This is the second, after phage
6 (Makeyev & Bamford, 2000
), dsRNA virus for which in vitro RdRp activity was shown to be associated with a single protein species. In contrast for the dsRNA rotavirus, two virus proteins (VP1 and VP2) were found to be necessary for polymerase activity (Patton et al., 1997
).
In our VP1-mediated RdRp assay we detected two types of product. Using the ss-specific RNase If, we proved that the slower-migrating reaction product was ssRNA template likely modified by cellular TNT. The faster-migrating RNA product, which was exclusively synthesized in the presence of VP1, was shown to be a hairpin RNA. The detected RdRp activity was due to VP1 rather than a contaminating cellular protein that was proved using the VP1-D416A mutant which carries an Ala replacement of the highly conserved Asp residue in the motif A responsible for divalent cation binding (Gorbalenya et al., 2002); with this mutant no polymerase activity was detected. The latter result provides a first evidence for the vital character of the RdRp motif A in the IBDV VP1 and it was recently extended by showing that the VP1-D416A mutant does not produce a viable progeny in vivo in cotransfection experiments using the reverse genetics system for IBDV (Mundt & Vakharia, 1996
) (data not shown).
Optimization of the reaction conditions for RdRp activity of VP1 showed that larger amounts of reaction product could be obtained at 37 °C. Spies & Müller (1990) stated that the polymerase activity detected within purified IBDV particles reached a peak at 40 °C. In our experiments VP1 activity decreased significantly at 40 °C, which may be due to the different reaction conditions. Using VP1-Bac at incubation temperatures above 22 °C, an additional faster-migrating reaction product was observed. The nature of this is unknown, but it is probably a reaction product of VP1 activity, as it was also dependent on the presence of Mg2+ ions. Mg2+ ions were essential for RdRp activity of VP1, but could be partially replaced by Mn2+ or Co2+ ions. This indicated that for IBDV VP1, like other polymerases, metal ion co-ordination is important in the polymerization reaction. Similar results were obtained for synthesis of dsRNA using purified IBDV particles (Spies & Müller, 1990
). In this system, use of Mn2+ instead of Mg2+ decreased polymerase activity by 80 % compared with the main reaction product consisting of hairpin RNA, in line with the results described here. For the related infectious pancreatic necrosis virus, it has been shown that absence of Mg2+ prevented synthesis of dsRNA using purified viral particles (Dobos, 1995
).
The finding that the template cRNA was labelled in the cell lysates even in the absence of VP1 (upper product) may be explained by the TNT activities that are present in Sf9 extracts. TNT activity has also been described for RdRps of RNA viruses including NS5B of HCV (Behrens et al., 1996; Ranjith-Kumar et al., 2001
), poliovirus 3Dpol RdRp (Neufeld et al., 1994
) and Q
phage replicase (Bausch et al., 1983
). Thus, the VP1 of IBDV itself may contain TNT activity whose analysis requires further experimental studies.
The data presented suggest that the mechanism of RNA synthesis by VP1 in vitro probably follows a copy-back mechanism, in which the template's 3' end primes the synthesis of an antisense strand that falls back to form an RNA hairpin. It is interesting that, in contrast to HCV and many other RNA viruses, the polymerase activity of VP1 in IBDV appeared to be strictly dependent on the 3' terminal sequences of genomic segments A and B. The molecular basis for this dependence remains to be elucidated, although it might be related to the permuted structural organization of VP1 polymerase. Thus, the IBDV polymerase in combination with the 3' end of the genomic RNA may define a certain specificity in the first replication step. A similar specificity was observed with the rotavirus system (Patton et al., 1997) where the very 3' end of the 3' NCR of gene 8 was shown to be necessary for RdRp activity of VP1+VP2, and thus it may be a special feature of segmented dsRNA viruses.
The data presented represent an important starting point for better understanding of the IBDV life cycle.
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ACKNOWLEDGEMENTS |
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Received 30 October 2003;
accepted 21 April 2004.