Characterisation of the RNA-Dependent RNA Polymerase from Rabbit Hemorrhagic Disease Virus Produced in Escherichia Coli

Arch Virol (2001) 146: 59–69

Characterisation of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli

A. López Vázquez, J. M. Mart´ın Alonso, and F. Parra Departamento de Bioqu´ımica y Biolog´ıa Molecular, Instituto Universitario de Biotecnolog´ıa de Asturias (CSIC), Universidad de Oviedo, Oviedo, Spain

Accepted July 10, 2000

Summary. All positive-strand RNA viruses encode a RNA-dependent RNA polymerase which in most cases has been only identiﬁed on the basis of its sequence conservation. Catalytic activity has been experimentally demonstrated in only a handful of these viral proteins, including that from Rabbit hemorrhagic disease virus. Studies from our laboratory have reported that RHDV RNA polymerase produced in Escherichia coli was enzymatically active showing poly(A)-dependent poly(U) polymerase as well as RNA polymerase activity on heteropolymeric substrates. In this work, we have investigated the in vitro activity of the recombinant 3Dpol from RHDV, including ion requirements, resistance to inhibitors, substrate speciﬁcity as well as data on the initiation mechanism of the template-linked products derived from heteropolymeric RNA substrates. Our study demonstrates that in an in vitro reaction recombinant RHDV RNA polymerase generated the minus strand of the heteropolymeric RNA substrates by a “copy-back” mechanism that initiated at the template 30-terminal OH.

Introduction Rabbit hemorrhagic disease virus (RHDV), a member of the Caliciviridae [2, 20, 21], has been recently designated as the type species of the genus Lagovirus [25]. The virions contain a polyadenilated plus-stranded RNA genome of approximately 7.4 kb [16] which has a virus encoded VPg [30], covalently at- tached to its 50-end [17]. Viral particles also encapsidate an abundant subgenomic RNA of about 2.2 kb which is also VPg-linked and polyadenilated. Both RNAs are coterminal at their 30-ends and have highly homologous 50- untranslated regions. The data obtained from in vitro translation studies [30], Escherichia coli expression [15] and detection of RHDV proteins after infection of cultured hepatocytes [9] revealed that the genomic RNA was translated into a polyprotein that 60 A. López Vázquez et al.

was subsequently cleaved to give rise to mature structural and non-structural proteins. The extensive sequence similarities between the 3D RNA-dependent RNA polymerase (RdRp) of picornaviruses and the RHDV polyprotein cleavage product p58 [9, 30] suggested that this polypeptide might have a similar role in RHDV genome replication. Our previous studies have reported the successful expression of enzymatically active RHDV RNA polymerase in Escherichia coli [13] supporting that this enzyme needed not to be synthesized as a precursor in order to become active, as reported by others for Poliovirus [23] and Encephalomyocarditis virus (EMCV) 3Dpol [27]. The recombinant RHDV 3Dpol showed a poly(U) polymerase activity resistant to rifampin and actinomycin D, which was dependent on the presence of both oligo(U) and poly(A). These results agreed with those found for other RdRps from members of the picornavirus-like superfamily of positive-sense single-stranded RNA viruses such as Poliovirus [4, 23], EMCV [27], and Potyvirus [7]. Similar results have been also reported for the Hepatitis C virus (HCV) RdRp [32], a member of the flavivirus superfamily. The purified recombinant RHDV enzyme also showed RdRp activity acting on synthetic RHDV subgenomic RNA, in the presence or absence of an oligo(U) primer. Template-sized products were synthesised in the oligo(U)-primed reactions, as described for EMCV 3Dpol [27]; whereas in the absence of added primer RNA products up to twice the length of the template were made, suggesting that the newly made minus-strand RNA was covalently linked to the plus-strand RNA template [13]. Several authors have reported the synthesis of double-length RNA products by Poliovirus RdRp, in the absence of primer or eukaryotic host factors [23], and in host factor-dependent reactions [5, 14, 31]; by HCV RdRp without an added exogenous primer or host factor [1, 3, 11]; and by purified Bovine viral diarrhea virus (BVDV) RdRp [33]. In this work we have investigated the substrate specificity of the recombinant enzyme as well as the optimum in vitro RdRp assay conditions. We have also used several heteropolymeric synthetic RNA templates in an attempt to understand the RHDV RdRp initiation mechanism giving rise to template-linked products.

Materials and methods Expression and purification of recombinant 3Dpol The recombinant RHDV enzyme was purified from bacterial lysates by affinity chromato- graphy using the Bulk GST Purification Module (Pharmacia), as previously described [13].

Preparation of oligo(U) Oligo(U) was made by alkali hydrolysis of poly(U) using a published protocol [23]. The size of the resulting oligo(U) was determined after end-labeling with [␥-32P]ATP (ICN) and electrophoresis on a 6% polyacrylamide gel. Recombinant RNA polymerase from RHDV 61

Synthetic RNA transcripts Three RNA transcripts (RNA-a, RNA-b and RNA-c) were made to be used as templates in RNA polymerase assays. RNA-a was synthesised by in vitro transcription of plasmid pRNA2.2T7R [13]. The resulting 2.2 kb RNA had a nucleotide sequence which was identical to the subgenomic RHDV mRNA (nt 5296 to 7437 from AST/89 RHDV isolate cDNA; EMBL Acc. Number Z49271) but lacked the 50-terminus VPg and had a shorter (18 nt) poly(A) tail. RNA-b (0.5 kb) was produced by in vitro transcription of nucleotide residues 2656 to 2978 from the RHDV cDNA cloned in a pRSET A (Invitrogen) vector. This nucleotide sequence coded for a fragment of the RHDV polyprotein cleavage product p43 [15]. RNA-c was made by in vitro transcription of a pRNA2.2T7R-derivative carrying an internal 828 nt deletion (nt 6024 to 6852 of RHDV cDNA). Consequently, the resulting 1.3 kb transcript had 50 and 30 sequences which were identical to the corresponding regions in RNA-a.

Gel electrophoresis RNA was analysed on 1.2% formaldehyde-agarose gels [26]. Protein samples were analysed by SDS-polyacrylamide gel electrophoresis (PAGE) as described elsewhere [10].

Enzymatic assays using homopolymeric templates These analyses were performed as previously described [13, 27] using the appropriate combi- nation of homopolymeric templates, primers and radioactive labels. Briefly, the reactions were carried out at 30 ◦C for 60 min in 50-␮l mixtures containing purified 3Dpol (2 ␮M), 50 mM HEPES (pH 8.0), 10 ␮M UTP or dTTP, 4 mM dithiothreitol, 3 mM magnesium acetate, 20 ␮g ml−1 of rifampin, 1 ␮g of oligo(U) or oligo(dT), 2.5 ␮g of poly(A) or poly(dA), and 1 ␮Ci of [␣-32P]UTP (Amersham, 400 Ci/mmol) or [␣-32P]dTTP (Amersham, 3000 Ci/mmol). The in vitro-synthesised products were precipitated with 10% trichloroacetic acid after addition of 100 ␮g of carrier tRNA in 0.2 M sodium pyrophosphate, collected onto 0.45- ␮m-pore-size Whatman GF/C filters, and vacuum dried. The radioactivity on the filters was measured in a scintillation counter.

Enzymatic assays using heteropolymeric templates RdRp activity was assayed in 50-␮l reaction mixtures containing puriﬁed 3Dpol (2 ␮M), 50 mM HEPES (pH 8.0), 10 ␮MATP,10␮M CTP, 10 ␮M GTP, 5 ␮M UTP, 4 mM dithiothreitol, 3 mM magnesium acetate, 50 U of ribonuclease inhibitor (Promega), 10 ␮Ci of [␣-32P]UTP and 0.5–1 ␮g of synthetic RNA template. After incubation at 30 ◦C for 60 min, the reaction mixtures were phenol-chloroform extracted and ethanol precipitated in the presence of 0.3 M sodium acetate (pH 6.0) and 20 ␮g of carrier tRNA. The sediments were dissolved in electrophoresis sample buffer, loaded onto 1.2% formaldehyde-agarose gels and analysed at 60 to 70 V. The gels were then dried and the 32P-labeled RNA products were detected by autoradiography.

Sodium metaperiodate treatment 0 The 3 -hydroxyl group oxidation using NaIO4 was performed as described previously [3]. For this purpose 10 ␮g of RNA-a transcript were dissolved in 100 ␮l of 50 mM sodium acetate (pH 6.0). After the addition of 25 ␮l of 100 mM NaIO4 the mixture was incubated for 1 h at room temperature, then phenol-chloroform extracted and ethanol precipitated. Residual NaIO4 was removed by several washes using 70% ethanol and ﬁnally by gel ﬁltration on a Sephacryl S400 column (Boehringer). 62 A. López Vázquez et al.

Results Substrate speciﬁcity A convenient means to investigate the template and primer preferences of the RHDV recombinant 3Dpol, was the use of reaction mixtures containing appropriate combinations of poly(A) or poly(dA) templates, oligo(U) or oligo(dT) primers and [␣-32P]UTP or [␣-32P]dTTP radioactive labels. The enzyme could efﬁciently incorporate UMP into nascent RNA using an oligo(U)-primed poly(A) template (Fig. 1), as have been demonstrated for other RdRps [3, 4, 27]. In this type of assay RHDV 3Dpol activity was completely dependent on the presence of both template and primer in the reaction mixture; omission of either one resulted in a complete loss of activity (Fig. 1). The requirements for primer and template ruled out the possibility that the observed activity was due to a terminal transferase-like reaction. In contrast, no UMP was incorporated into products using a DNA template, in the presence of an oligo(U) primer, indicating that the enzyme did not have DNA-dependent RNA polymerase (DdRp) activity. Similarly, the enzyme did not incorporated dTMP into oligo(dT)-primed poly(A) nor into oligo(dT)-primed poly(dA) reaction mixtures, thus demonstrating that the recombinant RHDV 3Dpol was devoid of

Fig. 1. Substrate speciﬁcity of the recombinant RHDV 3Dpol. The enzymatic activity of puriﬁed 3Dpol was measured in the presence (+) or absence (−) of the indicated RNA or DNA templates, primers or labelled nucleotide precursors Recombinant RNA polymerase from RHDV 63 reverse transcriptase (RNA-dependent DNA polymerase) and DNA-dependent DNA polymerase activities respectively (Fig. 1).

Ion and salt requirements of the in vitro RdRp assay Recombinant RHDV RdRp activity, using heteropolymeric RNA substrates was routinely assayed in the presence of all four NTPs and a full-length synthetic RHDV subgenomic RNA (RNA-a, 2.2 kb). Considering that the recombinant 3Dpol was puriﬁed using a buffer containing NaCl (see Materials and methods), the reaction mixture usually contained 6–15 mM NaCl, depending on the amount of enzyme used. Then, to optimise the RdRp assay we have ﬁrstly investigated the effects of increasing NaCl concentrations in the reaction mixtures. The results obtained indicated that the optimal NaCl concentration was in the range of 9 to 50 mM (Fig. 2A). The presence of divalent cations (specially Mg2+) is a well known require- ment for in vitro RNA polymerase activity. The standard RNA polymerase reaction mixtures used in previous studies [13] contained 3 mM magnesium acetate (MgAcO), as described by others for EMCV 3Dpol [27]. Accordingly, our data indicated that enzymatic activity was highest using 1–3 mM MgAcO concentrations (Fig. 2B) also showing that concentrations above 10 mM completely abolished 3D polymerase activity. We have also demonstrated that Mg2+ in the standard RHDV 3Dpol in vitro assays could be replaced by Mn2+ giving rise to comparable radioactive precursors incorporation into products (Fig. 2C). Under these assay conditions the optimal MnCl2 concentration was found to be 0.5 mM. In order to investigate whether recombinant 3Dpol was enzymatically active in the presence of alternative magnesium salts different from acetate, or divalent cations other than Mg2+ or Mn2+, MgAcO was substituted in the reaction mixture by 3 mM magnesium chloride, magnesium sulfate; calcium chloride, cobalt

Fig. 2. Effect of increasing concentrations (in mM) of NaCl (A); Mg2+ (B); Mn2+ (C) and 3 mM of other divalent cations (D) on the activity of recombinant RHDV 3Dpol 64 A. López Vázquez et al.

Fig. 3. Effect of rifampin (A) and actinomycin D (B) concentrations (␮gml−1)onthe polymerase activity of recombinant RHDV 3Dpol chloride or zinc chloride (Fig. 2D). The results showed that the enzymatic activity was highest in the presence of the acetate salt. It has been also found that Mg2+ could not be replaced by any of the alternative cations assayed.

Sensitivity of 3Dpol to inhibitors It was previously demonstrated that the recombinant RHDV 3Dpol exhibited poly(A)-dependent poly(U) polimerase activity that was rifampin- and actinomycin D-resistant [13]. In this work we have investigated the effects of these antibiotics on 3Dpol activity using heteropolymeric RNA templates in the absence of added primers. Our data indicated that the 3Dpol enzyme activity was not inhibited by 200 ␮g ml−1 of rifampin (Fig. 3A) an order of magnitude higher than the amount routinely used to exclude endogenous bacterial RNA polymerase activity [18, 27, 32]. Actinomycin D has been extensively used as a highly speciﬁc inhibitor of the synthesis of new RNA in both prokaryotic and eukaryotic cells. Our data indicate that 200 ␮gml−1 actinomycin D had no effect on RHDV 3Dpol activity (Fig. 3B).

RNA polymerase assay using a 30-blocked RNA template It has been previously shown that a host factor preparation that contained a low- level nuclease activity (and possibly a 30 phosphatase) was sufﬁcient to activate RNA templates for transcription by Poliovirus 3Dpol to generate dimer-length products [6]. To further determine whether the RHDV 3Dpol dimer-length products were in fact the result of a self-priming event that occurred at the 30-hydroxyl group of the template; or at a more internal 30-OH produced by an endonuclease activity, as described for Poliovirus, we blocked the 30-hydroxyl group of RNA- a by oxidation with sodium metaperiodate. No template-primed polymerisation was observed using this 30-blocked RNA (Fig. 4, lane 2), in contrast to the dimer- length product (4.4 kb) formed from the untreated RNA (Fig. 4, lane 1). However, the polymerisation blockage in the presence of a blocked template could be overcome by addition of 50 ng of oligo(U) primer (Fig. 4, lane 4). In this case, only a template-length product (2.2 kb) is formed. In contrast, the recombinant 3Dpol Recombinant RNA polymerase from RHDV 65

Fig. 4. 32P-labeled RNA products synthesised by recombinant 3Dpol from an untreated (1, 3)oran30-blocked heteropolymeric RNA template (2, 4) in the absence (1, 2)or presence (3, 4) of an oligo(U) primer synthesised template-size as well as double-length products from the untreated template in the presence of an oligo(U) primer (Fig. 4, lane 3). These data demonstrated that recombinant 3Dpol was not able to activate 30-blocked RNA templates and that under those conditions the polymerisation activity was strictly dependent on the addition of an external primer.

RNA polymerase assays using template RNA mixtures Two major hypothetical initiation mechanisms can be envisaged giving rise to the synthesis of double-length products from heteropolymeric RNA substrates (Fig. 5A). One of such mechanisms (self priming model) could be based on the 3Dpol-mediated formation of a hairpin self-priming structure at the template RNA 30-end, partly stabilised by conventional base-pairing between 30-neighbouring residues. A second mechanism (mixed template-primer model) might be consistent with the involvement of two RNA molecules in the initiation complex, each of them acting as template or primer of the other RNA molecule. Both models would equally explain the detection of labelled products larger than the starting template RNA, resulting from the addition of the newly synthesised RNA to the 30-end of the template. An approach to distinguish between both initiation mechanisms was the use of template mixtures containing different length synthetic RNAs whose 30 terminal sequences were unrelated (Fig. 5B and C) or identical (Fig. 5B and D). When RNA-a (2.2 kb) or RNA-b (0.5 kb) were separately used as templates, dimer-length products of 4.4 kb or 1 kb were observed respectively (Fig. 5C). The simultaneous inclusion of both templates in the reaction mixture gave rise to the same 4.4 kb and 1 kb products and no intermediate size products of 2.7 kb were detected. Considering that the nucleotide sequences of the two templates used in the preceding experiments were unrelated and their 30 regions were not identical, it could be proposed that the enzyme might require that the templates participating in the hypothetical bimolecular complex share an identical 30 region. For this purpose an RNA-a deletion derivative vas made giving rise to a smaller (1.3 kb) synthetic transcript (RNA-c) lacking of an RNA-a internal sequence of 828 nt (Fig. 5B). When RNA-a (2.2 kb) or RNA-c (1.3 kb) were used in separate reactions, dimer-length products of 4.4 kb or 2.6 kb were observed respectively (Fig. 5D). 66 A. López Vázquez et al.

Fig. 5. A Schematic representation of two hypotheses for the synthesis of template-linked RNA products by RHDV 3Dpol. B Schematic representation of the synthetic RNAs used and their localisation with respect to the RHDV genome. 32P-labeled RNA products synthesised by recombinant 3Dpol using mixtures of two RNA templates with different (C) or identical (D)30-end nucleotide sequences

The addition of both templates to the reaction mixture gave rise to the same 4.4 kb and 2.6 kb products and no intermediate 3.5 kb products were made.

Discussion The RNA viruses encode RdRps for their own genome replication. These proteins alone, or in conjunction with other host or virus-encoded proteins, carry out the synthesis of new RNA molecules. Despite the majority of RdRps were not identiﬁed by their RNA polymerase activities, but simply by sharing a number of well-conserved amino acid sequences [24], studies from our laboratory have demonstrated that the RHDV polyprotein cleavage product p58 [9, 30] was indeed an enzymatically active RNA-dependent RNA polymerase [13]. The substrate speciﬁcity of the recombinant RHDV 3Dpol clearly pointed out its role as an RdRp, as described also for HCV RNA polymerase [3]. These data were consistent with the RHDV 3Dpol resistance to high concentrations (200 ␮g ml−1) of antibiotic inhibitors such as rifampin or actinomycin D ruling out the presence of contaminating eukaryotic or prokaryotic DdRps activities. Recombinant RNA polymerase from RHDV 67

The 3Dpol polymerising activity was absolutely dependent on the presence of Mg2+, which could only be replaced by Mn2+. Similar ion requirements were found for Poliovirus [8] and HCV [12] RNA polymerases. The replication of a single-stranded RNA requires that the polymerase has either a primase activity or, alternatively, that the enzyme utilises a specific primer for extension. We have found that 3Dpol from RHDV can not act on 30-blocked heteropolymeric RNA templates, nor nucleolytically activate them to provide a 30-hydroxyl group which could be used to initiate polymerization. It can then be concluded that RNA synthesis catalysed by recombinant RHDV 3Dpol is strictly primer-dependent in the presence of homopolymeric or 30-blocked heteropolymeric templates which suggested that 3Dpol has no intrinsic primase activity. Nevertheless, a template self-primed reaction is preferred in the presence of 30- unblocked heteropolymeric templates. Similar requirements were described for the RdRp from HCV [3]. The results obtained using 30-blocked templates also supported that the in vitro synthesised dimer-length RNAs resulted from a covalent linkage between the orig- inal 30-OH of template RNA and the newly synthesised negative-polarity RNA product. Our data suggested that most likely the 30 terminus of the template RNA is held by the recombinant enzyme, looping back on itself, such that the RHDV RdRp reaction could initiate at the free 30-OH and extend toward the 50-end of the same RNA molecule, thus resulting in a hairpin-like RNA product (Fig. 5A). Nevertheless, from our results it could not be completely discarded that the initiation mechanism might involve two identical RNA molecules participating in a bimolecular mechanism (Fig. 5A). Sequence analysis of the template-product boundary, which will help to differentiate between these hypotheses, could not be performed after repeated attempts using PCR techniques, most likely due to the high stability of the double stranded RNA products. Several laboratories have found dimer-sized RNA products using viral RdRps in vitro. In the case of the Poliovirus 3Dpol, this finding has been described and dis- cussed by a number of authors [5, 14, 19, 23]. Interestingly, dimer-sized genomic RNA molecules have also been found in vivo [29, 31], which fuelled speculations that these molecules could represent replication intermediates. In view of these data, the priming at the 30-hydroxyl group of an input RNA and “copying-back” seems to be a common property of RNA polymerising enzymes. It should be mentioned that recent data have demonstrated that the Poliovirus VPg can be uridylylated by the 3Dpol in vitro and used to produce VPg-linked poly(U) in a poly(A) dependent reaction [22]. It could then be proposed that initiation of Poliovirus genome transcription might involve a protein primed mechanism. In spite of its demonstrated enzymatic capabilities, it seems likely that 3Dpol polymerase is only one of proteins involved in the replication of the RHDV genome. Further insights into the in vitro mechanism of RHDV RNA replication will require the expression and purification of other RHDV protein products, 68 A. López Vázquez et al. such as the VPg protein and its precursors, in addition to other required host cell proteins. Although we are far from a detailed knowledge of the mechanism underlying RHDV replication, and the cellular and viral factors required, the availability of sufficient amounts of purified 3Dpol should provide the basis for further studies in this direction and might be used as the starting point for the development of an in vitro replication system based on the reconstitution of a replicase complex from individual purified components. For this purpose it should be taken into account that our data indicates that the RHDV 3Dpol properties are very similar to those reported for Poliovirus [4, 8, 23], EMCV [28], and HCV [3, 12] polymerases. This might partly help to overcome the lack of a permissive tissue culture for RHDV, a fundamental tool for the study of virus replication.

Acknowledgements A. L. V.is recipient of a fellowship from Fundación Ramón Areces. This work was supported by grant PB96-0552-CO2-O1.

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