Abstract Increasing the intracellular Zn2+ concentration with zinc-ionophores like pyrithione (PT) can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn2+ and PT at low concentrations (2 µM Zn2+ and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV—thus eliminating the need for PT to transport Zn2+ across the plasma membrane—we show that Zn2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9) purified from E. coli subsequently revealed that Zn2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.

Author Summary Positive-stranded RNA (+RNA) viruses include many important pathogens. They have evolved a variety of replication strategies, but are unified in the fact that an RNA-dependent RNA polymerase (RdRp) functions as the core enzyme of their RNA-synthesizing machinery. The RdRp is commonly embedded in a membrane-associated replication complex that is assembled from viral RNA, and viral and host proteins. Given their crucial function in the viral replicative cycle, RdRps are key targets for antiviral research. Increased intracellular Zn2+ concentrations are known to efficiently impair replication of a number of RNA viruses, e.g. by interfering with correct proteolytic processing of viral polyproteins. Here, we not only show that corona- and arterivirus replication can be inhibited by increased Zn2+ levels, but also use both isolated replication complexes and purified recombinant RdRps to demonstrate that this effect may be based on direct inhibition of nidovirus RdRps. The combination of protocols described here will be valuable for future studies into the function of nidoviral enzyme complexes.

Citation: te Velthuis AJW, van den Worm SHE, Sims AC, Baric RS, Snijder EJ, van Hemert MJ (2010) Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture. PLoS Pathog 6(11): e1001176. https://doi.org/10.1371/journal.ppat.1001176 Editor: Raul Andino, University of California San Francisco, United States of America Received: May 17, 2010; Accepted: October 1, 2010; Published: November 4, 2010 Copyright: © 2010 te Velthuis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Netherlands Organization for Scientific Research (NWO) with grants from the Council for Chemical Sciences (NWO-CW grant 700.55.002 and 700.57.301) and an NWO Toptalent grant (021.001.037). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Zinc ions are involved in many different cellular processes and have proven crucial for the proper folding and activity of various cellular enzymes and transcription factors. Zn2+ is probably an important cofactor for numerous viral proteins as well. Nevertheless, the intracellular concentration of free Zn2+ is maintained at a relatively low level by metallothioneins, likely due to the fact that Zn2+ can serve as intracellular second messenger and may trigger apoptosis or a decrease in protein synthesis at elevated concentrations [1], [2], [3]. Interestingly, in cell culture studies, high Zn2+ concentrations and the addition of compounds that stimulate cellular import of Zn2+, such as hinokitol (HK), pyrrolidine dithiocarbamate (PDTC) and pyrithione (PT), were found to inhibit the replication of various RNA viruses, including influenza virus [4], respiratory syncytial virus [5] and several picornaviruses [6], [7], [8], [9], [10], [11]. Although these previous studies provided limited mechanistic information, this suggests that intracellular Zn2+ levels affect a common step in the replicative cycle of these viruses. In cell culture, PT stimulates Zn2+ uptake within minutes and inhibits RNA virus replication through a mechanism that has only been studied in reasonable detail for picornaviruses [11], [12]. In vitro studies with purified rhinovirus and poliovirus 3C proteases revealed that protease activity was inhibited by Zn2+ [13], [14], which is in line with the inhibition of polyprotein processing by zinc ions that was observed in cells infected with human rhinovirus and coxsackievirus B3 [11]. The replication of segmented negative-strand RNA viruses such as influenza virus, however, does not depend on polyprotein processing and the effect of PDTC-mediated Zn2+ import was therefore hypothesized to result from inhibition of the viral RNA-dependent RNA polymerase (RdRp) and cellular cofactors [4]. Moreover, an inhibitory effect of Zn2+ on the activity of purified RdRps from rhinoviruses and hepatitis C virus was noted, but not investigated in any detail [15], [16]. Details on the effect of zinc ions are currently largely unknown for nidoviruses. This large group of positive-strand RNA (+RNA) viruses includes major pathogens of humans and livestock, such as severe acute respiratory syndrome coronavirus (SARS-CoV), other human coronaviruses, the arteriviruses equine arteritis virus (EAV), and porcine reproductive and respiratory syndrome virus (PRRSV) [17], [18]. The common ancestry of nidoviruses is reflected in their similar genome organization and expression strategy, and in the conservation of a number of key enzymatic functions in their large replicase polyproteins [19]. A hallmark of the corona- and arterivirus replicative cycle is the transcription of a 5′- and 3′-coterminal nested set of subgenomic (sg) mRNAs from which the viral structural and accessory protein genes are expressed [20], [21]. Analogous to picornaviruses [13], [22], zinc ions were demonstrated to inhibit certain proteolytic cleavages in the processing of the coronavirus replicase polyproteins in infected cells and cell-free systems [23], [24]. In this study we report that the zinc-ionophore pyrithione (PT) in combination with Zn2+ is a potent inhibitor of the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. To assess whether - besides a possible effect on proteolytic processing - nidovirus RTC subunits and RNA synthesis are directly affected by Zn2+, we employed in vitro systems for SARS-CoV and EAV RNA synthesis that are based on membrane-associated RTCs isolated from infected cells (from here on referred to as RTC assays) [25], [26]. In addition, we used in vitro recombinant RdRp assays to directly study the effect of zinc ions on the RdRps of SARS-CoV and EAV [27], [28]. Using these independent in vitro approaches, we were able to demonstrate that Zn2+ directly impairs nidovirus RNA synthesis, since it had a strong inhibitory effect in both RTC and RdRp assays. Interestingly, the Zn2+-mediated inhibition could be reversed through the addition of a Zn2+ chelator (MgEDTA). We therefore applied this compound to stop and restart the in vitro RNA-synthesizing activity at will. This convenient tool allowed us to study various mechanistic aspects of arteri- and coronavirus RNA synthesis in more detail. Additionally, the zinc-mediated inhibition of nidovirus RNA synthesis described here may provide an interesting basis to further explore the use of zinc-ionophores in antiviral therapy.

Discussion Although a variety of compounds have been studied, registered antivirals are currently still lacking for the effective treatment of SARS and other nidovirus-related diseases [33]. RdRps are suitable targets for antiviral drug development as their activity is strictly virus-specific and may be blocked without severely affecting key cellular functions. Several inhibitors developed against the polymerases of e.g. human immunodeficiency virus (HIV) and hepatitis C virus are currently being used in antiviral therapy or clinical trials [34], [35], [36]. Therefore, advancing our molecular knowledge of nidovirus RdRps and the larger enzyme complexes that they are part of, and utilizing the potential of recently developed in vitro RdRp assays [25], [26], [27], [28] could ultimately aid in the development of effective antiviral strategies. Zinc ions and zinc-ionophores, such as PT and PDTC, have previously been described as potent inhibitors of various RNA viruses. We therefore investigated whether PT-stimulated import of zinc ions into cells also inhibited the replication of nidoviruses in cell culture. Using GFP-expressing EAV and SARS-CoV [29], [30], we found that the combination of 2 µM PT and 2 µM Zn2+ efficiently inhibited their replication, while not causing detectable cytoxicity (Fig. 1). Inhibition of replication by PT and Zn2+ at similar concentrations (2–10 µM) was previously observed for several picornaviruses such as rhinoviruses, foot-and-mouth disease virus, coxsackievirus, and mengovirus [6], [7], [8], [9], [10], [11]. The inhibitory effect of Zn2+ on the replication of picornaviruses appeared to be due to interference with viral polyprotein processing. In infections with the coronavirus mouse hepatitis virus (MHV), Zn2+ also interfered with some of the replicase polyproteins cleavages [24], albeit at a much higher concentration (100 µM Zn2+) than used in our studies. Since impaired replicase processing will indirectly affect viral RNA synthesis in the infected cell, we used two recently developed in vitro assays to investigate whether Zn2+ also affects nidovirus RNA synthesis directly. Our in vitro studies revealed a strong inhibitory effect of zinc ions on the RNA-synthesizing activity of isolated EAV and SARS-CoV RTCs. Assays with recombinant enzymes subsequently demonstrated that this was likely due to direct inhibition of RdRp function. The inhibitory effect could be reversed by chelating the zinc ions, which provides an interesting experimental (on/off) approach to study nidovirus RNA synthesis. Addition of Zn2+ following initiation of EAV RNA synthesis had little or no effect on NTP incorporation in molecules whose synthesis had already been initiated in the absence of Zn2+ (Fig. 6 and 7), indicating that Zn2+ does not affect elongation and does not increase the termination frequency, as was previously found for Mn2+ [25]. Therefore, Zn2+ appears to be a specific inhibitor of the initiation phase of EAV RNA synthesis. In contrast, Zn2+ inhibited SARS-CoV RdRp activity also during the elongation phase of RNA synthesis, probably by directly affecting template binding (Fig. 8). In coronaviruses, zinc ions thus appear to inhibit both the proper proteolytic processing of replicase polyproteins [23], [24] and RdRp activity (this study). Contrary to the RTC assays, millimolar instead of micromolar concentrations of ZnOAc 2 were required for a nearly complete inhibition of nucleotide incorporation in RdRp assays. It has been well established that DNA and RNA polymerases use a triad of conserved aspartate residues in motifs A and C to bind divalent metal ions like Mg2+, which subsequently coordinate incoming nucleotides during the polymerization reaction [37], [38]. Mg2+ is also the divalent metal ion that is required for the in vitro activity of isolated SARS-CoV and EAV RTCs and recombinant RdRps [25], [26], [27], [28], although de novo initiation of EAV nsp9 is primarily Mn2+-dependent. Zn2+ could not substitute for Mg2+ or Mn2+ as cofactor as it was incapable of supporting the polymerase activity of nidovirus RTCs and RdRps in the absence of Mg2+ (data not shown), as was also reported for the poliovirus RdRp [39]. Moreover, inhibition of nidovirus RdRp activity by Zn2+ was even observed at low concentrations and in the presence of a more than 25-fold excess of Mg2+, suggesting that either the affinity of the active site for Zn2+ is much higher or that Zn2+ does not compete for Mg2+-binding and binds to another zinc(-specific) binding site in the protein. Specific protein domains or pockets that contain zinc ions may be involved in protein-protein interactions, protein-RNA/DNA interactions, or conformational changes in enzyme structures. Zinc-binding domains commonly consist of at least three conserved cysteine and/or histidine residues within a stretch of ∼10–30 amino acids, such as in zinc-finger motifs and metalloproteases [2], [40], [41]. However, in RdRps there are only few precedents for the presence of zinc-binding pockets, such as those identified in the crystal structure of the Dengue RdRp [42]. Sequence analysis of the EAV nsp9 amino acid sequence revealed that it lacks patches rich in conserved cysteines and/or histidines. In contrast, inspection of the SARS-CoV nsp12 amino acid sequence revealed two such patches, namely H295-C301-C306-H309-C310 and C799-H810-C813-H816. A crystal structure for nsp12 is presently unavailable, but a predicted structure that represents the C-terminal two-thirds of the enzyme has been published [31]. Interestingly, in this model, C799, H810, C813 and H816 are in a spatial arrangement resembling that of the Zn2+ coordinating residues in the Zn2 zinc-binding pocket found in motif E of the Dengue virus RdRp (see Supplemental Fig. S3). Clearly, an in-depth analysis of nidovirus RdRps, e.g. through structural analysis and subsequent mutational studies targeting aforementioned cysteines and histidines, is required to provide further insight into and a structural basis for the Zn2+-induced inhibitory effects on RdRp activity documented in this study. Such studies may, however, be complicated when Zn2+ binding proves to be very transient in nature and not detectable with currently available methods. In summary, the combination of zinc ions and the zinc-ionophore PT efficiently inhibits nidovirus replication in cell culture. This provides an interesting basis for further studies into the use of zinc-ionophores as antiviral compounds, although systemic effects have to be considered [43], [44] and a water-soluble zinc-ionophore may be better suited, given the apparent lack of systemic toxicity of such a compound at concentrations that were effective against tumors in a mouse xenograft model [45]. In vitro, the reversible inhibition of the RdRp by Zn2+ has also provided us with a convenient research tool to gain more insight into the molecular details of (nido)viral RNA synthesis, and revealed novel mechanistic differences between the RdRps of SARS-CoV and EAV.

Materials and Methods Cells and viruses Vero-E6 cells were cultured and infected with SARS-CoV (strain Frankfurt-1; accession nr. AY291315) or SARS-CoV-GFP as described previously [46]. All procedures involving live SARS-CoV were performed in the biosafety level 3 facility at Leiden University Medical Center. BHK-21 or Vero-E6 cells were cultured and infected with EAV (Bucyrus strain; accession nr. NC_002532) or EAV-GFP [29] as described elsewhere [25]. Effect of zinc ions on nidovirus replication in cell culture One day prior to infection, Vero-E6 cells were seeded in transparent or black (low fluorescence) 96-well clusters at 10,000 cells per well. The next day, cells were infected with SARS-CoV-GFP or EAV-GFP with an m.o.i. of 4, and 1 h p.i. the inoculum was removed and 100 µl of medium containing 2% fetal calf serum (FCS) was added to each well. In some experiments 0–32 µM of pyrithione (Sigma) was added in addition to 0–2 µM ZnOAc 2 . Infected cells were fixed at 17 h p.i. by aspirating the medium and adding 3% paraformaldehyde in PBS. After washing with PBS, GFP expression was quantified by measuring fluorescence with a LB940 Mithras plate reader (Berthold) at 485 nm. To determine toxicity of ZnOAc 2 and PT, cells were exposed to 0–32 µM PT and 0–8 µM ZnOAc 2 . After 18 h incubation, cell viability was determined with the Cell Titer 96 AQ MTS assay (Promega). EC 50 and CC 50 values were calculated with Graphpad Prism 5 using the nonlinear regression model. RNA templates and oligonucleotides RNA oligonucleotides SAV557R (5′-GCUAUGUGAGAUUAAGUUAU-3′), SAV481R (5′-UUUUUUUUUUAUAACUUAAUCUCACAUAGC-3′) and poly(U) 18 (5′-UUUUUUUUUUUUUUUUUU-3′) were purchased from Eurogentec, purified from 7 M Urea/15% PAGE gels and desalted through NAP-10 columns (GE healthcare). To anneal the RNA duplex SAV557R/SAV481R, oligonucleotides were mixed at equimolar ratios in annealing buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl and 5 mM EDTA), denatured by heating to 90°C and allowed to slowly cool to room temperature after which they were purified from 15% non-denaturing PAGE gels. In vitro viral RNA synthesis assay with isolated RTCs SARS-CoV and EAV RTCs were isolated from infected cells and assayed for activity in vitro as described previously [25], [26]. To assess the effect of Zn2+, 1 µl of a ZnOAc 2 stock solution was added to standard 28-µl reactions, resulting in final Zn2+ concentrations of 10–500 µM. When Zn2+ had to be chelated in the course of the reaction, magnesium-saturated EDTA (MgEDTA) was added to a final concentration of 1 mM. After RNA isolation, the 32P-labeled reaction products were separated on denaturing 1% (SARS-CoV) or 1.5% (EAV) agarose formaldehyde gels. The incorporation of [α-32P]CMP into viral RNA was quantified by phosphorimaging of the dried gels using a Typhoon scanner (GE Healthcare) and the ImageQuant TL 7 software (GE Healthcare). Expression and purification of nidovirus RdRps SARS-CoV nsp12 and EAV nsp9 were purified essentially as described elsewhere [27], [28], but with modifications for nsp9. In short, E. coli BL21(DE3) with plasmid pDEST14-nsp9-CH was grown in auto-induction medium ZYM-5052 [47] for 6 hours at 37°C and a further 16 hours at 20°C. After lysis in buffer A (20 mM HEPES pH 7.4, 200 mM NaCl, 20 mM imidazole, and 0.05% Tween-20) the supernatant was applied to a HisTrap column (GE Healthcare). Elution was performed with a gradient of 20–250 mM imidazole in buffer A. The nsp9-containing fraction was further purified by gel filtration in 20 mM HEPES, 300 mM NaCl and 0.1% Tween-20 on a Superdex 200 column (GE Healthcare). The fractions containing nsp9-CH were pooled, dialyzed against 1000 volumes of buffer B (20 mM HEPES, 100 mM NaCl, 1 mM DTT and 50% glycerol) and stored at −20°C. RdRps with a D618A (SARS-CoV) or D445A (EAV) mutation were obtained by site-directed mutagenesis of the wild-type (wt) plasmid pDEST14-nsp9-CH [28] with oligonucleotides 5′-TACTGCCTTGAAACAGCCCTGGAGAGTTGTGAT-3′ and 5′-ATCACAACTCTCCAGGGCTGTTTCAAGGCAGTA-3′, and plasmid pASK3-Ub-nsp12-CHis 6 with oligonucleotides 5′-CCTTATGGGTTGGGCTTATCCAAAATGTG-3′ and 5′-CACATTTTGGATAAGCCCAACCCATAAGGA-3′, as described elsewhere [27]. Mutant proteins were purified parallel to the wt enzymes. RdRp assays with purified enzymes Standard reaction conditions for the RdRp assay with 0.1 µM of purified SARS-CoV nsp12 are described elsewhere [27]. To study the effect of Zn2+ in this assay, 0.5 µl of a dilution series of 0–80 mM ZnOAc 2 was added to the 5 µl reaction mixture, yielding final Zn2+ concentrations of 0–8 mM. The EAV RdRp assay contained 1 µM nsp9, 1 µM RNA template poly(U) 18 , 0.17 µM [α-32P]ATP (0.5 µCi/µl; Perkin-Elmer), 50 µM ATP, 20 mM Tris-HCl (pH 8.0), 10 mM NaCl, 10 mM KCl, 1 mM MnCl 2 , 4 mM MgOAc 2 , 5% glycerol, 0.1% Triton-X100, 1 mM DTT and 0.5 units RNaseOUT. ZnOAc 2 was added to the reaction to give a final concentration of 0–6 mM. To chelate Zn2+ during reactions, MgEDTA was added to a final concentration of 8 mM. Reactions were terminated after 1 hour and analyzed as described [27]. SARS-CoV nsp12 electrophoretic mobility shift assay SARS-CoV RdRp was incubated with 0.2 nM 5′ 32P-labeled SAV557R/SAV481R RNA duplex, for 10 minutes at 30°C either in presence or absence of 6 mM ZnOAc 2 . Reactions were analyzed as described previously [27].

Supporting Information Figure S1. Effect of various divalent cations on the RdRp activity of SARS-CoV nsp12. Purified recombinant SARS-CoV nsp12 was incubated with a primed template, ATP, and [α-32P]ATP in the presence of either 6 mM Mg2+ only (lane 1), and with increasing concentrations of a second divalent metal (M2+), specifically: 2–6 mM Ca2+ (lane 2–4), 2–6 mM Co2+ (lane 5–7), 2–6 mM Zn2+ (lane 8–10), or 2–6 mM Mn2+ (lane 11–13). The strongest inhibition was observed for Zn2+. For more details on the SARS-CoV nsp12 RdRp assay, see the main text. https://doi.org/10.1371/journal.ppat.1001176.s001 (1.55 MB TIF) Figure S2. Effect of Zn2+ on the dinucleotide extension activity of EAV nsp9. Purified recombinant EAV nsp9 was incubated with a U18 template in the presence of [α-32P]ATP, ATP, 4 mM Mg2+, 1 mM Mn2+, and 1 µM ApA. (A) Reaction mixtures were split into two aliquots, one of which was supplemented with 6 mM Zn2+, and samples were taken at the time points (minutes) indicated above the lanes. In the absence of Zn2+, EAV nsp9 initiates de novo and produces di- and trinucleotides, indicated with A2 and A3, respectively. A non-specific band, unrelated to RdRp activity, between A2 and A3 is indicated with an asterisk. In the presence of 6 mM Zn2+, the synthesis of dinucleotides and trinucleotides was blocked. (B) When performing the assay described under (A) in the absence of Zn2+, a full-length product of 18 nucleotides is formed. This product is not observed when the assay is performed in the presence of 6 mM Zn2+, but nsp9 was capable of elongating the provided dinucleotide primer ApA into tri- (ApA*pA) and tetranucleotide ((ApA*pA*pA) products (the asterisk indicates radiolabeled phosphates). Due to the absence of a 5′ triphosphate group, these reaction products migrate much slower in the 20% acrylamide and 7 M urea gel used for this analysis. See the main text for additional experimental details on the EAV nsp9 RdRp assay. https://doi.org/10.1371/journal.ppat.1001176.s002 (2.16 MB TIF) Figure S3. Putative zinc-binding residues in the predicted structure of SARS-CoV nsp12 and comparison with the structure of the zinc-containing Dengue virus RdRp domain. (A) Sequence alignment of coronavirus RdRps showing conservation of four potential zinc-binding residues amino acids (C799-H810-C813-H816 in SARS-CoV; indicated with asterisks) in the C-terminal region of coronavirus nsp12. Black shading indicates complete conservation among coronaviruses. The coronavirus RdRp sequences were aligned with Muscle 3.6. The aligned sequences and NCBI accession numbers are the following: mouse hepatitis virus strain A59 (MHV_A59; NP_068668), human CoV 229E (HCoV_229E; NP_068668), infectious bronchitis virus strain Beaudette (IBV_B; P0C6Y1), bovine coronavirus (BCoV; NP_742138.1), feline coronavirus (FeCoV; YP_239353.1), and SARS-CoV strain Frankfurt-1 (SARS_Fr1; AAP33696). (B) Crystal structure of the Dengue virus RdRp domain showing the position of four cysteine and histidine residues that form Zn2+-binding pocket Zn2, located close to motif E (depicted in red). A second Zn2+-binding pocket (Zn1) and the two zinc ions identified in the crystal structure are indicated in blue-gray. (C) Predicted three-dimensional structure model of SARS-CoV nsp12 (Xu et al., Nucl. Acids Res. 31: 7117–7130), based on PDB code 1O5S, rendered with Swiss-PdbViewer 4.01 and POV-Ray 3.6. The positions of the conserved cysteine and histidine residues indicated in panel A (C799-H810-C813-H816) close to motif E (depicted in red) and RdRp active-site residues (D618, D760 and D761) are indicated. The spatial arrangement of these cysteines and histidines in this model strikingly resembles the positioning of the metal ion-coordinating residues of Zn-binding pocket Zn2 in the Dengue virus RdRp domain (see panel B). https://doi.org/10.1371/journal.ppat.1001176.s003 (0.86 MB TIF)

Author Contributions Conceived and designed the experiments: AJWtV SHEvdW EJS MJvH. Performed the experiments: AJWtV SHEvdW MJvH. Analyzed the data: AJWtV SHEvdW ACS RSB EJS MJvH. Contributed reagents/materials/analysis tools: ACS RSB. Wrote the paper: AJWtV EJS MJvH.