Zinc ions are involved in many different cellular processes, but the concentration of free Zn²⁺ is maintained at a relatively low level by metallothioneins [1]. Zn²⁺ and compounds that stimulate import of Zn²⁺ into cells, such as PT, were previously found to inhibit replication of several picornaviruses, including rhinoviruses, foot-and-mouth disease virus, coxsackievirus, and mengovirus in cell culture [6], [7], [8], [9], [10], [11]. To determine whether Zn²⁺ has a similar effect on nidoviruses, we investigated the effect of PT and Zn²⁺ on the replication of EAV and SARS-CoV in Vero-E6 cells, using reporter viruses that express green fluorescent proteins (GFP), i.e., EAV-GFP [29] and SARS-CoV-GFP [30]. EAV-GFP encodes an N-terminal fusion of GFP to the viral nonstructural protein 2 (nsp2), one of the cleavage products of the replicase polyproteins, and thus provides a direct readout for translation of the replicase gene. In SARS-CoV-GFP, reporter expression occurs from sg mRNA 7, following the replacement of two accessory protein-coding genes (ORFs 7a and 7b) that are dispensable for replication in cell culture.

We first assessed the cytotoxicity of a range of PT concentrations (0–32 µM) in the presence of 0 to 8 µM ZnOAc₂. Treatment with PT of concentrations up to 32 µM in combination with <4 µM ZnOAc₂ did not reduce the viability of mock-infected cells after 18 h (Fig. 1A), as measured by the colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) viability assay. As elevated Zn²⁺ concentrations are known to inhibit cellular translation, we also used metabolic labeling with ³⁵S-methionine to assess the effect of PT and Zn²⁺ on cellular protein synthesis. Incubation of Vero-E6 cells for 18 h with the combinations of PT and Zn²⁺ mentioned above, followed by a 2-h metabolic labeling, revealed no change in overall cellular protein synthesis when the concentration of ZnOAc₂ was <4 µM (data not shown).

Using these non-cytotoxic conditions we subsequently tested the effect of PT and ZnOAc₂ on EAV-GFP and SARS-CoV-GFP replication. To this end, Vero-E6 cells in 96-well plates were infected with a multiplicity of infection (m.o.i.) of 4. One hour post infection (h p.i.), between 0 and 32 µM of PT and 0, 1, or 2 µM ZnOAc₂ were added to the culture medium. At 17 h p.i., a time point at which GFP expression in untreated infected cells reaches its maximum for both viruses, cells were fixed, and GFP fluorescence was quantified.

The reporter gene expression of both SARS-CoV-GFP and EAV-GFP was already significantly inhibited in a dose-dependent manner by the addition of PT alone (Fig. 1B and C). This effect was significantly enhanced when 2 µM of Zn²⁺ was added to the medium. We found that addition of ZnOAc₂ alone also reduced virus replication, but only at levels that were close to the 50% cytotoxicity concentration (CC₅₀) of ZnOAc₂ in Vero-E6 cells (∼70 µM, data not shown). This is likely due to the poor solubility of Zn²⁺ in phosphate-containing medium and the inefficient uptake of Zn²⁺ by cells in the absence of zinc-ionophores. The combination of 2 µM PT and 2 µM ZnOAc₂ resulted in a 98±1% and 85±3% reduction of the GFP signal for EAV-GFP and SARS-CoV-GFP, respectively. No cytotoxicity was observed for this combination of PT and ZnOAc₂ concentrations. From the dose-response curves in Fig. 1, a CC₅₀ value of 82 µM was calculated for PT in the presence of 2 µM zinc. Half maximal inhibitory concentrations (IC₅₀) of 1.4 µM and 0.5 µM and selectivity indices of 59 and 164 were calculated for SARS-CoV and EAV, respectively.

We previously developed assays to study the in vitro RNA-synthesizing activity of RTCs isolated from cells infected with SARS-CoV or EAV [25], [26]. In these RTC assays [α-³²P]CMP is incorporated into both genomic (replication) and sg mRNA (transcription) (Fig. 2). This allowed us to monitor the synthesis of the same viral RNA molecules that can be detected by hybridization of RNA from nidovirus-infected cells. A benefit of these assays is that the activity does not depend on continued protein synthesis and that it allows us to study viral RNA synthesis independent of other aspects of the viral replicative cycle [26]. To investigate whether the inhibitory effect of PT and zinc ions on nidovirus replication in cell culture is reflected in a direct effect of Zn²⁺ on viral RNA synthesis, we tested the effect of Zn²⁺ addition on RTC activity. For both EAV (Fig. 2A) and SARS-CoV (Fig. 2B), a dose-dependent decrease in the amount of RNA synthesized was observed when ZnOAc₂ was present. For both viruses, a more than 50% reduction of overall RNA-synthesis was observed at a Zn²⁺ concentration of 50 µM, while less than 5% activity remained at a Zn²⁺ concentration of 500 µM. Both genome synthesis and sg mRNA production were equally affected.

To test whether the inhibition of RTC activity by Zn²⁺ was reversible, RTC reactions were started in the presence or absence of 500 µM Zn²⁺. After 30 min, these reactions were split into two aliquots and magnesium-saturated EDTA (MgEDTA) was added to one of the tubes to a final concentration of 1 mM (Fig. 3A). We used MgEDTA as Zn²⁺ chelator in these in vitro assays, because it specifically chelates Zn²⁺ while releasing Mg²⁺, due to the higher stability constant of the ZnEDTA complex. Uncomplexed EDTA inhibited RTC activity in all reactions (data not shown), most likely by chelating the Mg²⁺ that is crucial for RdRp activity [27], [28], whereas MgEDTA had no effects on control reactions without Zn²⁺ (Fig. 3B, compare lane 1 and 2). As shown in Fig. 2, the EAV RTC activity that was inhibited by Zn²⁺ (Fig. 3B&C, lane 3) could be restored by the addition of MgEDTA (Fig. 3B, lane 4) to a level observed for control reactions without Zn²⁺ (Fig. 3B, lane 1). Compared to the untreated control, the EAV RTC assay produced approximately 30% less RNA, which was consistent with the 30% shorter reaction time after the addition of the MgEDTA (100 versus 70 min for lanes 1 and 4, respectively). Surprisingly, SARS-CoV RTC assays that were consecutively supplemented with Zn²⁺ and MgEDTA incorporated slightly more [α-³²P]CMP compared to untreated control reactions (Fig. 3C; compare lane 1 and 4). This effect was not due to chelation of the Zn²⁺ already present in the post-nuclear supernatant (PNS) of SARS-CoV-infected cells, as this increase was not observed when MgEDTA was added to a control reaction without additional Zn²⁺ (Fig. 3C, lane 2).

To establish whether inhibition of RTC activity might be due to a direct effect of Zn²⁺ on nidovirus RdRp activity, we tested the effect of Zn²⁺ on the activity of the purified recombinant RdRps of SARS-CoV (nsp12) and EAV (nsp9) using previously developed RdRp assays [27], [28]. Using an 18-mer polyU template, the EAV RdRp incorporated [α-³²P]AMP into RNA products of up to 18 nt in length (Fig. 4A). Initiation was de novo, which is in line with previous observations and the presence of a conserved priming loop in the nsp9 sequence [28]. Unlike the EAV RdRp nsp9, the in vitro activity of the SARS-CoV RdRp nsp12 - which lacks a priming loop - was shown to be strictly primer-dependent [27]. Thus, to study the RdRp activity of SARS-CoV nsp12, a primed polyU template was used (Fig. 4B), thereby allowing us to sample [α-³²P]AMP incorporation as described previously [27]. As specificity controls, we used the previously described SARS-CoV nsp12 mutant D618A [27], which contains an aspartate to alanine substitution in motif A of the RdRp active site, and EAV nsp9-D445A, in which we engineered an aspartate to alanine substitution at the corresponding site of EAV nsp9 [28], [31]. Both mutant RdRps showed greatly reduced [α-³²P]AMP incorporation in our assays (Fig. 4), confirming once again that the radiolabeled RNA products derived from nidovirus RdRp activity.

Addition of ZnOAc₂ to RdRp assays resulted in a strong, dose-dependent inhibition of enzymatic activity for both the EAV and SARS-CoV enzyme (Fig. 5A and B, respectively), similar to what was observed in RTC assays. In fact, compared to other divalent metal ions such Co²⁺ and Ca²⁺, which typically bind to amino acid side chains containing oxygen atoms rather than sulfur groups, Zn²⁺ was the most efficient inhibitor of SARS-CoV nsp12 RdRp activity (Supplemental Fig. S1). To test whether, as in the RTC assay, the RdRp inhibition by zinc ions was reversible, RdRp assays were pre-incubated with 6 mM Zn²⁺, a concentration that consistently gave >95% inhibition. After 30 min, 8 mM MgEDTA was added to both a control reaction and the reaction inhibited with ZnOAc₂, and samples were incubated for another 30 min (Fig. 5C). As shown in Fig. 5D, the inhibition of EAV RdRp activity by Zn²⁺ could be reversed by chelation of Zn²⁺ (Fig. 5D; compare lanes 3 and 4). The amount of product synthesized was consistently 60±5% of that synthesized in a 60-min control reaction (Fig. 5D; compare lanes 1 and 4), which was within the expected range given the shorter reaction time. The inhibition of the SARS-CoV RdRp was reversible as well. During the 30-min incubation after the addition of MgEDTA, SARS-CoV nsp12 incorporated 40±5% of the label incorporated during a standard 60-min reaction (Fig. 5E). This was slightly lower than the expected yield and may be caused by the elevated Mg²⁺ concentration, which was shown to be suboptimal for nsp12 activity [27] and results from the release of Mg²⁺ from MgEDTA upon chelation of Zn²⁺.

For EAV, close inspection of the RdRp assays revealed a less pronounced effect of Zn²⁺ on the generation of full-length 18-nt products than on the synthesis of smaller reaction intermediates (Fig. 5A). This suggested that Zn²⁺ specifically inhibited the initiation step of EAV RNA synthesis. To test this hypothesis, an RTC assay was incubated for 30 min with unlabeled CTP (initiation), after which the reaction was split in two. Then, [α-³²P]CTP was added to both tubes (pulse), 500 µM Zn²⁺ was added to one of the tubes, and samples were taken at different time points during the reaction (Fig. 6A). Fig. 6B shows that in the presence of Zn²⁺ [α-³²P]CMP was predominantly incorporated into nascent RNA molecules that were already past the initiation phase at the moment that Zn²⁺ was added to the reaction. No new initiation occurred, as was indicated by the smear of short radiolabeled products that progressively shifted up towards the position of full-length genomic RNA. This suggested that Zn²⁺ does not affect the elongation phase of EAV RNA synthesis and that it specifically inhibits initiation. This also explains the relatively weak signal intensity of the smaller sg mRNA bands (e.g., compare the relative change in signal of RNA2 to RNA7) produced in the presence of Zn²⁺, since multiple initiation events are required on these short molecules to obtain signal intensities similar to those resulting from a single initiation event on the long genomic RNA, e.g., 16 times more in the case of RNA7. In contrast to EAV, the effect of Zn²⁺ on RNA synthesis by SARS-CoV RTCs was not limited to initiation, but appeared to impair the elongation phase as well, given that the addition of Zn²⁺ completely blocked further incorporation of [α-³²P]CMP when added 40 min after the start of the reaction (Fig. 6C).

In the RdRp assays, the short templates used made it technically impossible to do experiments similar to those performed with complete RTCs. However, we previously noticed that at low concentrations of [α-³²P]ATP (∼0.17 µM) SARS-CoV nsp12 RdRp activity was restricted to the addition of only a single nucleotide to the primer [27]. EAV nsp9 mainly produced very short (2–3 nt long) abortive RNA products and only a fraction of full length products, as is common for de novo initiating RdRps [28]. This allowed us to separately study the effect of Zn²⁺ on initiation and elongation by performing an experiment in which a pulse with a low concentration of [α-³²P]ATP was followed by a chase in the presence of 50 µM of unlabeled ATP, which increased processivity and allowed us to study elongation (Fig. 7A and C) as described previously [27]. The results of these experiments were in agreement with those obtained with isolated RTCs. For EAV, with initiation and dinucleotide synthesis completely inhibited by the presence of 6 mM Zn²⁺ (Supplemental Fig. S2A), the amount of reaction intermediates shorter than 18 nt diminished with time, while products from templates on which the RdRp had already initiated were elongated to full-length 18-nt molecules (Fig. 7B, right panel). This was consistent with the observation that the EAV RdRp remained capable of extending the synthetic dinucleotide ApA to trinucleotides in the presence of Zn²⁺ (Supplemental Fig. S2B). Likely due to the absence of reinitiation in the reactions shown in Fig. 7B, the low processivity of the EAV RdRp, and the substrate competition between the remaining [α-³²P]ATP and the >200 fold excess of unlabeled ATP, the differences between the 5- and 30-min time points were small. In the absence of Zn²⁺, the RdRp continued to initiate as indicated by the ladder of smaller-sized RNA molecules below the full-length product (Fig. 7B, left panel) and the time course shown in Supplemental Fig. S2A. In contrast, the addition of Zn²⁺ to a SARS-CoV RdRp reaction also blocked elongation, since extension of the radiolabeled primer as observed in the absence of Zn²⁺ (Fig. 7D, left panel) no longer occurred (Fig. 7D, right panel).

To assess whether Zn²⁺ affects the interaction of recombinant SARS-CoV nsp12 with the template used in our assays, we performed electromobility shift assays (EMSA) in the presence and absence of Zn²⁺ (Fig. 8A). To measure the binding affinity of the RdRp for the template, we determined the fraction of bound template at various protein concentrations and observed a 3–4 fold reduction in RNA binding when Zn²⁺ was present in the assay (Fig. 8B). We also assessed whether pre-incubation of the RdRp or RNA with Zn²⁺ was a requirement for this drop in binding affinity, but found no significant difference with experiments not involving such a preincubation (data not shown).

No binding was observed when a similar RNA binding assay was performed with purified EAV RdRp. Likewise, nsp9 did not bind RNA in pull-down experiments with Talon-beads, His₆-tagged nsp9, and radiolabeled EAV genomic RNA or various short RNA templates including polyU, whereas we were able to detect binding of a control protein (SARS-CoV nsp8, which has demonstrated RNA and DNA binding activity [32]) using this assay. It presently remains unclear why we are not able to detect the binding of recombinant EAV nsp9 to an RNA template.

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].

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 ZnOAc₂. 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 ZnOAc₂ and PT, cells were exposed to 0–32 µM PT and 0–8 µM ZnOAc₂. After 18 h incubation, cell viability was determined with the Cell Titer 96 AQ MTS assay (Promega). EC₅₀ and CC₅₀ values were calculated with Graphpad Prism 5 using the nonlinear regression model.

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.

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 ZnOAc₂ 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).

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-CHis₆ with oligonucleotides 5′-CCTTATGGGTTGGGCTTATCCAAAATGTG-3′ and 5′-CACATTTTGGATAAGCCCAACCCATAAGGA-3′, as described elsewhere [27]. Mutant proteins were purified parallel to the wt 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 ZnOAc₂ 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 MnCl₂, 4 mM MgOAc₂, 5% glycerol, 0.1% Triton-X100, 1 mM DTT and 0.5 units RNaseOUT. ZnOAc₂ 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 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 ZnOAc₂. Reactions were analyzed as described previously [27].