PLoS Pathogens – November 2010:

Zn Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

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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.

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 co-factor 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 co-factors [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 59- and 39- 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.

Results

Zinc and pyrithione inhibit nidovirus replication in vivo 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 mM) in the presence of 0 to 8 mMZnOAc2. Treatment with PT of concentrations up to 32 μm in combination with <4 mM ZnOAc2 did not reduce the viability of mock-infected cells after 18 h (Fig. 1A), as measured by the colorimetricMTS (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 mM (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 mM of PT and 0, 1, or 2 mM 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 mM 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 mM, 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 mM PT and 2 mM ZnOAc2 resulted in a 9861% and 8563% 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 mM was calculated for PT in the presence of 2 mM zinc. Half maximal inhibitory concentrations (IC50) of 1.4 mM and 0.5 mM and selectivity indices of 59 and 164 were calculated for SARS-CoV and EAV, respectively.

Zn2+ reversibly inhibits the RNA-synthesizing activity of isolated nidovirus RTCs We previously developed assays to study the in vitro RNAsynthesizing activity of RTCs isolated from cells infected with SARS-CoV or EAV [25,26]. In these RTC assays [a-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 mM, while less than 5% activity remained at a Zn2+ concentration of 500 mM. 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 mM 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 [a-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).

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