【病毒外文文獻】2010 Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replicatio

Zn 2 Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture Aartjan J W te Velthuis 1 Sjoerd H E van den Worm 1 Amy C Sims 2 Ralph S Baric 2 Eric J Snijder 1 Martijn J van Hemert 1 1Molecular Virology Laboratory Department of Medical Microbiology Center of Infectious Diseases Leiden University Medical Center Leiden The Netherlands 2Departments of Epidemiology and Microbiology and Immunology University of North Carolina at Chapel Hill Chapel Hill North Carolina United States of America Abstract Increasing the intracellular Zn 2 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 Zn 2 and PT at low concentrations 2 mMZn 2 and 2 mM 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 Zn 2 across the plasma membrane we show that Zn 2 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 Zn 2 directly inhibited the in vitro activity of both nidovirus polymerases More specifically Zn 2 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 Zn 2 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 Citation te Velthuis AJW van den Worm SHE Sims AC Baric RS Snijder EJ et al 2010 Zn 2 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 doi 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 C223 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 E mail e j snijder lumc nl ES m j van hemert lumc nl MJvH 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 Zn 2 is probably an important cofactor for numerous viral proteins as well Neverthe less the intracellular concentration of free Zn 2 is maintained at a relatively low level by metallothioneins likely due to the fact that Zn 2 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 Zn 2 concentrations and the addition of compounds that stimulate cellular import of Zn 2 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 Zn 2 levels affect a common step in the replicative cycle of these viruses In cell culture PT stimulates Zn 2 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 Zn 2 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 Zn 2 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 Zn 2 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 PLoS Pathogens www plospathogens org 1 November 2010 Volume 6 Issue 11 e1001176 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 demon strated 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 Zn 2 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 Zn 2 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 Zn 2 directly impairs nidovirus RNA synthesis since it had a strong inhibitory effect in both RTC and RdRp assays Interestingly the Zn 2 mediated inhibition could be reversed through the addition of a Zn 2 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 Zn 2 is maintained at a relatively low level by metallothioneins 1 Zn 2 and compounds that stimulate import of Zn 2 into cells such as PT were previously found to inhibit replication of several picornaviruses including rhinovirus es foot and mouth disease virus coxsackievirus and mengovirus in cell culture 6 7 8 9 10 11 To determine whether Zn 2 has a similar effect on nidoviruses we investigated the effect of PT and Zn 2 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 inthepresenceof0to8 mMZnOAc 2 TreatmentwithPT ofconcentrations up to32 mM incombination with 4 mMZnOAc 2 did not reduce the viability of mock infected cells after 18 h Fig 1A asmeasured bythe colorimetricMTS 3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2H tetrazolium viabili tyassay AselevatedZn 2 concentrationsareknowntoinhibitcellular translation we also used metabolic labeling with 35 S methionine to assess the effect of PT and Zn 2 on cellular protein synthesis IncubationofVero E6cellsfor18 hwiththecombinationsofPTand Zn 2 mentioned above followed by a 2 h metabolic labeling revealed no change in overall cellular protein synthesis when the concentration of ZnOAc 2 was 4 mM data not shown Using these non cytotoxic conditions we subsequently tested the effect of PT and ZnOAc 2 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 ZnOAc 2 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 mMofZn 2 was added to the medium We found that addition of ZnOAc 2 alone also reduced virus replication but only at levels that were close to the 50 cytotoxicity concentration CC 50 of ZnOAc 2 in Vero E6 cells 70 mM data not shown This is likely due to the poor solubility of Zn 2 in phosphate containing medium and the inefficient uptake of Zn 2 by cells in the absence of zinc ionophores The combination of 2 mM PT and 2 mM ZnOAc 2 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 ZnOAc 2 concentrations From the dose response curves in Fig 1 a CC 50 value of 82 mM was calculated for PT in the presence of 2 mM zinc Half maximal inhibitory concentrations IC 50 of 1 4 mM and 0 5 mM and selectivity indices of 59 and 164 were calculated for SARS CoV and EAV respectively Zn 2 reversibly inhibits the RNA synthesizing activity of isolated nidovirus RTCs 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 a 32 P 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 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 Zn 2 concentrations are known to efficiently impair replication of a number of RNA viruses e g by interfering with correct proteolytic pro cessing of viral polyproteins Here we not only show that corona and arterivirus replication can be inhibited by increased Zn 2 levels but also use both isolated replication complexes and purified recombinant RdRps to demon strate 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 Zn 2 Inhibits Nidovirus Replication PLoS Pathogens www plospathogens org 2 November 2010 Volume 6 Issue 11 e1001176 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 Zn 2 on viral RNA synthesis we tested the effect of Zn 2 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 ZnOAc 2 was present For both viruses a more than 50 reduction of overall RNA synthesis was observed at a Zn 2 concentration of 50 mM while less than 5 activity remained at a Zn 2 concentration of 500 mM Both genome synthesis and sg mRNA production were equally affected To test whether the inhibition of RTC activity by Zn 2 was reversible RTC reactions were started in the presence or absence of 500 mMZn 2 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 Zn 2 chelator in these in vitro assays because it specifically chelates Zn 2 while releasing Mg 2 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 Mg 2 that is crucial for RdRp activity 27 28 whereas MgEDTA had no effects on control reactions without Zn 2 Fig 3B compare lane 1 and 2 As shown in Fig 2 the EAV RTC activity that was inhibited by Zn 2 Fig 3B compare lane 1 and 4 This effect was not due to chelation of the Zn 2 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 Zn 2 Fig 3C lane 2 Figure 1 The zinc ionophore pyrithione inhibits nidovirus replication in cell culture A Cytotoxicity of PT in Vero E6 cells in the absence blue circles or presence of 2 black squares 4 red triangles or 8 mM gray diamonds ZnOAc 2 as determined by the MTS assay after 18 hours of exposure B Dose response curves showing the effect of PT and Zn 2 on the GFP fluorescence in Vero E6 cells infected with a GFP expressing EAV reporter strain at 17 h p i Data were normalized to GFP expression in infected untreated control cultures 100 The different Zn 2 concentrations added to the medium were 0 blue circles 1 green triangles or 2 mM ZnOAc 2 black squares C Effect of PT and Zn 2 on the GFP fluorescence in Vero E6 cells infected with a GFP expressing SARS CoV reporter strain at 17 h p i Data were normalized to GFP expression in infected untreated control cells 100 Colors for different Zn 2 concentrations as in Fig 1B Error bars indicate the standard deviation n 4 doi 10 1371 journal ppat 1001176 g001 Figure 2 Inhibition of the in vitro RNA synthesizing activity of isolated RTCs by Zn 2 Incorporation of a 32 P CMP into viral RNA by EAV A and SARS CoV B in RTC assays in the presence of various Zn 2 concentrations as indicated above each lane doi 10 1371 journal ppat 1001176 g002 Zn 2 Inhibits Nidovirus Replication PLoS Pathogens www plospathogens org 3 November 2010 Volume 6 Issue 11 e1001176 Zinc ions affect the in vitro activity of recombinant nidovirus RdRps To establish whether inhibition of RTC activity might be due to a direct effect of Zn 2 on nidovirus RdRp activity we tested the effect of Zn 2 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 a 32 P 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 a 32 P 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 a 32 P AMP incorporation in our assays Fig 4 confirming once again that the radiolabeled RNA products derived from nidovirus RdRp activity Addition of ZnOAc 2 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 Co 2 and Ca 2 which typically bind to amino acid sidechainscontainingoxygenatomsratherthansulfurgroups Zn 2 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 werepre incubated with 6 mM Zn 2 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 ZnOAc 2 andsampleswereincubatedforanother30 min Fig 5C Asshown in Fig 5D the inhibition of EAV RdRp activity by Zn 2 could be reversed by chelationof Zn 2 Fig 5D compare lanes 3 and 4 The amount of product synthesized was consistently 6065 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 Figure 3 Inhibition of nidovirus RTC activity by Zn 2 can be reversed by chelation A Schematic representation of the in vitro assays with isolated RTCs which were initiated with a 32 P CTP either in the absence sample 1 and 2 or presence of 500 mMZn 2 After a 30 min incubation at 30uC both the untreated and Zn 2 treated samples were split into two aliquots and 1 mM of the Zn 2 chelator MgEDTA was added to samples 2 and 4 All reactions were subsequently incubated for another 70 min before termination B Analysis of RNA products synthesized in assays with EAV RTCs Numbers above the lanes refer to the sample numbers described under A C In vitro activity assay with SARS CoV RTCs doi 10 1371 journal ppat 1001176 g003 Figure 4 EAV and SARS CoV RdRp assays with wild type enzyme and active site mutants A The EAV polymerase was incapable of primer extension and required a free 39 end and poly U residues to initiate Nucleotide incorporating activity of the wild type enzyme and D445A mutant of nsp9 on an 18 mer poly U template confirmed the specificity of our assay B SARS CoV nsp12 RdRp assays were performed with an RNA dupl