These Viruses in Cell Culture
Published November 4, 2010

Aartjan J W Te Velthuis
Department of Medical Microbiology
Center of Infectious Diseases
Molecular Virology Laboratory
Leiden University Medical Center
LeidenThe Netherlands

Sjoerd H E Van Den Worm
Department of Medical Microbiology
Center of Infectious Diseases
Molecular Virology Laboratory
Leiden University Medical Center
LeidenThe Netherlands

Amy C Sims
Departments of Epidemiology and Microbiology and Immunology
University of North Carolina at Chapel Hill
Chapel Hill

North Carolina
United States of America

Ralph S Baric
Departments of Epidemiology and Microbiology and Immunology
University of North Carolina at Chapel Hill
Chapel Hill

North Carolina
United States of America

Eric J Snijder email@example.com
Department of Medical Microbiology
Center of Infectious Diseases
Molecular Virology Laboratory
Leiden University Medical Center
LeidenThe Netherlands

Martijn J Van Hemert email@example.com
Department of Medical Microbiology
Center of Infectious Diseases
Molecular Virology Laboratory
Leiden University Medical Center
LeidenThe Netherlands

These Viruses in Cell Culture

PLoS Pathog
6111001176Published November 4, 2010Received May 17, 2010; Accepted October 1, 2010;Editor: Raul Andino, University of California San Francisco, United States of America 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. *
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 mM Zn 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. (

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. Nevertheless, 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 large replicase polyproteins [19]. A hallmark of the corona-and arterivirus replicative cycle is the transcription of a 59-and 39coterminal 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 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 rhinoviruses, 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) in the presence of 0 to 8 mM ZnOAc 2 . Treatment with PT of concentrations up to 32 mM in combination with ,4 mM ZnOAc 2 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 Zn 2+ concentrations are known to inhibit cellular translation, we also used metabolic labeling with 35 S-methionine to assess the effect of PT and Zn 2+ on cellular protein synthesis. Incubation of Vero-E6 cells for 18 h with the combinations of PT and 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 mM of Zn 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 Zn 2+ 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-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 processing 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 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.

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 mM Zn 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&C, lane 3) could be restored by the addition of MgEDTA (Fig. 3B, lane 4) to a level observed for control reactions without Zn 2+ (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 Zn 2+ and MgEDTA incorporated slightly more [a-32 P]CMP compared to untreated control reactions ( Fig. 3C; 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).

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, dosedependent 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 side chains containing oxygen atoms rather than sulfur groups, 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 were preincubated 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 , and samples were incubated for another 30 min (Fig. 5C). As shown in Fig. 5D, the inhibition of EAV RdRp activity by Zn 2+ could be reversed by chelation of 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  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 4065% 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 Mg 2+ concentration, which was shown to be suboptimal for nsp12 activity [27] and results from the release of Mg 2+ from MgEDTA upon chelation of Zn 2+ .

Differential effect of Zn 2+ on the initiation and elongation phase of nidovirus RNA synthesis For EAV, close inspection of the RdRp assays revealed a less pronounced effect of Zn 2+ on the generation of full-length 18-nt products than on the synthesis of smaller reaction intermediates (Fig. 5A). This suggested that Zn 2+ 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, [a-32 P]CTP was added to both tubes (pulse), 500 mM Zn 2+ 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 Zn 2+ [a-32 P]CMP was predominantly incorporated into nascent RNA molecules that were already past the initiation phase at the moment that Zn 2+ 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 Zn 2+ 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 Zn 2+ , 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 Zn 2+ 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 Zn 2+ completely blocked further incorporation of [a-32 P]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 [a-32 P]ATP (,0.17 mM) 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 Zn 2+ on initiation and elongation by performing an experiment in which a pulse with a low concentration of [a-32 P]ATP was followed by a chase in the presence of 50 mM 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 Zn 2+ (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 Zn 2+ (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 [a-32 P]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 Zn 2+ , 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 Zn 2+ to a SARS-CoV RdRp reaction also blocked elongation, since extension of the radiolabeled primer as observed in the absence of Zn 2+ (Fig. 7D, left panel) no longer occurred (Fig. 7D, right panel).

Zinc affects SARS-CoV RdRp template binding

To assess whether Zn 2+ 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 Zn 2+ (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 Zn 2+ was present in the assay (Fig. 8B). We also assessed whether pre-incubation of the RdRp or RNA with Zn 2+ 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, His 6tagged 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.

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 mM PT and 2 mM Zn 2+ efficiently inhibited their replication, while not causing detectable cytoxicity (Fig. 1). Inhibition of replication by PT and Zn 2+ at similar concentrations (2-10 mM) 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 Zn 2+ on the replication of picornaviruses appeared to be due to interference with viral polyprotein processing. In infections with the coronavirus mouse hepatitis virus (MHV), Zn 2+ also interfered with some of the replicase polyproteins cleavages [24], albeit at a much higher concentration (100 mM Zn 2+ ) 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 Zn 2+ also affects nidovirus RNA synthesis directly. Our in vitro studies revealed a strong inhibitory effect of zinc ions on the RNAsynthesizing 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 Zn 2+ 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 Zn 2+ (Fig. 6 and 7), indicating that Zn 2+ does not affect elongation and does not increase the termination frequency, as was previously found for Mn 2+ [25]. Therefore, Zn 2+ appears to be a specific inhibitor of the initiation phase of EAV RNA synthesis. In contrast, Zn 2+ 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 Mg 2+ , which subsequently coordinate incoming nucleotides during the polymerization reaction [37,38]. Mg 2+ 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 Mn 2+ -dependent. Zn 2+ could not substitute for Mg 2+ or Mn 2+ as cofactor as it was incapable of supporting the polymerase activity of nidovirus RTCs and RdRps in the absence of Mg 2+ (data not shown), as was also reported for the poliovirus RdRp [39]. Moreover, inhibition of nidovirus RdRp activity by Zn 2+ was even observed at low concentrations and in the presence of a more than 25-fold excess of Mg 2+ , suggesting that either the affinity of the active site for Zn 2+ is much higher or that Zn 2+ does not compete for Mg 2+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 Zn 2+ Figure 7. The effect of Zn 2+ on initiation and elongation activity of purified EAV and SARS-CoV RdRps. (A) An EAV RdRp reaction was initiated in the presence of [a-32 P]ATP under conditions that do not allow elongation, i.e., low ATP concentration. After 20 min, the reaction was split into two equal volumes, and Zn 2+ was added to one of the tubes. A chase with 50 mM unlabeled ATP, which allows elongation, was performed on both reactions and samples were taken after 5 and 30 min. (B) EAV RdRp reaction products that accumulated in the presence and absence of Zn 2+ (indicated above the lanes) after a 5-and 30-min chase with unlabeled ATP. The length of the reaction products in nt is indicated next to the gel. (C) A SARS-CoV RdRp reaction was initiated in the presence of 0.17 mM [a-32 P]ATP, which limits elongation. After 10 min, the reaction was split into two equal volumes, and Zn 2+ was added to one of the tubes. A chase with 50 mM unlabeled ATP was performed on both reactions and samples were taken after 5, 10, 15, and 30 min. (D) SARS-CoV RdRp reaction products formed at the chase times indicated above the lanes in the presence and absence of Zn 2+ . The length of the reaction products in nt is indicated next to the gel (p is the primer length). doi:10.1371/journal.ppat.1001176.g007 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 Zn 2+ -induced inhibitory effects on RdRp activity documented in this study. Such studies may, however, be complicated when Zn 2+ 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 zincionophores 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 Zn 2+ 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 ml of medium containing 2% fetal calf serum (FCS) was added to each well. In some experiments 0-32 mM of pyrithione (Sigma) was added in addition to 0-2 mM 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 mM PT and 0-8 mM 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 (59-GCUAUGUGAGAU-UAAGUUAU-39), SAV481R (59-UUUUUUUUUUAUAACUU-AAUCUCACAUAGC-39) and poly(U) 18 (59-UUUUUUUUU-UUUUUUUUU-39) 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 90uC 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 Zn 2+ , 1 ml of a ZnOAc 2 stock solution was added to standard 28-ml reactions, resulting in final Zn 2+ concentrations of 10-500 mM. When Zn 2+ 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 32 P-labeled reaction products were separated on denaturing 1% (SARS-CoV) or 1.5% (EAV) agarose formaldehyde gels. The incorporation of [a-32 P]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 37uC and a further 16 hours at 20uC. 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 220uC. RdRps with a D618A (SARS-CoV) or D445A (EAV) mutation were obtained by site-directed mutagenesis of the wildtype (wt) plasmid pDEST14-nsp9-CH [28] with oligonucleotides 59-TACTGCCTTGAAACAGCCCTGGAGAGTTGTGAT-39 and 59-ATCACAACTCTCCAGGGCTGTTTCAAGGCAGTA -39, and plasmid pASK3-Ub-nsp12-CHis 6 with oligonucleotides 59-CCTTATGGGTTGGGCTTATCCAAAATGTG-39 and 59-CACATTTTGGATAAGCCCAACCCATAAGGA-39, 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 mM of purified SARS-CoV nsp12 are described elsewhere [27]. To study the effect of Zn 2+ in this assay, 0.5 ml of a dilution series of 0-80 mM ZnOAc 2 was added to the 5 ml reaction mixture, yielding final Zn 2+ concentrations of 0-8 mM. The EAV RdRp assay contained 1 mM nsp9, 1 mM RNA template poly(U) 18 , 0.17 mM [a-32 P]ATP (0.5 mCi/ml; Perkin-Elmer), 50 mM 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 Zn 2+ 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 59 32 P-labeled SAV557R/SAV481R RNA duplex, for 10 minutes at 30uC either in presence or absence of 6 mM ZnOAc 2 . Reactions were analyzed as described previously [27]. Reaction mixtures were split into two aliquots, one of which was supplemented with 6 mM Zn 2+ , and samples were taken at the time points (minutes) indicated above the lanes. In the absence of Zn 2+ , EAV nsp9 initiates de novo and produces di-and trinucleotides, indicated with A2 and A3, respectively. A nonspecific band, unrelated to RdRp activity, between A2 and A3 is indicated with an asterisk. In the presence of 6 mM Zn 2+ , the synthesis of dinucleotides and trinucleotides was blocked. (B) When performing the assay described under (A) in the absence of Zn 2+ , 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 Zn 2+ , 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 59 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.

Supporting Information

, 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.

Figure 1 .
1The 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 .
2Inhibition 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

Figure 3 .
3Inhibition 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 mM Zn 2+ . After a 30min 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 .
4EAV 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 duplex with a 59 U 10 overhang as template. The bar graph shows the nucleotide incorporating activities of wild-type and D618A nsp12. Error bars represent standard error of the mean (n = 3). doi:10.1371/journal.ppat.1001176.g004

Figure 5 .
5The activity of the RdRps of EAV and SARS-CoV is reversibly inhibited by Zn 2+ . RdRp activity of purified EAV nsp9 (A) and SARS-CoV nsp12 (B) in the presence of various Zn 2+ concentrations, as indicated above the lanes. (C) Schematic representation of the experiment to test whether Zn 2+ -mediated inhibition of RdRp activity could be reversed with MgEDTA. RdRp reactions, either untreated controls (sample 1 and 2) or reactions containing 6 mM Zn 2+ (samples 3 and 4) were incubated for 30 min. Both Zn 2+ -containing and control samples were split into two aliquots and 6 mM MgEDTA was added to sample 2 and 4. All reactions were incubated for an additional 30 min and then terminated. Reaction products of the RdRp assays with EAV nsp9 and SARS-CoV nsp12 are shown in (D) and (E), respectively. Numbers above the lanes refer to the sample numbers described under (C). doi:10.1371/journal.ppat.1001176.g005

Figure 6 .
6Effect of Zn 2+ on initiation and elongation in in vitro assays with isolated EAV and SARS-CoV RTCs. (A) An in vitro RTC assay with isolated EAV RTCs was allowed to initiate with unlabeled NTPs (initiation). After 30 min, [a-32 P]CTP was added (pulse), the reaction was split into two equal volumes, and Zn 2+ was added to a final concentration of 0.5 mM to one of the tubes. At the time points indicated, samples were taken and incorporation of [a-32 P]CMP into viral RNA was analyzed. (B) Radiolabeled EAV RNA synthesized at the time points indicated above the lanes in the presence and absence of Zn 2+ . (C) Radiolabeled RNA synthesized by isolated SARS-CoV RTCs in reactions terminated after 100 (lane 1) and 40 (lane 2) min. Reaction products of a reaction to which 500 mM Zn 2+ was added after 40 min, and that was terminated at t = 100 are shown in lane 3. doi:10.1371/journal.ppat.1001176.g006

Figure 8 .
8The effect of Zn 2+ on SARS-CoV nsp12 template binding. (A) Electrophoretic mobility shift assay with radiolabeled dsRNA and nsp12 in the presence and absence of Zn 2+ (indicated above the lanes). The position of unbound and nsp12-bound RNA in the gel is marked on the left of the panel. (B) Determination of the nsp12 affinity for RNA in the presence and absence of Zn 2+ . A fixed amount of RNA was incubated with an increasing amount of nsp12. This revealed a 3-4 fold reduction in the percentage of bound RNA in the presence of zinc ions (grey) relative to the percentage of bound RNA in the absence of zinc ions (black). Error bars represent standard error of the mean (n = 3). doi:10.1371/journal.ppat.1001176.g008

Figure S1
S1Effect 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 [a-32 P]ATP in the presence of either 6 mM Mg 2+ only (lane 1), and with increasing concentrations of a second divalent metal (M 2+ ), specifically: 2-6 mM Ca 2+ (lane 2-4), 2-6 mM Co 2+ (lane 5-7), 2-6 mM Zn 2+ (lane 8-10), or 2-6 mM Mn 2+ (lane 11-13). The strongest inhibition was observed for Zn 2+ . For more details on the SARS-CoV nsp12 RdRp assay, see the main text. Found at: doi:10.1371/journal.ppat.1001176.s001 (1.55 MB TIF) Figure S2 Effect of Zn 2+ on the dinucleotide extension activity of EAV nsp9. Purified recombinant EAV nsp9 was incubated with a U 18 template in the presence of [a-32 P]ATP, ATP, 4 mM Mg 2+ , 1 mM Mn 2+ , and 1 mM ApA. (A)

Found at: doi: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 Zn 2+ -binding pocket Zn2, located close to motif E (depicted in red). A second Zn 2+ -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). Found at: doi:10.1371/journal.ppat.1001176.s003 (0.86 MB TIF)
November 2010 | Volume 6 | Issue 11 | e1001176
PLoS Pathogens | www.plospathogens.org
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Anticipatory prescribing in terminal care at home: what challenges do community health professionals encounter?
Published Online First 27 September 2012

Christina Faull email@example.com
LOROS
The Leicestershire and Rutland Hospice
LeicesterUK

Kate Windridge
Department of Health Sciences
Dr Christina Faull, LOROS, The Leicestershire and Rutland Hospice, Groby Road
University of Leicester
NIHR RDS East Midlands
LE3 9QELeicester, LeicesterUK, UK

Elizabeth Ockleford
Department of Health Sciences
Dr Christina Faull, LOROS, The Leicestershire and Rutland Hospice, Groby Road
University of Leicester
NIHR RDS East Midlands
LE3 9QELeicester, LeicesterUK, UK

Michael Hudson
LOROS
The Leicestershire and Rutland Hospice
LeicesterUK

Anticipatory prescribing in terminal care at home: what challenges do community health professionals encounter?
Published Online First 27 September 201210.1136/bmjspcare-2012-000193Received 4 January 2012 Revised 23 July 2012 Accepted 7 August 2012▸ Additional data are published online only. To view these files please visit the journal online Correspondence to To cite: Faull C, Windridge K, Ockleford E, et al. BMJ Supportive & Palliative Care 2013;3:91-97.
Background The prompt availability of medications to manage symptoms is key to high quality end-of-life care and anticipatory prescribing of these drugs is thought good practice. This study explored the challenges encountered by primary and community health professionals in Leicestershire and Rutland related to anticipatory prescribing when caring for terminally ill patients who wish to remain at home to die. Method A qualitative study was conducted using eight focus groups (54 participants) and nine individual interviews with a purposively sampled range of health professionals providing care for people who wished to die at home. Themes were identified iteratively via constant comparison.Results Challenges fell into four categories: resourcing concerns, professional expertise/ experience, professionals' relationships with patients, and professionals' relationships with other professionals. The latter included the most serious perceived challenges. Links between community and hospital care providers and between 'usual' hours and 'out-of-hours' care providers were seen as particularly unstable.Conclusions These findings suggest that building and maintaining trusting, responsive, personal links between professionals, both within and between teams, is essential when implementing good practice guidelines about anticipatory end-of-life prescribing in the community. The need for good communication and relationships between patients and professionals and maintaining expertise and confidence in end-of-life care are also key factors in the effective use of anticipatory prescribing for symptom management for dying patients.ResearchFaull C, et al. BMJ Supportive & Palliative Care 2013;3:91-97.

BACKGROUND

Most terminally ill people wish to die at home. 1 It has been observed that a cluster of symptoms is experienced commonly by patients (at least in those with cancer) in the last days of life, namely anxiety, delirium and agitation, pain, nausea, dyspnoea and troublesome respiratory secretions. 2 The distress caused by these symptoms may be a factor in preventing people staying at home, 3 4 and the management of these symptoms is a major consideration in quality of care at the end of life 5 and is a component of the new quality standards for end-of-life care for adults in England. 6 The anxiolytic, midazolam and the opioids, diamorphine and morphine, both controlled drugs, have a well established place in managing some of these symptoms and their use is best practice in hospices. 7 The Liverpool Care Pathway for care of the dying (LCP) is an explicit framework for the care of people in the last days of their lives and is well established across much of the UK. 8 The pathway, which is constructed as a series of goals of care, incorporates management of the common end-of-life symptoms. 9 10 To achieve best care at the end of life requires anticipatory clinical planning for a range of potential situations and patient needs. 11 In the community setting, the planning of symptom management must include consideration of how appropriate medications can be accessed and administered as promptly as possible once the patient develops a problem. One solution is the anticipatory prescribing and administration authorisations of drugs, to be placed in the home in advance of the patient actually requiring them 10 (Goal 4.1 of the LCP V.12). The drugs are then readily available to any healthcare practitioner who is visiting the patient at the time when the patient's situation changes and intervention is required. This has been anecdotally shown to have potential in keeping patients out of hospital if they wish to remain at home. 12 The specific area of anticipatory prescribing in terminal care in the community is largely unresearched, 13 but there are several reasons for thinking that successful terminal care at home must take account of complexity and unpredictability. First, patient safety is a cornerstone of the NHS plan and of health professional training, 14 but patients' homes are more diverse and less controllable (organisationally, socially and physically) environments for prescribing, dispensing and drug administration than hospitals or hospices. The Shipman enquiry 15 has focussed minds on issues related to the safe and audited use in the community of controlled drugs and there is some evidence to suggest it has changed practice to the detriment of patient care. 16 Second, the number of individual professionals involved introduces complexity. The practitioner who actually administers the drug to manage a patient's symptoms may know the patient well, or may be visiting for the first time. They may have a well developed relationship with the prescriber or the parties may be completely unknown to each other with no direct clinical accountability.

Third, the number of disciplines involved also introduces complexity. The roles of nurses have been extended [17][18][19] and this means there is now the potential for controlled drug prescribing by district nurses, palliative care nurses, disease-specific clinical nurse specialists, community matrons, general practitioners (GPs), and hospital or hospice specialists, all or any of whom may be involved in the care of a given patient.

Fourth, community healthcare practitioners are increasingly caring for dying people with a wider range of terminal conditions than has been the case until recently. 20 Lastly, the frequent re-organisation of local community services is a source of complexity.

Aim

The aim of the study was to explore the issues that arise for all practitioners working in the community, in relation to the prescribing, dispensing and administering of subcutaneous midazolam and diamorphine or morphine for the anticipated need of dying patients to have timely and effective symptom management.

In this report we focus on professionals' experiences of the challenges encountered.

METHODS

Participants

The study took place in Leicestershire, Leicester and Rutland. Information about the project and invitation letters were sent out by email and post (see online supplementary appendix 1). Purposive sampling was used to ensure that there was at least one participant from each of the following areas: district nursing, Marie Curie nursing, 'Hospice at Home' nursing, specialist nursing in palliative care and heart failure, community matrons, nursing home nursing, pharmacy and general practice, and at least one from each of a range of levels of responsibility, experience, practice size, age, experience of providing out-of-hours care, nurse prescribers and non-prescribers, rural and city practitioners, and males and females (online supplementary appendix 2 gives sampling frame criteria). Participants were asked about these characteristics via a checklist at the time of consent, but in order to maintain anonymity their responses were not linked in any way to the content of individual interviews. Consent was sought from participants before the start of the interview, at least a week after they received full information about the research.

Data collection

Focus groups and individual interviews were used so the research process could benefit from the advantages of each approach and to provide participants with choice, given the potential sensitivity of the area.

The topic guide (see online supplementary appendix 3) was developed utilising research team and steering group discussions, clinical and qualitative interviewing experience, significant event analysis, educational interactions with primary care professionals, and analysis of available 'best practice' guidance developed by some services. Guides were used flexibly so that unanticipated issues of importance to individual participants could be explored. The topic guide evolved in response to new data. Data collection took place in 2007.

Data analysis

The interviews and focus groups were audio-recorded and interviewers (KW, MH and EO) maintained reflexive diaries. Data from transcripts were analysed by constant comparison based on grounded theory to identify themes. 21 22 Open coding (KW, MH and CF) summarised the ways that participants talked about the processes that mattered. These codes were progressively focussed into broad categories, forming the initial coding frame, further shaped by steering group discussion. The coding frame was systematically applied by KW, using QSR N6 software, and continuously developed in response to new information. In situations where there was uncertainty about how to categorise a particular section of text, EO provided an alternative perspective. No new issues were elicited after eight individual interviews (n=8 participants) and six focus group (n=51 participants) transcripts had been coded. Independent coding by MH checked allocation of text to categories.

RESULTS

A total of 63 participants were recruited (online supplementary appendix 2): 22 GPs, four 'Hospice at Home' nurses, four community matrons, five Marie Curie nurses, 16 community nurses, four specialist palliative care nurses, one nursing home matron, three community pharmacists, three heart-failure nurses and one student nurse. Six GPs, two pharmacists and one nursing professional chose one-to-one interviews (n=9), while the remaining 54 participants chose focus groups. Thus 63 participants yielded 17 transcripts for analysis.

(In quoting from participants we use 'NP' as a generic term for all professionals with nursing qualifications, 'Ph' refers to pharmacists and 'I' indicates an interviewer or focus group facilitator. FG01 to FG08 refer to the eight focus groups and INT01 to INT09 to the nine interviews. The term transcript is used where needed to be inclusive of both individual and multiple participant interviews.)

There was no disagreement among participants about the principle of ensuring that distressed patients did not have to wait for relief. Only one interview, however, described no challenges in writing a prescription for controlled injectable drugs in advance of need, obtaining such drugs from a pharmacy in advance of need, or administering drugs ( prescribed and dispensed in advance) to a patient so that they could remain at home. For this GP it seemed that the absence of challenges was due to the fact that such situations rarely occurred in their practice.

The nature of the challenges The challenges described in the remaining 16 transcripts fell into four categories.

Most transcripts contained multiple examples of all four types of challenge, but there were two interviews where only one challenge arose per transcript, seemingly because the participants said they had little personal involvement with anticipatory prescribing. A perceived lack of resources and the associated need to avoid waste, were seen as challenging in 12 transcripts. Interviewees described instances:

▸ where prescriptions were not written in advance of need because of concern about waste since some had found that patients did not actually require them; ▸ where there was delay in dispensing because of limited availability of drugs in pharmacies (see quotation below); and ▸ where delay in administering drugs arose through lack of syringe drivers.

NP, FG05: … every single time I've had to take prescriptions to my local chemist's it's 'next day'or you phone round and you have to split the prescription … so the GP has to do single prescriptions and you're all over the place, and it can take you hours, hours, to sort it outor days even.

Dissenting views: was resourcing always seen as a problem?

One further transcript referred to resourcing problems in the past which were now resolved. Three transcripts contained no indication that lack of resources or concern about waste had hindered prescribing or dispensing in advance in their own experience, although they mentioned that these issues could be a concern for some. There were 10 transcripts in which participants emphasised the importance of learning, both by formal education and from experience. There were different types of concern within this category. These were:

▸ knowing when to prescribe, or administer, medication:

GP, FG03: … actually there isn't … a formula to predict these people … it is gut feel.

NP, FG02: I think it's just that 'fear factor', isn't it? … it's recognising symptoms and realising when the right time is to give it … if … there's nobody to get back to and say, 'right, well the midazolam's there, but shall I give it?

More accuracy and confidence in this was perhaps a feature of experience, emphasising that this is a learnt skill. NP, FG05: I've been involved in a few cases where … I'm covering the weekend and I've gone in and I've thought 'Oh my God, look how ill this person is'. Maybe it was the district nursing team that was going in before or it was a junior nurse that went in a few days before, they can't recognise the signs, they don't realise how poorly these people are.

▸ knowing what should be prescribed, or administered:

Ph, INT09: … if there was a list of things that were really commonly used, which I know there is of sorts, then if we could know what that list was and keep those in stock then that would help.

▸ concern about accountability: GP, INT03: You get a bit concerned about prescribing some of these medicines. And if you start pre-emptive prescribing, then that's another ball-game if it happens to be mis-used, for example. Who would take the responsibility?

Harold Shipman was mentioned in six transcripts as having an effect on confidence.

▸ and concern about a lack of emphasis on conditions other than cancer: NP, FG04: GPs … accept that cancer patients are dying but with all the other (non-cancer, terminal conditions) the care just isn't there for them … I think it's just the fear of prescribing drugs that they don't prescribe that often for that group of patients.

Category 3: Patient-professional links (see online supplementary box 6)

In 12 transcripts, lack of opportunity to build and maintain patient-professional links was seen as contributing to failure to prescribe sufficiently in advance. Having enough contact with patients to develop longer term, trusting relationships was seen as important because it enabled sensitive communication and provided a way of ensuring that past, present and future treatment was timely and coherent and that care felt 'human' and personal. 'Going in blind' was a huge challenge in making care effective, in the justification of prescribing decisions and in the stress it caused professionals. GPs felt they were less likely to admit their own patients than those of their colleagues, especially with the confidence that they could review the situation the following day.

NP, FG07: … it is very hard to prescribe for someone you don't know … you have got responsibilities to your patients, relatives, the GP whose patient it is … so I am not sure I would prescribe for someone I didn't know. In fact I know I wouldn't.

Dissenting views: did a trusting patient-professional relationship always facilitate pre-emptive prescribing?

There were four transcripts in which participants described how getting to know patients and their family had prevented pre-emptive prescribing because that knowledge gave rise to grave concerns about placing controlled drugs in a house where there were reasons to think they might be misused. This did not necessarily give rise to serious problems with symptom relief but did mean that strategies other than placing drugs in the home in advance, or at all, tended to be used. Only one transcript highlighted that an established trusting professional-patient relationship was not always necessary so long as the professional involved had knowledge of and could trust other professionals' judgements and communications about previous medical history. Participants had experienced many occasions when the success of anticipatory prescribing or dispensing, with its ultimate aim of enabling a patient to stay at home, had been threatened by the failure of reliable links between or within professional teams or disciplines. The importance of this issue is illustrated by that fact that the only two transcripts in which it did not arise were interviews in which the participant had almost no direct experience of pre-emptive prescribing.

The challenges arising from not knowing or trusting other professionals, whether within teams or between teams, tended to be those that caused greatest concern and promoted most discussion among participants. There were three areas where links were seen as particularly vulnerable.

Firstly, the links between out-of-hours care and usual care providers presented considerable challenges in joined-up decision making and care planning. The very limited information available to out-of-hours doctors, the inadequate communication processes for even these small amounts of information, coupled with the severe time pressures, often lead to admissions to hospitals. Participants gave detailed stories of very poor patient outcomes which reflected the behaviours of professionals working across services and teams. They illustrate a fragility in the continuity of the plan of care despite anticipatory medications being in the home. NP, FG03: I offered to meet the out-of-hours doctor, it was a … patient who was very, very terminally ill and he said he would, but didn't, and he … admitted that patient and she died in the ambulance on the way to hospital and you know, that was-I was quite happy to meet him, to give whatever, but he wasn't interested.

NP, FG08: I've written in notes saying 'this person's swallowing has deteriorated overnight and would it be a good idea to have a look at what medication is being given … and see if they can have it in a different form'. Sometimes you'll come back again and they're still trying to shove in tablets … GP, INT02: We had a patient who was requiring an awful lot of morphine … the breakthrough dosages were very high and were continually being adjusted and because [specialist palliative nurses] were involved I used to give quite a big range actually, because they were women that I worked with … and I know and trust, so you give a big range … but when you have a [different] nurse going in at night, they were very, very reluctant to give the dose that they had been having and they would tend to go to the lowest dose on the range, which caused difficulty with pain control … not knowing the patient, not knowing the family, not knowing me, not knowing the team, and being asked to give what seemed to be a lethal dose of morphine.

Secondly, links between community professionals and hospital professionals were seen as a challenge in anticipatory planning for care at home with patients and their families. This was especially so when the more 'trusted' relationship for the patients was with hospital providers. It was very difficult for community providers to change the direction of care and prepare and plan with the patient and family for deterioration.

Thirdly, links between specialist and generalist teams could also pose a challenge in anticipatory prescribing. A professional's title or role was not sufficient in itself for others to trust their advice.

Participants gave examples of where advise and guidance had been ignored to the detriment of patients. NP, FG07: … the GP said to me 'what would this patient have in the syringe driver' and when I gave a suggestion he said 'I don't think we will go with that'. And he gave something, what I would say was inappropriate, and the next day, which was a Saturday, it was then deemed necessary that this patient needed the drug that I had said the previous day and by the time we got it from the chemist, the patient had died. So it is … building up a relationship with your particular GPs to the point that they actually trust you and trust your judgement.

DISCUSSION AND CONCLUSIONS

The study suggests that although anticipatory prescribing for terminally ill patients in the community was welcomed by GPs, community nurses and pharmacists as a way of providing good care and avoiding distressing symptoms, achieving this can pose challenges for the professionals involved. There is no current literature on this area of care and our findings suggest significant complexities in what may on the surface seem a straightforward element of best practice.

The interviews brought to light a range of interpretations of what was meant by prescribing in advance of need. For some, there was an assumption that anticipatory prescribing means obtaining medication in case patients need to start it over the weekend/bank holiday. It was less common to hear accounts of getting drugs in the house more than a day or two ahead of anticipated need. This may have been a reflection of local circumstances, in that for some, it was easy to obtain medication within 24-48 h and it was therefore not seen as important to have it in the house further in advance. However, those who habitually placed drugs in the home well before this did not describe any drawbacks so long as interprofessional communications were good. They saw it as part of the process of ensuring that they were communicating well with their patients and thereby increasing their chances of providing excellent care.

There were uncertainties among pharmacists, GPs and nursing professionals about which drugs should be prescribed, and some resistance to having all the drugs that might be needed 'just in case' symptoms develop. Some professionals distinguish between prescribing in advance for symptoms that already exist but that might get worse, versus prescribing in advance for symptoms that have not yet appeared. They are happy to do the former, but some worry about waste in connection with the latter.

Anticipatory prescribing is a process, not a one-off event and evidently there is scope for it to fail at any of three stages. The reported difficulties occur at the prescription-writing stage (eg, no-one writes the prescription in advance of the patient needing it), at the dispensing stage (eg, a prescription is written but the health professional on duty cannot exchange it for drugs in time for them to be useful because the pharmacy does not have the drugs in stock) and at the administering stage (eg, the drugs in the home are not administered at the correct level because of fears about accountability).

Participants described the importance that trusting, responsive relationships between patients and healthcare professionals have in facilitating anticipatory prescribing. The absence of these can make care planning, including discussing deterioration and therefore the anticipatory provision of medications, much more difficult to achieve. However, the findings go beyond this and highlighted the central importance in the effectiveness of anticipatory prescribing of building relationships between professionals, both within and between teams. Despite the diversity (of experience, profession and locality) represented by the participants, this issue was a significant one in every data collection session. It was seen as crucial that wherever possible there should be personal links with other professionals-a chance to get to know and trust each other-in addition to 'structural' links. Even when structures were in place, for example, that were designed to ensure that information was passed between out-of-hours and usual care, this did not necessarily work in the way it was intended to unless professionals personally knew a member of the corresponding team and found ways of communicating with that particular individual. The data we collected provided a plethora of specific, everyday examples of when not knowing and trusting other professionals led to delay in relief of symptoms. The issue of professional links should therefore be taken seriously.

Although this is a geographically local study, given the essentially uniform characteristics of the NHS, these findings are likely to be generalisable to England and much of the UK. Furthermore, given that the findings are as much about human relationships as healthcare systems, it is likely that the findings have some resonance for practitioners more globally. Research looking at other situations where there are high levels of multidisciplinarity has highlighted the importance of avoiding breakdowns in communication, 23 and of high quality team processes such as shared vision, plenty of interaction, and trust and reflexivity. 24 A literature review of interdisciplinary team working in community care identified that both team structure and team processes have an impact on teamworking. 25 Regular team meetings and audit appear to foster effective teamworking. The National Institute for Health and Clinical Excellence (NICE) guidelines for palliative care, 26 and the earlier Calman-Hine report on cancer care, 27 have been important documents in driving better team working in cancer and palliative care through multidisciplinary meetings. However, effective care in the community requires teamworking to cross organisational boundaries and be inclusive of out-of-hours services. Our findings in relation to anticipatory prescribing are most likely illustrative of the challenge in providing many elements of effective end-of-life care at home for patients.

The study also highlights perhaps specific challenges in the care of people dying from conditions other than cancer. Participants emphasised that one size does not fit all in terms of trajectories or predictability. A related issue that emerged was that currently, for some participants, very few of their patients stayed at home to die and some expressed concern that they would not have the confidence or experience to deal with the wish to die at home as and when it might arise. This is an important topic for further exploration given the emphasis on end-of-life care planning and choice for people with a wider range of terminal conditions. 28

Category 1 :
1Resourcing problems (see online supplementary box 1) Perceived lack of resources

Category 2 :
2Reflecting on expertise and experience (see online supplementary boxes 2-5)

Category 4 :
4Failing to build or maintain trusting and responsive links between professionals (see online supplementary boxes 7 and 8)
Faull C, et al. BMJ Supportive & Palliative Care 2013;3:91-97. doi:10.1136/bmjspcare-2012-000193
AcknowledgementsThe authors would like to thank all the participants both for giving up time and for their level of engagement and openness while contributing; the multi-professional steering group, including patients and carers, whose contributions have guided this project and provided an excellent sounding board and quality assurance framework; and Clayton Research Support for the excellent transcribing service. We would also like to acknowledge the support of the NIHR Research Design Service for the East Midlands.Contributors CF conceived and designed the study, wrote the funding bid and obtained ethics approval. KW led the data collection and analysis with contributions from CF, EO and MH. MH facilitated participant recruitment. KW, EO, CF and MH contributed to and commented on the paper. CF and KW act as guarantors for the paper.Funding Leicestershire, Northamptonshire and Rutland Primary Care Research Alliance provided funding for this study.Competing interests MH was also a part of the 'Hospice at Home' team.
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