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      Zn 2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

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          Abstract

          Increasing the intracellular 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 µM Zn 2+ and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV—thus eliminating the need for PT to transport 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.

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          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.

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          Protein production by auto-induction in high density shaking cultures.

          Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).
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            SARS: Systematic Review of Treatment Effects

            Introduction The severe acute respiratory syndrome (SARS) is a febrile respiratory illness primarily transmitted by respiratory droplets or close personal contact. A global outbreak of SARS between March 2003 and July 2003 caused over 8,000 probable or confirmed cases and 774 deaths [1]. The causative organism has been identified as a novel coronavirus (SARS-CoV) [2–4]. The overall mortality during the outbreak was estimated at 9.6% [5,6]. The overriding clinical feature of SARS is the rapidity with which many patients develop symptoms of acute respiratory distress syndrome (ARDS). This complication occurred in approximately 16% of all patients with SARS, and when it occurred was associated with a mortality rate of 50% [7,8]. At the time of the SARS epidemic it was not known what treatments would reduce SARS-related illness and deaths. Because the urgency of the international outbreak did not allow time for efficacy studies, physicians in Canada and Hong Kong treated the earliest patients with intravenous ribavirin, based on its broad-spectrum antiviral activity [9,10]. Corticosteroids and immune-modulating agents were often prescribed empirically. Soon after SARS-CoV was identified as the causative agent, antiviral screening programs were initiated; these programs reported several antiviral agents that inhibited SARS-CoV replication in vitro. These results led to the experimental use of protease inhibitors and interferon alpha (IFN-α) in the treatment of patients. The most commonly used treatments for SARS are associated with adverse effects when used for other conditions (Table S1). In October 2003, the WHO established an International SARS Treatment Study Group, consisting of experts experienced in managing SARS. The group recommended a systematic review of potential treatment options to identify the targets for proper evaluation in trials should the disease recur [11]. This paper reports on this systematic review designed to summarise available evidence on the effects of ribavirin, lopinavir and ritonavir (LPV/r), corticosteroids, type I IFN, intravenous immunoglobulin (IVIG), or convalescent plasma in relation to (1) SARS-CoV replication inhibition in vitro; (2) mortality or morbidity in SARS patients; and (3) effects on ARDS in adult patients. Methods We prepared a protocol that defined our scope, inclusion criteria, and outcomes to be assessed. The interventions we included were defined by the WHO: ribavirin, LPV/r, corticosteroids, type I IFN, convalescent plasma, or IVIG. The types of study we included were: (1) in vitro studies, in which the authors examined inhibition of SARS-CoV viral replication, and data from an assay in human or animal cell line; (2) in vivo studies, which included randomised controlled trial (RCT), or prospective uncontrolled study design, or retrospective cohort design, or case-control design, or a case series, and patients treated for SARS, and ten or more patients; and (3) studies of ARDS that included RCT, or systematic review, and treatment for ARDS or acute lung injury, and 20 or more patients. In February 2005, we systematically searched the literature databases MEDLINE, EMBASE, BIOSIS, and the Cochrane Central Register of Controlled Trials (CENTRAL) for articles that included the selected treatments (Table S2). The full text of each identified study was retrieved and each was independently reviewed by two authors (LS and RB). Publications in Chinese were selected after review of the English abstract. Unpublished data were not sought, as the task of summarising existing published data was extensive and the International SARS Treatment Group indicated that much of the clinical data had already been published. We used the QUOROM checklist to help ensure the quality of this review (Table S3). Data from the full text of studies in English were extracted independently by two authors (LS and RB). Data from the Chinese literature were extracted with the assistance of a translator. Because the Chinese articles were reviewed by only one author, the consistency of the translated information with that from English articles was maintained by subsequent discussion with the translator to verify the extracted data. We established explicit criteria to assess the level of evidence for each human treatment study (Box 1). Since the treatments chosen for evaluation were often given in combination, evidence was classified by the treatment that was given to all patients in the cohort or given to some with the author's intention of studying its effects. If putative effects within a study included several drugs, then we extracted data for each intervention. The level of evidence was independently classified by two authors (LS and RB). Chinese studies were appraised and classified in the same way using translated information extracted from each report. Discrepancies were resolved by consensus. Box 1. Categories of Evidence Defined for In Vivo Studies of Treatments in SARS Patients “Inconclusive” if a study could not be used to inform a decision about treatment efficacy due to having either outcomes which were not reported consistently, an inconsistent treatment regimen, no control group or a control group which was a likely source of bias. A control group was considered a likely source of bias if there were differences in co-morbidities, sex, age and markers of severe disease compared to the treatment group. “Possible harm” if a study reported adverse effects of treatment that were consistent with adverse effects reported with the use of the drug in the treatment of other conditions. Evidence of direct causality was not required. A study could be classified as suggesting possible harm from the drug even if the study had methodological weaknesses. “Possible benefit” if a study had evidence of benefit for an important outcome measure which was recorded consistently (e.g., case fatality, need for mechanical ventilation, duration of hospitalization, frequency of ARDS) in patients treated in a defined way compared to a valid control group. A control group was considered valid if randomized, or if patient characteristics and illness severity were comparable to the treatment group. Evidence of direct causality was not required. “Definite harm” if a study contained statistically significant evidence of harm demonstrated in a double-blind randomized trial, which did not contain serious methodological weaknesses. “Definite benefit” if a study contained statistically significant evidence of harm demonstrated in a double-blind randomized trial, which did not contain serious methodological weaknesses. Results In vitro evidence was available in 15 studies. Clinical evidence of SARS treatment in humans was reported in 54 studies (37 in English, 17 in Chinese). Three studies addressed treatment of ARDS (Figure 1). Ribavirin In vitro. We found six studies that described the antiviral effect of ribavirin in vitro (Table S4); four showed an antiviral effect (Table S5). A synergistic antiviral effect between ribavirin and type I IFN (IFN-β1a or leukocytic IFN-α) was described in two studies performed in human cell lines and Vero cell lines [12,13]. In SARS patients. We found 24 studies that described ribavirin treatment in cohorts larger than ten patients (Table S6). Our formal assessment classified 20 studies as “inconclusive,” due to study design or because the effect of ribavirin could not be distinguished from the effects of other treatments (such as steroids and antiviral drugs). Four publications presented evidence of possible harm (14–17). Three of these studies, each of which included over 100 patients, documented a fall in haemoglobin levels after ribavirin treatment when compared to levels in patients before treatment [14–16]. Of patients treated with ribavirin, 49/138 to 67/110 (36%–61%) developed haemolytic anaemia, a recognised complication with this drug, although it is not possible to rule out the possibility that SARS-CoV infection caused the haemolytic anaemia, as there is no control group. One study noted that over 29% of SARS patients had some degree of liver dysfunction indicated by ALT levels higher than normal, and the number of patients with this complication increased to over 75% after ribavirin treatment (Table S7) [17]. In the Chinese literature six additional reports described patients with SARS treated with ribavirin (often with steroids). These six reports were determined to be inconclusive in the evaluation of treatment for SARS (Tables S8 and S9). LPV/r In vitro. Of three studies, two demonstrated that lopinavir inhibits cytopathic effects of SARS-CoV in fetal rhesus monkey kidney cells (Table S4). One study showed detectable but reduced activity in Vero-E6 cells [13], and one study concluded that neither lopinavir nor ritonavir had an effect [18]. A synergistic effect of lopinavir with ribavirin has been reported (Table S5). In SARS patients. We found two studies of LPV/r (lopinavir 400 mg with ritonavir 100 mg orally every 12 h) in cohorts larger than ten patients (Table S6). Patients also received ribavirin and corticosteroids. LPV/r use was compared among three groups of patients: those who received it as an early SARS treatment, those who received it as a late treatment, and those who did not receive it at all. When LPV/r was added as an initial treatment to ribavirin and corticosteroid therapy, the death rate was lower than among those who received ribavirin and corticosteroids (1/44 [2.3%] versus 99/634 [15.6%]; p < 0.05) [19]. A second study of this regimen reported fewer episodes of ARDS or death compared with historical controls who had not received LPV/r (1/41 [2.4%] versus 32/111 [28.8%]; p < 0.001) (Table S7) [20]. Both studies were determined to be inconclusive due to possible bias in the selection of control group or treatment allocation. No additional studies were identified from the Chinese literature. Corticosteroids In vitro. No studies were found on the cytopathic effect of corticosteroids alone against SARS-CoV. Corticosteroids act as immunomodulatory agents, and therefore studies to measure direct antiviral effects in vitro were not expected. In SARS patients. Fifteen articles examined corticosteroid treatment in ten or more patients. Of these cohorts 13 were also treated with ribavirin (Table S6). We determined that 13 of the 15 studies were inconclusive. Of these, in an uncontrolled and nonrandomised study, 95/107 (89%) of patients treated with high-dose methylprednisolone (0.5–1 mg/kg prednisolone on day 3 of illness, followed by hydrocortisolone 100 mg every 8 h, and pulse-doses of methylprednisolone 0.5 g IV for 3 d) after the first week of illness recovered from progressive lung disease (Table S7) [16]. Two studies contained evidence of possible harm from corticosteroids [21,22]. One measured SARS-CoV plasma viral load across time after fever onset in a randomized, double-blind, placebo-controlled trial; corticosteroid use within the first week of illness was associated with delayed viral clearance. The other study, which was case-controlled, found that patients with psychosis received higher cumulative doses of steroids than patients without psychosis (10,975 mg versus 6,780 mg; p = 0.017) [22]. In the Chinese literature, we found 14 reports in which steroids were used (Table S8 and Table S9). Twelve studies were inconclusive and two showed possible harm. One study reported diabetes onset associated with methylprednisolone treatment [23]. Another study (an uncontrolled, retrospective study of 40 SARS patients) reported avascular necrosis and osteoporosis among corticosteroid-treated SARS patients [24]. In ARDS patients. Three clinical trials examined the effect of corticosteroids on mortality in patients with established ARDS (Table S10). In two trials, high-dose methylprednisolone given for approximately 2 d was not effective for early ARDS [25,26]. One small RCT that used a regimen of lower dose methylprednisolone (2 mg/kg per day), tapered after 2 wk, showed possible evidence of ARDS improvement (Table S11) [27]. IFN Type I In vitro. Twelve in vitro studies with data on the antiviral effect of IFN type I have been reported, and all demonstrated an antiviral effect against SARS-CoV (six for IFN-α and ten for IFN-β) (Tables S4 and S5). Antiviral effects have been demonstrated in monkey (Vero; Vero-E6), fetal rhesus monkey kidney (fRhK-4), and human (Caco2, CL14, and HPEK) cell lines. Three reports presented evidence that IFN-β was superior against SARS-CoV compared to IFN-α and found rIFN-α2 virtually ineffective against SARS-CoV compared to other IFNs [28]. Synergistic effects were reported for leukocytic IFN-α with ribavirin [13], IFN-β with ribavirin [12,13] and IFN-β with IFN-γ [28,29]. In SARS patients. Two studies of IFN-α given with steroids and/or ribavirin were reported (Table S6). No significant difference was seen in outcome between IFN-α treatment group and those treated with other regimens. Results of both studies were inconclusive due to a lack of a consistent treatment regimen or suitable control group (Table S7). In the Chinese literature, one additional study reported the use of IFN-α as part of a regimen that included ribavirin and steroids [30]. We determined this study to be inconclusive because a variety of treatments given masked the effect of IFN-α alone (Table S8 and Table S9). Convalescent Plasma or Immunoglobulin In vitro. No studies were found on the cytopathic effect of this treatment on SARS-CoV. Convalescent plasma and IVIG act as immunomodulatory agents and therefore studies to measure direct antiviral effects in vitro were not expected. In SARS patients. Five studies of either IVIG or convalescent plasma treatment given in addition to steroids and ribavirin were reported for treatment of SARS (Table S6). These studies were inconclusive, because the effect of convalescent plasma or IVIG could not be discerned from effects of patient comorbidities, stage of illness, or effect of other treatments (Table S7). In the Chinese literature, two additional studies reported evidence on the effect of convalescent plasma as a treatment for SARS [30,31]. These studies were inconclusive (Table S8 and Table S9). Evidence collected on the benefit or harm of drugs used to treat SARS is summarized in Table 1. Discussion The rapid spread and subsequent control of SARS precluded controlled clinical treatment trials during the outbreak of 2002–2003. In this report we summarize the results of a systematic evaluation of the findings from published reports of treatments used for SARS during the epidemic. Publications from the Chinese literature were included to capture as much evidence as possible. We developed specific criteria (Box 1) to look for large, obvious effects of benefit, adverse or poor outcomes, or evidence of potential benefit that could be used to prioritise future research of SARS treatments. A summary of this evidence in SARS patients is shown in Table 1. Despite thirty reports of SARS-infected patients treated with ribavirin, there is no convincing evidence that it led to recovery. Haemolytic anaemia, a recognized side effect of this treatment, was observed in three studies. We would infer from these findings that any future use of ribavirin for SARS should be within the context of a controlled trial with close attention given to adverse effects. Corticosteroids were commonly prescribed to SARS patients with worsening pulmonary disease or progressing abnormalities on chest X-rays. Treatment regimens varied widely but can be classified into two groups, early treatment and rescue treatment given at a later stage of illness. It is difficult to make a clear recommendation about whether corticosteroids should be used to treat SARS-associated lung injury in any stage of illness, particularly as the drug is immunosuppressive and may delay viral clearance if given before viral replication is controlled [21]. Of added concern are infectious complications, avascular necrosis, and steroid-induced psychosis—recognized adverse effects of corticosteroid use. Fungal superinfection and aspergillosis have been noted in case reports and autopsy findings of SARS patients given corticosteroids at high doses or for prolonged periods [32,33]. This review has found evidence of avascular necrosis and steroid-induced psychosis in SARS patients. Seven studies of treatment with convalescent plasma or IVIG, three with IFN type I, and two with LPV/r were inconclusive by the criteria used in our analyses. Authors of four of the IVIG studies commented that patients seemed to improve upon treatment, but that more controlled trials of this approach are needed to provide evidence of an effect for SARS. Important caveats should be considered in this review. Most of the studies of SARS patients were descriptions of the natural course of the disease and had not been designed to reliably assess the effects of the treatments used. Patient characteristics such as age and presence of diabetes mellitus have been associated with severe disease and can confound treatment effects. A diagnostic test for early SARS illness was not validated or widely available, and in general, treatment was initiated once patients fulfilled a clinical and epidemiological case definition. It is possible that the inclusion of patients without laboratory confirmation of SARS-CoV infection in this review could cause an underestimate of any true effect of antiviral treatment on SARS. The variation in treatment regimens—particularly the wide range in doses, duration of therapy, and route of administration of ribavirin and corticosteroids—is a major obstacle to a clear interpretation of the data in this review. The nonstandardised collection of clinical information limits the conclusions that can be drawn from a retrospective analysis. We suggest that, in the event of a future outbreak of SARS-CoV or another novel agent, attempts be made to develop treatment protocols and to collect and contribute information for a standardized minimum dataset that could facilitate analysis of treatment outcomes among different settings. As observational studies pose problems of interpretation, the need is great for good-quality randomised trials, despite the difficulties in organising such trials. Supporting Information Table S1 Rationale for Treatments and Recognized Adverse Effects (55 KB DOC) Click here for additional data file. Table S2 Method of Systematic Review (A) Search strategy, step 1: Select the treatments. (B) Search strategy, step 2: Narrow the scope. (C) Inclusion criteria and information sought from each study. (45 KB DOC) Click here for additional data file. Table S3 QUOROM Statement (50 KB DOC) Click here for additional data file. Table S4 Description of SARS-CoV Replication Studies: Assay Type and Outcomes Measured (79 KB DOC) Click here for additional data file. Table S5 Results from SARS-CoV Replication Studies: Inhibition of SARS-CoV Replication (84 KB DOC) Click here for additional data file. Table S6 Description of Studies within SARS Patients (186 KB DOC) Click here for additional data file. Table S7 Results of Treatment within SARS Patients (English literature) (183 KB DOC) Click here for additional data file. Table S8 Description of Studies of SARS Patients (Chinese Literature) (108 KB DOC) Click here for additional data file. Table S9 Results of Treatment within SARS Patients (Chinese Literature) (93 KB DOC) Click here for additional data file. Table S10 Description of Studies of ARDS or ALI (50 KB DOC) Click here for additional data file. Table S11 Results of Treatment of ARDS or ALI (48 KB DOC) Click here for additional data file.
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              Coronaviruses post-SARS: update on replication and pathogenesis

              Key Points Coronaviruses are positive strand RNA viruses that cause disease in humans, and domestic and companion animals. They are most notorious for causing severe acute respiratory syndrome (SARS) outbreaks in 2002–2003. All coronaviruses follow the same basic strategy of replication. All coronaviruses encode 15 or 16 replicase related proteins, 4 or 5 structural proteins and 1–8 group-specific or accessory proteins. Many of the replicase proteins are assembled into replication machinery in double-membrane vesicles (DMVs) and on a reticular network of membranes that are derived from the endoplasmic reticulum. Coronaviruses are readily transmitted across species. This phenomenon was illustrated when the SARS-coronavirus crossed species from bats to intermediate hosts, such as palm civets, and then to humans. It also explains the large number of species, including humans, that are infected with viruses closely related to bovine coronavirus. In many coronavirus infections, disease severity increases during virus clearance, suggesting that the host immune response is both protective and pathogenic. Furthermore, inhibition of specific aspects of the immune response results in less severe disease and less tissue destruction, without diminishing the kinetics of virus clearance. Like all successful viruses, coronaviruses have evolved both passive and active mechanisms to evade the interferon response. Replication in DMVs may contribute to passive evasion of the innate immune response by making double-stranded RNA inaccessible to cellular sensors.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                November 2010
                November 2010
                4 November 2010
                : 6
                : 11
                : e1001176
                Affiliations
                [1 ]Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
                [2 ]Departments of Epidemiology and Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                University of California San Francisco, United States of America
                Author notes

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

                Article
                10-PLPA-RA-3358R3
                10.1371/journal.ppat.1001176
                2973827
                21079686
                76d44a65-4152-4610-b075-aa6ea6df37d9
                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.
                History
                : 17 May 2010
                : 1 October 2010
                Page count
                Pages: 10
                Categories
                Research Article
                Biochemistry/Replication and Repair
                Molecular Biology/RNA-Protein Interactions
                Virology
                Virology/Antivirals, including Modes of Action and Resistance
                Virology/Viral Replication and Gene Regulation

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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