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

DOI:10.1371/journal.ppat.1001176
Authors:
Aartjan te Velthuis at Princeton University
Aartjan te Velthuis
Sjoerd van den Worm at Oregon Health and Science University
Sjoerd van den Worm
Amy C Sims
Amy C Sims
Amy C Sims
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Ralph S Baric at University of North Carolina at Chapel Hill
Ralph S Baric
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Abstract and Figures

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.
The zinc ionophore pyrithione inhibits nidovirus replication in cell culture. ( A ) Cytotoxicity of PT in Vero-E6 cells in the absence (blue circles) or presence of 2 (black squares), 4 (red triangles), or 8 m M (gray diamonds) ZnOAc 2 as determined by the MTS
The zinc ionophore pyrithione inhibits nidovirus replication in cell culture. ( A ) Cytotoxicity of PT in Vero-E6 cells in the absence (blue circles) or presence of 2 (black squares), 4 (red triangles), or 8 m M (gray diamonds) ZnOAc 2 as determined by the MTS
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Inhibition of the in vitro RNA-synthesizing activity of isolated RTCs by Zn 2 + . Incorporation of [ a - 32 P]CMP into viral RNA by EAV ( A ) and SARS-CoV ( B ) in RTC assays in the presence of various Zn 2 +
Inhibition of the in vitro RNA-synthesizing activity of isolated RTCs by Zn 2 + . Incorporation of [ a - 32 P]CMP into viral RNA by EAV ( A ) and SARS-CoV ( B ) in RTC assays in the presence of various Zn 2 +
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Inhibition of nidovirus RTC activity by Zn 2 + can be reversed by chelation. ( A ) Schematic representation of the in vitro assays with isolated RTCs, which were initiated with [ a - 32 P]CTP, either in the absence (sample 1 and 2) or presence of 500 m M Zn 2 + . After a 30- min incubation at 30 u C, both the untreated and Zn 2 + -treated samples were split into two aliquots, and 1 mM of the Zn 2 + chelator MgEDTA
Inhibition of nidovirus RTC activity by Zn 2 + can be reversed by chelation. ( A ) Schematic representation of the in vitro assays with isolated RTCs, which were initiated with [ a - 32 P]CTP, either in the absence (sample 1 and 2) or presence of 500 m M Zn 2 + . After a 30- min incubation at 30 u C, both the untreated and Zn 2 + -treated samples were split into two aliquots, and 1 mM of the Zn 2 + chelator MgEDTA
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EAV and SARS-CoV RdRp assays with wild-type enzyme and active-site mutants. ( A ) The EAV polymerase was incapable of primer extension and required a free 3 9 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 5 9 U 10 overhang as template. The bar graph shows the nucleotide incorporating activities
EAV and SARS-CoV RdRp assays with wild-type enzyme and active-site mutants. ( A ) The EAV polymerase was incapable of primer extension and required a free 3 9 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 5 9 U 10 overhang as template. The bar graph shows the nucleotide incorporating activities
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The 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,
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The 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,
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Supplementary resources (3)

Figure S2
Data
November 2010
Aartjan te Velthuis · Sjoerd van den Worm · Amy C. Sims · Ralph S Baric · Martijn J van Hemert
Figure S3
Data
November 2010
Aartjan te Velthuis · Sjoerd van den Worm · Amy C. Sims · Ralph S Baric · Martijn J van Hemert
Figure S1
Data
November 2010
Aartjan te Velthuis · Sjoerd van den Worm · Amy C. Sims · Ralph S Baric · Martijn J van Hemert
... Antiviral effect of zinc have been demonstrated in vivo and in vitro in several viruses, including corona viruses, picornaviruses, papilloma viruses, metapneumoviruses, rhinoviruses, herpes simplex viruses, varicella-zoster viruses, respiratory syncytial viruses, retroviruses, SARS-CoV and SARS-CoV-2 etc. (Bracha and Schlesinger, 1976;Gupta and Rapp, 1976;Polatnick and Bachrach, 1978;Katz and Margalith, 1981;Kümel et al., 1990;Hulisz, 2004;Krenn et al., 2009;Te Velthuis et al., 2010;Liu and Kielian, 2012;Wei et al., 2012;Antoine et al., 2016;Liu et al., 2021;Samad et al., 2021). Based on the available literature, mechanism underlying the antiviral properties of zinc may be broadly classified into two categories: (a) direct inhibitory effect on the different stages of the life cycle of the virus and (b) indirect effect of zinc attributed to its ability to modulate various host cellular processes and immune response. ...
... RNA dependent RNA polymerase (RdRp) produced by proteolytic processing of the viral nonstructural proteins plays the central role in the viral replication process. Zinc inhibits RdRp activity of many viruses, including TGEV (Transmissible gastroenteritis virus), SARS-CoV (Severe acute respiratory syndrome coronavirus), EAV (Equine arteritis virus), Rhinovirus and HEV (Hepatitis E virus; Korant et al., 1974;Hung et al., 2002;Te Velthuis et al., 2010;Wei et al., 2012;Kaushik et al., 2017). Different steps in the replication process have been shown to be targeted by zinc for inhibiting RdRp activity. ...
... Different steps in the replication process have been shown to be targeted by zinc for inhibiting RdRp activity. In case of SARS-CoV, zinc treatment reduced template binding and elongation by the RdRp whereas in case of EAV, initiation step of RNA synthesis was inhibited (Te Velthuis et al., 2010). Zinc also inhibits Rhinovirus RdRp activity in vitro although the mechanism remains to be understood (Korant et al., 1974;Hung et al., 2002). ...
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Viral hepatitis is a major public health concern globally. World health organization aims at eliminating viral hepatitis as a public health threat by 2030. Among the hepatitis causing viruses, hepatitis B and C are primarily transmitted via contaminated blood. Hepatitis A and E, which gets transmitted primarily via the feco-oral route, are the leading cause of acute viral hepatitis. Although vaccines are available against some of these viruses, new cases continue to be reported. There is an urgent need to devise a potent yet economical antiviral strategy against the hepatitis-causing viruses (denoted as hepatitis viruses) for achieving global elimination of viral hepatitis. Although zinc was known to mankind for a long time (since before Christ era), it was identified as an element in 1746 and its importance for human health was discovered in 1963 by the pioneering work of Dr. Ananda S. Prasad. A series of follow up studies involving zinc supplementation as a therapy demonstrated zinc as an essential element for humans, leading to establishment of a recommended dietary allowance (RDA) of 15 milligram zinc [United States RDA for zinc]. Being an essential component of many cellular enzymes and transcription factors, zinc is vital for growth and homeostasis of most living organisms, including human. Importantly, several studies indicate potent antiviral activity of zinc. Multiple studies have demonstrated antiviral activity of zinc against viruses that cause hepatitis. This article provides a comprehensive overview of the findings on antiviral activity of zinc against hepatitis viruses, discusses the mechanisms underlying the antiviral properties of zinc and summarizes the prospects of harnessing the therapeutic benefit of zinc supplementation therapy in reducing the disease burden due to viral hepatitis.
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... This resultant de ciency in Retinoic acid induced by infection could potentially hinder the lung's capability to restore impaired epithelial surfaces, conceivably contributing to pulmonary brosis and subsequent lung capacity reduction51 . Zinc ions serve to inhibit coronavirus RNA polymerase activity, effectively thwarting virus replication52 . A meta-analysis unveiled that zinc supplementation substantially impacted the reduction of mortality in COVID-19 patients 53 . ...
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Background The emergence of severe coronavirus disease 2019 (COVID-19) and its ensuing complications presents a substantial challenge to human safety. Osteoarthritis (OA) stands as the most common degenerative joint disease, while the intricate molecular relationship between OA and COVID-19 remains enigmatic. In this investigation, we employed systematic bioinformatics analysis to uncover the underlying molecular mechanisms associated with these two diseases. Additionally, we identified potential therapeutic drugs with the potential to aid in the treatment of patients afflicted with both COVID-19 infection and osteoarthritis (OA). Methods Datasets for both COVID-19 and OA were sourced from the GEO database. Subsequently, a differential expression analysis was executed to procure Differentially Expressed Genes (DEGs). Co-expressed genes shared between OA and COVID-19 were identified through the intersection of differential gene sets, employing a Venn diagram. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed by Metascape. The hub genes were identified through protein-protein interaction (PPI) analysis carried out in Cytoscape, and their validity was subsequently affirmed through brief experiment. Finally, transcription factor-gene interactions, microRNA (miRNA) candidate identification and drug candidate identification were identified by co-expression of genes. Results A total of 94 co-expressed DEGs were obtained. GO and KEGG enrichment analysis of DEGs showed that they mainly affect inflammation, cytokine and immune-related functions, and inflammation-related signaling pathways. Through the analysis of the PPI network, we obtained 9 hub genes, and validated them with brief experiments. In addition, the top ten drug candidates ranked by P-value were screened, which may exhibit potential for providing therapeutic benefits in the context of treating individuals affected by both COVID-19 infection and OA. Conclusion This study reveals a shared molecular mechanism between osteoarthritis (OA) and neocoronary pneumonia. Additionally, it clarifies potential mechanisms linked to synovial lesions in both neocoronary pneumonia and osteoarthritis. These shared pathways and hub genes might offer insights for future investigations.
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... Além de seu papel nas funções imunológicas, o zinco tem sido aplicado em atividade antiviral direta para uma série de RNA viral, sendo demonstrado que este micromineral inibe de forma eficiente a replicação de SARS-CoV em cultura celular, mostrando o papel crucial do zinco intracelular na inibição da replicação do vírus (Velthuis et al., 2010). ...
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... Despite the interest in zinc sulfate as a prevention therapy, their findings do not support a prophylactic benefit in the absence of a zinc ionophore. To help answer this question, a zinc sulfate prophylactic strategy should be evaluated [35,[62][63][64][65]. ...
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Although coronaviruses were first identified nearly 60 years ago, they only received notoriety in 2003 when one of their members was identified as the aetiological agent of severe acute respiratory syndrome. Previously these viruses were known to be important agents of respiratory and enteric infections of domestic and companion animals and to cause approximately 15% of all cases of the common cold. This Review focuses on recent advances in our understanding of the mechanisms of coronavirus replication, interactions with the host immune response and disease pathogenesis. It also highlights the recent identification of numerous novel coronaviruses and the propensity of this virus family to cross species barriers.
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Antiviral Activity of the Zinc Ionophores Pyrithione and Hinokitiol against Picornavirus Infections
Article
We have discovered two metal ion binding compounds, pyrithione (PT) and hinokitiol (HK), that efficiently inhibit human rhinovirus, coxsackievirus, and mengovirus multiplication. Early stages of virus infection are unaffected by these compounds. However, the cleavage of the cellular eukaryotic translation initiation factor eIF4GI by the rhinoviral 2A protease was abolished in the presence of PT and HK. We further show that these compounds inhibit picornavirus replication by interfering with proper processing of the viral polyprotein. In addition, we provide evidence that these structurally unrelated compounds lead to a rapid import of extracellular zinc ions into cells. Imported Zn2+ was found to be localized in punctate structures, as well as in mitochondria. The observed elevated level of zinc ions was reversible when the compounds were removed. As the antiviral activity of these compounds requires the continuous presence of the zinc ionophore PT, HK, or pyrrolidine-dithiocarbamate, the requirement for zinc ions for the antiviral activity is further substantiated. Therefore, an increase in intracellular zinc levels provides the basis for a new antipicornavirus mechanism.
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