ArticlePDF Available

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

Authors:

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.
Content may be subject to copyright.
A preview of the PDF is not available
... Zinc deficiency leads to a wide range of disorders, including metabolic diseases, compromised immune system, developmental imbalances, neurodegenerative problems, and inflammatory pathologies (Simmer and Thompson, 1985;Fabris and Mocchegiani, 1995;Prasad, 2008). Importantly, it is known to possess antiviral properties against many different viruses, namely, herpes simplex virus (HSV), severe acute respiratory syndrome coronavirus (SARS CoV), rhinovirus, respiratory syncytial virus (RSV), equine arteritis virus (EAV), human papilloma virus (HPV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis E virus (HEV) (Korant et al., 1974;Haraguchi et al., 1999;Suara and Crowe Jr, 2004;Te Velthuis et al., 2010;Kaushik et al., 2017). Multiple mechanisms underlie the antiviral activity of zinc, which includes inhibition of virus entry and viral polyprotein processing or inhibition of viral RNA dependent RNA polymerase activity (Haraguchi et al., 1999;Krenn et al., 2009;Te Velthuis et al., 2010;Kaushik et al., 2017). ...
... Importantly, it is known to possess antiviral properties against many different viruses, namely, herpes simplex virus (HSV), severe acute respiratory syndrome coronavirus (SARS CoV), rhinovirus, respiratory syncytial virus (RSV), equine arteritis virus (EAV), human papilloma virus (HPV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis E virus (HEV) (Korant et al., 1974;Haraguchi et al., 1999;Suara and Crowe Jr, 2004;Te Velthuis et al., 2010;Kaushik et al., 2017). Multiple mechanisms underlie the antiviral activity of zinc, which includes inhibition of virus entry and viral polyprotein processing or inhibition of viral RNA dependent RNA polymerase activity (Haraguchi et al., 1999;Krenn et al., 2009;Te Velthuis et al., 2010;Kaushik et al., 2017). Zinc also contributes toward modulating the host immune response to restrict viral replication. ...
Article
Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis. The disease takes a severe form in pregnant women, leading to around 30% mortality. Zinc is an essential micronutrient that plays a crucial role in multiple cellular processes. Our earlier findings demonstrated the antiviral activity of zinc salts against HEV infection. Zinc oxide (ZnO) and its nanostructures have attracted marked interest due to their unique characteristics. Here we synthesized ZnO nanoparticles [ZnO(NP)] and tetrapod-shaped ZnO nanoparticles [ZnO(TP)] and evaluated their antiviral activity. Both ZnO(NP) and ZnO(TP) displayed potent antiviral activity against hepatitis E and hepatitis C viruses, with the latter being more effective. Measurement of cell viability and intracellular reactive oxygen species levels revealed that both ZnO(NP) and ZnO(TP) are noncytotoxic to the cells even at significantly higher doses, compared to a conventional zinc salt (ZnSO4). Our study paves the way for evaluation of the potential therapeutic benefit of ZnO(TP) against HEV and HCV.
... RNA polymerase) activity (36)(37)(38)(39)(40)(41)(42)(43). The infection of virus and other pathogens, Zn is directly or indirectly triggering the host immune response. ...
Article
Full-text available
In the present Review Article, we describe the potential of three herbal products as Turmeric (Haldi), Heart-leaved moonseed (Giloy), and Black cumin (Kalonji) that can be used for preventative or nutritional therapy of Covid-19.
... A strong interrelationship of Zn status with survival odds and mortality risk of patients affected by the ongoing Coronavirus Disease-2019 (COVID- 19) pandemic has been described in recent observational studies (18)(19)(20)(21)(22)(23). This finding was also supported by in vitro studies assessing the effects of Zn2 + on SARS-CoV replication and RNA polymerase activity (24). In addition, a potential direct interrelation of Zn was also proposed for SARS-CoV-2 maturation (25,26). ...
Article
Full-text available
Background Zinc (Zn) is an essential trace element with high relevance for the immune system, and its deficiency is associated with elevated infection risk and severe disease course. The association of Zn status with the immune response to SARS-CoV-2 vaccination is unknown. Methods A cohort of adult health care workers (n=126) received two doses of BNT162B2, and provided up to four serum samples over a time course of 6 months. Total SARS-CoV-2 IgG and neutralizing antibody potency was determined, along with total as well as free Zn concentrations. Results The SARS-CoV-2 antibodies showed the expected rise in response to vaccination, and decreased toward the last sampling point, with highest levels measured three weeks after the second dose. Total serum Zn concentrations were relatively stable over time, and showed no significant association with SARS-CoV-2 antibodies. Baseline total serum Zn concentration and supplemental intake of Zn were both unrelated to the antibody response to SARS-CoV-2 vaccination. Time resolved analysis of free Zn indicated a similar dynamic as the humoral response. A positive correlation was observed between free Zn concentrations and both the induced antibodies and neutralizing antibody potency. Conclusion While the biomarkers of Zn status and supplemental Zn intake appeared unrelated to the humoral immune response to SARS-CoV-2 vaccination, the observed correlation of free Zn to the induced antibodies indicates a diagnostic value of this novel biomarker for the immune system.
... [53] Zinc inhibits Coronavirus replication. [54] Hydroxychloroquine and chloroquine potentiate uptake of zinc into lysosomes. [55] The role of monoclonal and polyclonal antibodies and teicoplanin (which inhibits the viral genome exposure in cytoplasm) is not clear. ...
Article
Full-text available
Coronavirus disease 2019 (COVID-19) was first identified in December 2019 and declared a pandemic in March 2020 by the World Health Organization. Countries with advanced technology and huge financial base are finding it difficult to curtail the COVID-19 spread and its attendant morbidity and mortality. Spread of the disease to the African Sub-Region became fearful because poor health-care facilities and manpower needs due to the majorly dilapidated health-care infrastructure, considering the mortality tables from other more advanced nations. To adequately prepare the manpower and avoid strain on the workforce before the incidence of disease peaks, there was a massive re-training of health-care personnel and the workforce routine schedule re-designed. This paper reviews the disease and highlights the challenges in our setting toward combating the coronavirus 2019 disease.
... Zinc supplement reduces the mortality rate caused due to lower respiratory tract infections (Awotiwon et al., 2017). It has been seen that zinc along with zinc ionospheres pyrithione can impair a range of RNA viruses, and these at low concentrations impede replication of SARS corona (TeVelthuis et al., 2010). So, zinc is effective not only on symptoms like low respiratory infection and diarrhea, but also for COVID treatment . ...
... 87 Zinc may inhibit coronavirus RNA polymerase activity in vitro and viral replication in cultured cells. 88 Zinc has also an immunomodulatory, antioxidant, and antiinflammatory role that could influence the trajectory of SARS-CoV-2 infection, and its supplementation could ameliorate lung function, enhance mucociliary clearance, and reduce ventilator-induced lung injury in critical patients with COVID-19. 85 Lower serum zinc levels were found in patients with severe COVID-19 with acute respiratory distress syndrome and associated with a higher rate of complications and in-hospital length of stay. ...
Article
Long COVID affects a large share of persons of all age groups but may be especially burdensome for vulnerable older adults. Long COVID has a complex pathophysiology encompassing inflammatory and autoimmune processes, perturbations in metabolic pathways, and alterations in endothelial function and redox homeostasis. The management of long COVID requires a multidimensional approach that should include a comprehensive nutritional assessment. Several food bioactive compounds, nutraceuticals, and supplements may target specific pathways involved in long COVID and may, therefore, be used as an adjunctive therapy to manage the condition.
... Zinc plays a key role in modulating the immune system by promoting the development and function of macrophages, neutrophils, NK cells, inducing CD8+ T-cells proliferation, and assisting in the inflammatory process [120]. This micronutrient shows important antiviral properties, counteracting the replication of RNA viruses and inhibiting the replication of SARS-CoV at low concentrations [136]. Zinc deficiency is related to an altered phagocytic function, and reduced production of macrophages, DCs, and B and T cells [137]. ...
Article
Full-text available
The immune system is highly dynamic and susceptible to many alterations throughout pregnancy. Since December 2019, a pandemic caused by coronavirus disease 19 (COVID-19) has swept the globe. To contain the spread of COVID-19, immediate measures such as quarantine and isolation were implemented. These containment measures have contributed to exacerbate situations of anxiety and stress, especially in pregnant women, who are already particularly anxious about their condition. Alterations in the psychological state of pregnant women are related to alterations in the immune system, which is more vulnerable under stress. COVID-19 could therefore find fertile soil in these individuals and risk more severe forms. Normally a controlled dietary regimen is followed during pregnancy, but the use of particular vitamins and micronutrients can help counteract depressive-anxiety states and stress, can improve the immune system, and provide an additional weapon in the defense against COVID-19 to bring the pregnancy to fruition. This review aims to gather data on the impact of COVID-19 on the immune system and psychological condition of pregnant women and to assess whether some micronutrients can improve their psychophysical symptoms.
... Doxycycline is the most potent tetracycline derivatives inhibitor of MMPs, even at low doses (25 mg) (Castro et al., 2011). It may act as an ionophore by increasing intracellular Zinc concentrations, suppressing viral replication and strengthening the immune system (Griffin et al., 2010;te Velthuis et al., 2010).In addition to Doxycycline effect, Zinc have also a benefic role to protect against COVID-19 infection. In fact, many reports have shown that Zinc can inhibit the enzymatic activity and replication of SARS-CoV-2's RNA polymerase, and can inhibit angiotensin converting enzyme ACE2 activity (Skalny et al., 2020;Ratia et al., 2006). ...
Article
Full-text available
Background SARS-CoV-2 is a novel virus that causes coronavirus disease-19 (COVID-19). Many antiviral and immunomodulatory drugs can be used as a potential treatment. Doxycycline combined to Zinc can play a pivotal role to protect against SARS-CoV-2 infection Objective This study aims to assess the efficacy of a combination treatment of Doxycycline and Zinc, in primary prevention of COVID-19 infection in Tunisian Health Care Workers (HCWs) compared to two control groups. Methods We conducted a prospective randomized double-blind clinical trial over five months to determine the efficacy of a combination preventive treatment dose of Doxycycline (100 mg/day) and Zinc (15 mg/day), compared to a single-dose treatment with Doxycycline versus a placebo. The effectiveness of preventive treatment was measured by the significant decline in the number of cases of COVID-19 infection and/or a decrease in the viral load determined by SARS-CoV-2 cycle threshold (Ct) value using RT-PCRs test. Results We detected a significant decrease of SARS-CoV-2 infection in group who received both Doxycycline and Zinc compared to other participants. We are also demonstrated that COVID-19 infection was not associated with diabetes (p=0.51), nor with hypertension (p=0.99), asthma (p=0.52) and chronic obstructive pulmonary disease (p=0.27). Conclusions Our finding indicated that preventive therapy reduced the risk of SARS-CoV-2. These results suggest that the combination of Doxycycline and Zinc has a protective effect in SARS-CoV-2 infection.
Article
Full-text available
The COVID-19 threatens nearly most of the world's population with a significant mortality rate over the last 2 years. The main cause of death in those infected with SARS-CoV-2 is acute lungs infection, which is brought about by increased inflammatory activity, oxidative anxiety, and inflammatory reactions in the respiratory tract. To date, there is no clear and conclusive treatment or cure and affecting elderly adults heavily across the world. In this regard, assessing the effectiveness of current pharmacological agents, which can be used to resolve or attenuate the seriousness of this pulmonary complication that has threatened the lives of so many people globally, is of particular importance. For instance, evidence reveals that vita-D (VD) and Zinc supplementation may have a positive effect in treating COVID-19 patients. Moreover, numerous trials and studies have already started to look into the consequence of VD and zinc supplements on individuals with SARS-COVID-19 infection.
Article
Full-text available
An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the ∼30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8.
Article
Full-text available
Metalloproteases cleave proteins and peptides, and deregulation of their function leads to pathology. An understanding of their structure and mechanisms of action is necessary to the development of strategies for their regulation. Among metallopeptidases are the metzincins, which are mostly multidomain proteins with ∼130–260-residue globular catalytic domains showing a common core architecture characterized by a long zinc-binding consensus motif, HEXXHXXGXX(H/D), and a methionine-containing Met-turn. Metzincins participate in unspecific protein degradation such as digestion of intake proteins and tissue development, maintenance, and remodeling, but they are also involved in highly specific cleavage events to activate or inactivate themselves or other (pro)enzymes and bioactive peptides. Metzincins are subdivided into families, and seven such families have been analyzed at the structural level: the astacins, ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases, snapalysins, leishmanolysins, and pappalysins. These families are reviewed from a structural point of view.
Article
Full-text available
Current therapy for chronic hepatitis C virus (HCV) infection is effective in less than 50% of genotype 1-infected patients. Antiviral agents specifically targeting either the HCV protease or polymerase, or other targets, are now in clinical development. In general, direct antivirals are potent inhibitors of HCV replication and can result in rapid declines in serum HCV RNA levels. Yet these agents drive selection pressure for mutant viruses that can reduce susceptibility to any given drug. Using pegylated interferon (PEG-IFN) and ribavirin (RBV) in combination with direct antivirals can suppress viral breakthrough and increase the likelihood of sustained virologic response. Direct antivirals also result in adverse events in a proportion of patients, adding to concerns of tolerability that exist with PEG-IFN and RBV. Direct antivirals are very likely to become an integral part of treatment within the next decade, and already their use in clinical trials has raised important issues related to duration of treatment, early stopping rules, retreatment of previously treated patients, and how or when direct antivirals should be combined. Here, we provide current information regarding the effectiveness of direct antivirals in treating chronic HCV infection and discuss the key questions and challenges now facing the field.
Article
Full-text available
The involvement of Zn(2+) ions in the structure of ubiquitous protein zinc fingers gives this metal a particular position in the biology of the cell. ...
Article
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).
Article
DNA and a large proportion of RNA are antiparallel duplexes composed of an unvarying phosphosugar backbone surrounding uniformly stacked and highly similar base pairs. How do the myriad of enzymes (including ribozymes) that perform catalysis on nucleic acids achieve exquisite structure or sequence specificity? In all DNA and RNA polymerases and many nucleases and transposases, two Mg2+ ions are jointly coordinated by the nucleic acid substrate and catalytic residues of the enzyme. Based on the exquisite sensitivity of Mg2+ ions to the ligand geometry and electrostatic environment, we propose that two-metal-ion catalysis greatly enhances substrate recognition and catalytic specificity.
Article
Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusionn roteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of >320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.
Article
As health care providers, we find ourselves on the verge of a new era in the treatment of chronic hepatitis C virus (HCV) infection. A number of directly acting antiviral agents are now in the latter stages of clinical development. The more promising candidates include direct inhibitors of the HCV nonstructural 3 protease, as well as both nucleoside and non-nucleoside inhibitors of the NS5B RNA-dependent RNA polymerase. Although these agents have demonstrated potent antiviral effect, monotherapy has been complicated by rapid virological breakthrough due to the selection of drug-resistant mutants. As for HIV and HBV, combination therapy will therefore be necessary. This brief review summarizes the current literature concerning resistance and directly acting antiviral agents, and identifies key challenges facing this emerging field.
Article
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.
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.