Zn2+ inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture
| Autor: | Te Velthuis, AJW, van den Worm, SH, Sims, AC, Baric, RS, Snijder, EJ, van Hemert, MJ, Andino, R |
|---|---|
| Přispěvatelé: | Andino, R |
| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Arterivirus
viruses Electrophoretic Mobility Shift Assay Severe Acute Respiratory Syndrome Virus Replication chemistry.chemical_compound 0302 clinical medicine Transcription (biology) RNA polymerase Chlorocebus aethiops 030212 general & internal medicine lcsh:QH301-705.5 Polymerase Biochemistry/Replication and Repair 0303 health sciences Arterivirus Infections biology Reverse Transcriptase Polymerase Chain Reaction 3. Good health Virology/Viral Replication and Gene Regulation Severe acute respiratory syndrome-related coronavirus RNA Viral dependent rna pyrrolidine dithiocarbamate escherichia-coli sars-coronavirus transcription protein ions rhinoviruses pathogenesis translation Molecular Biology/RNA-Protein Interactions Research Article inorganic chemicals lcsh:Immunologic diseases. Allergy Blotting Western Immunology RNA-dependent RNA polymerase In Vitro Techniques Microbiology Virus 03 medical and health sciences Virology Escherichia coli Genetics Animals RNA Messenger Vero Cells Molecular Biology 030304 developmental biology Virology/Antivirals including Modes of Action and Resistance Ionophores RNA RNA-Dependent RNA Polymerase biology.organism_classification Molecular biology Viral replication chemistry lcsh:Biology (General) Zinc Compounds biology.protein Parasitology lcsh:RC581-607 |
| Zdroj: | PLoS Pathogens, Vol 6, Iss 11, p e1001176 (2010) PLoS Pathogens PLoS Pathogens, 6(11) |
| Popis: | Increasing the intracellular Zn2+ 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 Zn2+ and PT at low concentrations (2 µM Zn2+ 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 Zn2+ across the plasma membrane—we show that Zn2+ 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 Zn2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn2+ 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 Zn2+ 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. 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 Zn2+ 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 Zn2+ 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. |
| Databáze: | OpenAIRE |
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