Zn2+ 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
• Publication Date: 2010-11-04T19:49:17Z
• Authors: Aartjan J. W. te ...; Sjoerd H. E. van ...; Amy C. Sims; Ralph S. Baric; Eric J. Snijder; Martijn J. van He...
• Keywords: 0301 basic medicine; Arterivirus; QH301-705.5; Blotting, Western; Electrophoretic Mobility Shift Assay; In Vitro Techniques; Severe Acute Respiratory Syndrome; Virus Replication; 03 medical and health sciences; Chlorocebus aethiops; Escherichia coli; Animals; RNA, Messenger; Biology (General); Vero Cells; Arterivirus Infections; Ionophores; Reverse Transcriptase Polymerase Chain Reaction; RC581-607; RNA-Dependent RNA Polymerase; 3. Good health; Severe acute respiratory syndrome-related coronavirus; Zinc Compounds; RNA, Viral; dependent rna pyrrolidine dithiocarbamate escherichia-coli sars-coronavirus transcription protein ions rhinoviruses pathogenesis translation; Immunologic diseases. Allergy; Research Article

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.