Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

TLDR

Intracellular Zn²⁺ elevation via ionophores such as pyrithione impairs replication of diverse RNA viruses, partly by disrupting viral polyprotein processing, and for nidoviruses like SARS‑CoV and EAV the RNA‑dependent RNA polymerase (RdRp) is the core enzyme of the replication‑transcription complex. This study aimed to show that low concentrations of Zn²⁺ and pyrithione together inhibit SARS‑CoV and EAV replication in cell culture. By isolating RTCs from infected cells, the authors demonstrated that Zn²⁺ directly suppresses the RNA‑synthesizing activity of both viruses’ RdRp enzymes, independent of ionophore‑mediated transport. Zn²⁺ at 2 µM with 2 µM pyrithione blocks SARS‑CoV and EAV replication, directly inhibits their RdRp activity—preventing initiation in EAV and elongation/template binding in SARS‑CoV—and the effect is reversible by MgEDTA.

Abstract

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.