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

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. 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.