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.

Antiviral activity of zinc salts against transmissible gastroenteritis virus in vitro

“…Additionally or alternatively, Zn could also impair stages of viral replication that follow the release of TGEV into the cytoplasm. A possible intracellular target for Zn-mediated inhibition is the viral RNA polymerase which was reportedly inhibited by increasing intracellular Zn ions in case of the SARS-Coronavirus (SARS-CoV) and other viruses (te Velthuis et al, 2010). In our study, the relative amount of viral Sprotein RNA detectable in infected cells was significantly reduced by Zn treatment for 1-4 hpi without previous inhibition of virus penetration as shown in Fig.…”

Anti-SARS coronavirus agents: a patent review (2008 – present)

“…In addition to the recent patents, the focus of this review article, there are papers describing potential therapeutic agents such as nucleic acids aptamers targeting helicase [94,95] and inhibitors of RdRp [96], which should not be ignored. However, most of the patents describe the agents for treatment or prevention of SARS based on in vitro and/or cell-based experiments, but few of them have been actually tested in animals and none in humans.…”

Tilmicosin Reduces PRRSV Loads in Pigs in vivo

“…Therefore, successful supplementary strategies should include antiviral drugs and immune-modulation therapies (Benfield et al, 2002;Du et al, 2011;Han et al, 2009;Jiang et al, 2010;Karuppannan et al, 2012;Keirstead et al, 2008;Kreutz & Ackermann, 1996;Li et al, 2009;Opriessnig et al, 2011;Patel et al, 2008;te Velthuis et al, 2010;van der Meer et al, 2007;Wei et al, 2011;Yang et al, 2013;Zhuge et al, 2012). When evaluating the antiviral effect of tilmicosin against PRRSV, our data suggested that tilmicosin would be effective in reducing the PRRSV loads in vivo.…”

“…These data indicate that reducing the PRRSV load in the serum may prevent pigs from developing clinical signs after PRRSV infection. Several studies have attempted to identify effective anti-PRRSV treatments in vitro and in vivo (Benfield, Chase, Moore, Wagner, & Zeman, 2002;Du, Yoo, Paradis, & Scherba, 2011;Han, Fan, Patel, & Zhang, 2009;Jiang, Fang, Luo, Xiao, & Chen, 2010;Karuppannan, Wu, Qiang, Chu, & Kwang, 2012;Keirstead, Lee, Yoo, Brooks, & Hayes, 2008;Kreutz & Ackermann, 1996;Li et al, 2009;Opriessnig et al, 2011;Patel et al, 2008;te Velthuis et al, 2010;van der Meer et al, 2007;Wei et al, 2011;Yang et al, 2013;Zhuge et al, 2012). However, there are no effective commercial drugs available to prevent PRRSV infection in pigs in vivo.…”