Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections.

We have discovered two metal ion binding compounds, pyrithione (PT) and hinokitiol (HK), that efficiently inhibit human rhinovirus, coxsackievirus, and mengovirus multiplication. Early stages of virus infection are unaffected by these compounds. However, the cleavage of the cellular eukaryotic translation initiation factor eIF4GI by the rhinoviral 2A protease was abolished in the presence of PT and HK. We further show that these compounds inhibit picornavirus replication by interfering with proper processing of the viral polyprotein. In addition, we provide evidence that these structurally unrelated compounds lead to a rapid import of extracellular zinc ions into cells. Imported Zn(2+) was found to be localized in punctate structures, as well as in mitochondria. The observed elevated level of zinc ions was reversible when the compounds were removed. As the antiviral activity of these compounds requires the continuous presence of the zinc ionophore PT, HK, or pyrrolidine-dithiocarbamate, the requirement for zinc ions for the antiviral activity is further substantiated. Therefore, an increase in intracellular zinc levels provides the basis for a new antipicornavirus mechanism.

PDTC inhibits picornavirus polyprotein processing and RNA replication by transporting zinc ions into cells.

Previously, it was shown that pyrrolidine dithiocarbamate (PDTC) inhibits proteolytic polyprotein processing and replication of human rhinovirus by transporting metal ions into cells. Here, it is shown that PDTC also inhibits replication of two other picornaviruses: coxsackievirus B3 (CVB3), a closely related virus that belongs to the genus Enterovirus, and mengovirus, an encephalomyocarditis virus strain that belongs to the genus Cardiovirus, and that this inhibition is due to the dithiocarbamate moiety of the compound. Making use of subgenomic replicons, evidence is provided that PDTC inhibits replication of these two viruses by disturbing viral RNA synthesis. Furthermore, it is shown that PDTC transports zinc ions into cells and that these zinc ions play an important role in the antiviral activity mediated by PDTC. Finally, it is shown that PDTC interferes with proteolytic processing of the polyproteins of both CVB3 and mengovirus, but that the underlying mechanism between these two viruses differs. In CVB3-infected cells, PDTC interferes strongly with the proteolytic activity of 3CD(pro), as shown by the impaired production of the mature capsid proteins as well as the autocleavage of 3CD(pro) into 3C(pro) and 3D(pol). In mengovirus-infected cells, however, PDTC had no effect on the proteolytic production of capsid proteins or the autocleavage of 3CD(pro). Instead, PDTC caused the accumulation of a high-molecular-mass precursor protein, due to an impairment in the primary 'break' that normally occurs at the 2A-2B junction. Thus, PDTC disturbs polyprotein processing and replication of two groups of picornaviruses, enteroviruses and cardioviruses, but the underlying mechanism is different.