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.Curing virus infections harbors an enormous economic potential, and the search for new antiviral substances is of great interest for worldwide health. We have previously described the commonly used NF-B inhibitor and metal ion chelator pyrrolidine-dithiocarbamate (PDTC) to significantly inhibit the replication of several picornaviruses such as human rhinovirus (HRV), poliovirus, coxsackievirus, and mengovirus (9,22). These examples suggest that a common step in the life cycle of these picornaviruses is the target for the antiviral drug.In particular, we have demonstrated that PDTC has negative effects on picornavirus replication by influencing the processing of the viral polyprotein (21,22).The antiviral activity of PDTC is not restricted to the family Picornaviridae, since PDTC was shown to prevent the multiplication of human influenza virus, a member of the Orthomyxoviridae (33, 34). However, due to strong differences in the life cycle and host-cell interaction between human influenza virus and picornaviruses, it is likely that entirely different mechanisms might be relevant for the antiviral action of PDTC against these viruses.Currently, the precise mode of the antiviral action of PDTC is unknown, although several theories have been substantiated with experimental evidence. Antioxidative properties of PDTC are postulated to be the reason for antiviral effects against influenza virus infections (33), which is not the case for human rhinovirus multiplication (9).We have demonstrated that the antiviral effects of PDTC are metal ion dependent, and, in particular, Zn 2ϩ ions play a pivotal role. To underline the hypothesis that influx of zinc into the cells has antiviral capacity, pyrithione (PT) and hinokitiol (HK) were examined. PT is known to be a zinc ionophore that leads to a rapid increase in intracellular zinc levels (27), and HK is a ch...

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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.