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...
Human rhinoviruses (HRVs) are the predominant cause of the common cold. The frequency of HRV infections in industrial countries and the lack of effective therapeutical treatment underline the importance of research for new antiviral substances. As viral infections are often accompanied by the generation of oxidative stress inside the infected cells, several redox-active substances were tested as potential antivirals. In the course of these studies it was discovered that pyrrolidine dithiocarbamate (PDTC) is an extremely potent compound against HRV and poliovirus infection in cell culture. Besides the ability to dramatically reduce HRV production by interfering with viral protein expression, PDTC promotes cell survival and abolishes cytopathic effects in infected cells. PDTC also protects cells against poliovirus infection. These effects were highly specific, as several other antioxidants (vitamin C, Trolox, 2-mercaptoethanol, and N-acetyl-L-cysteine) are inactive against HRV infection. Synthesis of HRV proteins and cleavage of eucaryotic initiation factor 4G responsible for host cell shutoff of cellular protein synthesis are severely inhibited in the presence of PDTC.
Pyrrolidine dithiocarbamate (PDTC) is an antiviral compound that was shown to inhibit the replication of human rhinoviruses (HRVs), poliovirus, and influenza virus. To elucidate the mechanism of PDTC, the effects on the individual steps of the infection cycle of HRV were investigated. PDTC did not interfere with receptor binding or internalization by receptor mediated endocytosis of HRV2 particles into HeLa cells. But we demonstrate that the processing of the viral polyprotein was prevented by PDTC treatment in HeLa cells infected with HRV2. Furthermore, PDTC inhibited the replication of the viral RNA, even when added four hours post infection. As PDTC is described as a metal ion binding agent, we investigated the effect of other metal chelators on the multiplication of HRV2. We show that EDTA, -phenanthroline, and bathocuproine disulfonic acid do not exhibit any antiviral properties. Surprisingly, these substances, coadministered with PDTC, abolished the antiviral effect of PDTC, suggesting that metal ions play a pivotal role in the inhibition of virus multiplication. These results suggest that PDTC inhibits the activity of the viral proteases in a metal ion dependent way.Human rhinoviruses (HRVs) are the most frequent cause of the common cold and are implicated in more than 50% of upper respiratory tract infections (45). Although not life threatening, infection with HRV can prepare the ground for more serious diseases, such as acute exacerbation of asthma (20, 30) or otitis media (5). As there are more than 100 HRV serotypes, vaccine development is unfeasible. Apart from symptomatic medication no causative treatment for HRV infections is currently available. Therefore, analysis of functions of new antiviral substances is of great interest and might also shed light on novel aspects of virus-cell interactions.HRVs belong to the genus Picornaviridae. These viruses have a single-stranded positive-sense RNA genome of approximately 7,400 nucleotides. The viral RNA encodes four capsid proteins (VP1 through VP4) and seven nonstructural proteins that are involved in viral RNA replication and polyprotein processing. The HRV serotypes can be classified into A and B groups based on sequence alignments (36). Alternatively, HRVs are divided into two groups according to their receptor specificity (1, 49). Members of the major group HRVs bind to the intercellular adhesion molecule 1 (ICAM-1) (15, 44), whereas serotypes of the minor group use various members of the low-density lipoprotein receptor family (18).Upon receptor binding, the viral particle is internalized by receptor-mediated endocytosis. After acidification of the late endosome and subsequent uncoating, the RNA of minor group HRVs is released into the cytoplasm (32). Then the viral RNA is translated into a single large polyprotein from an internal ribosome entry site (IRES), which is located in the 5Ј untranslated region of the HRV genome. The polyprotein is processed into the mature viral proteins through a sequence of cleavages performed by two virus-encoded prot...
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