Inhibition of Polyprotein Processing and RNA Replication of Human Rhinovirus by Pyrrolidine Dithiocarbamate Involves Metal Ions

Abstract

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...

Copper accumulation and lipid oxidation precede inflammation and myelin lesions in N,N-diethyldithiocarbamate peripheral myelinopathy

“…In addition to the use of disulfiram in alcohol aversion therapy (Eneanya et al, 1981), and N,Ndiethyldithiocarbamate in the treatment of nickel carbonyl intoxication (Sunderman, 1979), a wide range of new medical applications for dithiocarbamates are currently being explored. Investigations have presented support for potential uses of dithiocarbamates in treating cocaine addiction (Sofuoglu and Kosten, 2005), inflammation (Fang et al, 2005), viral infections (Krenn et al, 2005;Si et al, 2005), and as adjuncts for chemotherapy (Bach et al, 2000). In vitro mechanistic studies have demonstrated the ability of dithiocarbamates to modulate numerous biological processes including apoptosis, oxidative stress, and transcription, providing the molecular basis for these proposed medical applications (Kang et al, 2001;Kimoto-Kinoshita et al, 2004).…”

Antioxidant Therapy as a Potential Approach to Severe Influenza-Associated Complications

“…It has been demonstrated that copper and zinc ions inhibit influenza virus RNA-dependent RNA polymerase activity, and that the inhibitory effect of bathocuproine-copper and bathocuproine-zinc complexes is greater than that of bathocuproine itself [40]. Moreover, it has been known that PDTC-copper and PDTC-zinc complexes (9 and 10, respectively, Figure 3) inhibited the replication of Coxsackie virus [41] and rhinovirus [42]. Conceivably, PDTC may inhibit influenza virus gene replication and transcription through the inhibition of viral RNA-dependent RNA polymerase activity by increasing the amount of intracellular copper and zinc ions or intracellular PDTC-copper and PDTC-zinc complexes, but further study is needed to elucidate the precise mechanism of inhibitory effect of PDTC on influenza virus gene replication and transcription.…”

Cellular kinase inhibitors that suppress enterovirus replication have a conserved target in viral protein 3A similar to that of enviroxime

“…Calpain inhibitors inhibit the replication of echovirus 1, possibly by targeting the activities of calpain 1 and 2 (Upla et al, 2008). Pyrrolidine dithiocarbamate (PDTC) inhibits polyprotein processing of entero-and cardioviruses by transporting zinc ions into the cells with different mechanisms depending on the virus species (Krenn et al, 2005;Lanke et al, 2007). For PDTC, a mechanism via the ubiquitin-proteasome pathway is also proposed for the inhibitory effect on coxsackievirus B3 (CVB3) infection (Si et al, 2005).…”