title: Gene- and protein-delivered zinc finger-staphylococcal nuclease hybrid for inhibition of DNA replication of human papillomavirus.
abstract: Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.
authors: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan.
authors: Takashi
authors: T
authors: Mino
authors: Tomoaki
authors: T
authors: Mori
authors: Yasuhiro
authors: Y
authors: Aoyama
authors: Takashi
authors: T
authors: Sera
mesh_terms: D004261:DNA Replication / Q000187:drug effects* / Q000235:genetics; D004279:DNA, Viral / Q000187:drug effects / Q000235:genetics; D016679:Genome, Viral; D052161:Human papillomavirus 18 / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008836:Micrococcal Nuclease / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*; D027383:Papillomaviridae / Q000187:drug effects* / Q000235:genetics; D018741:Replication Origin; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004279:DNA, Viral; D008836:Micrococcal Nuclease