Recently, we designed artificial zinc-finger proteins (AZPs) that prevent a viral replication protein, E2, of human papillomavirus type 18 (HPV-18) from binding to its replication origin and demonstrated that the gene-delivered AZPs inhibited HPV-18 DNA replication in mammalian cells. In the present study, we examined another approach to delivery of AZPs. We constructed cell-permeable AZPs by fusing an AZP previously generated for inhibition of HPV-18 DNA replication with a cell-penetrating peptide (CPP), and confirmed that these CPP-AZP fusions reduced the replication rate in transient replication assays when added to the culture medium. In particular, 250 nM CPP-AZP reduced HPV-18 DNA replication to 3% of that of a control experiment. Western blot analysis detected 7% of the CPP-AZP added to the culture medium in the cell lysates, and demonstrated that greater internalization of CPP-AZP into mammalian cells causes greater inhibition of viral DNA replication. Furthermore, CPP-AZP did not show any significant cytotoxixity in MTT assays. Thus, our results demonstrate that cell-permeable AZPs could serve as potent protein-based antiviral drugs.
|Number of pages||2|
|Journal||Nucleic acids symposium series (2004)|
|Publication status||Published - 2007|
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