Stepanyan, V.; Finnemann, S. C.; Meneses, P. I.

High-risk Human Papillomaviruses (HR-HPVs) are responsible for 5% of global cancers. While vaccines against HR-HPVs exist, there are no treatments available for individuals already infected. Cell penetrating peptides (CPPs) have demonstrated antiviral properties against viruses by blocking viral entry and delivering antivirals into infected cells. Developing CPP-based therapies faces challenges including inefficient delivery of macromolecules and endosomal entrapment, which must be overcome for effective clinical application. This study identifies an HPV16 major capsid protein L1 derived cationic peptide as a potent CPP. Peptide uptake depended on both a cluster of cationic residues and the specific peptide sequence. Mechanistic studies showed peptide entry occurred via cell surface heparan sulfate-mediated, lipid-raft dependent endocytosis. The peptide efficiently delivered GFP into HaCaT keratinocytes, and associated with the Golgi apparatus, demonstrating endosomal escape. GFP fusion protein endocytosis relied on binding of the cationic peptide to cell surface heparan sulfates. Cell-penetrating ability was conserved among homologous regions of various HPV types. The peptide showed potent antiviral activity by inhibiting infection of HaCaT cells by several HR-HPV types collectively responsible for nearly all HPV-associated cancers. Excitingly, HPV18 L1-derived peptide from the homologous region exhibited potent antiviral activity against HPV16 by preventing viral internalization. Our findings characterize HPV-derived peptides as highly efficient CPPs with potential to deliver therapeutic agents into cells and assist in development of treatments for high-risk HPVs.