A Cholesterol Tag at the N Terminus of the Relatively Broad-Spectrum Fusion Inhibitory Peptide Targets an Earlier Stage of Fusion Glycoprotein Activation and Increases the Peptide's Antiviral Potency In Vivo

In previous work, we designed peptides that showed potent inhibition of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) infections in chicken embryos. In this study, we demonstrate that peptides modified with cholesterol or 3 U of polyethylene glycol (PEG₃) conjugated to the peptides'' N termini showed even more promising antiviral activities when tested in animal models. Both cholesterol- and cholesterol-PEG₃-tagged peptides were able to protect chicken embryos from infection with different serotypes of NDV and IBV when administered 12 h prior to virus inoculation. In comparison, the untagged peptides required intervention closer to the time of viral inoculation to achieve a similar level of protection. Intramuscular injection of cholesterol-tagged peptide at 1.6 mg/kg 1 day before virus infection and then three times at 3-day intervals after viral inoculation protected 70% of the chickens from NDV infection. We further demonstrate that the cholesterol-tagged peptide has an in vivo half-life greater than that of untagged peptides. It also has the potential to cross the blood-brain barrier to enter the avian central nervous system (CNS). Finally, we show that the cholesterol-tagged peptide could play a role before the viral fusion peptide''s insertion into the host cell and thereby target an earlier stage of fusion glycoprotein activation. Our findings are of importance for the further development of antivirals with broad-spectrum protective effects.