A General Strategy to Endow Natural Fusion-protein-Derived Peptides with Potent Antiviral Activity

Abstract

Fusion between the viral and target cell membranes is an obligatory step for the infectivity of all enveloped virus, and blocking this process is a clinically validated therapeutic strategy.Viral fusion is driven by specialized proteins which, although specific to each virus, act through a common mechanism, the formation of a complex between two heptad repeat (HR) regions. The HR regions are initially separated in an intermediate termed “prehairpin”, which bridges the viral and cell membranes, and then fold onto each other to form a 6-helical bundle (6HB), driving the two membranes to fuse. HR-derived peptides can inhibit viral infectivity by binding to the prehairpin intermediate and preventing its transition to the 6HB.The antiviral activity of HR-derived peptides differs considerably among enveloped viruses. For weak inhibitors, potency can be increased by peptide engineering strategies, but sequence-specific optimization is time-consuming. In seeking ways to increase potency without changing the native sequence, we previously reported that attachment to the HR peptide of a cholesterol group (”cholesterol-tagging”) dramatically increases its antiviral potency, and simultaneously increases its half-life in vivo. We show here that antiviral potency may be increased by combining cholesterol-tagging with dimerization of the HR-derived sequence, using as examples human parainfluenza virus, Nipah virus, and HIV-1. Together, cholesterol-tagging and dimerization may represent strategies to boost HR peptide potency to levels that in some cases may be compatible with in vivo use, possibly contributing to emergency responses to outbreaks of existing or novel viruses.

Prevention of Measles Virus Infection by Intranasal Delivery of Fusion Inhibitor Peptides

“…The HPIV3-derived dimeric peptide [VG PEG4 ] 2 -chol, despite being the most potent of the HPIV3-derived peptides at inhibiting MV fusion, did not block infection in vivo. These results suggest that, unlike NiV and HPIV3 infections, which can both be inhibited by the same HPIV3 HRC peptides (51,58), inhibition of MV requires a sequence specifically derived from its own F protein. In the future, "cocktails" of antiviral peptides may be a useful approach to achieving a broad-spectrum coverage.…”

“…Since we expect our proposed antiviral strategy to be host factor independent, it will fill a specific need for immunocompromised people at risk for MV infection, who cannot be vaccinated or do not respond adequately to vaccine. We have previously shown that HPIV3 HRC-derived peptides are effective inhibitors of viral entry for other paramyxoviruses as well, including Hendra virus, Nipah virus, and simian virus 5 (SV5 or PIV5) (51,58,59), raising the possibility of broad-spectrum antivirals. We therefore tested their efficacy against the MV fusion machinery in vitro and against MV infection in vivo.…”

“…All peptides were produced by standard 9-fluorenylmethoxy carbonyl (Fmoc) solid-phase methods. The cholesterol moieties were attached to the peptides via chemoselective reaction between the thiol group of an extra cysteine residue, added C terminally to the sequence, and a bromoacetyl derivative of cholesterol or a bis-maleimide functionalized cholesterol core, as previously described (58)(59)(60). Peptides were stored as powder, and before use they were dissolved in dimethyl sulfoxide (DMSO) to 5 mM stock solutions that were kept at Ϫ80°C for the in vitro experiment.…”

“…We tested the three peptides whose biodistribution was assessed as described above (VIKI PEG4 -chol, MV HRC2, and MV HRC4), and we added MV HRC1, MV HRC3, VG, [VG-PEG11 ] 2 , VG PEG4 -chol, and [VG-PEG4 ] 2 -chol peptides as additional controls (51,58). The animals were given the fusion-inhibitory peptides i.n.…”

Fatal Measles Virus Infection Prevented by Brain-Penetrant Fusion Inhibitors

“…We used structure-based design to improve the strength of the peptide-target interaction, and we conjugated a cholesterol group to the peptides to increase the inhibitor concentration at the location of receptor binding (30,31); in this way, we obtained a potent fusion inhibitor that prevented and treated lethal Nipah virus (NiV) encephalitis in vivo (30). Recently, we showed that peptide efficacy against NiV, human parainfluenza virus type 3 (HPIV3), and human immunodeficiency virus type 1 (HIV-1) can also be improved by combining cholesterol conjugation with dimerization of the HRC peptide (32). Here, we report that cholesterolconjugated dimeric HRC peptides derived from MV F can effectively inhibit MV fusion, block viral spread, and prevent MV infection both ex vivo in brain explants and in vivo in an established animal model of MV encephalitis.…”