Mutations in the Fusion Protein of Measles Virus That Confer Resistance to the Membrane Fusion Inhibitors Carbobenzoxy- d -Phe- l -Phe-Gly and 4-Nitro-2-Phenylacetyl Amino-Benzamide

Abstract

The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

Measles Virus Bearing Measles Inclusion Body Encephalitis-Derived Fusion Protein Is Pathogenic after Infection via the Respiratory Route

“…The IC 50 dropped in the presence of mocktransfected target cells to ϳ50,000 nM. Thus, susceptibility to HRC-derived fusion inhibitors was higher for the F L454W than for the wt F. For 3G, we confirmed the previous findings that the L454W mutation in F confers resistance to this fusion inhibitor (12,31,35,36).…”

“…The N462K F was inherently destabilized but was retained in the prefusion state in the presence of inhibitor (35,36). FIP was reported to have a mechanism of action similar to 3G (12,31), suggesting that MeV with L454W F may emerge under the selective pressure of a fusion inhibitor (31), and MeV bearing the L454W F was resistant to the inhibitory activity of 3G ( Fig. 3 and 4; see also Fig.…”

“…The virus's origin could not be determined. A recent report showed that a virus bearing L454W F emerges under the selective pressure of certain small-molecule fusion inhibitors, including fusion inhibitory peptide (FIP) (31). These findings raised the question of whether a virus bearing this neuropathogenic F protein can be found outside the CNS and spread within an individual.…”

Measles Encephalitis: Towards New Therapeutics

“…These inhibitors are known to stabilize the prefusion state of the F protein. Nevertheless the use of these inhibitors leads to the emergence of mutations in the HRC of the F that can evade their efficacy leading to the selection of MeV hyperfusogenic variants [254].…”

Hijacking the Fusion Complex of Human Parainfluenza Virus as an Antiviral Strategy

“…However, resistance to this inhibitor arose quickly (50)(51)(52)(53)(54), with a mutated F that was destabilized (50,51). The resistant F proteins were inherently destabilized (55) and became neuropathogenic in vivo (56)(57)(58), and their stability was increased in the presence of inhibitor (50,51). Several small molecules that prevent RSV F refolding have been identified (59); however, resistance emerges readily (60).…”