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

Abstract

The receptor binding protein of parainfluenza virus, hemagglutinin-neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein prior to receptor engagement of HN at the host cell membrane inactivate the virus. We previously identified small molecules that interact with HN and induce it to activate F in an untimely fashion, validating a new antiviral strategy. To obtain highly active pretriggering candidate molecules we carried out a virtual modeling screen for molecules that interact with sialic acid binding site II on HN, which we propose to be the site responsible for activating F. To directly assess the mechanism of action of one such highly effective new premature activating compound, PAC-3066, we use cryo-electron tomography on authentic intact viral particles for the first time to examine the effects of PAC-3066 treatment on the conformation of the viral F protein. We present the first direct observation of the conformational rearrangement induced in the viral F protein. IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), upon binding to its cell receptor, triggers conformational changes in the fusion protein (F). This action of HN activates F to reach its fusion-competent state. Using small molecules that interact with HN, we can induce the premature activation of F and inactivate the virus. To obtain highly active pretriggering compounds, we carried out a virtual modeling screen for molecules that interact with a sialic acid binding site on HN that we propose to be the site involved in activating F. We use cryo-electron tomography of authentic intact viral particles for the first time to directly assess the mechanism of action of this treatment on the conformation of the viral F protein and present the first direct observation of the induced conformational rearrangement in the viral F protein.

Host cell membrane capture by the SARS CoV-2 spike protein fusion intermediate

“…The first task of the unleashed FI is thought to be capture of the host target membrane by insertion of the fusion peptides at the protomer N-termini. Due to its flexibility the FI extension ranged from ~ 21-30 nm (mean ~ 26 nm), its orientation varied over angles ~ ±60° to the membrane normal, and the FPs in consequence swept out a volume ~ 25,000 nm 1 at rate ~ 750 nm 1 µs 23 (Figures 3d,e, 95% of sampled locations).…”

“…@A 9: = ℎ 2 >? @A ≈ 0.7 nm µs 23 where the factor of 2 reflects the two membranes. Importantly, binding was mediated by the N-terminal helix of the FP, which provided first contact with the membrane during a binding event (Figure 5b and Supplementary Movie 8).…”

“…The spike protein is an important target for vaccines, therapeutic antibodies and other antivirals. Most current recombinant neutralizing or vaccine-elicited antibodies to CoV-2 bind the S1 receptor binding domain [23][24][25][26] , while class I viral fusion inhibitors are thought to bind the fusionexecuting subunit 14,[27][28][29] , including one FDA-approved drug against HIV 30 . Importantly, two recently emerged CoV-2 variants harboring spike protein mutations, B.1.351 (South African variant) and P.1 (Brazilian variant), escaped from two antibodies granted FDA emergency authorization, whereas the efficiency of S2-targeting antivirals was unaffected 31 .…”

“…Most current recombinant neutralizing or vaccine-elicited antibodies to CoV-2 bind S1 [17][18][19][20] . Viral fusion inhibitors targeting S2 12,21 and the fusion-executing subunits of other class I fusion proteins have been developed, including one FDA-approved drug against HIV [22][23][24] . Importantly, recently emerged CoV-2 variants harboring spike protein mutations, B.1.351 (South African variant) and P.1 (Brazilian variant), escaped from two S1-targeting antibodies one of which has FDA emergency authorization, whereas the efficiency of S2-targeting antivirals was unaffected 25 .…”

Effects of Single α-to-β Residue Replacements on Recognition of an Extended Segment in a Viral Fusion Protein

“…[27][28][29] This study began with a previously reported peptide derived from the HRC domain of HPIV3, designated VIQKI ( Figure 1C), which blocks infection by both HPIV3 and respiratory syncytial virus (RSV). [30][31][32] These two viruses are leading causes of fatal lower respiratory infections in young children. [33][34][35] A crystal structure of VIQKI co-assembled with HPIV3-HRN revealed a six-helix bundle assembly similar to the HRC/HRN assembly in the post-fusion conformation of the HPIV3 F protein ( Figure 1B).…”