Data from: How a paramyxovirus fusion/entry complex adapts to escape a neutralizing antibody

Marcink, Tara 1 ; Zipursky, Gillian1 ; Moscona, Anne1

Published Sep 11, 2024 on Dryad. https://doi.org/10.5061/dryad.g1jwstqz6

Data files

Sep 11, 2024 version files 4.10 MB

Hats_Off_Paper_Raw_Data_Final.xlsx

855.52 KB
README.md

5.23 KB
Sequences.xlsx

36.24 KB
uncropped_gels.pptx

3.20 MB

Abstract

Paramyxoviruses including measles, Nipah, and parainfluenza viruses are public health threats with pandemic potential. Human parainfluenza virus type 3 (HPIV3) is a leading cause of illness in pediatric, elderly, and immunocompromised populations. There are no approved vaccines or therapeutics for HPIV3. Neutralizing antibodies that target viral fusion are a potential strategy for mitigating paramyxovirus infection, however their utility may be curtailed by viral evolution that leads to resistance. Paramyxoviruses enter cells by fusing with the cell membrane in a process mediated by a complex consisting of a receptor binding protein (HN) and a fusion protein (F). Existing atomic resolution structures fail to reveal physiologically relevant interactions during viral entry. We present cryo-ET structures of pre-fusion HN-F complexes in situ on the surfaces of authentic virions that evolved resistance to an anti-HPIV3 F neutralizing mAb. Single mutations in F abolish mAb binding and neutralization. In these complexes, the HN protein that normally restrains F triggering has shifted to uncap the F apex. These complexes are more readily triggered to fuse. These structures shed light on the adaptability of the pre-fusion HN-F complex and mechanisms of paramyxoviral resistance to mAbs, and help define potential barriers to resistance for the design of mAbs.

https://doi.org/10.5061/dryad.g1jwstqz6

Excel file including the raw data for the article "How a Paramyxovirus Fusion/Entry Complex Adapts to Escape a Neutralizing Antibody."

Description of the data and file structure

We have submitted our raw data for figures 1,2,3, and 5 (Hats_Off_Paper_Raw_Data_Final.xlsx), plasmid sequences encoding HN, HA, and F (Sequences.xlsx), and uncropped western blot surface expressions of F variants using anti-HPIV3 F HRC

Descriptions

Hats_Off_Paper_Raw_Data_Final.xlsx

In Hats_Off_Paper_Raw_Data_Final.xlsx each tab contains the raw data and is labelled with the figure panels used in the manuscript: How a Paramyxovirus fusion/entry complex adapts to escape a neutralizing antibody.

· HN: hemagglutinin-neuraminidase protein

· F: Fusion protein

· HA: Uncleaved influenza hemagglutinin

· EV1: HPIV3 virus Escape Variant 1

· EV2: HPIV3 virus Escape Variant 2

· Parental: HPIV3 virus used as the input virus for viral evolution.

· Exp: Experiment using 3 biological replicates of technical triplets.

· Rep: A single biological replicate consisting of technical triplets.

· Avg: Average of technical triplets.

· Fab: Antibody fragment

· Uncleaved: F protein that has not been cleaved and cannot insert into a host membrane.

· Stapled: The engineered stabilized F bearing 4 cysteine mutations; one pair that connect the central helix (and A194) to the domain III helix-turn-helix that contains the L234F mutation, and another pair in the N-terminal heptad repeat domain containing the 3x1 Ab epitope.

· A194T: An alanine to threonine mutation at residue 194 in the F protein at precisely the site on the F apex that the viral complex’s structure predicts to be the site of HN/F interaction.

· L234F: A leucine to phenylalanine mutation at residue 234 in the F protein at the side of F, distant from the trimeric F apex but adjacent to the central helix of the F trimer.

· D216R: An aspartic acid to arginine HN mutation at position 216 that makes HN sialidase-deficient to maximize the HN-receptor contact and HN’s fusion promotion.

· T193A: A threonine to alanine HN mutation at position 193 that confers increased avidity.

· PIA174: HPIV3 neutralizing monoclonal antibody that binds at the F apex.

· 3x1: HPIV3 neutralizing monoclonal antibody that binds at the side of F.

Sequences.xlsx

Sequences.xlsx contains a list of each plasmid used in the manuscript: How a Paramyxovirus fusion/entry complex adapts to escape a neutralizing antibody.

· HN: hemagglutinin-neuraminidase protein

· F: Fusion protein

· HA: Hemagglutinin

· A194T: An alanine to threonine F protein mutation at residue at precisely the site on the F apex that the viral complex’s structure predicts to be the site of HN/F interaction.

· L234F: A leucine to phenylalanine F protein mutation at residue 234 at the side of F, distant from the trimeric F apex but adjacent to the central helix of the F trimer.

· D216R: An aspartic acid to arginine HN mutation at residue 216 that makes HN sialidase-deficient to maximize the HN-receptor contact and HN’s fusion promotion.

· T193A: A threonine to alanine HN mutation at residue 193 that confers increased avidity.

· H552Q: A histidine to glutamine HN mutation at residue 552 with enhanced F activation properties.

· I243V: An isoleucine to valine HN mutation at residue 243 that arose during viral evolution.

· Stabilized: The engineered stabilized F bearing 4 cysteine mutations; one pair that connect the central helix (and A194) to the domain III helix-turn-helix that contains the L234F mutation, and another pair in the N-terminal heptad repeat domain containing the 3x1 Ab epitope.

Uncropped_gels.pptx

This file contains scans of uncropped gels used in supplementary figure 1A-B of the manuscript: How a Paramyxovirus fusion/entry complex adapts to escape a neutralizing antibody.

Key Information Sources

· Recombinant viruses were generated by reverse genetics using an HPIV3 virus sequence (modified from a gift from Ursula Buchholz and Peter Collins, NIAID). All recombinant viruses were sequenced and the sequencing reads are available in NCBI BioProject PRJNA1083633 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1083633).

· Plasmids encoding HPIV3 HN and F were generated through site-directed mutagenesis of a previously constructed pCAGGS mammalian expression vector and sequenced via Sanger sequencing prior to experimental use.

Code/Software

· Excel or Prism is required for data visualization for files Hats_Off_Paper_Raw_Data_Final.xlsx and Sequences.xlsx

· Powerpoint is required to visualize western blot data for the file Uncropped_gels.pptx.