DOI: 10.5061/dryad.g1jwstqz6
title: Data from: How a paramyxovirus fusion/entry complex adapts to escape a neutralizing antibody

description: 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.
description: # Data from: How a paramyxovirus fusion/entry complex adapts to escape a
 neutralizing antibody
 [https://doi.org/10.5061/dryad.g1jwstqz6](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](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.