A morphological transformation in respiratory syncytial virus leads to enhanced complement deposition

  1. Jessica P Kuppan
  2. Margaret D Mitrovich
  3. Michael D Vahey  Is a corresponding author
  1. Department of Biomedical Engineering and Center for Science & Engineering of Living Systems (CSELS), Washington University in St. Louis, United States
7 figures, 1 table and 2 additional files

Figures

Figure 1 with 4 supplements
Complement activation and C3 deposition vary across antigenic sites of RSV F.

(A) A fluorescence-based approach to measuring opsonization of RSV particles with mAbs and C3. RSV particles with site-specifically labeled F are immobilized on coverslips and incubated with normal …

Figure 1—figure supplement 1
A fluorescence imaging-based approach to study complement deposition on RSV.

(A) Top: Schematic of the RSV genome highlighting specific modifications: an mTagBFP2 reporter expressed from an IRES following NS1, and tags for site-specific labeling on G (C-terminal ybbR tag) …

Figure 1—figure supplement 2
Determining the linearity of fluorescence measurements.

(A) Left: RSV F bound in a 3:3 ratio with the D25 Fab (PDB ID 4JHW). Coloring of the D25 Fab indicates the two populations used for these experiments: one labeled 1:1 (Fab:dye) with AF488 and a …

Figure 1—figure supplement 3
Estimating the efficiency of enzymatic labeling.

(A) Procedure for calibrating the relative intensities of AF488 and Sulfo-Cy5 (‘Cy5’) dyes. Distributions to the left show data for RSV particles bound with saturating concentrations of D25(AF488) …

Figure 1—figure supplement 4
Effects of IgG/IgM depletion on complement deposition and binding of RSV-specific mAbs.

(A) Comparison of C3 deposition in complete normal human serum (5%) and IgG/IgM-depleted serum (5%), with and without supplemental mAbs. Images are displayed at matching contrast across each …

Complement activation and C1q binding varies with Fc position.

(A) Modeling Fc positions for F-specific mAbs. Structures for RSV F (or portions thereof) with bound antibodies (PDB IDs 6OE4, 5W23, 4ZYP, 3IXT, 6APD, 6APB, and 3O45) were aligned with human IgG1 …

CD55 is packaged into RSV particles and increases thresholds for C3 opsonization.

(A) Images of RSV particles with enzymatically labeled F and CD55 or CD46 labeled via fluorescent antibodies. Panels show representative images of virus released from wildtype (wt) A549 cells and …

Globular particles are preferentially and rapidly opsonized with C3 and C4.

(A) RSV particles opsonized with antibody (CR9501 IgG1) and C3. White arrows in merged panel indicate globular particles with high levels of C3. (B) Comparison of particle aspect ratios across RSV …

Figure 5 with 1 supplement
Globular particles are enriched in post-F, but remain sensitive to pre-F-mediated complement deposition.

(A) Three-color labeling strategy to detect total F (via enzymatic labeling with Sortase A [SrtA]), post-F (via the post-F-specific mAb ADI-14359), and pre-F (via the pre-F-specific mAb 5C4). …

Figure 5—figure supplement 1
Pre-F and post-F containing RSV particles occur naturally in cell culture.

Images of live RSV-infected A549 cells at 48 hpi showing mTagBFP2 reporter (indicating infected cells) along with pre- and postfusion F, labeled using 5C4 (Alexa Fluor 488) and ADI-14359 (Alexa …

Figure 6 with 3 supplements
Membrane detachment from the viral matrix increases complement activation.

(A) Process for detaching the RSV matrix from the viral membrane using osmotic swelling. Fluorescence images show details of the transformation, with t = 0 s approximately corresponding to the …

Figure 6—figure supplement 1
Osmotic swelling detaches the RSV matrix from the viral membrane with no loss of infectivity.

(A) Comparison of infectivity (quantified as single-round infectious units per ml) of virus shed from A549 cells during a 2 hr period starting at 60 hpi. RSV samples were divided into control …

Figure 6—figure supplement 2
C3 deposition on RSV particles increases with decreasing particle curvature.

(A) Model of an idealized morphological transformation in RSV. Detachment of the viral matrix leads to the rounding of virus particles. Mean curvature can be predicted from particle geometry and the …

Figure 6—figure supplement 3
A conceptual model for the changing surface of RSV.

RSV particles emerge from cells as filaments. Detachment of the viral matrix (either spontaneously or through physical perturbations) alters virus morphology, transforming particles into globular …

RSV curvature constrains docking of IgG hexamers.

(A) Schematic of RSV F on virion surfaces with varying curvature (left) and a structure (PDB ID 1HZH) and simplified model of an IgG1 hexamer (right). The curved membrane, F, and hexameric IgG are …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)HEK 293TATCCATCC Cat# CRL-3216, RRID:CVCL_0063
Cell line (Homo sapiens)A549ATCCATCC Cat# CCL-185, RRID:CVCL_0023
Cell line (Melanochromis auratus)BHK-21ATCCATCC Cat# CCL-10, RRID:CVCL_1915
Strain, strain background (Escherichia coli)SW102NCI Preclinical RepositoryUsed for BAC recombineering
Recombinant DNA reagentRSV BACBEI ResourcesNR-36460This BAC has been further modified for this work as described in Materials and methods
Recombinant DNA reagentpA2-LoptBEI ResourcesNR-36461
Recombinant DNA reagentpA2-NoptBEI ResourcesNR-36462
Recombinant DNA reagentpA2-PoptBEI ResourcesNR-36463
Recombinant DNA reagentpA2-M2-1optBEI ResourcesNR-36464
Recombinant DNA reagentpCAGGS-T7 RNAPThis workPlasmid encoding T7 RNAP
Recombinant DNA reagentpmAb-ADI-14353 HCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-ADI-14353 LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-ADI-14359 HCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-ADI-14359 HC FabThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-ADI-14359 LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-ADI-19425 HCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-ADI-19425 LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-5C4 HC (IgG1)This workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-5C4 HC (IgM)This workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-5C4 LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-CR9501 HCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-CR9501 LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-Motavizumab HCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-Motavizumab LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-101F HCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-101F LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-D25 HC FabThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-D25 LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-3D3 HCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-3D3 LCThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentpmAb-J chainThis workPlasmid for expressing recombinant antibody
Recombinant DNA reagentLenti CRISPRv2AddgeneRef. [70]
Biological sample (Homo sapiens)IgG/IgM-depleted normal human serumPelfreeze34,014
Biological sample (Homo sapiens)C3Complement Technology, IncA113
Biological sample (Homo sapiens)C4Complement Technology, IncA105
Biological sample (Homo sapiens)C1Complement Technology, IncA098
AntibodyMouse anti-CD46 monoclonal (TRA-2–10)BioLegendBioLegend Cat# 352404, RRID:AB_10900243IF (1:500)
AntibodyMouse anti-CD55 monoclonal (JS11)BioLegendBioLegend Cat# 311301, RRID:AB_314858IF (1:500)
AntibodyMouse anti-C1q monoclonal (1A4)Santa Cruz Biotech.Santa Cruz Biotechnology Cat# sc-53544, RRID:AB_1119798IF (1:500)
Peptide, recombinant proteinStreptavidinThermo Fisher Scientific434,302
Peptide, recombinant proteinCLPMTGG peptideGenscriptPeptide for SrtA-based labeling
Peptide, recombinant proteinSortase AThis workRecombinant SrtA for labeling
Peptide, recombinant proteinSfp synthaseThis workRecombinant Sfp for labeling
Chemical compound, drugSulfo-Cy5 maleimideLumiprobeCat# 13,380
Chemical compound, drugAF-488 NHS EsterLumiprobeCat# 11,820
Chemical compound, drugAlexa Fluor 488 C5 maleimideThermo Fisher ScientificCat# A10254
Chemical compound, drugAlexa Fluor 555 C2 maleimideThermo Fisher ScientificCat# A20346
Chemical compound, drugNH2-PEG-BiotinRapp PolymereCat# 133000-25-20
Chemical compound, drugCH3O-PEG-NH2Rapp PolymereCat# 122000–2
Chemical compound, drugCoenzyme A trilithium saltSigma-AldrichCat# C3019
Software, algorithmMatlab R2020aMathworksUsed for data analysis, plotting, and simulations
Software, algorithmNikon ElementsNikonUsed for image acquisition
Software, algorithmPyMOLSchrödinger, IncUsed for structure alignment

Additional files

Supplementary file 1

This document contains the genomic sequence of the modified respiratory syncytial virus (RSV) strain used in this work as well as antibody sequences.

https://cdn.elifesciences.org/articles/70575/elife-70575-supp1-v1.pdf
Transparent reporting form
https://cdn.elifesciences.org/articles/70575/elife-70575-transrepform1-v1.pdf

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