(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 …
Matlab code and data for Figure 1, Figure 1—figure supplement 1, Figure 1—figure supplement 2, Figure 1—figure supplement 3, and Figure 1—figure supplement 4.
(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) …
(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 …
(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) …
(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 …
(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 …
Matlab code and data for Figure 2.
(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 …
Matlab code and data for Figure 3.
(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 …
Matlab code and data for Figure 4.
(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). …
Matlab code and data for Figure 5.
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 …
(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 …
Matlab code and data for Figure 6, Figure 6—figure supplement 1, and Figure 6—figure supplement 2.
(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 …
(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 …
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 …
(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 …
Matlab code for running simulations for Figure 7.
Matlab code and data for Figure 7.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Homo sapiens) | HEK 293T | ATCC | ATCC Cat# CRL-3216, RRID:CVCL_0063 | |
Cell line (Homo sapiens) | A549 | ATCC | ATCC Cat# CCL-185, RRID:CVCL_0023 | |
Cell line (Melanochromis auratus) | BHK-21 | ATCC | ATCC Cat# CCL-10, RRID:CVCL_1915 | |
Strain, strain background (Escherichia coli) | SW102 | NCI Preclinical Repository | Used for BAC recombineering | |
Recombinant DNA reagent | RSV BAC | BEI Resources | NR-36460 | This BAC has been further modified for this work as described in Materials and methods |
Recombinant DNA reagent | pA2-Lopt | BEI Resources | NR-36461 | |
Recombinant DNA reagent | pA2-Nopt | BEI Resources | NR-36462 | |
Recombinant DNA reagent | pA2-Popt | BEI Resources | NR-36463 | |
Recombinant DNA reagent | pA2-M2-1opt | BEI Resources | NR-36464 | |
Recombinant DNA reagent | pCAGGS-T7 RNAP | This work | Plasmid encoding T7 RNAP | |
Recombinant DNA reagent | pmAb-ADI-14353 HC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-ADI-14353 LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-ADI-14359 HC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-ADI-14359 HC Fab | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-ADI-14359 LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-ADI-19425 HC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-ADI-19425 LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-5C4 HC (IgG1) | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-5C4 HC (IgM) | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-5C4 LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-CR9501 HC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-CR9501 LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-Motavizumab HC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-Motavizumab LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-101F HC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-101F LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-D25 HC Fab | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-D25 LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-3D3 HC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-3D3 LC | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | pmAb-J chain | This work | Plasmid for expressing recombinant antibody | |
Recombinant DNA reagent | Lenti CRISPRv2 | Addgene | Ref. [70] | |
Biological sample (Homo sapiens) | IgG/IgM-depleted normal human serum | Pelfreeze | 34,014 | |
Biological sample (Homo sapiens) | C3 | Complement Technology, Inc | A113 | |
Biological sample (Homo sapiens) | C4 | Complement Technology, Inc | A105 | |
Biological sample (Homo sapiens) | C1 | Complement Technology, Inc | A098 | |
Antibody | Mouse anti-CD46 monoclonal (TRA-2–10) | BioLegend | BioLegend Cat# 352404, RRID:AB_10900243 | IF (1:500) |
Antibody | Mouse anti-CD55 monoclonal (JS11) | BioLegend | BioLegend Cat# 311301, RRID:AB_314858 | IF (1:500) |
Antibody | Mouse anti-C1q monoclonal (1A4) | Santa Cruz Biotech. | Santa Cruz Biotechnology Cat# sc-53544, RRID:AB_1119798 | IF (1:500) |
Peptide, recombinant protein | Streptavidin | Thermo Fisher Scientific | 434,302 | |
Peptide, recombinant protein | CLPMTGG peptide | Genscript | Peptide for SrtA-based labeling | |
Peptide, recombinant protein | Sortase A | This work | Recombinant SrtA for labeling | |
Peptide, recombinant protein | Sfp synthase | This work | Recombinant Sfp for labeling | |
Chemical compound, drug | Sulfo-Cy5 maleimide | Lumiprobe | Cat# 13,380 | |
Chemical compound, drug | AF-488 NHS Ester | Lumiprobe | Cat# 11,820 | |
Chemical compound, drug | Alexa Fluor 488 C5 maleimide | Thermo Fisher Scientific | Cat# A10254 | |
Chemical compound, drug | Alexa Fluor 555 C2 maleimide | Thermo Fisher Scientific | Cat# A20346 | |
Chemical compound, drug | NH2-PEG-Biotin | Rapp Polymere | Cat# 133000-25-20 | |
Chemical compound, drug | CH3O-PEG-NH2 | Rapp Polymere | Cat# 122000–2 | |
Chemical compound, drug | Coenzyme A trilithium salt | Sigma-Aldrich | Cat# C3019 | |
Software, algorithm | Matlab R2020a | Mathworks | Used for data analysis, plotting, and simulations | |
Software, algorithm | Nikon Elements | Nikon | Used for image acquisition | |
Software, algorithm | PyMOL | Schrödinger, Inc | Used for structure alignment |
This document contains the genomic sequence of the modified respiratory syncytial virus (RSV) strain used in this work as well as antibody sequences.