Antibody escape by polyomavirus capsid mutation facilitates neurovirulence
- Department of Microbiology and Immunology, Penn State College of Medicine, United States
- Department of Biochemistry and Molecular Biology, Pennsylvania State University, United States
- Huck Institutes of the Life Sciences, Pennsylvania State University, United States
- Department of Pathology, Penn State College of Medicine, United States
- The Jake Gittlen Laboratories for Cancer Research, Penn State College of Medicine, United States
- Department of Medicine, Penn State College of Medicine, United States
Figures
Figure 1 with 2 supplements
The V296F VP1 mutation in MuPyV impairs kidney, but not brain, infection.
(A) Structural comparison of JCPyV.S268 (PDB 3NXG) and MuPyV.V296 (PDB 5CPU) VP1 residues (Buch et al., 2015; Neu et al., 2010). (B) A2 and A2.V296F LT mRNA levels 4 dpi in the kidneys of mice …
Figure 1—figure supplement 1
The A2.V296F VP1 mutant virus retains infectivity in vitro.
(A) Single-cycle replication assay in A31 fibroblasts. Cells were infected with an multiplicity of infection (MOI) of 0.1 and virus was collected 60 hpi and measured by plaque assay. Data are from …
Figure 1—figure supplement 2
Discrimination of A2 and A2.V296F DNA by PCR.
PCR products after amplification of A2 or A2.V296F DNA with a combination of PCR primers for LT or the V296F-VP1 mutation.
Figure 2
Persistent infection with either A2 or A2.V296F results in CNS pathology.
(A) Left: LFB-PAS-stained brain sections 30 dpi with A2 or A2.V296F. Right: Hydrocephalus was quantified as the size of the lateral ventricle compared to total brain size. Data are from three …
Figure 3
The V296F VP1 mutation confers resistance to a neutralizing mAb.
(A) LT mRNA levels in NMuMG cells 24 hr pi with A2 or V296 mutant viruses preincubated with 8A7H5 or control IgG. Data are from two independent experiments, n = 12. For A2 p<0.0001, A2.V296F …
Figure 4 with 5 supplements
Cryo-EM image sub-particle refinement reconstructions showing architecture of MuPyV-Fab complexes.
(A) Micrographs of virus and virus–Fab complex (shown left and right, throughout figure) illustrate particle diameter difference due to bound Fab. (B) Surface rendered icosahedrally averaged maps. (C…
Figure 4—figure supplement 1
8A7H5 Fab neutralizes A2 but not A2.V296F.
(A) LT mRNA levels in NMuMG cells 24 hpi with A2 or A2.V296F preincubated with 8A7H5 Fab or control Fab. Data are from two independent experiments, n = 6. For A2 p<0.0001, A2.V296F p<0.0001. (B) A31 …
Figure 4—figure supplement 2
Fourier Shell Correlation (FSC).
Gold standard FSC curves were generated in cryoSPARC from half-maps, displayed as central sections with scale bars for the MuPyV-Fab complex (A, top), pentavalent capsomer complex (A, middle), and …
Figure 4—figure supplement 3
Asymmetric Unit.
(A) The surface rendered MuPyV capsid map overlaid with an asymmetric unit (ROYGBV) consisting of one VP1 chain from the pentavalent capsomer (red), together with five VP1 chains from a neighboring …
Figure 4—figure supplement 4
Refinement workflow.
(A) Icosahedral refinement produces a moderate resolution map of the full capsid and allows for designation of x,y,z coordinates of pentavalent (red) and hexavalent capsomers (blue). (B) Coordinates …
Figure 4—figure supplement 5
Local resolution.
(A and B) Equivalent zoomed views with density colored according to local resolution at the interface for the pentavalent (A) and hexavalent (B) capsomers show resolution improved after subvolume …
Figure 5 with 2 supplements
Cryo-EM reconstruction of MuPyV identifies mechanism of VP1 antibody escape by the V296F mutation.
(A and B) The Fab epitope bridges adjacent copies of VP1 on the pentavalent capsomer (A, shades of red) and hexavalent capsomer (B, OYGBV). Neighboring epitopes abut directly against each other. …
Figure 5—figure supplement 1
Heavy and light chain interactions with the capsid surface.
(A) The heavy chain (cyan) interacts with one copy of VP1 (yellow residues) with the light chain (purple) making additional contacts with the neighboring VP1 (orange residues). Symmetry-related VP1 …
Figure 5—figure supplement 2
8A7H5 Fab blocks A2 attachment and fails to bind A2.V296F.
(A) Increasing concentrations of 8A7H5 Fab prevent the attachment of A2, but not A2.V296F, to NMuMG cells. Data from two independent experiments, n = 5–6. (B) Binding of 8A7H5 Fab to VP1 is …
Figure 6 with 2 supplements
Additional JCPyV-PML mutations in MuPyV impair kidney, but not brain infection, and have varying effects on VP1 mAb neutralization.
(A) Structural comparison of PML mutation sites in JCPyV VP1 (PDB 3NXG) with MuPyV VP1 (PDB 5CPU) residues (Buch et al., 2015; Neu et al., 2010). (B) Kidney LT mRNA levels in mice 4 dpi with A2 or …
Figure 6—figure supplement 1
Relation of other VP1 mutations to virus–Fab interface.
(A) Cryo-EM density at the interface between the pentavalent capsomer (red) and Fab (cyan, purple). Location of the residues mutated to mimic those found in JCPyV-PML (H139, T291, N293, V296, R77) …
Figure 6—figure supplement 2
Resistance of escape mutations to 8A7H5 neutralization.
LT mRNA levels in NMuMG cells 24 hpi with A2, A2.N80K, A2.∆294, A2.∆295 preincubated with 8A7H5 or control IgG. Data are from two independent experiments, n = 6 (A2 p<0.0001, A2.N80K p=0.2374, …
Tables
Table 1
VP1 contact residues within −0.4 Å van der Waal’s overlap.
The conformational epitope spans three loops over two copies of VP1. Contributions from the adjacent VP1 are denoted with `.
| Loop | Residue | |
|---|---|---|
| BC | THR | 67 |
| GLU | 68 | |
| ARG | 77 | |
| GLY | 78 | |
| ASN | 80 | |
| THR | 83 | |
| GLU | 91 | |
| DE | PHE | 141’ |
| LYS | 151’ | |
| HI | ARG | 292’ |
| ASN | 293 | |
| TYR | 294 | |
| VAL | 296 | |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-VP1 (Rat Clone 8A7H5) | Swimm et al., 2010 | Clone 8A7H5 | See Materials and methods for concentrations |
| Antibody | ChromPure Rat IgG | Jackson ImmunoResearch | Cat#012-000-003 | See Materials and methods for concentrations |
| Antibody | Anti-CD8β (Rat monoclonal) | Pierres et al., 1982 | Clone H35-17.2 | 250 μg per injection |
| Antibody | Anti-VP1 (Rabbit polyclonal) | Provided by Robert Garcea (University of Colorado Boulder) | IF(1:1000) | |
| Antibody | Anti-Vimentin (Rat monoclonal) | R & D Systems | Cat#MAB2105 | IF(1:100) |
| Antibody | Anti-GFAP (Goat polyclonal) | Abcam | Cat#ab53554 | IF(1:1000) |
| Antibody | Anti-Iba1 (Rabbit polyclonal) | FUJIFILM Wako | Cat#019–19741 | IF(1:500) |
| Antibody | Anti-CD3 (Rabbit monoclonal) | Abcam | Cat#ab16669 | IF(1:100) |
| Antibody | Anti-Goat IgG AF488 (Bovine polyclonal) | Jackson ImmunoResearch | Cat#805-545-180 | IF(1:500) |
| Antibody | Anti-Rat IgG AF568 (Donkey polyclonal) | Abcam | Cat#ab175475 | IF(1:500) |
| Antibody | Anti-Rabbit IgG AF647 (Donkey polyclonal) | Jackson ImmunoResearch | Cat#711-605-152 | IF(1:500) |
| Antibody | Anti-CD8α-AF700 (Rat monoclonal) | Biolegend | Cat#100730 | FC(1:200) |
| Antibody | Anti-CD44-FITC (Rat monoclonal) | Biolegend | Cat#103006 | FC(1:200) |
| Antibody | Anti-Rat IgG-APC (Goat polyclonal) | BD | Cat#551019 | FC(1:200) |
| Antibody | Anti-Mouse IgG-HRP (Goat polyclonal) | Biolegend | Cat#405306 | ELISA(1:2800) |
| Antibody | Anti-Mouse IgG-HRP (Goat polyclonal) | Bethyl Laboratories INC | Cat#A90-116P | ELISA(1:7000) |
| Antibody | Biotinylated Anti-Rabbit (Goat Polyclonal) | Vector Laboratories | Cat#BA-1000 | IHC(1:500) |
| Other | Mouse Polyomavirus (Strain A2) | N/A | N/A | |
| Strain, strain background (Escherichia coli) | BL21 | Agilent | Cat#200133 | |
| Recombinant DNA reagent | PyVP1-pGEX-4T-2 (plasmid) | Provided by Robert Garcea | N/A | |
| Recombinant DNA reagent | H2B-GFP (plasmid) | Kanda et al., 1998, Addgene | Plasmid #11680 | |
| Peptide, recombinant protein | Benzonase Nuclease | Sigma | Cat#E1014 | Virus Purification (1:3333) |
| Other | Db-LT359 Tetramer | NIH Tetramer Core | N/A | FC(1:400) |
| Peptide, recombinant protein | Neuraminidase from Vibrio cholerae (Type II) | Sigma | Cat#N6514 | Virus Purification (1:2000) |
| Peptide, recombinant protein | RevertAid H Minus Reverse Transcriptase | ThermoFisher | Cat#EP0451 | |
| Chemical compound, drug | OptiPrep | STEMCELL Technologies | Cat#07820 | |
| Chemical compound, drug | Glutathione Sepharose 4B | GE Healthcare | Cat#17075601 | |
| Chemical compound, drug | TRIzol Reagant | ThermoFisher | Ref#15596018 | |
| Chemical compound, drug | Lipofectamine 2000 Transfection Reagent | ThermoFisher | Cat#11668030 | |
| Commercial assay, kit | TBP PrimeTime XL qPCR Assay | IDT | Mm.PT.39a.22214839 | |
| Other | ProLong Gold antifade reagent with DAPI | ThermoFisher | Ref#P36931 | |
| Other | Fixable Viability Dye eFluor780 | ThermoFisher | Cat# 65-0865-14 | FC(1:1000) |
| Commercial assay, kit | Avidin/Biotin Blocking Kit | Vector Laboratories | Cat#SP-2001 | |
| Commercial assay, kit | VECTASTAIN Elite ABC-HRP Kit | Vector Laboratories | Cat#PK-6100 | |
| Commercial assay, kit | NovaRED Substrate Kit | Vector Laboratories | Cat#SK-4800 | |
| Commercial assay, kit | 1-Step Ultra TMB-ELISA | ThermoFisher | Ref#34028 | |
| Commercial assay, kit | PerfectCTa SYBR Green FastMix | Quantabio | P/N 84069 | |
| Commercial assay, kit | PerfectCTa FastMix II ROX | Quantabio | P/N 84210 | |
| Commercial assay, kit | PureLink Viral RNA/DNA mini Kit | ThermoFisher | Ref#12280–050 | |
| Commercial assay, kit | Pierce Fab Micro Preparation Kit | ThermoFisher | Ref#44685 | |
| Commercial assay, kit | Nab Protein G Spin Columns | ThermoFisher | Ref#89953 | |
| Commercial assay, kit | Wizard Genomic DNA Purification Kit | Promega | Ref#A1120 | |
| Commercial assay, kit | QuikChange II Site-Directed Mutagenesis Kit | Agilent | Cat#200523 | |
| Commercial assay, kit | QIAquick PCR Purification Kit | Qiagen | Cat#28104 | |
| Cell Line (Mus musculus) | BALB/3T3 Clone A31 | ATCC | CCL-163, RRID:CVCL_0184 | |
| Cell Line (M. musculus) | NMuMG | ATCC | CRL-1636, RRID:CVCL_0075 | |
| Cell Line (M. musculus) | mIMCD-3 | ATCC | CRL-2123, RRID:CVCL_0429 | |
| Cell Line (M. musculus) | C57BL/6 MEF | This paper | Primary murine embryonic fibroblasts | |
| Strain, strain background (M. musculus) | C57BL/6 | National Cancer Institute | Cat#OIC55 | |
| Genetic reagent (M. musculus) | Stat1–/– | Jackson Laboratory | Cat#012606 | |
| Genetic reagent (M. musculus) | μMT | Jackson Laboratory | Cat#002288 | |
| Software, algorithm | Prism | Graphpad | RRID:SCR_002798 | |
| Software, algorithm | FlowJo | BD | RRID:SCR_008520 | |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | |
| Software, algorithm | Leica LAS X | Leica | RRID:SCR_013673 | |
| Software, algorithm | Photoshop | Adobe | RRID:SCR_014199 | |
| Software, algorithm | Relion | Scheres et al., 2009 | RRID:SCR_016274 | |
| Software, algorithm | cryoSPARC | Structura Biotechnology | RRID:SCR_016501 | |
| Software, algorithm | ISECC | See Data and code availability | v 2019.09 | |
| Software, algorithm | PHENIX | phenix-online.org | RRID:SCR_014224 | |
| Software, algorithm | Coot | Emsley et al., 2010 | RRID:SCR_014222 |
Additional files
-
Supplementary file 1
Collection statistics.
Independent datasets were collected for MuPyV and the MuPyV-Fab complex.
- https://cdn.elifesciences.org/articles/61056/elife-61056-supp1-v3.docx
-
Supplementary file 2
Refinement statistics.
Local refinement allowed models to be built into the higher resolution capsomer maps.
- https://cdn.elifesciences.org/articles/61056/elife-61056-supp2-v3.docx
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Supplementary file 3
Fab contact residues.
The majority of Fab contacts are through the heavy chain, with minor contributions from the light chain.
- https://cdn.elifesciences.org/articles/61056/elife-61056-supp3-v3.docx
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Supplementary file 4
Statistics of existing and new cryo-EM polyomavirus maps.
Capsomer-based local refinement allowed rapid refinement of polyomavirus to high resolution, using only a modest particle number.
- https://cdn.elifesciences.org/articles/61056/elife-61056-supp4-v3.docx
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Supplementary file 5
ISECC source code.
Archive of the ISECC source code used in sub-particle generation. The most current version of the software is maintained at https://github.com/goetschius/isecc.
- https://cdn.elifesciences.org/articles/61056/elife-61056-supp5-v3.zip
-
Supplementary file 6
Oligonucleotide sequences.
Sequences of oligonucleotides used for site-directed mutagenesis, qPCR, sequencing.
- https://cdn.elifesciences.org/articles/61056/elife-61056-supp6-v3.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/61056/elife-61056-transrepform-v3.docx
