Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity
- Division of Viral Transformation Mechanisms, Research Program 'Infection, Inflammation and Cancer', German Cancer Research Center, Germany
- Division of Functional Genome Analysis, Research Program 'Functional and Structural Genomics', German Cancer Research Center, Germany
- Research Group Tumorvirus-specific Vaccination Strategies, Research Program 'Infection, Inflammation and Cancer', German Cancer Research Center, Germany
Figures
Figure 1
Alignment of L1 sequences from different PV types.
N-terminal sequences of L1 proteins from 29 PV types were aligned using Clustal Omega. The highly conserved motif (Wx7YLPP) is marked in pink boxes. The first methionine of L1 is marked in red. The last methionine upstream of the Wx7YLPP motif is shown in green and methionines between the first and the last one are depicted in blue. In the case of MmuPV and McPV2, both methionines upstream of the conserved motif fit to the consensus sequence of L1SHORT and are therefore depicted in green.
Figure 2 with 2 supplements
Seroreactivity against viral proteins in naturally MnPV-infected animals.
Seroresponses of 682 sera from 60 animals measured by GST-ELISA against (A) L1SHORT, (B) L1LONG, (C) L1MIDDLE, (D) E2 and (E) L2 GST-fusion proteins and F) VLP-ELISA. Dashed lines represent the methods’ cut-off (OD450 = 0.2 for GST-ELISA or titer of 300 for VLP-ELISA) (Mean ± SEM; 1-Way-ANOVA test, *p<0.05, **p<0.01, ***p<0.001).
Figure 2—figure supplement 1
Rate of L1LONG and L1SHORT positive animals.
Rate of L1LONG and L1SHORT positive animals at different time points of the follow-up study. The 2 × 2 contingency table used for the two-tailed McNemar’s test shows the 68 week time point, which was used for the main comparison, since here, most animals were still alive.
Figure 2—figure supplement 2
Additional correlation of seroreactivities measured by GST-ELISA.
Correlation of seroreactivities against GST-L1LONG with seroreactivities against GST-L1MIDDLE (correlation coefficient, R2 = 0.4723).
Figure 3
Correlation of GST- and VLP-ELISAs.
(A) Correlation of seroreactivities against GST-L1LONG with seroreactivities against GST-L1SHORT (correlation coefficient, R2 = 0.0261). (B) Correlation of GST-L1LONG ELISA with VLP-ELISA (R2 = 0.0062). (C) Correlation of GST-L1SHORT ELISA with VLP-ELISA (R2 = 0.8394). All graphs include all 682 sera taken during the study. Dashed lines indicate the methods’ cut-offs.
Figure 4
Neutralizing capacity of anti-L1LONG and anti-L1SHORT antibodies.
(A) Neutralization assay for all L1LONG-/L1SHORT-positive sera (n = 294) from 60 naturally infected animals. (B) Correlation of VLP-ELISA titers and neutralizing titers of all L1LONG-/L1SHORT-positive sera (correlation coefficient, R2 = 0.9883). The regression line represents a linear regression fit (Please note, that for both assays all sera were diluted in three-fold dilution steps. Since the titers are calculated from the dilution, data points of 294 different sera overlay when having the same titer in both assays). (C) Correlation of GST-L1LONG ELISA and neutralization assay for 234 L1LONG-positive/L1SHORT-negative sera (R2 = 0.0000). Correlation analyses contain sera from animals representing the complete age range. Dashed lines indicate the methods’ cut-offs (OD450 = 0.2 for GST-ELISA or titer of 300 for neutralization assay).
Figure 5 with 1 supplement
Peptide arrays identify known immunogenic epitopes in L1.
(A) Synthetic 15-mer peptides with residue overlaps of 14 residues were spotted on microarrays and incubated with serum mix from five tumor-bearing animals with high titers against both L1 isoforms. Bound serum antibodies were detected with fluorophore-conjugated secondary antibodies. Positive regions (ITGHPLY, DYLGMSK and KRSLPASRN) are indicated and were mapped to their position in L1LONG (B). (C) Two of these regions (ITGHPLY and DYLGMSK) coincide with the DE and the FG loop, respectively (scheme shows MnPV L1SHORT; see Supplementary file 1).
Figure 5—figure supplement 1
Immunogenicity prediction of L1LONG.
Prediction of antigenic determinants within L1LONG suggests an immunogenic epitope between residues 11 and 34 of the N-terminus of L1LONG. Also the DE loop (determinant no. 8) and partially the FG loop (last 2 residues of determinant no. 12) are predicted.
Figure 6 with 1 supplement
Seroreactivity against the N-terminus of L1LONG measured by GST-ELISA.
(A) Seroreactivity against the 31 aa exclusive for L1LONG (60 animals, 682 sera) and (B) Against 41 aa of the N-terminus of L1LONG (39 L1LONG-positive animals, 297 sera). Dashed lines represent the cut-off (0.11) based on virus-free animals. (C) Sera from MnPV-free animals (14 sera, black dots) vaccinated with VLPs (made from L1SHORT) and six pre-immune sera (grey squares) from a previous study (Vinzón et al., 2014) measured in the different L1 GST-ELISAs. Dashed lines indicate the cut-offs (grey: OD450 = 0.2 for L1SHORT and L1LONG; black: OD450 = 0.11 for L1LONGaa1-31 and L1LONGaa1-41). (D) Correlation of ELISAs for GST-L1LONG and GST-L1LONGaa1-41 (correlation coefficient, R2 = 0.0996).
Figure 6—figure supplement 1
Additional correlation of seroreactivities measured by GST-ELISA.
Correlation of GST-L1SHORT with GST-L1MIDDLE (R2 = 0.0106). Dashed lines indicate the cut-offs (OD450 = 0.2 for GST-L1SHORT, GST-L1MIDDLE and GST-L1LONG; OD450 = 0.11 for L1LONGaa1-41).
Figure 7
L1 denaturation abolishes recognition by naturally-raised antibodies.
Binding efficacy of five monoclonal antibodies against (A) native VLPs and (B) VLPs denatured by heating (Mean ± SD, n = 3). (C) Seroresponse of Mastomys sera (306 sera positive for L1LONG and L1SHORT) against denatured VLPs. (D) Seroresponse of Mastomys sera against denatured GST-L1LONG. (E) Seroresponse of Mastomys sera against denatured GST-L1SHORT. For (E and D), 281 sera from 40 animals positive for L1LONG and L1SHORT were measured. Dashed lines indicate the cut-offs (OD450 = 0.3 for denatured VLP-ELISA; OD450 = 0.15 for denatured GST-ELISA).
Figure 8 with 2 supplements
VLP and pseudovirion formation capacity of L1SHORT, L1MIDDLE and L1LONG.
EM micrographs of peak fractions and the respective fractions of lowest densities of (A) L1SHORT (B) L1MIDDLE and (C) L1LONG at 16,000x magnification. Capacity to form infectious pseudovirions in presence of L2 was analyzed by infectivity assay and EM for (D) L1SHORT (E) L1MIDDLE and (F) L1LONG. Note that high signals with unpurified lysate from PsV-producing cells result from co-expressed luciferase reporter protein (Mean ± SD, n = 3).
Figure 8—figure supplement 1
Production of L1 isoforms with the MultiBac baculovirus expression system.
(A) The CsCl density (refractive index) of each fraction was determined via refractometer at RT. (B) Analysis of fractions from PsV production with L1SHORT, L1MIDDLE and L1LONG analyzed by Coomassie blue staining (upper panels) and Western blot (lower panels). Immunoblots were incubated with a serum mix from five MnPV-infected tumor-bearing animals.
Figure 8—figure supplement 2
Pseudoatomic modeling of L1 proteins in the viral capsid.
Model of HPV16 capsid (cryo-electron microscopy, structure ID: 3J6R, view from inside of the capsid, display: left: ball and stick, middle: trace, right: cartoon). The chain starts at residue 9. The first three residues ATV of the different L1 proteins are indicated.
Figure 9
L1SHORT and L1LONG protein expose different epitopes when expressed in Mastomys cells.
Mastomys-derived fibroblasts were transfected with HA-tagged L1SHORT, L1MIDDLE or L1LONG (humanized codons and artificial Kozak sequences), or the polycistronic plasmid vL1 (encodes all three viral L1 ORFs and Kozak sequences). (A) Expression of L1 isoforms was visualized with mAb 2D11 (only recognizing conformational L1 epitopes), serum mix from five tumor-bearing animals and anti-HA as a control. (B) Western blotting reveals two high-MW bands only in L1SHORT-expressing cells corresponding to L1 dimers (#) and trimers (§). Transfection with vL1 results in synthesis of both L1LONG as well as L1SHORT and its multimer. (C) Upon harsher denaturation of cell lysates, L1SHORT multimer bands disappear. Vinculin served as loading control.
Figure 10
L1LONG synthesis occurs much earlier than capsid formation in vivo.
(A) EM micrograph of MnPV particles (p) in the stratum corneum (s.c.) of a MnPV-induced papilloma. The nearly shed cell shown here is strongly degraded and only tonofilaments (f) are left. (B) Immunohistochemical analysis reveals MnPV capsids (detected with serum of a MnPV-VLP-immunized Mastomys) in the uppermost layers of the stratum corneum while L1LONG (detected by serum from a mouse immunized with the N-terminal 31 residues of MnPV-L1LONG) appears throughout the whole epidermis. Pre-immune sera were used as controls. (C) Tissue sections were stained with cross-reactive anti-HPV-L2 antibody (K18L2, red) or cross-reactive anti-HPV-VLP guinea pig serum (green) as controls. Consistent with capsid formation, L2 only appears in the uppermost layers of the tissue shortly prior to VLP formation (d: dermis, e: epidermis, k: keratin).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mastomys coucha) | African Multimammate rodent | DKFZ, Prof. F. Rösl | Mastomys coucha | Used asexperimental model, Hasche and Rösl, 2019 |
| Gene (Mastomys natalensis Papillomavirus1) | MnPV | GenBank | NC_001605.1 | Tan et al., 1994 |
| Biological sample (Mastomys coucha) | Sera | This paper | Sera tested for seroconversionagainst different MnPV proteins | |
| Biological sample (Mastomys coucha) | Sera | Vinzón et al., 2014 | Sera from VLP-vaccinated animals | |
| Cell line (Mastomys coucha) | MaFi132; Mastomys coucha- derived fibroblasts | DKFZ, Prof. F. Rösl | Hasche et al., 2016 | |
| Cell line (Homo sapiens) | HeLaT | DKFZ, Prof. M. Müller, Sehr et al., 2002 | HeLaT clone-4, | Used for pseudovirion--based neutralization assay |
| Cell line (Homo sapiens) | 293TT | DTP, DCTD TUMOR REPOSITORY | NCI-293TT; RRID:CVCL_1D85 | Used forpseudovirion production |
| Cell line (Spodoptera frugiperda) | Sf9 | DKFZ, Prof. M. Müller | RRID:CVCL_0549 | Insect cells,used for VLPproduction |
| Cell line (Trichoplusia ni) | TN-High Five | Gibco | BTI-TN-5B1-4; RRID:CVCL_C190 | Insect cells, used for VLPproduction |
| Strain, strain background (Escherichia coli) | TOP10 (DH10B) | Invitrogen | Cat#: C404010 | Chemically competent cells |
| Strain, strain background (Escherichia coli) | DH10MultiBacCre | Geneva Biotech | Electrocompetent cells | Fitzgerald et al., 2006 |
| Transfected construct (Mastomys natalensis Papillomavirus1) | pFBDM_L1SHORT, pFBDM_L1MIDDLE , pFBDM_L1LONG | This paper | Backbone RRID:Addgene_110738 | Multibac constructs to transfect andexpress MnPVL1 variants ininsect cells for VLPproduction |
| Transfected construct (Mastomys natalensis Papillomavirus1) | pPK-CMV-E3_L1SHORT, pPK-CMV-E3_L1MIDDLE, pPK-CMV-E3_L1LONG | This paper | Constructs totransfect humanized ORFs andexpress MnPVL1 variants inMaFi132 cells | |
| Transfected construct (Mastomys natalensis Papillomavirus1) | pPK-CMV-E3_vL1 | This paper | Construct totransfect and express all L1 ORFs as found in the genuineMnPV genomein MaFi132 cells | |
| Biological sample (Mastomys natalensis Papillomavirus1) | MnPV VLPs | This paper | MnPV virus-likeparticles usedfor VLP-ELISAand assemblystudies | |
| Biological sample (Mastomys natalensis Papillomavirus1) | MnPV PsVs | This paper | MnPV pseudovirions for infectivity assay andPBNA | |
| Antibody | anti-L1 (Mouse monoclonal) | This paper | mAb 2E2, 2D11, 3H8, 2D6, 5E5 | ELISA (1:20-1:14,580), IF(1:5), WB(2D11, 1:1000) |
| Antibody | Mastomys serum mix (Mastomys coucha polyclonal serum) | This paper | IF (1:1000),Peptide Array(1:300) | |
| Antibody | Anti-HA clone 3F10 (Rat monoclonal) | Sigma-Aldrich | Cat#: 11867423001; RRID:AB_390918 | IF (1:1000), WB (1:1000) |
| Antibody | Anti-Vinculin clone 7F9 (Mouse monoclonal) | Santa Cruz | Cat#: sc-73614; RRID:AB_1131294 | WB (1:4000) |
| Antibody | Anti-L1LONGaa1-31 serum (Mouse polyclonal serum) | This paper | IHC (1:100) | |
| Antibody | Anti-L2 serum (Guinea pig polyclonal serum) | DKFZ, Prof. M. Müller | IHC (1:200) | |
| Antibody | Anti-L2 clone K18L2 (Guinea pig polyclonal serum) | DKFZ, Prof. M. Müller, Rubio et al., 2011 | IHC (1:200) | |
| Antibody | Anti-Mouse IgG (H+L), HRP Conjugate (Goat polyclonal) | Promega | Cat#: W4021; RRID:AB_430834 | ELISA (1:10,000), WB (1:10,000) |
| Antibody | Peroxidase AffiniPure Goat Anti-Rat IgG (H+L) | Jackson ImmunoResearch | Cat#: 112-035-003; RRID:AB_2338128 | WB (1:10,000) |
| Antibody | Goat anti-Mouse IgG (H+L), Alexa Fluor 488 (Goat polyclonal) | Invitrogen | Cat#: A11029; RRID:AB_138404 | IF (1:1000), IHC (1:1000) |
| Antibody | Goat anti-Guinea Pig IgG (H+L), Alexa Fluor 488 (Goat polyclonal) | Invitrogen | Cat#: A11073; RRID:AB_2534117 | IHC (1:1000) |
| Antibody | Donkey anti-Rat IgG (H+L), Alexa Fluor 488, (Donkey polyclonal) | Invitrogen | Cat#: A21208; RRID:AB_141709 | IF (1:1000) |
| Antibody | Mouse IgG (H and L) Antibody DyLight 680 Conjugated | Rockland Immunochemicals | Cat#: 610-144-121; RRID:AB_1057546 | Peptide Array (1:5000) |
| Recombinant DNA reagent | pPK-CMV-E3 | Promocell | Cat#: PK-MB-P003300 | |
| Peptide, recombinant protein | GST-L1SHORT, GST-L1MIDDLE, GST-L1LONG, | This paper, Schäfer et al., 2010 | GST proteinfused to thedifferent MnPV L1 variants | |
| Peptide, recombinant protein | GST-L1LONGaa1-31, GST-L1LONGaa1-41 | This paper | GST proteinfused to the N-terminus of MnPV L1 | |
| Peptide, recombinant protein | GST-E2, GST-L2 | This paper, Schäfer et al., 2010 | GST proteinfused to MnPVE2 or L2 | |
| Peptide, recombinant protein | L1 peptide array | PEPperPRINT GmbH | Stadler et al., 2008 | |
| Commercial assay or kit | Dako REAL Detection System, Peroxidase/AEC, Rabbit/Mouse | Agilent | Cat#: K5007 | IHC chromogenicdetection Kit |
| Commercial assay or kit | Gaussia glow juice | PJK Biotech | Cat#: 102542 | Luciferase activity detection kit |
| Chemical compound, drug | DAPI | Sigma-Aldrich | Cat#: D9542-5MG | |
| Software, algorithm | GraphPad Prism 6.0 | GraphPad |
Additional files
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Supplementary file 1
Alignment of L1 amino acid sequences of MnPV and HPV6, 16 and 18.
- https://cdn.elifesciences.org/articles/57626/elife-57626-supp1-v2.docx
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Supplementary file 2
Binding properties of monoclonal antibodies against L1 isoforms.
- https://cdn.elifesciences.org/articles/57626/elife-57626-supp2-v2.docx
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Supplementary file 3
Prediction of ATG usage in MnPV late transcript Q.
- https://cdn.elifesciences.org/articles/57626/elife-57626-supp3-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/57626/elife-57626-transrepform-v2.pdf
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Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity
eLife 9:e57626.
https://doi.org/10.7554/eLife.57626
