Figures and data in Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity

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Tables
Additional files

10 figures, 1 table and 4 additional files

Figures

Figure 1

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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 L1_SHORT and are therefore depicted in green.

Figure 2 with 2 supplements

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Seroreactivity against viral proteins in naturally MnPV-infected animals.

Seroresponses of 682 sera from 60 animals measured by GST-ELISA against (A) L1_SHORT, (B) L1_LONG, (C) L1_MIDDLE, (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

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Rate of L1_LONG and L1_SHORT positive animals.

Rate of L1_LONG and L1_SHORT 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

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Additional correlation of seroreactivities measured by GST-ELISA.

Correlation of seroreactivities against GST-L1_LONG with seroreactivities against GST-L1_MIDDLE (correlation coefficient, R² = 0.4723).

Figure 3

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Correlation of GST- and VLP-ELISAs.

(A) Correlation of seroreactivities against GST-L1_LONG with seroreactivities against GST-L1_SHORT (correlation coefficient, R² = 0.0261). (B) Correlation of GST-L1_LONG ELISA with VLP-ELISA (R² = 0.0062). (C) Correlation of GST-L1_SHORT 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

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Neutralizing capacity of anti-L1_LONG and anti-L1_SHORT antibodies.

(A) Neutralization assay for all L1_LONG-/L1_SHORT-positive sera (n = 294) from 60 naturally infected animals. (B) Correlation of VLP-ELISA titers and neutralizing titers of all L1_LONG-/L1_SHORT-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-L1_LONG ELISA and neutralization assay for 234 L1_LONG-positive/L1_SHORT-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

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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 L1_LONG (B). (C) Two of these regions (ITGHPLY and DYLGMSK) coincide with the DE and the FG loop, respectively (scheme shows MnPV L1_SHORT; see Supplementary file 1).

Figure 5—figure supplement 1

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Immunogenicity prediction of L1_LONG.

Prediction of antigenic determinants within L1_LONG suggests an immunogenic epitope between residues 11 and 34 of the N-terminus of L1_LONG. 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

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Seroreactivity against the N-terminus of L1_LONG measured by GST-ELISA.

(A) Seroreactivity against the 31 aa exclusive for L1_LONG (60 animals, 682 sera) and (B) Against 41 aa of the N-terminus of L1_LONG (39 L1_LONG-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 L1_SHORT) 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 L1_SHORT and L1_LONG; black: OD₄₅₀ = 0.11 for L1_LONGaa1-31 and L1_LONGaa1-41). (D) Correlation of ELISAs for GST-L1_LONG and GST-L1_LONGaa1-41 (correlation coefficient, R² = 0.0996).

Figure 6—figure supplement 1

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Additional correlation of seroreactivities measured by GST-ELISA.

Correlation of GST-L1_SHORT with GST-L1_MIDDLE (R² = 0.0106). Dashed lines indicate the cut-offs (OD₄₅₀ = 0.2 for GST-L1_SHORT, GST-L1_MIDDLE and GST-L1_LONG; OD₄₅₀ = 0.11 for L1_LONGaa1-41).

Figure 7

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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 L1_LONG and L1_SHORT) against denatured VLPs. (D) Seroresponse of *Mastomys* sera against denatured GST-L1_LONG. (E) Seroresponse of *Mastomys* sera against denatured GST-L1_SHORT. For (E and D), 281 sera from 40 animals positive for L1_LONG and L1_SHORT 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

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VLP and pseudovirion formation capacity of L1_SHORT, L1_MIDDLE and L1_LONG.

EM micrographs of peak fractions and the respective fractions of lowest densities of (A) L1_SHORT (B) L1_MIDDLE and (C) L1_LONG at 16,000x magnification. Capacity to form infectious pseudovirions in presence of L2 was analyzed by infectivity assay and EM for (D) L1_SHORT (E) L1_MIDDLE and (F) L1_LONG. 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

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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 L1_SHORT, L1_MIDDLE and L1_LONG 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

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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

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L1_SHORT and L1_LONG protein expose different epitopes when expressed in *Mastomys* cells.

*Mastomys*-derived fibroblasts were transfected with HA-tagged L1_SHORT, L1_MIDDLE or L1_LONG (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 L1_SHORT-expressing cells corresponding to L1 dimers (#) and trimers (§). Transfection with vL1 results in synthesis of both L1_LONG as well as L1_SHORT and its multimer. (C) Upon harsher denaturation of cell lysates, L1_SHORT multimer bands disappear. Vinculin served as loading control.

Figure 10

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L1_LONG 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 L1_LONG (detected by serum from a mouse immunized with the N-terminal 31 residues of MnPV-L1_LONG) 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)	DH10MultiBac^Cre	Geneva Biotech	Electrocompetent cells	Fitzgerald et al., 2006
Transfected construct (Mastomys natalensis Papillomavirus1)	pFBDM_L1_SHORT, pFBDM_L1_MIDDLE , pFBDM_L1_LONG	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_L1_SHORT, pPK-CMV-E3_L1_MIDDLE, pPK-CMV-E3_L1_LONG	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-L1_LONGaa1-31 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-L1_SHORT, GST-L1_MIDDLE, GST-L1_LONG,	This paper, Schäfer et al., 2010		GST proteinfused to thedifferent MnPV L1 variants
Peptide, recombinant protein	GST-L1_LONGaa1-31, GST-L1_LONGaa1-41	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