Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity

  1. Yingying Fu
  2. Rui Cao
  3. Miriam Schäfer
  4. Sonja Stephan
  5. Ilona Braspenning-Wesch
  6. Laura Schmitt
  7. Ralf Bischoff
  8. Martin Müller
  9. Kai Schäfer
  10. Sabrina E Vinzón
  11. Frank Rösl  Is a corresponding author
  12. Daniel Hasche  Is a corresponding author
  1. Division of Viral Transformation Mechanisms, Research Program 'Infection, Inflammation and Cancer', German Cancer Research Center, Germany
  2. Division of Functional Genome Analysis, Research Program 'Functional and Structural Genomics', German Cancer Research Center, Germany
  3. Research Group Tumorvirus-specific Vaccination Strategies, Research Program 'Infection, Inflammation and Cancer', German Cancer Research Center, Germany
10 figures, 1 table and 4 additional files


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).

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.

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).

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.

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.

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).


Key resources table
Reagent type
(species) or
DesignationSource or
Genetic reagent (Mastomys coucha)African Multimammate rodentDKFZ, Prof. F. RöslMastomys couchaUsed asexperimental model, Hasche and Rösl, 2019
Gene (Mastomys natalensis Papillomavirus1)MnPVGenBankNC_001605.1Tan et al., 1994
Biological sample (Mastomys coucha)SeraThis paperSera tested for seroconversionagainst different MnPV proteins
Biological sample (Mastomys coucha)SeraVinzón et al., 2014Sera from VLP-vaccinated animals
Cell line (Mastomys coucha)MaFi132; Mastomys coucha- derived fibroblastsDKFZ, Prof. F. RöslHasche et al., 2016
Cell line (Homo sapiens)HeLaTDKFZ, Prof. M. Müller, Sehr et al., 2002HeLaT clone-4,Used for pseudovirion--based neutralization assay
Cell line (Homo sapiens)293TTDTP, DCTD TUMOR REPOSITORYNCI-293TT; RRID:CVCL_1D85Used forpseudovirion production
Cell line (Spodoptera frugiperda)Sf9DKFZ, Prof. M. MüllerRRID:CVCL_0549Insect cells,used for VLPproduction
Cell line (Trichoplusia ni)TN-High FiveGibcoBTI-TN-5B1-4; RRID:CVCL_C190Insect cells, used for VLPproduction
Strain, strain background (Escherichia coli)TOP10 (DH10B)InvitrogenCat#: C404010Chemically competent cells
Strain, strain background (Escherichia coli)DH10MultiBacCreGeneva BiotechElectrocompetent cellsFitzgerald et al., 2006
Transfected construct (Mastomys natalensis Papillomavirus1)pFBDM_L1SHORT, pFBDM_L1MIDDLE , pFBDM_L1LONGThis paperBackbone RRID:Addgene_110738Multibac 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_L1LONGThis paperConstructs totransfect humanized ORFs andexpress MnPVL1 variants inMaFi132 cells
Transfected construct (Mastomys natalensis Papillomavirus1)pPK-CMV-E3_vL1This paperConstruct totransfect and express all L1 ORFs as found in the genuineMnPV genomein MaFi132 cells
Biological sample (Mastomys natalensis Papillomavirus1)MnPV VLPsThis paperMnPV virus-likeparticles usedfor VLP-ELISAand assemblystudies
Biological sample (Mastomys natalensis Papillomavirus1)MnPV PsVsThis paperMnPV pseudovirions for infectivity assay andPBNA
Antibodyanti-L1 (Mouse monoclonal)This papermAb 2E2, 2D11, 3H8, 2D6, 5E5ELISA (1:20-1:14,580), IF(1:5), WB(2D11, 1:1000)
AntibodyMastomys serum mix (Mastomys coucha polyclonal serum)This paperIF (1:1000),Peptide Array(1:300)
AntibodyAnti-HA clone 3F10 (Rat monoclonal)Sigma-AldrichCat#: 11867423001; RRID:AB_390918IF (1:1000), WB (1:1000)
AntibodyAnti-Vinculin clone 7F9 (Mouse monoclonal)Santa CruzCat#: sc-73614; RRID:AB_1131294WB (1:4000)
AntibodyAnti-L1LONGaa1-31 serum (Mouse polyclonal serum)This paperIHC (1:100)
AntibodyAnti-L2 serum (Guinea pig polyclonal serum)DKFZ, Prof. M. MüllerIHC (1:200)
AntibodyAnti-L2 clone K18L2 (Guinea pig polyclonal serum)DKFZ, Prof. M. Müller, Rubio et al., 2011IHC (1:200)
AntibodyAnti-Mouse IgG (H+L), HRP Conjugate (Goat polyclonal)PromegaCat#: W4021; RRID:AB_430834ELISA (1:10,000), WB (1:10,000)
AntibodyPeroxidase AffiniPure Goat Anti-Rat IgG (H+L)Jackson ImmunoResearchCat#: 112-035-003; RRID:AB_2338128WB (1:10,000)
AntibodyGoat anti-Mouse IgG (H+L), Alexa Fluor 488 (Goat polyclonal)InvitrogenCat#: A11029; RRID:AB_138404IF (1:1000), IHC (1:1000)
AntibodyGoat anti-Guinea Pig IgG (H+L), Alexa Fluor 488 (Goat polyclonal)InvitrogenCat#: A11073; RRID:AB_2534117IHC (1:1000)
AntibodyDonkey anti-Rat IgG (H+L), Alexa Fluor 488, (Donkey polyclonal)InvitrogenCat#: A21208; RRID:AB_141709IF (1:1000)
AntibodyMouse IgG (H and L) Antibody DyLight 680 ConjugatedRockland ImmunochemicalsCat#: 610-144-121; RRID:AB_1057546Peptide Array (1:5000)
Recombinant DNA reagentpPK-CMV-E3PromocellCat#: PK-MB-P003300
Peptide, recombinant proteinGST-L1SHORT, GST-L1MIDDLE, GST-L1LONG,This paper, Schäfer et al., 2010GST proteinfused to thedifferent MnPV L1 variants
Peptide, recombinant proteinGST-L1LONGaa1-31, GST-L1LONGaa1-41This paperGST proteinfused to the N-terminus of MnPV L1
Peptide, recombinant proteinGST-E2, GST-L2This paper, Schäfer et al., 2010GST proteinfused to MnPVE2 or L2
Peptide, recombinant proteinL1 peptide arrayPEPperPRINT GmbHStadler et al., 2008
Commercial assay or kitDako REAL Detection System, Peroxidase/AEC, Rabbit/MouseAgilentCat#: K5007IHC chromogenicdetection Kit
Commercial assay or kitGaussia glow juicePJK BiotechCat#: 102542Luciferase activity detection kit
Chemical compound, drugDAPISigma-AldrichCat#: D9542-5MG
Software, algorithmGraphPad Prism 6.0GraphPad

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  1. Yingying Fu
  2. Rui Cao
  3. Miriam Schäfer
  4. Sonja Stephan
  5. Ilona Braspenning-Wesch
  6. Laura Schmitt
  7. Ralf Bischoff
  8. Martin Müller
  9. Kai Schäfer
  10. Sabrina E Vinzón
  11. Frank Rösl
  12. Daniel Hasche
Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity
eLife 9:e57626.