Journal: Frontiers in Immunology

Article Title: ErbB2/HER2 receptor tyrosine kinase regulates human papillomavirus promoter activity

doi: 10.3389/fimmu.2024.1335302

Figure Lengend Snippet: ErbB2 receptor tyrosine kinase enriches with internalized HPV16 PsVs in endosomes. (A) Western blot analysis of Rab5, major capsid protein L1 (anti-L1 312F Ab) and CD151 in sucrose flotation density gradient fractions. Rab5 serves as a marker for early endosomes (red frame) and CD151 as positive control for HPV co-internalized receptor component. Note: Rab5 exhibits a dual presence in cytosolic complexes and a membrane-associated state. It is noteworthy that only a small fraction of Rab5, specifically that which is associated with intact endosomes, has the capability to enter the gradient and subsequently accumulates in fraction 6 (endosomes). The remaining Rab5, which does not form complexes with intact endosomes e.g., cytosolic complexes, remains in the loaded post-nuclear supernatant within fractions 11-13. NHEK cells were treated with PsVs for 7 hours (7 h) or left untreated (0 h) and endosomes were isolated by density gradient centrifugation. (B) Label-free quantitative mass spectrometry (MS) analysis in NHEK and HaCaT detected enrichment of cellular proteins in endosomal fractions after cells’ exposure to HPV16 PsVs (see also Supplementary Table S1 ). Upper panel: Venn diagram of cellular proteins which were detected as > 2-fold enriched at 7 h compared to the untreated control are displayed for NHEK and HaCaT. Lower panel: String-db analysis ( https://string-db.org/ ) of > 2-fold enriched proteins found in endosomal fractions of both NHEK and HaCaT cells at 7 h when compared to untreated cells: Arf6 (ARF6_HUMAN), Cldn3 (CLD3_HUMAN), CHC22 (CLH2_HUMAN), ErbB2/HER2/neu (ERBB2_HUMAN), Gα olf (GNAL_HUMAN), Gnat2 (GNAT2_HUMAN), HLA-A (1A34_HUMAN), HSPA7/HSP70B (HSP77_HUMAN), Lano/LRRC1 (LRRC1_HUMAN), Rab39B (RB39B_HUMAN), Rab3A (RAB3A_HUMAN), Rab3B (RAB3B_HUMAN) and Rap2A (RAP2A_HUMAN). Lines indicate protein interaction networks with an interaction score of 0.700 (high confidence) for thick lines, 0.400 (medium confidence) for medium lines, and 0.150 (low confidence) for thin lines. (C) Graphs with parts per million (ppm) values of total protein for ErbB2 (in red), EGFR (in black), and LAMP1 (in grey) determined by MS of endosomal fractions taken at 0, 4 and 7 h post virus addition (depicted as 0 h, 4 h and 7 h, respectively). ErbB2 is enriched in endosomes during the time course of HPV16 entry in NHEK (left panel) and HaCaT (right panel) cells. As expected, LAMP1 shows comparable endosome content as it is located in all stages of endosomes . (D) Representative CLSM image of ErbB2 and L1 co-localization in NHEK. Cells were fixed at 7 h and stained with monoclonal anti-ErbB2 antibody (green) and polyclonal rabbit anti-L1 antiserum K75 (red). Nucleus is depicted as a dotted line. Box in the upper left overview indicates the area shown as magnified views.

Article Snippet: A rabbit pAb raised against CD151 (ab185684) used for STED experiments was purchased from (Abcam, Amsterdam, Netherlands) and CD151-specific mouse mAb antibody (11G5a) for WB from Bio-Rad (Munich, Germany). β-actin (A5441) and α-tubulin (B-5-1-2) specific mouse mAbs were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Western Blot, Marker, Positive Control, Membrane, Isolation, Gradient Centrifugation, Mass Spectrometry, Control, Virus, Staining

Journal: Frontiers in Immunology

Article Title: ErbB2/HER2 receptor tyrosine kinase regulates human papillomavirus promoter activity

doi: 10.3389/fimmu.2024.1335302

Figure Lengend Snippet: ErbB2 depletion neither affects HPV16 PsVs binding to the cell-surface nor the density of CD151-maxima at PsV-binding sites. (A, B) HaCaT cells were treated either with a control siRNA (contr.) or a mixture of two ErbB2-specific siRNAs, siRNA #1 and #3 (- ErbB2). 48 h later control cells were either left untreated or were treated with PEI (contr. + PEI) for 1 h. All cells were exposed to the HPV16 PsVs for 1 h at 4°C to prevent virus endocytosis, washed and processed for Western blot (WB). (A) WB showing surface-bound L1 detected using anti-L1 (312-F) Ab. β-actin was used as loading control. (B) Quantification of the surface-bound L1 from WBs as shown in (A) . Values (n = 8 – 11) are given as means ± SEM and the mean for contr. was set to 100%. The statistical difference between the two groups was analyzed with the Mann-Whitney test (p = 0.1014 for contr. vs. ErbB2; p < 0.0001 for contr. vs. contr. + PEI and ErbB2 vs. contr. + PEI). (C) HaCaT cells were transfected either with control or ErbB2 targeting siRNA (- ErbB2) and after 48 h incubated with HPV16 PsVs for 3 h. Then membrane sheets were generated, fixed, permeabilized and stained. EdU-PsVs (red) were visualized by click-labeling of the plasmid DNA in the confocal channel, whereas CD151 (green) was visualized by antibody labelling in the STED-channel. Images from the same channels are scaled equally and are displayed using a linear lookup table. (D) An image algorithm detects local maxima in the PsV- images. At the maxima positions of the PsVs, 925 nm diameter circular ROIs (37-pixel-diameter) were placed, in which the number of CD151-maxima was counted. The percentage of PsVs is plotted versus the number of their neighbored CD151-maxima. Values (n = 3) are given as means ± SD. The statistical difference between the two groups was analyzed with the Welch’s t test and showed no significant effect. p ≤ 0.001 ***, ns (not significant).

Article Snippet: A rabbit pAb raised against CD151 (ab185684) used for STED experiments was purchased from (Abcam, Amsterdam, Netherlands) and CD151-specific mouse mAb antibody (11G5a) for WB from Bio-Rad (Munich, Germany). β-actin (A5441) and α-tubulin (B-5-1-2) specific mouse mAbs were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Binding Assay, Control, Virus, Western Blot, MANN-WHITNEY, Transfection, Incubation, Membrane, Generated, Staining, Labeling, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: ErbB2/HER2 receptor tyrosine kinase regulates human papillomavirus promoter activity

doi: 10.3389/fimmu.2024.1335302

Figure Lengend Snippet: ErbB2-targeting inhibitors tucatinib and CP-724714 inhibit ErbB2 phosphorylation but do not affect the density of CD151-maxima at PsVs binding-sites. (A, B) HaCaT cells were treated with different concentration (in µM) of either control (contr.) or ErbB2-targeting inhibitors for 1 h and then processed for Western blot (WB) analysis (upper panels). Contr. stands for control (DMSO solvent; the medium of control-treated cells and inhibitor-treated cells contained equal amount of DMSO), T for tucatinib, CP for CP-724714, and Y for tyrosine phosphorylation sites. Values are shown as means ± SEM with the mean for the contr. set to 100% (lower panels). β-actin was used as a loading control as indicated. (A) WBs show p-ErbB2 (Y877) and total ErbB2 for the indicated contr. and inhibitor concentrations. Phosphorylated ErbB2 was detected using anti-p-ErbB2 (Y877) and total ErbB2 with anti-ErbB2 Abs. Ratio of p-ErbB2 (Y877) to total ErbB2, related to actin. The statistical analysis between the two groups of interest (n = 4) was analyzed with the Mann-Whitney test (p = 0.0286 for contr. 1 vs. T 1) and the Welch’s t test (p = 0.0098 for contr. 2.5 vs. T 2.5, p = 0.0090 for contr. 2.5 vs CP 2.5, p = 0.0020 for contr. 5 vs. CP 5). (B) WBs show p-ErbB2 (Y1221/1222) and total ErbB2. Phosphorylated ErbB2 was detected using anti-p-ErbB2 (Y1221/1222) and total ErbB2 with anti-ErbB2 Abs. β-actin was used as a loading control. Ratio of p-ErbB2 (Y1221/1222) to total ErbB2, related to actin. The statistical difference between the two groups (n = 4 – 5) was analyzed with the Welch’s t test (p = 0.2172 for contr. 1 vs. T 1, p = 0.0023 for contr. 5 vs. CP 5) and with the Mann-Whitney test (p = 0.0079 for contr. 2.5 vs. T 2.5, p = 0.1058 for contr. 2.5 vs. CP 2.5). (C) HaCaT cells were treated with control buffer (contr.) or 1 µM or 2.5 µM of the ErbB2-specific inhibitor tucatinib (T) for 1 h prior to PsVs addition for 3h. Then, membrane sheets were generated, fixed, permeabilized and stained. EdU-PsVs (red) were visualized by click-labeling of the plasmid DNA in the confocal channel, whereas CD151 (green) was visualized by antibody staining in the STED-channel. Images from the same channels are scaled equally and are displayed using a linear lookup table. (D) An image algorithm detects local maxima in the PsV- images. At the maxima positions of the PsVs, 925 nm diameter circular ROIs (37-pixel-diameter) were placed, in which the number of CD151-maxima was counted. The percentage of PsVs is plotted versus the number of their neighbored CD151-maxima. Values (n = 3) are given as means ± SD. The statistical difference between the two groups was analyzed with the Welch’s t test and showed no significant effect. p ≤ 0.05 *, p ≤ 0.01 **, ns (not significant).

Article Snippet: A rabbit pAb raised against CD151 (ab185684) used for STED experiments was purchased from (Abcam, Amsterdam, Netherlands) and CD151-specific mouse mAb antibody (11G5a) for WB from Bio-Rad (Munich, Germany). β-actin (A5441) and α-tubulin (B-5-1-2) specific mouse mAbs were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Binding Assay, Concentration Assay, Control, Western Blot, Solvent, MANN-WHITNEY, Membrane, Generated, Staining, Labeling, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion

doi: 10.1371/journal.ppat.1007471

Figure Lengend Snippet: ( A ) Sequence analysis of ORF3 protein. Amino acid sequences of ORF3 from HEV genotypes 1–8 (GenBank accession numbers AB740232, AF444002, M74506, AJ272108, AB573435, AB856243, KJ496144 and KX387866) were aligned using ClustalW . Segment aa 30–53 predicted as transmembrane passage by TMPred is boxed in grey . A consensus secondary structure was predicted using algorithms MLRC, DSC and PHD (available at https://npsa-prabi.ibcp.fr ) and is shown below the alignment (c, random coil; h, α-helix; e, extended strand;?, discrepant prediction). The degree of aa physicochemical conservation at each position is shown on the bottom line and can be inferred with the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) . ( B ) ORF3 protein oligomerization was analyzed by FLAG immunoprecipitation. Lysates (Input) of U-2 OS cells transfected with pCMVORF3-HA and/or pCMVORF3-FLAG as well as immunoprecipitates (IP: FLAG) were subjected to immunoblot with either specific anti-FLAG or anti-HA antibodies. The presence of a strong signal for ORF3-HA after pull-down of ORF3-FLAG indicates oligomerization of ORF3 protein in cells. ( C ) FRET analyses reveal oligomerization of HEV ORF3. CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein) fused to the C-termini of HEV ORF3 segments aa 1–113, 1–94, 1–70, 1–53, 28–113 or 53–113 were co-expressed in U-2 OS cells. FRET analyses were performed by the acceptor photobleaching method as described in the Materials and Methods section. The CFP-YFP fusion protein and cotransfection of unfused CFP and YFP served as positive and negative controls, respectively. Box-and-whisker plots represent the median FRET efficiency (FRETeff) values of 20 measurements (middle line), the values from the lower to the upper quartile (central box), and the minimum and maximum values (vertical line). The significance of the observed differences was assessed as described in Materials and Methods (*, P<0.0001).

Article Snippet: Polyclonal antibody (pAb) against HA tag (Y-11) and mouse mAbs 11G5a against CD151 were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Sequencing, Immunoprecipitation, Transfection, Western Blot, Cotransfection, Whisker Assay

Journal: PLoS Pathogens

Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion

doi: 10.1371/journal.ppat.1007471

Figure Lengend Snippet: ( A ) N- and C-terminal ends of HEV ORF3 protein are intracellularly exposed. S10-3 cells were transfected with pCMV_FLAG-ORF3-HA and subjected to immunfluorescence detection of HA and FLAG tags, using rabbit pAb anti-HA (Y-11) and mouse mAb anti-FLAG M2, respectively, after permeabilization with 0.5% saponin (Perm. Sap. 0.5%) or in the absence of permeabilization (No Perm.). ( B ) Similarly, S10-3 cells transfected with pCMVORF3 were subjected to immunofluorescence to detect the plasma membrane tetraspanin CD151 (mouse mAb 11G5a), the cytoplasmic protein MAVS (rabbit pAb anti-MAVS) or HEV ORF3 protein using mAb MRB198. Nuclei were stained by DAPI. ( C ) Selective membrane permeabilization. S10-3 cells were transfected with pCMVORF3 or co-transfected with pUHD15-1 and pUHD-Cp7 allowing the expression of the hepatitis C virus (HCV) core-p7 region (top panel) and cultured for 24 h. S10-3 cells were transfected with the HEV p6 infectious clone (middle panel) and cultured for 5 d. All cells were fixed and permeabilized with either 0.2% or 0.01% digitonin. Immunofluorescence detection of the cytoplasmic HCV core with mouse mAb C7-50 or the endoplasmic reticulum luminal HCV E1 glycoprotein with mouse mAb A4 served as controls for selective permeabilization of intracellular membranes. HEV ORF3 protein is detected using anti-ORF3 mAb MRB198. The lower panel shows histograms summarizing fluorescence intensities, as determined by using ImageJ software in 10 to 35 cells per condition, obtained after immunofluorescence with total (Dig. 0.2%) or selective (Dig. 0.01%) membrane permeabilization of S10-3 cells replicating the HEV p6 infectious clone. The asterisk (*) indicates statistically significant results with p<0.001. Scale bars indicate 10 μm.

Article Snippet: Polyclonal antibody (pAb) against HA tag (Y-11) and mouse mAbs 11G5a against CD151 were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Transfection, Immunofluorescence, Clinical Proteomics, Membrane, Staining, Expressing, Virus, Cell Culture, Fluorescence, Software

Journal: Journal of Virology

Article Title: Recombinant Hepatitis E Viruses Harboring Tags in the ORF1 Protein

doi: 10.1128/JVI.00459-19

Figure Lengend Snippet: HA-tagged ORF1 protein partially colocalizes with exosomal markers. Hep293TT cells transfected with full-length B2HA or parental (wild type [wt]) HEV RNA were fixed 5 days posttransfection and subjected to immunofluorescence using rabbit MAb C29F4 against HA (red) and antibodies against markers for different cell compartments (green), including protein disulfide-isomerase (PDI) for the endoplasmic reticulum (ER), ERGIC-53 for the ER-Golgi intermediate compartment, GM130 for the Golgi apparatus, MAVS for mitochondria, and the tetraspanins CD63 and CD151 for exosomes (see Materials and Methods for abbreviations and the list of antibodies used). Cell nuclei were counterstained with DAPI (blue). White squares indicate fields shown in higher magnification on the right. Arrowheads denote examples of colocalization. The scale bar represents 20 μm. Means ± SEM of Pearson's correlation coefficients (coeff.) determined for each condition ( n ≥ 10 cells each) are represented as histograms in the bottom panel.

Article Snippet: Mouse MAbs against ERGIC-53 (OTI108), PDI (1D3), and CLIMP63 (G1/296) were from Enzo Life Sciences (Farmingdale, NY), and MAbs against CD63 (MX491295) and CD151 (11G5a) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Transfection, Immunofluorescence

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Characteristics of CD151 and integrin maxima . CD151-GFP was transfected into HaCaT cells. CD151-GFP and endogenous CD151 localize to the same domains (Fig. ) and CD151-GFP is as functional as non-tagged CD151 (Fig. ). One day after transfection, cells were treated for 5 h without or with PsVs (Fig. ), washed, membrane sheets were generated, stained and analyzed by three channel STED microscopy. Green lookup table, CD151-GFP visualized by nanobodies; red and cyan lookup tables, integrin α6 and integrin α3 stained by antibody labeling, respectively. Images are displayed at arbitrary intensity scalings (linear lookup tables). Staining with the integrin antibodies is highly specific (Fig. ). ( A ) Large panel, membrane sheet (channel overlay). The white box marks an area from which magnified views of the individual channels are shown. From regions of interest (ROIs) we measured ( B ) maxima size and signal overlap ( C ). For ( C ), for all three channels within a ROI the pixels with an intensity higher than the average ROI intensity were selected. Then, the number of pixels positive in all three channels were related to the number of all positive pixels in one specific channel as indicated. ( D ) Shortest inter-maxima distances of CD151-GFP to CD151-GFP, integrin α3 or integrin α6. Values are given as means ± SD (n = 60 membrane sheets collected from three biological replicates).

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Functional Assay, Membrane, Generated, Staining, Microscopy, Antibody Labeling

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Viral particle distance to CD151 and integrin maxima . ( A – C ) Membrane sheets were generated from HaCaT cells treated for 5 h with EdU-PsVs. They were stained for EdU-PsVs (cyan lookup table; click-labelled with fluorescein), CD151 (green lookup table; immunostaining) and integrin α6 (red lookup table; immunostaining), followed by STED microscopy imaging. ( A ) Images from the three channels and an overlay are shown and displayed at arbitrary intensity scalings (linear lookup tables). Circles mark identical pixel locations. ( B ) Pearson correlation coefficient between PsVs and CD151 (left) and integrin α6 (right). A control value for randomized distribution (‘flipped’) was calculated after flipping of one channel. ( C ) For individual PsVs, we plotted their nearest distance to a CD151 and an integrin α6 maximum (the analysis includes 4452 PsVs from 57 membrane sheets collected from three biological replicates). ( D – F ) As in ( A – C ) but cells were analysed and we stained for integrin α3 (the plot in F includes 8519 analyzed PsVs from 60 cells collected from three biological replicates). The pattern of PsV binding to the cell membrane looks the same on cells and membrane sheets (Fig. ). Values are given as means ± SD (n = 57 membrane sheets or 60 cells, each collected from three biological replicates). Statistical analysis was performed employing the unpaired Student’s t-test comparing the original to the flipped images (***p < 0.001). Red boxes frame viral particles potentially used for further analysis of the platform area (Fig. ). Only PsVs were considered that were closer than 250 nm to bright CD151/integrin maxima.

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Membrane, Generated, Staining, Immunostaining, Microscopy, Imaging, Control, Binding Assay

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Local crowding of clusters at virus attachment sites . To characterize the anatomy of a possibly forming viral platform area, we selected those PsVs from Fig. with a distance smaller than 250 nm to both a bright CD151 and integrin maximum (for examples see centrally located PsVs in the left panels in A and B). In a PsV-centred 1 µm × 1 µm ROI the number of bright maxima (see methods) in the CD151 and integrin channels were counted. The same was performed after flipping the CD151/integrin channels, causing a randomized relationship between PsV-attachment sites and maxima (A and B, right panels). In the original images more closely associated maxima are present at sites of PsV-attachment. Images are displayed in cyan (PsV) green (CD151) and red (integrin) at arbitrary intensity scalings (linear lookup tables). ( C , D ), quantification of the bright maxima in the 1 µm × 1 µm ROI. Values are given as means ± SD (n = 678 ( C ) and 283 ( D ) 1 µm × 1 µm ROIs, respectively). Statistical analysis was performed employing the unpaired Student’s t-test comparing the original to the randomized condition (***p < 0.001). Please note, that the number of clusters per attachment site was highly variable. For a histogram illustrating the variability see Fig. .

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Virus

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Integrin α3 and integrin α6 knockdown differentially affect the CD151 level of the cell membrane and PsV-binding . HaCaT cells were transfected with siRNA and two days later incubated without or with HPV16 PsVs for 5 h. Cells were fixed, permeabilized, immunostained for CD151 (green) and L1 (red), and imaged by STED microscopy. Images are displayed employing linear lookup tables. For A and D different scalings were applied, although all panels in A or D have the same arbitrary scaling. ( A ) Integrin α6 knockdown. For each condition, overlays from an overview and a magnified view are shown. ( B ) Average CD151 immunostaining intensity and ( C ) the PsVs particle density (100% correspond to 1.4 particles/μm 2 ). ( D – F ) As ( A – C ), showing the integrin α3 knockdown (100% PsV density correspond to 1.2 particles/μm 2 ). Values are shown as means ± SD (n = 60 cells collected from three biological replicates). The unpaired Student’s t-tests compare the conditions as indicated by the bars ( B , E ) or PsV to PsV and knockdown ( C , F ) (***p < 0.001; **p < 0.01; *p < 0.05).

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Membrane, Binding Assay, Transfection, Incubation, Microscopy, Immunostaining

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Large CD151 patches coincide with intracellular actin accumulations . CD151-GFP transfected HaCaT cells were treated for 3 h without or with PsVs. Membrane sheets were generated, stained, and imaged by confocal microscopy. Green (CD151-GFP; GFP signal was enhanced by nanobodies), red (PsVs visualized by L1 antibody labeling) and cyan (filamentous actin; fluorescently labelled phalloidin). Images are displayed using a linear lookup table. For each channel, the same arbitrary scaling was applied. ( A ) For each condition a membrane sheet is shown. Magnified views from the white boxes are shown, illustrating the individual channels. ( B ) For the CD151 and the actin channels, within a freehand ROI excluding membrane edges, the pixels with an intensity higher than the average ROI intensity were selected. Then, the number of pixels positive in both channels were related to the number of all positive pixels in the CD151 channel. Values are given as means ± SD (n = 45 membrane sheets collected from three biological replicates). Unpaired Student’s t-test (***p < 0.001).

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Membrane, Generated, Staining, Confocal Microscopy, Antibody Labeling

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Integrin α3 or integrin α6 knockdown inhibits viral capsid processing and infection . Integrin α3 or integrin α6 were knocked down in HaCaT cells by siRNA transfection (for knockdown efficiency see Fig. ). ( A ) Two days after transfection, cells were incubated for 24 h with HPV16 PsVs, washed, lysed and analyzed by Western blot for the viral protein L1 and its ~25 kDa cleavage product. For clarity, lanes were cropped from original blots shown in full in Fig. (L1) and (actin). Values were related to the control which was set to 100% and are given as means ± SD (n = 3 independent experiments). ( B , C ) Two days after transfection, cells were incubated for 5 h with HPV16 PsVs, washed fixed, stained with an antibody that recognizes L1 after capsid disassembly, and imaged by confocal microscopy taking an optical section from the cell body. ( B ) Actin (cyan) and L1–7 (inverted greyscale) each are displayed at the same arbitrary scaling (linear lookup tables). From the optical section ( B ), an image analysis algorithm counted the number of detected vesicles per cell ( C ) and quantified the vesicle staining intensity (Fig. ). Values are given as means ± SD (n = 60 analysed cells collected from three biological replicates). ( D ) HaCaT cells were transfected and incubated with PsVs as in ( A ) with the difference that on the encapsidated plasmid luciferase expression is under the control of the HPV16 promoter instead of the CMV promoter. One day after adding PsVs, cells were lysed and the infection rate was assessed by analysing the luciferase activity. For normalization to cell number, luciferase activity was related to the dehydrogenase activity. Values are expressed as percent of control (average of control was set to 100%). Values are given as means ± SD (n = 20–21 technical replicates collected from five biological replicates). Unpaired Student’s t-test, comparing control to knockdown conditions (***p < 0.001; **p < 0.01).

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Infection, Transfection, Incubation, Western Blot, Control, Staining, Confocal Microscopy, Plasmid Preparation, Luciferase, Expressing, Activity Assay

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Knockdown of integrin α6 but not of integrin α3 strongly inhibits PsV binding. Two days after siRNA transfection, cells were incubated for 1 h at 0 °C with HPV16 PsVs, scraped from the substrate, washed, lysed and analyzed by Western blot. Full Western blots are shown in Fig. (L1) and (actin). Binding of viral particles was assayed by quantification of the L1 ~55 kDa band. The control value was set to 100% and used for normalization. Values are given as means ± SD (n = 4 independent experiments). Unpaired Student’s t-test, comparing control to knockdown conditions (***p < 0.001; *p < 0.05).

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Binding Assay, Transfection, Incubation, Western Blot, Control

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Characteristics of CD151 and integrin maxima . CD151-GFP was transfected into HaCaT cells. CD151-GFP and endogenous CD151 localize to the same domains (Fig. ) and CD151-GFP is as functional as non-tagged CD151 (Fig. ). One day after transfection, cells were treated for 5 h without or with PsVs (Fig. ), washed, membrane sheets were generated, stained and analyzed by three channel STED microscopy. Green lookup table, CD151-GFP visualized by nanobodies; red and cyan lookup tables, integrin α6 and integrin α3 stained by antibody labeling, respectively. Images are displayed at arbitrary intensity scalings (linear lookup tables). Staining with the integrin antibodies is highly specific (Fig. ). ( A ) Large panel, membrane sheet (channel overlay). The white box marks an area from which magnified views of the individual channels are shown. From regions of interest (ROIs) we measured ( B ) maxima size and signal overlap ( C ). For ( C ), for all three channels within a ROI the pixels with an intensity higher than the average ROI intensity were selected. Then, the number of pixels positive in all three channels were related to the number of all positive pixels in one specific channel as indicated. ( D ) Shortest inter-maxima distances of CD151-GFP to CD151-GFP, integrin α3 or integrin α6. Values are given as means ± SD (n = 60 membrane sheets collected from three biological replicates).

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Functional Assay, Membrane, Generated, Staining, Microscopy, Antibody Labeling

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Viral particle distance to CD151 and integrin maxima . ( A – C ) Membrane sheets were generated from HaCaT cells treated for 5 h with EdU-PsVs. They were stained for EdU-PsVs (cyan lookup table; click-labelled with fluorescein), CD151 (green lookup table; immunostaining) and integrin α6 (red lookup table; immunostaining), followed by STED microscopy imaging. ( A ) Images from the three channels and an overlay are shown and displayed at arbitrary intensity scalings (linear lookup tables). Circles mark identical pixel locations. ( B ) Pearson correlation coefficient between PsVs and CD151 (left) and integrin α6 (right). A control value for randomized distribution (‘flipped’) was calculated after flipping of one channel. ( C ) For individual PsVs, we plotted their nearest distance to a CD151 and an integrin α6 maximum (the analysis includes 4452 PsVs from 57 membrane sheets collected from three biological replicates). ( D – F ) As in ( A – C ) but cells were analysed and we stained for integrin α3 (the plot in F includes 8519 analyzed PsVs from 60 cells collected from three biological replicates). The pattern of PsV binding to the cell membrane looks the same on cells and membrane sheets (Fig. ). Values are given as means ± SD (n = 57 membrane sheets or 60 cells, each collected from three biological replicates). Statistical analysis was performed employing the unpaired Student’s t-test comparing the original to the flipped images (***p < 0.001). Red boxes frame viral particles potentially used for further analysis of the platform area (Fig. ). Only PsVs were considered that were closer than 250 nm to bright CD151/integrin maxima.

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Membrane, Generated, Staining, Immunostaining, Microscopy, Imaging, Control, Binding Assay

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Local crowding of clusters at virus attachment sites . To characterize the anatomy of a possibly forming viral platform area, we selected those PsVs from Fig. with a distance smaller than 250 nm to both a bright CD151 and integrin maximum (for examples see centrally located PsVs in the left panels in A and B). In a PsV-centred 1 µm × 1 µm ROI the number of bright maxima (see methods) in the CD151 and integrin channels were counted. The same was performed after flipping the CD151/integrin channels, causing a randomized relationship between PsV-attachment sites and maxima (A and B, right panels). In the original images more closely associated maxima are present at sites of PsV-attachment. Images are displayed in cyan (PsV) green (CD151) and red (integrin) at arbitrary intensity scalings (linear lookup tables). ( C , D ), quantification of the bright maxima in the 1 µm × 1 µm ROI. Values are given as means ± SD (n = 678 ( C ) and 283 ( D ) 1 µm × 1 µm ROIs, respectively). Statistical analysis was performed employing the unpaired Student’s t-test comparing the original to the randomized condition (***p < 0.001). Please note, that the number of clusters per attachment site was highly variable. For a histogram illustrating the variability see Fig. .

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Virus

Journal: Scientific Reports

Article Title: Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

doi: 10.1038/s41598-020-62202-9

Figure Lengend Snippet: Integrin α3 and integrin α6 knockdown differentially affect the CD151 level of the cell membrane and PsV-binding . HaCaT cells were transfected with siRNA and two days later incubated without or with HPV16 PsVs for 5 h. Cells were fixed, permeabilized, immunostained for CD151 (green) and L1 (red), and imaged by STED microscopy. Images are displayed employing linear lookup tables. For A and D different scalings were applied, although all panels in A or D have the same arbitrary scaling. ( A ) Integrin α6 knockdown. For each condition, overlays from an overview and a magnified view are shown. ( B ) Average CD151 immunostaining intensity and ( C ) the PsVs particle density (100% correspond to 1.4 particles/μm 2 ). ( D – F ) As ( A – C ), showing the integrin α3 knockdown (100% PsV density correspond to 1.2 particles/μm 2 ). Values are shown as means ± SD (n = 60 cells collected from three biological replicates). The unpaired Student’s t-tests compare the conditions as indicated by the bars ( B , E ) or PsV to PsV and knockdown ( C , F ) (***p < 0.001; **p < 0.01; *p < 0.05).

Article Snippet: A mouse monoclonal antibody raised against GFP was obtained from Abcam (diluted 1:100 in IF; clone 9F9.F9, cat# ab1218, Cambridge, UK), a mouse monoclonal antibody against CD151 from Bio-Rad (diluted 1:100 in IF; clone 11G5a, cat# MCA1856, Hercules, CA), a mouse monoclonal against integrin α3 from Santa Cruz (diluted 1:100 in IF and 1:1,000 in Western blot (WB); clone A-3, cat# sc-374242, Dallas, TX), a rabbit polyclonal against integrin α6 from Thermo Fisher (diluted 1:200 in IF and 1:1,000 in WB; cat# PA5-12334, Waltham, MA), a mouse monoclonal against L1 from Novus Biologicals (diluted 1:2,000 in WB; CamVir 1, cat# NB100-2732, Centennial, CO) and rabbit monoclonal antibodies against β-Actin (diluted 1:4,000 in WB; clone 13E5, cat# 4970) and CD151 (diluted 1:500 in IF; clone E9M8T, cat# 81626) from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Membrane, Binding Assay, Transfection, Incubation, Microscopy, Immunostaining