Journal: bioRxiv

Article Title: A chimeric Japanese encephalitis vaccine protects against lethal yellow fever virus infection without inducing neutralizing antibodies

doi: 10.1101/717181

Figure Lengend Snippet: Serological analysis of serum of JE-CVax-vaccinated and YFV-17D- or ZIKV-MR766-challenged animals. (A) Detection of nAbs against JEV and YFV. CPE neutralization tests (CPENT) for JE-CVax (•) and YFV-17D (■) were performed on sera d0 prior to vaccination (pre-immune, red), d28 after vaccination (blue) and after challenge (study endpoint, orange) for samples of JE-CVax-vaccinated AG129 mice, of JE-CVax-vaccinated mice after subsequent YFV-17D-challenge (n = 34), of mice hyperimmunized with JE-CVax (n = 13, first bleed two weeks post last booster immunization, blue) and of mice vaccinated with Ixiaro ® (green). Limit of detection (LOD) for virus neutralization was log 10 20 (1.3). Data presented as log 10 CPENT 50 (mean ± SD). The data presented are from n ≥ 3 independent experiments. Statistical significance was determined using one-way ANOVA analysis. ****p-value ≤ 0.0001 to mean log 10 CPENT 50 titers against JEV or YFV compared to mean log 10 CPENT 50 titers pre JE-CVax vaccination and pre YFV-17D-challenge, respectively. (B) Quantitation of anti-YFV NS1 binding antibodies by direct ELISA. Serum from naïve, non-vaccinated mice (red) or mice that had been vaccinated with either 10 3-5 PFU JE-CVax (blue), or that had been infected with 10 4 PFU YFV-17D (orange) or with 10 5 PFU ZIKV-MR766 (pink) were collected either 28 days post immunization or when euthanized at the humane endpoint (n ≥ 5). The data shown are means of two independent analyses. Statistical significance was determined using one-way ANOVA analysis. ** p-value ≤ 0.01 compared to YFV-17D. (C) Binding of serum antibodies to NS1 expressing cells. HEK-293 cells were transfected with a plasmid expressing YFV-17D NS1 as a transcriptional fusion to GFP (top), or infected with the YFV-17D-mCherry reporter virus (bottom). Either 48 h after transfection or 72 h after infection, cells were stained with the anti-YFV NS1-specific mAb 1A5 (mAb, left), with serum from mice that were vaccinated with JE-CVax (center), or with serum from naïve, non-vaccinated mice (right). Graph showing flow cytometric analysis of GFP or mCherry fluorescence and visualization of anti-YFV NS1 antibody binding using a PE-Cy7 conjugated goat anti-mouse IgG secondary antibody. The fraction of NS1 positive cells (GFP or mCherry) stained by mAb 1A5 or serum of JE-CVax-immunized mice (a-mouse IgG) is given in percentage in the upper right quadrant. Data from one representative experiment out of four independent experiments.

Article Snippet: Anti-NS1 antibody binding was detected by a biotinylated goat anti-mouse IgG secondary antibody solution (ThermoFisher Scientific, cat # A16076; 1:200 dilution).

Techniques: Neutralization, Quantitation Assay, Binding Assay, Direct ELISA, Infection, Expressing, Transfection, Plasmid Preparation, Staining, Fluorescence

Journal: bioRxiv

Article Title: A chimeric Japanese encephalitis vaccine protects against lethal yellow fever virus infection without inducing neutralizing antibodies

doi: 10.1101/717181

Figure Lengend Snippet: Gating strategy for flow cytometry analysis. (A, B) Exclusion of debris was achieved by gating out the FSC-low population in a FSC-A vs SSC-A plot. Then, only single cells were retained by elimination of the high SSC-W population, in a SSC-W vs SSC-H plot. In a subsequent step, live cells were selected by gating out the Zombie Aqua-positive population as shown here in an NS1-eGFP/FSC-A vs ZA plot. Finally for the detection of anti-NS1 antibodies (A) cells were gated based on positivity for NS1-eGFP and positivity/negativity of a-NS1 Ab PE-Cy7. (B) For intracellular cytokine staining, based on positivity for CD3e (eFluor450-conjugated) and negativity (CD4) or positivity for CD8a (APC/Cy7-conjugated), CD4 + and CD8 + T cell populations were defined as CD3 + CD8 − and CD3 + CD8 + populations, respectively. Finally, cells were gated based on positivity/negativity for IFN-γ and positivity/negativity of TNF-α. Samples from non-vaccinated mice were used to set the boundaries that define cells positive and negative for intracellular markers.

Article Snippet: Anti-NS1 antibody binding was detected by a biotinylated goat anti-mouse IgG secondary antibody solution (ThermoFisher Scientific, cat # A16076; 1:200 dilution).

Techniques: Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Schematic view of TBEV viral proteins. b. Comprehensive interactome analysis was conducted to study interactions between TBEV and host proteins. c. Viral baits expressed in HEK293T cells and analyzed by immunoblot assay using anti-Flag antibody. d, e. Enrichment analysis of TBEV-interacted host proteins (d) and the proteins (e) significantly deregulated by TBEV infection. f. UpSet plot showing the overlap of flavivirus-host protein–protein interactions from other studies.( , ) Each bar shows the interactions shared by only the marked studies at the bottom.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Western Blot, Infection, Protein-Protein interactions

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. An integrated protein-protein interaction network showed high-confidence TBEV-host interactions. Viral proteins are represented as rhombus nodes. Edge intensity reflects the MIST value associated with the interaction. Solid thick edges represent the most significant interactions, while light edges correspond to low MIST (Molecular Interaction Search Tool) value (0.5 < MIST < 0.99). b-f. Validation of virus-host interactions using co-immunoprecipitation. Flag-tagged empty vector (EV) or viral proteins were overexpressed in HEK293T cells, and CHUK co-precipitated with prM ( b ), SYVN1 co-precipitated with NS2B ( c ), SPCS1 co-precipitated with NS4A ( d ), endogenous Ephrin B1(EFNB1) co-precipitated with NS4B ( e ), along with PAF1 and LEO1 co-precipitated with NS5( f ), were detected using flag beads and immunoblot assay.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Biomarker Discovery, Virus, Immunoprecipitation, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. The schematic illustration outlines the network diffusion analysis process. The proteome results were initially integrated with the virus-host interactome, and subsequently mapped to the global human protein-protein interactome to identify affected host pathways. b. Subnetworks derived from network diffusion predict the correlation of TBEV NS5 with spliceosome (| Log 2 FC|>0.1, P value < 0.05). c. Subnetworks resulting from network diffusion depict the correlation of TBEV prM with factors involved in autophagy (|Log 2 FC|>0.1, P value < 0.05). d, e. Additional subnetworks derived from network diffusion predict the correlation of TBEV NS5 with DNA damage response ( d ) and cell cycle ( e ) (|Log 2 FC|>0.1, P value < 0.05). Black edges denote connections present in ReactomeFI. The thickness of the line reflects the combined score provided by STRING. The thickness of directed edges is proportional to the random-walk transition probability.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Diffusion-based Assay, Virus, Derivative Assay

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Vero cells were either mock-infected or infected with TBEV at various MOI (0.1, 1.0 and 10), cells were collected at 48 hours post infection (hpi) and their distribution was analyzed by flow cytometry. b. The experiments in ( a ) were repeated for three times, and the proportions of cells at different cell cycle phases were shown in column graph. c. Vero cells were mock-infected or infected with TBEV at the MOI of 1.0, cells were collected at 12, 18, 24, 36 and 48 hpi, and the percentages of cells in G0/G1 ( up ), S ( middle ) and G2/M ( down ) phase were analyzed by flow cytometry. d-i . Vero cells were synchronized to G0/G1 ( d ), S ( f ) and G2/M ( h ) phases via no-serum starvation, adding 0.85mM Thymi or 25 ng/mL Noco for 20 h. Subsequently, the cells were either mock-infected or infected with TBEV at an MOI of 10, cells were collected and analyzed using flow cytometry. The experiments were repeated for three times, and the proportion of cells in were shown in column graphs. j-l. Vero cells were synchronized to different stages and subsequently infected with TBEV, the mRNA level of TBEV envelope (E) gene was analyzed at 48 hpi by probe qPCR ( j ). The protein level of TBEV NS1 was analyzed at 48 hpi by immunoblot ( k, l ).

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Cells were transfected with the plasmids of Flag-tagged empty vector (EV) and ten TBEV viral proteins, the cell distribution was analyzed by flow cytometry. b. The Flag-EV, 0.5 and 1.0 μg Flag-NS5 were transfected into A549 cells, the cell distribution was analyzed by flow cytometry. The experiment was repeated for three times, and the percentage of cells were shown in column graph. c. Cells were transfected with Flag-EV or Flag NS5 together with HA-P300, the interaction between NS5 and P300 was analyzed by co-immunoprecipitation analysis. d. The colocalization of P300 (green) and NS5 (red) were examined by immunofluorescence analysis. Scale bar: 10 μm. e-g . Cells transfected with NS5 were collected after 48 h, the expression of CDK4, CDK6 and P16 was analyzed by immunoblot ( e ) and qPCR analysis ( f ). The relative expression of CDK4, CDK6 and P16 was shown in column graph ( g ). h. A549 cells transfected with HA-EV, HA P300 and P300 Hm were further transfected with NS5, the distribution of cells was analyzed by flow cytometry. The experiments were repeated for three times, and the proportions of cells were shown in column graph, the expression of indicated proteins was assessed by immunoblot assay. i. Cells transfected with P300 siRNA and NC siRNA (siNC) were further transfected with NS5, the distribution of cells was analyzed by flow cytometry, and the proportions of cells and the relative expression of P300 mRNA were shown in column graph. j. Cells transfected with HA-EV, HA P300 and P300 Hm were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. k. Cells transfected with P300 siRNA and NC siRNA were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. l. Outline of G0/G1 cell cycle arrest regulated by TBEV NS5.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Immunoprecipitation, Immunofluorescence, Expressing, Western Blot, Infection

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Cells were either mock-infected or infected with TBEV, and then collected at 12, 24, 36 and 48 hpi, the expression levels of cell cycle regulators were detected by immunoblot using indicated antibodies. M: mock, I: infection. b. Representative images of brain organoid generation from iPSCs. Scale bar, 500 µm. c. Microscopic images of brain organoids, either mock-infected or infected with TBEV at 0- and 3-days post-infection (dpi). Scale bar, 200 µm. d. Microscopic images depicting 42-day-old iPSC-derived organoids, featuring TUJ1 (neurons), SOX2 (progenitors), Olig2 (oligodendrocytes), and NeuN (neuronal nuclei) markers. Scale bar, 500 µm. e, f . Brain organoids were either mock-infected or infected with TBEV at the MOI of 2.0, the expression of CDK4 ( e ) and P16 ( f ) in brain organoids were examined using immunofluorescence analysis. CDK4 and P16 were visualized in red, TBEV NS1was visualized in green, and the nuclei were stained in blue. Scale bar: 50 μm.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Expressing, Western Blot, Derivative Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a, b . A549 cells were either mock-infected or infected with TBEV, the cells were then treated with indicated concentration of C646 ( a ) and CPI-637 ( b ), the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. c-f . Cells mock-infected or infected with TBEV at the MOI of 10 were treated with 10 μM C646 and CPI-637, the expression of TBEV NS1 protein was detected by immunoblot ( c, d ) and immunofluorescence analysis ( e ), and the cell cycle distribution was analyzed by flow cytometry ( f ). Scale bar: 50 μm. g. Cells transfected with HA-EV, HA P300 and P300Hm were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. h. Cells transfected with P300 siRNA and NC siRNA were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. i. A549 cells were treated with the indicated concentrations of chrysin for 2 h, the cells were then infected with TBEV, the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. j-l. Cells were treated with 10 μM chrysin for 2 h, the cells were then mock-infected or infected with TBEV at the MOI of 10, the expression of TBEV NS1 protein was detected by immunoblot ( j ), and the cell distribution was analyzed by flow cytometry ( k ). The percentage of cells ( l ) upon chrysin treatment was shown in column graphs. Scale bar: 50 μm.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Concentration Assay, Expressing, Western Blot, Immunofluorescence, Flow Cytometry, Transfection

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Virus-protein interaction network and predicted drug targets. Potential drug-protein interactions were predicted by NeDREx using the TrustRank algorithm, integrating data from various biomedical databases and experimentally validated targets. Nodes represent proteins and edges indicate their interactions. b-d. Cells were pretreated with indicated inhibitors targeting RIOK1/BRD9 ( b ), XIAP ( c ) and KAT6A ( d ) before TBEV infection for 48 h. The IC 50 value was determined using probe qPCR. Cell viability was assessed using cell counting (CCK-8) assay in the absence of viral infection. The percentage of viral titer compared with DMSO treatment (red) and cell viability (black) depicted. Error bars represent the mean ± SEM of three biological replicates.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Virus, Infection, Cell Counting, CCK-8 Assay

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: APPL1 is a part of the β-catenin-Reptin-HDAC1/2 complex. A , HEK293 cell lysates were subjected to a coimmunoprecipitation assay ( IP ) using anti-Reptin or control IgG, and the resultant precipitates were subjected to immunoblotting ( IB ) with the indicated antibodies. TCL , total cell lysate. B , a 2D BN/SDS-PAGE analysis of HEK293 cell extracts. Multiprotein complexes were resolved on 6–13% acrylamide gradient gel, followed by Tris-glycine-SDS-PAGE (8% gel). Immunoblotting was performed with specific antibodies recognizing the indicated proteins.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: Overexpression of APPL proteins reduces the amounts of HDACs bound to Reptin. A , HEK293 cells were transiently transfected with APPL1-Myc and APPL2-Myc. Forty-eight hours post-transfection, cell extracts were prepared, and a coimmunoprecipitation assay ( IP ) was performed with either anti-Reptin or control IgG. The resultant precipitates were analyzed by immunoblotting ( IB ) against the indicated antibodies ( A ) or were subjected to fluorometric assays for measuring the respective HDAC activities ( C ). Each HDAC activity assay was performed in duplicate, and relative fluorescence unit ( RFU ) values shown are the averages ± S.D. from which the RFU values of assay buffer were subtracted. B , HEK293 cells were cotransfected with plasmids encoding YFP-tagged APPL1 or empty vector. Cell extracts were prepared and immunoprecipitated using anti-HDAC2. Bound proteins were separated and visualized by immunoblotting using anti-HDAC2, anti-HDAC1, anti-Reptin, and anti-APPL1 antibodies.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Western Blot, HDAC Activity Assay, Fluorescence, Plasmid Preparation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: Recruitment of β-catenin, Reptin, and HDAC1 to the promoters of Wnt target genes is affected upon overexpression of APPL proteins. A , overexpression of APPL proteins reduces the amount of Reptin bound to β-catenin. HEK293 cells were transiently transfected with APPL1 or APPL2. Thirty-six hours post-transfection, cells were treated with Wnt3a-conditioned medium for 4 h. Subsequently, cell extracts were prepared, and a coimmunoprecipitation assay ( IP ) was performed with anti-β-catenin or control IgG. The resulting precipitates were analyzed by immunoblotting ( IB ) with the indicated antibodies. B , APPL proteins increase the recruitment of β-catenin and reduce the amounts of Reptin and HDAC1 at the Wnt target gene promoters. HEK293 cells were transfected with either empty pcDNA 3.1 ( lane 1 ) or plasmids expressing APPL1 ( lane 2 ) and APPL2 ( lane 3 ). Thirty-six hours post-transfection, cells were treated with Wnt3a-conditioned medium for 4 h and then subjected to chromatin immunoprecipitation using anti-β-catenin, anti-HDAC1, and anti-Reptin antibodies. PCR was performed from these immunoprecipitates by using the primer pair covering the β-catenin binding sites at the promoters of cyclin D1 and Axin2. PCR products were resolved by agarose gel and stained with ethidium bromide. Lane c , PCR mixture without template.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, Agarose Gel Electrophoresis, Staining