pglyrp2  (Novus Biologicals)

Journal: The Journal of Clinical Investigation

Article Title: PGLYRP2 drives hepatocyte-intrinsic innate immunity by trapping and clearing hepatitis B virus

doi: 10.1172/JCI188083

Figure Lengend Snippet: ( A ) Schematic overview of screening for HBV promoter–binding proteins. Diagram illustrating the experimental design for identifying proteins that bind to HBV promoter regions. ( B ) Identification of HBV promoter–binding host proteins. Proteins bound to HBV promoter DNA were isolated using a biotin-streptavidin affinity pull-down assay and visualized on a 12% SDS-PAGE gel with silver staining to confirm purity and presence. ( C ) ChIP assays were conducted to examine the interaction between PGLYRP2 and HBV promoter DNA. Anti-PGLYRP2 antibodies were used to pull down the relevant DNA-protein complexes from HBV + and HBV – tissue lysates (Input). ( D ) Single-cell transcriptomic analysis by t-distributed stochastic neighbor embedding (t-SNE). Left: t-SNE plots illustrating the distribution of hepatocytes sampled at 4 distinct time points, displayed in 4 different colors. Right: A separate t-SNE plot highlights expression levels of PGLYRP2 across these cells. D, day; W, week. ( E ) Bar plots showing expression levels of PGLYRP2 in hepatocytes, as derived from the t-SNE analysis. The y axis represents log-normalized expression levels, emphasizing differences across time points. ( F ) Age-related expression of Pglyrp2 in mouse liver. Real-time PCR and Western blot analyses were used to measure PGLYRP2 levels across various age groups in mouse liver, illustrating an age-dependent expression pattern. ( G ) Dnmt3a expression analysis in mouse liver by age group. Real-time PCR was used to assess the expression of Dnmt3a across different age groups, revealing a decline in expression with age. Dots indicate biological replicates ( n = 3 independent experiments). ( H ) Correlation between DNMT3A and PGLYRP2 expression. The relationship between Dnmt3a and Pglyrp2 mRNA levels was quantitatively analyzed in mouse liver tissues, highlighting a significant negative correlation. Data are represented as mean ± SD. Kruskal-Wallis with Dunn’s post hoc multiple-comparison test ( E ) and 1-way ANOVA with post hoc Bonferroni’s test ( F and G ) were used for statistical analysis. Pearson’s correlation coefficient was used in H . * P < 0.05; ** P < 0.001.

Article Snippet: The assay began with an overnight incubation at 4°C, where mouse anti-HBc antibody (NB110-7396, Novus) and rabbit anti-PGLYRP2 antibody (NBP2-32042, Novus) were applied to the samples, followed by the Streptavidin PLA-PLUS probe (Merck).

Techniques: Binding Assay, Isolation, Pull Down Assay, SDS Page, Silver Staining, Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Comparison

Journal: The Journal of Clinical Investigation

Article Title: PGLYRP2 drives hepatocyte-intrinsic innate immunity by trapping and clearing hepatitis B virus

doi: 10.1172/JCI188083

Figure Lengend Snippet: ( A ) Schematic representation depicts the HBV promoter–luciferase reporter construct containing BCP/Enh II, Enh I, and NRE regions, alongside a CMV promoter–driven luciferase reporter for control purposes (top). The cotransfection setup in C3A cells (bottom) involves varying concentrations of PGLYRP2 expression plasmid (0–400 ng) or a control vector, alongside a Renilla luciferase reporter to assess transfection efficiency. Dots indicate biological replicates ( n = 3–12; independent experiments). ( B ) Truncated HBV promoter constructs assessed for luciferase activities with PGLYRP2 cotransfection. Dots indicate biological replicates (right, n = 3). ( C ) Schematic representation of HBV genome (1.1 copies) under a Tet-off CMV promoter in HepAD38 cells. ( D – G ) Monitoring HBV replication in modified HepAD38 cell lines. HepAD38 lines (control, PGLYRP2 expressing,or PGLYRP2 knockdown) either control vector, PGLYRP2, or shRNA against PGLYRP2 were cultured in Tet-free conditions for 9 days. Intracellular pgRNA ( D ), extracellular/ intracellular HBV DNA ( E and F ), and supernatant HBeAg/HBsAg ( G , ELISA) were analyzed. ( H and I ) HBV infection assay in modified Huh7-NTCP and PHH cells (control or PGLYRP2-suppressed). Levels of intracellular pgRNA and HBV DNA after infection were analyzed (day 9; n = 3). ( J ) cccDNA quantification in HepAD38 cells treated with ExoI/ExoIII/T5 nuclease (day 9 in Tet-free medium). PCR ( J ; n = 3) and Southern blot ( K , normalized to control). Western blot shows PGLYRP2/GAPDH levels. ( L ) Evaluation of cccDNA binding activity by EMSA. Purified PGLYRP2 or control peptides were incubated with Mfei-cut cccDNA, resolved via native agarose gel, and analyzed by Southern and Western blotting. Data are represented as mean ± SD. Two-tailed Student’s t test ( A , left), 1-way ANOVA with post hoc Bonferroni’s test ( A , right, and G ), 2-way ANOVA with post hoc Bonferroni’s test ( D – F ), and 2-tailed Student’s t test ( B and H – J ) were used for statistical analysis. * P < 0.05; ** P < 0.001.

Article Snippet: The assay began with an overnight incubation at 4°C, where mouse anti-HBc antibody (NB110-7396, Novus) and rabbit anti-PGLYRP2 antibody (NBP2-32042, Novus) were applied to the samples, followed by the Streptavidin PLA-PLUS probe (Merck).

Techniques: Luciferase, Construct, Control, Cotransfection, Expressing, Plasmid Preparation, Transfection, Modification, Knockdown, shRNA, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Southern Blot, Western Blot, Binding Assay, Activity Assay, Purification, Incubation, Agarose Gel Electrophoresis, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: PGLYRP2 drives hepatocyte-intrinsic innate immunity by trapping and clearing hepatitis B virus

doi: 10.1172/JCI188083

Figure Lengend Snippet: ( A – C ) Schematic representation and functional assays of PGLYRP2 variants. ( A ) Top: Schematic representation of full-length (FL) and truncated forms of PGLYRP2. Bottom: Cotransfection assays in C3A cells used HBV promoter–luciferase reporter constructs with either FL or truncated PGLYRP2 plasmids, alongside HBV or HBc expression constructs. ( B ) ELISA and PCR analysis in stable HepAD38 cell lines for supernatant levels of HBsAg and HBeAg. ( C ) Real-time PCR determined the levels of intracellular HBV DNA. ( D ) Prediction of intrinsically disordered regions in PGLYRP2 using the PONDR tool. ( E ) Expression and phase separation analysis of DsRed-tagged PGLYRP2 IDR/209–377 and DsRed-tagged PGLYRP2 IDR . DsRed-PGLYRP2 IDR/209–377 facilitated the formation of membraneless condensates that colocalized with HBV DNA FAM–Enh II. Scale bars: 10 μm. ( F ) FRAP analysis of HBV DNA FAM–Enh II within PGLYRP2 IDR/209–377 -induced condensates. Right: Quantification of fluorescence intensity. Scale bars: 5 μm. ( G ) Model of PGLYRP2-mediated phase separation as a platform for trapping HBV DNA. ( H ) Relative luciferase activity analysis of PGLYRP2 variants with missense SNPs. ( I ) Structural and protein-DNA docking analyses. Conformations of PGLYRP2 and HBV DNA Enh II were predicted using AlphaFold3 and UNAFold, respectively. Protein-DNA docking with HDOCK elucidated specific interactions between the bent structure of Enh II and the PGLYRP2 209–377 pocket. ( J ) HBV promoter DNA pull-down assay. Cell lysates from HEK293 cells transfected with FL PGLYRP2, WT, or SNP-containing PGLYRP2 209–377 constructs were incubated with 5′ biotinylated HBV promoter DNA probes or control DNA and streptavidin-agarose beads. Data are represented as mean ± SD. One-way ANOVA with post hoc Bonferroni’s test was used for statistical analysis ( A – C and H ). * P < 0.05; ** P < 0.001.

Article Snippet: The assay began with an overnight incubation at 4°C, where mouse anti-HBc antibody (NB110-7396, Novus) and rabbit anti-PGLYRP2 antibody (NBP2-32042, Novus) were applied to the samples, followed by the Streptavidin PLA-PLUS probe (Merck).

Techniques: Functional Assay, Cotransfection, Luciferase, Construct, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Fluorescence, Activity Assay, Pull Down Assay, Transfection, Incubation, Control

Journal: The Journal of Clinical Investigation

Article Title: PGLYRP2 drives hepatocyte-intrinsic innate immunity by trapping and clearing hepatitis B virus

doi: 10.1172/JCI188083

Figure Lengend Snippet: ( A ) Localization of PGLYRP2 (red) and HBc (green) in HBV or HBc expression construct–transfected Huh7/PGLYRP2 cells was analyzed by immunofluorescence staining. Scale bars: 20 μm. ( B ) HBV – or HBV + human liver tissue lysates were prepared for co-IP using anti-PGLYRP2 and blotted using the indicated antibodies. ( C ) Schematic representation of WT and mutants of HBc (C61G and Y132A). ( D ) PGLYRP2 expression construct was cotransfected with Con vector, HBc WT, HBc-C61G, or HBc-Y132A expression construct into HEK293 cells. After 48 hours, cell lysates were harvested for co-IP using anti-FLAG antibody and blotted using the indicated antibodies. The high-order complex in the whole-cell lysates (WCLs) was separated by native PAGE and subjected to immunoblot. ( E ) Nucleocytoplasmic translocation of PGLYRP2 (red) in WT HBc, HBc-C61G, or HBc-Y132A mutant (green) expression construct–transfected Huh7/PGLYRP2 cells was analyzed by immunofluorescence staining. Scale bars: 20 μm. ( F ) Left: Representative confocal images showing PLA + and PLA – signal in HBV + liver tissue or HBV – liver tissue, respectively. Top right: A PLA signal corresponds to PGLYRP2/HBV capsid proximity, whereas the absence of HBV capsid resulted in the lack of a PLA fluorescent signal. Bottom right: Quantification of percentage of PLA + cells from the total number of detected cells. Dots indicate biological replicates ( n = 5 for HBV – tissues and n = 9 for HBV + tissues; independent experiments). Scale bars: 20 μm. Data are represented as mean ± SD. Two-tailed Student’s t test was used for statistical analysis. ** P < 0.001. ( G ) Our proposed model of PGLYRP2 NLS masking. The AlphaFold3-predicted structure model of PGLYRP2 and the 3D structure of PGLYRP2/HBV capsid (Protein Data Bank: 6VZP) complex were visualized by PyMOL software ( https://pymol.org ).

Article Snippet: The assay began with an overnight incubation at 4°C, where mouse anti-HBc antibody (NB110-7396, Novus) and rabbit anti-PGLYRP2 antibody (NBP2-32042, Novus) were applied to the samples, followed by the Streptavidin PLA-PLUS probe (Merck).

Techniques: Expressing, Construct, Transfection, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Plasmid Preparation, Clear Native PAGE, Western Blot, Translocation Assay, Mutagenesis, Two Tailed Test, Software

Journal: The Journal of Clinical Investigation

Article Title: PGLYRP2 drives hepatocyte-intrinsic innate immunity by trapping and clearing hepatitis B virus

doi: 10.1172/JCI188083

Figure Lengend Snippet: ( A ) Top: Flow cytometry was used to assess HBV clearance in Huh7-NTCP/Con and Huh7-NTCP/PGLYRP2 cells infected with HBV at a multiplicity of infection of 1:2,500. Bottom: Quantification of HBc fluorescence intensity. Dots indicate biological replicates ( n = 3 independent experiments). ( B ) Immunohistochemical staining for PGLYRP2 and HBc in HBV + human liver tissues from distal non-tumor liver tissues of hepatocellular carcinoma patients. The correlation between HBc and PGLYRP2 expression was statistically analyzed. Scale bars: 200 μm. ( C ) Stable cell lines HepAD38/Con and HepAD38/PGLYRP2 were cultured in the medium without Tet for 15 days. Top: Supernatant levels of PGLYRP2 were detected by ELISA. Dots indicate biological replicates ( n = 3 independent experiments). Bottom: Levels of PGLYRP2 and HBc in the cell lysates were analyzed by Western blot at the indicated times. ( D ) Dot blot analysis was used to measure extracellular PGLYRP2 levels in serum samples from healthy individuals and chronic hepatitis B patients. Each serum sample (10 μL) was applied onto a nitrocellulose membrane, followed by detection using a rabbit anti-PGLYRP2 antibody and an HRP-labeled anti-rabbit IgG. Ponceau S staining served as the loading control, with dot intensities quantified using Multi Gauge software v3.0 (Fujifilm). ( E ) Cotransfection of a PGLYRP2 expression plasmid with an HBV plasmid into Huh7 cells resulted in enhanced secretion of naked capsids. At day 5 after transfection, extracellular HBV particles were isolated by ultracentrifugation and analyzed via 1% native agarose gel electrophoresis, followed by Western blot to assess both capsid and PGLYRP2 levels. Data are represented as mean ± SD. Two-tailed Student’s t test ( A ), Pearson’s correlation coefficient ( B ), and 1-way ANOVA with post hoc Bonferroni’s test ( C ) were used for statistical analysis. * P < 0.05; ** P < 0.001.

Article Snippet: The assay began with an overnight incubation at 4°C, where mouse anti-HBc antibody (NB110-7396, Novus) and rabbit anti-PGLYRP2 antibody (NBP2-32042, Novus) were applied to the samples, followed by the Streptavidin PLA-PLUS probe (Merck).

Techniques: Flow Cytometry, Infection, Fluorescence, Immunohistochemical staining, Staining, Expressing, Stable Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Dot Blot, Membrane, Labeling, Control, Software, Cotransfection, Plasmid Preparation, Transfection, Isolation, Agarose Gel Electrophoresis, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: PGLYRP2 drives hepatocyte-intrinsic innate immunity by trapping and clearing hepatitis B virus

doi: 10.1172/JCI188083

Figure Lengend Snippet: ( A ) Coimmunoprecipitation analysis to confirm the specificity of interaction. ( B ) Left: Size-exclusion chromatography was performed to discern the molecular complexes formed between PGLYRP2 and HBV capsid. Right: The collected fractions corresponding to elution peaks were further analyzed to validate the protein compositions. ( C ) Multiplex immunofluorescence staining was used to visualize the colocalization of PGLYRP2, HBc, and CD68 in HBV-infected non-tumor liver tissues from HCC patients. ( D ) Conditioned media were prepared from Tet-off HepAD38 cells expressing either control vector or PGLYRP2. THP-1 M0 macrophages were treated with CM for 4 or 16 hours, and changes in the immune profile were assessed. ( E ) Hierarchical clustering analysis revealed distinct cytokine and chemokine profiles in THP-1 M0 macrophages following 4-hour treatment with CM. ( F ) Flow cytometry analysis of THP-1 macrophages treated with CM for 16 hours identified a subset of ITGAM + cells producing CXCL9/10. ( G ) Multiplex immunofluorescence staining in HBV-infected liver tissues corroborated the presence of PGLYRP2, HBc, CD68, and CD8. Scale bar: 20 μm. ( H ) Coculture experiments of CD8 + T cells from healthy donors with CM-treated macrophages for 48 hours revealed significant changes in the activation status of these T cells, analyzed by flow cytometry and a Seahorse extracellular flux analyzer. ( I and J ) The Seahorse MitoStress Test was conducted to measure the complete oxygen consumption rate (OCR) trace ( I ), basal OCR, and ATP-linked respiration ( J ). ( K ) Intracellular cytokine staining for IFNG and TNFA in cocultured CD8 + T cells was performed to evaluate their effector functions. Dots indicate biological replicates ( n = 3 independent experiments). Data are represented as mean ± SD. One-way ANOVA with post hoc Bonferroni’s test ( F , J , and K ) was used for statistical analysis. * P < 0.05; ** P < 0.001.

Article Snippet: The assay began with an overnight incubation at 4°C, where mouse anti-HBc antibody (NB110-7396, Novus) and rabbit anti-PGLYRP2 antibody (NBP2-32042, Novus) were applied to the samples, followed by the Streptavidin PLA-PLUS probe (Merck).

Techniques: Size-exclusion Chromatography, Multiplex Assay, Immunofluorescence, Staining, Infection, Expressing, Control, Plasmid Preparation, Flow Cytometry, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: PGLYRP2 drives hepatocyte-intrinsic innate immunity by trapping and clearing hepatitis B virus

doi: 10.1172/JCI188083

Figure Lengend Snippet: ( A ) Multiple sequence alignment of PGLYRP2 proteins from various species. ( B ) Cotransfection of HBV promoter–luciferase reporter constructs with human PGLYRP2 (hPGLYRP2), mouse PGLYRP2 (mPGLYRP2), or mutant mPGLYRP2 Q268R into C3A cells. Renilla luciferase was used as a control for transfection efficiency. Dots indicate biological replicates ( n = 4 for Con, mPGLYRP2 WT, and mPGLYRP2 Q268R , n = 5 for hPGLYRP2 WT; independent experiments). ( C ) Purification of HBV promoter–binding proteins (hPGLYRP2, mPGLYRP2, mPGLYRP2 Q268R ) via DNA pull-down assays followed by Western blot and agarose gel analysis. ( D ) Pglyrp2- WT, Pglyrp2- knockout ( Pglyrp2 –/– ), and Pglyrp2 -knockout replaced with human PGLYRP2 ( Pglyrp2 –/– / PGLYRP2 ) C57BL/6J mice ( n = 8) were injected with pAAV vectors expressing HBV. The vectors used were pAAV-CMV/HBV1.2 (left) or pAAV-HBV core promoter/HBV1.2 (right). Serum HBV titers were quantitatively measured by real-time PCR at indicated times after injection. ( E ) Comparison of HBV serum titers in WT, Pglyrp2 –/– , and Pglyrp2 –/– / PGLYRP2 mice at 10 weeks after injection. Dots indicate biological replicates ( n = 5 independent experiments). ( F ) Immunohistochemical analysis of HBc expression in mouse liver sections after injection with pAAV-HBV promoter/HBV1.2, complemented by quantitative evaluation of HBc staining in regions active or inactive in HBV replication. Dots indicate biological replicates ( n = 3 independent experiments). ( G ) Left: Immunofluorescence staining for IFNG (red) and CD8 (green) in HBV-infected WT, Pglyrp2 –/– , or Pglyrp2 –/– / PGLYRP2 liver tissues from HBV mouse model at 10 weeks after AAV-HBV promoter/1.2×HBV injection. Right: Statistical analysis of CD8 + T and IFNG + CD8 + T cell counts in HBV-infected mouse liver tissues. Dots indicate biological replicates ( n = 3 independent experiments). ( H ) Schematic depiction of PGLYRP2-mediated HBV clearance. Data are represented as mean ± SD. One-way ANOVA with post hoc Bonferroni’s test ( B and E – G ) and 2-way ANOVA with post hoc Bonferroni’s test ( D ) were used for statistical analysis. * P < 0.05; ** P < 0.001.

Article Snippet: The assay began with an overnight incubation at 4°C, where mouse anti-HBc antibody (NB110-7396, Novus) and rabbit anti-PGLYRP2 antibody (NBP2-32042, Novus) were applied to the samples, followed by the Streptavidin PLA-PLUS probe (Merck).

Techniques: Sequencing, Cotransfection, Luciferase, Construct, Mutagenesis, Control, Transfection, Purification, Binding Assay, Western Blot, Agarose Gel Electrophoresis, Knock-Out, Injection, Expressing, Real-time Polymerase Chain Reaction, Comparison, Immunohistochemical staining, Staining, Immunofluorescence, Infection