Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication

doi: 10.3389/fcimb.2023.1195063

Figure Lengend Snippet: HBV Core Particles, But Not HBc Dimers or Monomers, Interact with Pin1. (A) Schematic diagram and amino acid sequence of the HBV HBc (adw). The positions of the helices ( Wynne et al., 1999 ) are shown by arrows. The CTD-truncated (at amino acid 149) HBc used for this study is denoted as ΔCTD. The S/TP motifs are in bold, italicized, and underlined. Y132 and other phosphoacceptor sites are marked by arrowheads and asterisks, respectively. (B) Myc-HBc WT and Pin1 WT can be coimmunoprecipitated. HEK293T cells were (co)transfected with mock (lane 1), Myc-HBc WT (lane 2), Myc-HBc WT plus Pin1 WT (lane 3), or Pin1 WT (lane 4). Two days after transfection, cell lysates were immunoprecipitated with an in-house rabbit polyclonal anti-HBc antibody ( Jung et al., 2012 ) and then immunoblotted with a mouse monoclonal anti-Pin1 antibody, or vice versa. As a negative control, normal rabbit IgG or normal mouse IgG was used to examine lysates from Myc-HBc WT plus Pin1 WT cotransfected cells (lane 5). Immunoprecipitated proteins were subjected to SDS-PAGE, followed by immunoblotting with anti-HBc and anti-Pin1 antibodies. Lysates were also subjected to SDS-PAGE, followed by immunoblotting with anti-HBc and anti-Pin1 antibodies. Coimmunoprecipitated proteins are marked with a white arrowhead. (C) Pin1 interacts with the core particle. HEK293T cells were (co)transfected as above. Two days after transfection, cell lysates were prepared and subjected to NAGE; transferred to PVDF membranes; and immunoblotted with anti-Pin1, anti-HBc, or mouse monoclonal anti-Myc antibodies. Lysates were also subjected to SDS-PAGE, followed by immunoblotting with anti-HBc, anti-Myc, anti-Pin1, or mouse monoclonal anti-GAPDH antibodies, as described in B. GAPDH was used as a loading control. (D) The interaction between Pin1 and the core particle is more specific than that between the core particle and other PPIases. HEK293T cells were transfected with mock (lane 1), Myc-HBc WT (lane 2), or HA-HBc WT (lane 3), and cell lysates were subjected to NAGE plus immunoblotting and SDS-PAGE plus immunoblotting, as described in (B, C) . Additionally, blots were probed with rabbit monoclonal anti-CypA and mouse monoclonal anti-FKBP12 antibodies. (E) The core particle, not a dimer or monomer of HBc, binds to Pin1 through the CTD phosphoacceptor S/TP motifs of HBc. HEK293T cells were cotransfected with Pin1 WT plus Myc-HBc WT (SST-STSSSS) (lane 1), or with the Myc-HBc-Y132A (lane 2), Myc-HBc-AAA-STSSSS (lane 3), or Myc-HBc-SST-AAAAAA (lane 4) mutants. Lysates were immunoprecipitated and then subjected to SDS-PAGE and immunoblotting as described in B. Core particle immunoblotting after NAGE was also performed as described in (C) . (F) Pin1 does not interact with core particle-defective, dimer-positive HBc mutants. HEK293T cells were (co)transfected with mock (lane 1), Myc-HBc WT (lane 2), Pin1 WT (lane 3), Pin1 WT plus Myc-HBc WT (lane 4), Myc-HBc-R133D (lane 5), Myc-HBc-R133E (lane 6), Myc-HBc-AAA-AAASAS (lane 7), Myc-HBc-AAA-AAAAAA (lane 8), or Myc-HBc-Y132A (lane 9). Lysates were immunoprecipitated and then subjected to SDS-PAGE and immunoblotting as described in (B) . Representative data are shown. Relative levels of core particles and HBc proteins were calculated using ImageJ 1.50b software. Statistical significance was evaluated using Student’s t test. ns , not significant; * P < 0.05; ** P < 0.005, relative to the corresponding control.

Article Snippet: Core particles, virions, Pin1-bound-core particles, and/or subviral particles were detected using rabbit polyclonal anti-HBc (produced in-house; 1:1,000) , mouse monoclonal anti-PreS1 (1:1,000; Santa Cruz), rabbit polyclonal anti-HBs (for NAGE; 1:1,000; Virostat), mouse monoclonal anti-HBs (for SDS-PAGE; 1:1,000; Santa Cruz), or mouse monoclonal anti-Pin1 (1:1,000; Santa Cruz) primary antibodies, followed by HRP–conjugated anti-rabbit (1:5,000; Thermo Fisher Scientific) or anti-mouse (1:5,000; Seracare) secondary antibodies.

Techniques: Sequencing, Transfection, Immunoprecipitation, Negative Control, SDS Page, Western Blot, Control, Software

Journal: Scientific Reports

Article Title: Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers

doi: 10.1038/s41598-017-09965-w

Figure Lengend Snippet: Effects of substitutions in ORF-S on HBsAg antigenicity and its detection. ( A ) Antigenicity plot of “a” determinant (aa.124–147) of HBsAg showing decreased antigenicity due to substitutions T125M and P127T as obtained by Kolaskar and Tongaonkar Antigenicity Prediction method. The relative ratio of PreS2/S and PreS1 mRNA determined by quantitative real-time PCR ( B ), the expression of HBV envelope proteins by immunoblot assay with mouse monoclonal anti-HBs primary antibody [the cropped gels are shown here for clarity while the full-length blots are presented in Supplementary Figure ( A ); α-Tubulin served as the loading control] ( C ) and HBsAg level [Signal(S)/Cutoff(CO)] in culture supernatant estimated by ELISA ( D ), following transfection of wt-HBV (HBV/D-wt) and mutant construct [HBV/D-mt(HBsAg)], having T125M and P127T substitutions in Huh7 cells. Paired t-test p values; ***p < 0.0005, ns; non-significant.

Article Snippet: Additionally, the levels of intracellular HBV envelope proteins were determined by immunoblotting with mouse monoclonal anti-HBs primary antibody (anti-Ad/Ay; MyBioSource) while the extracellular HBsAg in culture supernatant was determined by ELISA using BeneSphera HBsAg Microwell ELISA kit.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection, Mutagenesis, Construct

Journal: Scientific Reports

Article Title: Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers

doi: 10.1038/s41598-017-09965-w

Figure Lengend Snippet: Effects of substitutions in Enh-II on the envelope protein expression. Relative ratio of PreS2 and PreS1-mRNA as determined by quantitative real-time PCR ( A ), expression of HBV envelope proteins by immunoblot assay with mouse monoclonal anti-HBs primary antibody [the cropped gels are shown here for clarity while the full-length blots are presented in Supplementary Figure ( B ); α-Tubulin served as the loading control] ( B ) and HBsAg level [Signal(S)/Cutoff(CO)] in culture supernatant estimated by ELISA ( C ), following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding Enh-II-mutated HBV [HBV/D-mt(Enh-II)] in Huh7 cells. Paired t-test p values; *p < 0.05.

Article Snippet: Additionally, the levels of intracellular HBV envelope proteins were determined by immunoblotting with mouse monoclonal anti-HBs primary antibody (anti-Ad/Ay; MyBioSource) while the extracellular HBsAg in culture supernatant was determined by ELISA using BeneSphera HBsAg Microwell ELISA kit.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Human Vaccines & Immunotherapeutics

Article Title: Immune response to the hepatitis B antigen in the RTS,S/AS01 malaria vaccine, and co-administration with pneumococcal conjugate and rotavirus vaccines in African children: A randomized controlled trial

doi: 10.1080/21645515.2018.1442996

Figure Lengend Snippet: Results of the inferential analyses for HBV, PHiD-CV, HRV and pertussis vaccine antigens administered as three priming doses during infancy (according to protocol immunogenicity cohort).

Article Snippet: Anti-HBs antibodies were measured using a Chemiluminescence Immunoassay (CLIA) ( Centaur ™, Siemens) with a cut-off of 6.2 mIU/ml.

Techniques:

Journal: Human Vaccines & Immunotherapeutics

Article Title: Immune response to the hepatitis B antigen in the RTS,S/AS01 malaria vaccine, and co-administration with pneumococcal conjugate and rotavirus vaccines in African children: A randomized controlled trial

doi: 10.1080/21645515.2018.1442996

Figure Lengend Snippet: Anti-HBs seroprotection rates and geometric mean antibody titers one month post-dose 3 (ATP immunogenicity cohort).

Article Snippet: Anti-HBs antibodies were measured using a Chemiluminescence Immunoassay (CLIA) ( Centaur ™, Siemens) with a cut-off of 6.2 mIU/ml.

Techniques: