Journal: bioRxiv

Article Title: Multiomics Interrogation into HBV (Hepatitis B Virus)-Host Interaction Reveals Novel Coding potential in Human Genome, and Identifies Canonical and Non-canonical Proteins as Host Restriction Factors against HBV

doi: 10.1101/2021.03.19.436126

Figure Lengend Snippet: ( A ) RNA-sequencing was conducted with Huh7.5.1 cells transfected with Vector (Control) or prcccDNA and pCMV-Cre. Heatmap of 11 transcriptional DEGs, which have transcription corepressor activity, three biological replicates were shown for HBV and control group. ( B ) RT-qPCR confirmed that HBV down-regulated the transcriptional level of endogenous SIRT6 transcription. ( C ) Network analysis of SIRT6-associated genes whose transcription was altered by HBV. Red and blue nodes indicate the up- and down-regulated genes, respectively. The color intensity indicates the fold change level of the gene. Nodes with * are DEGs (P-adjust <0.05, |log 2 FC|>=1, also see methods). ( D ) RT-qPCR validation of some of the SIRT6-associated genes that were shown in C. ( E ) Representative profiles of ribosome footprints of human SIRT6 ORF upon HBV, translation initiation of endogenous SIRT6 was down-regulated by HBV in Huh7.5.1 cells. ( F ) Endogenous SIRT6 was down-regulated by HBV in Huh7.5.1 cells. ( G ) De novo infection of HBV down-regulated endogenous SIRT6 level in HepG2-NTCP cells, HepG2 cells stably expressing NTCP (sodium taurocholate cotransporting polypeptide), the functional receptor of HBV. The results of two independent biological replicates were shown. ( H ) HBV down-regulated SIRT6 in mouse livers infected with adenovirus harboring HBV genome. ( I ) Total proteins were extracted from the normal liver tissues of 12 patients who were diagnosed with HBV positive and negative, respectively. For patient information see . Endogenous SIRT6 or GAPDH proteins were visualized with IB using anti-SIRT6 or anti-GAPDH, with their relative abundances calculated using ImageJ. ( J ) The reverse correlation between the serum levels of HBsAg and the relative abundances of endogenous SIRT6 protein in liver tissues of the 12 patients. *, p<0.05, **, p<0.01, ***, p<0.001. qPCR results are presented as bar chart (For SIRT6, n=2; for other genes, n = 3); ELISA data are presented as bar chart (n = 3).

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: RNA Sequencing Assay, Transfection, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Infection, Stable Transfection, Expressing, Functional Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Multiomics Interrogation into HBV (Hepatitis B Virus)-Host Interaction Reveals Novel Coding potential in Human Genome, and Identifies Canonical and Non-canonical Proteins as Host Restriction Factors against HBV

doi: 10.1101/2021.03.19.436126

Figure Lengend Snippet: ( A and B )The cDNA of sirtuins-family were each co-transfected with HBV system into Huh7.5.1 cells and the protein levels of HBc, GAPDH and SIRTUINS were detected using indicated antibodies (A). The HBsAg and HBeAg level in supernatants were measured by ELISA. (B). ( C ) HepAD38 cells chromosomally integrated with the Tet-controlled HBV expression system were infected with lenti-virus vector expressing SIRT6, Cells were harvested and lysates subjected to IB using the indicated antibodies, while the supernatants from the cell culture were collected and subjected to ELISA using anti-HBsAg or anti-HBeAg. ( D ) HepG2 cells were co-transfected with HBV cccDNA system (H+C, prCCCDNA and pCMV-Cre) and pCDNA3.0-Sirt6-FLAG or pCDNA3.0 using Polyetherimide. Cells were harvested 72 h.p.t., with lysates collected and subjected to IB analyses using the indicated antibodies. The HBsAg and HBeAg in supernatants were determined by ELISA. ( E and F ) Experiments similar to those in D were performed with Huh7.5.1 cells transfected with 1.1mer- (E) or 1.3mer- (F) HBV linear genomes, For ELISA, n=3. ( G ) HepAD38 cells were transduced with lenti-virus vector (NC) or those encoding shRNAs that targeted endogenous SIRT6 (shSIRT6 1,2,3,4), After tetracyclin withdrawn for 4 days. HBcAg was measured using western blot, and ( H ) the level of HBsAg and HBeAg in supernatants was determined by ELISA. ( I ) The effect of SIRT6 on HBV genome replication was measured via southern blotting in Huh7 cells. RC, relaxed circular DNA; DSL, double strand linear DNA; SS, single strand DNA. *, p<0.05, **, p<0.01, ***, p<0.001.

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Infection, Plasmid Preparation, Cell Culture, Transduction, Western Blot, Southern Blot

Journal: bioRxiv

Article Title: Multiomics Interrogation into HBV (Hepatitis B Virus)-Host Interaction Reveals Novel Coding potential in Human Genome, and Identifies Canonical and Non-canonical Proteins as Host Restriction Factors against HBV

doi: 10.1101/2021.03.19.436126

Figure Lengend Snippet: ( A and B ) Introduction of Sirt6 suppressed the expression of HBc (A) and HBs and HBe (B) antigens in Huh7.5.1 cells. ( C and D ) Knocking-down endogenous Sirt6 promoted the expression of HBc (C) and HBs and HBe (D) antigens in Huh7.5.1 cells. ( E and F ) Effect of SIRT6 enzymatic mutants on HBc (E) and HBs and HBe (F) expression in huh7.5.1 cells. ( G ) The effect of SIRT6 wild type or enzymatic deficient mutant on HBV genome replication was measured via southern blotting in HepG2 cells. ( H ) The chemical formula and a close-up view of MDL-800 in complex with 2′-O-acyl-ADP ribose (2′-O-acyl-ADPR) motif of human SIRT6 (X-ray structure, PDB number 5Y2F). ELISA shows that MDL800 suppressed the expression of HBc ( I ) and HBs and HBe ( J ) antigens in dose-dependent manner in Tet-controlled HBV in HepAD38 cells. ( K ) MDL-800 treatment significantly suppressed HBV genome replication intermediates in Huh7 cells. RC, relaxed circular DNA; DSL, double strand linear DNA; SS, single strand DNA. For ELISA, for panel B, n=2; For others, n=3. *, p<0.05, **, p<0.01, ***, p<0.001.

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: Expressing, Mutagenesis, Southern Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Multiomics Interrogation into HBV (Hepatitis B Virus)-Host Interaction Reveals Novel Coding potential in Human Genome, and Identifies Canonical and Non-canonical Proteins as Host Restriction Factors against HBV

doi: 10.1101/2021.03.19.436126

Figure Lengend Snippet: ( A-D ) Huh7.5.1 (A, B) or HepG2(C, D) cells were treated with increasing doses of MDL-800 after HBV transfection, the endogenous proteins were visualized with the indicated antibodies, while the levels of HBsAg or HBeAg in supernatants of cell cultures were determined with ELISA using anti-HBsAg or anti-HBeAg, n=3 for each group. ( E ) The effectiveness of MDL800 on HBV was tested in SIRT6 knock-down cell line or control cell line. For ELISA, n=3. *, p<0.05, **, p<0.01, ***, p<0.001.

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Multiomics Interrogation into HBV (Hepatitis B Virus)-Host Interaction Reveals Novel Coding potential in Human Genome, and Identifies Canonical and Non-canonical Proteins as Host Restriction Factors against HBV

doi: 10.1101/2021.03.19.436126

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with HA-tagged HBc and FLAG-tagged Sirtuins family proteins, and co-immunoprecipitation was performed using anti-FLAG beads. ( B and C ) HEK293T cells were transfected with plasmids encoding HA-tagged HBx and FLAG-tagged SIRT6, with cells harvested at 48 h.p.t (hours post transfection), and lysates were subjected to Co-immunoprecipitation (Co-IP) followed by IB with indicated antibodies. ( B ) SIRT6 did not interact with HBx. ( C ) 293T cells were co-transfected with plasmids encoding HA tagged HBc and FLAG-tagged full-length SIRT6 or the indicated fragments, after 48 hours, Co-immunoprecipitation (Co-IP) was performed. ΔN, SIRT6 deleted the N terminal domain (1-48Aa); ΔC , SIRT6 deleted the C terminal domain (272-328Aa), ΔCore , SIRT6 with the catalytic core domain (49-271 Aa) deleted. WCE: whole cell extracts. WCL: whole cell lysates.

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Multiomics Interrogation into HBV (Hepatitis B Virus)-Host Interaction Reveals Novel Coding potential in Human Genome, and Identifies Canonical and Non-canonical Proteins as Host Restriction Factors against HBV

doi: 10.1101/2021.03.19.436126

Figure Lengend Snippet: ( A ) SIRT6 co-immunoprecipitated with HBcAg in 293FT cells. ( B ) SIRT6 and HBcAg co-localized in the nuclei of Huh7.5.1 cells during HBV replication. Scale bars, 20 μm. ChIP assays were performed using indicated antibodies with cells upon either HBV transfection alone ( C ), or in combination with SIRT6 overexpression ( D ) or knockdown ( E ) or MDL800 treatment ( F ). *, p<0.05, **, p<0.01, ***, p<0.001. ChIP data were acquired in Huh7.5.1 cells and were presented as bar chart (n=3 per group).

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: Immunoprecipitation, Transfection, Over Expression