Additional file 1 of Hepatitis B virus virion secretion is a CRM1-spike-mediated late event
Creators: name: Su, Pei-Yi
name: Yen, Shin-Chwen Bruce
name: Yang, Ching-Chun
name: Chang, Chih-Hsu
name: Lin, Wen-Chang
name: Shih, Chiaho
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Pharmacology
subject: Biotechnology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Cancer
subject: Hematology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: 60506 Virology

description: Additional file 1: Fig S1. A cartoon illustration for the analysis methods of extracellular and intracellular HBV viral and subviral particles. (A) The extracellular HBV particles in the media were first precipitated by ultracentrifugation through a sucrose cushion before native agarose gel electrophoresis. Viral and subviral particles were separated and characterized by Southern and Western blot analyses. Anti-HBc and anti-HBs antibodies were used consecutively for the same Western blot filters. Southern blot was performed in a separate agarose gel which allows the differentiation of genome-containing (G +) from genome-free (G-) viral and subviral particles. (B) The intracellular core-associated viral RNA and DNA were purified by PEG precipitation and nucleic acid extraction. Then, the RNA/DNA signals were analyzed by Northern blot and Southern blot. 3.5 kb pgRNA: full-length 3.5 kb pre-genomic RNA. RT RNA: reverse transcripted RNA. (Related to Fig. 1. Fig. S2. HBc amino acid L68 is required for secretion of naked capsids. (A) Alanine substitutions at L68, L72, V74, L84, V86 and M93 (blue colored) exhibited no effect on HBc nuclear accumulation [29]. (B) Intracellular core-associated RNA and DNA genomes were examined by Northern and Southern blot analyses. Single mutant M93A was defective in RNA packaging and DNA synthesis. Capsid assembly was normal by native agarose gel and Western blot analysis. (C) Extracellular HBV particles were analyzed by the method in Fig. S1A. HBc mutant Single mutant M93A appeared to have lost genome-containing virion secretion. HBc mutant L68A completely lost naked capsid secretion. Both mutant V86A and M93A exhibited strongly decreased signal of genome-containing naked capsids. Red asterisk * indicates reproducibly enhanced signal of secreted virions (see text for discussion). (D) A hexameric version of Fig. 2E. Red: secretion of genome-containing virions. Blue: secretion of naked capsids. Merge: purple. Cyan: a previous map of key residues involved in the secretion of HBV genome-containing virions [37]. (Related to Fig. 2). Fig S3. No significant anti-proliferation effect by SINE treatment in HuH-7 cells. Cell proliferation was monitored by (A) alamarBlue and (B) CCK-8 assays after treatment with CRM1 inhibitors. No apparent side effect was detected below 0.25 μM SINE compound. (Related to Fig. 3). Fig S4. HBV capsid particles accumulated in the nucleus by SINE compound treatment. Three major patterns of HBc subcellular distribution (Cy &gt; Nu, Nu &gt; Cy, Nu&amp;Cy) were scored [26] in an IFA assay using a rabbit polyclonal anti-HBc antibody (A) and a capsid-specific mouse monoclonal antibody (B). (Related to Fig. 4). Fig S5. Cotransfection with a drug-resistant mutant CRM1 rescued HBsAg secretion and shifted nuclear HBc to the cytoplasm in the presence of a SINE compound. (A) In an HBV transient transfection system, HuH-7 cells were treated with or without SINE compound. Cotransfection with a drug-resistant mutant mtCRM1 could significantly rescue the secreted HBsAg by the ELISA assay. (B) Nuclear HBc accumulation induced by the SINE compound treatment can be redirected to the cytoplasm by cotransfection with a drug-resistant mtCRM1 plasmid (see Methods). (C) SINE compound reduced the level of cytoplasmic HBc in HBV transfected HuH-7 cells by subcellular fractionation and Western blot analysis. Cytoplasmic HBc was increased after cotransfection with a drug-resistant mtCRM1 plasmid. (Related to Fig. 5). Fig S6. Colocalization of CRM1, microtubule and HBc protein at the perinuclear region by confocal immunofluorescence microscopy. Left panel: Serial sectioning of confocal microscopy was analysed by the ZEISS ZEN Microscope Software Profile. Right panel: Colocalization was revealed by the superimposed colors: tubulin (green), CRM1-mCherry (red) and HBc (cyan) at the perinuclear area. DAPI (blue) (Related to Fig. 6)