Modulation of hepatitis B virus infection by epidermal growth factor secreted from liver sinusoidal endothelial cells

Abstract

HBV attachment assay. HBV derived from the culture supernatant of HepAD38 cells. Cells were incubated with the virus at 10,000 GEq/cell in the presence of 4% PEG8000 and 2% DMSO at 4 °C for 3 h. After washing out the free virus, HBV DNA was detected by qPCR. Primers are listed in Supplementary information. HBV internalization assay. Following HBV attachment assay, the cells were washed to remove free virus and incubated at 37 °C for 24 h. The attached virus was removed by 0.25% trypsin/EDTA and HBV DNA was detected by qPCR. Cytokine array analysis. Human Cytokine Antibody Array Membrane (Abcam) was used to detect cytokines according to the manufacturer's protocol. iPSC-derived LSECs were suspended in NPC maintenance medium and seeded at the density of 40,000 cells/cm 2 in the upper chamber of trans-well. Control wells contained NPC maintenance medium only. HepG2-NTCP maintenance medium was added to the lower chamber and changed to fresh medium every day. The samples were prepared from the lower chamber at day 5, and the data were analyzed by ImageJ quantitatively. The pictures were taken in ImageQuant LAS 4000 version1.2. Cross-linking assay. In cross-linking assay, the cells were incubated with the cross-linker BS 3 at 4 °C for 30 min, and the cross-linking reaction was terminated using 20 mM of glycine (final concentration) as previously described 11. Collected and isolated the cell surface lysates using Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Sciences). Samples were then separated on SDS-PAGE, performed western blotting. Antibodies are listed in Supplementary information. Knockdown of EGFR. Sequences of siRNAs used for knockdown EGFR are: si-EGFR: 5′-GGA ACU GGA UAU UCU GAA A TT-3′ and 5′-GAU CUU UCC UUC UUA AAG A TT-3′. Cells were transfected with siRNAs at a final concentration of 30 nM using Lipofectamine RNAiMAX Reagent (Life Technologies). The Mission siRNA Universal Negative control were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were used for experiments 48 h post-transfection.

Microfilaments and Microtubules Alternately Coordinate the Multistep Endosomal Trafficking of Classical Swine Fever Virus

“…In addition, EGFR is a coreceptor for many viruses that help to invade the host cells (48,49). Once activated, EGFR can regulate the rearrangements of F-actin in the host cells following clathrin-mediated viral endocytosis (50). Therefore, we speculate that EGFR is also related to CSFV entry into PK-15 cells.…”

Multiomics Analysis of Endocytosis upon HBV Infection and Identification of SCAMP1 as a Novel Host Restriction Factor against HBV Replication

“…The overexpression of SCAMP1 reduced, whereas the suppression of endogenous SCAMP1 increased HBV transcripts and viral proteins (Figures 6 and 7). Previous studies showed that EGF modulated HBV infection dose-dependently via EGFR-mediated endocytosis pathways [52,53]. HBsAg expression was found to be more than 10-folds suppressed upon SCAMP1 over-expression (Figure 6A and Supplementary Figure S2A).…”

Anti-EGFR Single-Chain Fv Antibody Fragment Displayed on the Surface of Ferritin H-Chain Protein Nanoparticle for Asthma Therapy

“…How anti-EGFR scFv-FTH1/FTH1 nanoparticles down-regulate EGFR and MUC5AC expression remain unknown. However, it could be found that once binding to EGFR-positive cells, both EGFR and anti-EGFR scFv-FTH1/FTH1 nanoparticles were internalized into cells and degraded in lysosomes, resulting in down-regulation of EGFR, a mechanism similar to that caused by high concentrations of EGF ligands. , Moreover, patients with asthma produce excessive mucus rich in MUC5AC . It was also reported that EGFR ligands could induce the expression of MUC5AC in both human airway epithelial cells and a chronic murine asthma model, and a positive correlation existed between EGFR and MUC5AC expresstion .…”