Fig 1: The five host factors affect the abundance of multiple HBV molecules(A) Kinetics of the decrease in extracellular HBsAg produced by HepG2.2.15 cells transfected with siRNAs. The amount of HBsAg produced was measured in 12-h intervals after siRNA transfection and normalized to the level produced by mock-treated cells. The siRNAs were transfected at a final concentration of 10 nM. REP 2139 was at 20 nM. (B) siRNAs that knock down the five host factors reduced the levels of intracellular HBsAg. The western blot image contains the signal for the large S antigen. The LC is of the β-actin protein. The knockdown of the host factors is shown in Figure S3. (C) The five host factor siRNAs can reduce the HBV polymerase levels. The western blot image shows the amounts of the HBV polymerase identified by a monoclonal antibody specific to the HBV polymerase. The L.C. shows the amount of β-actin protein. Additional western blots of cellular proteins are shown in Figure S6. HepG2.2.15 cells were transfected with a final concentration of10 nM siRNA, 5 nM ALG-20002, 8 nM ALG-10000, or 20 nM REP 2139. (D) The host factors have differential effects on the amounts of HBsAg, HBeAg, and core. ALG-10000 was transfected into HepG2.2.15 cells at 8 nM and the siRNAs at 10 nM. (E) Total HBV RNAs are modestly reduced by the knockdown of SRSF1 and HNRNPA2B1, but not by knockdown of RPLP1 or RPLP2. The cells were harvested 76 h after transfection. The amount of HBV RNA was normalized to the amount of GAPDH in the cells. In the graphs on panel A and D, each error bar represents one standard deviation of uncertainty.
Fig 2: RPLP1 and RPLP2 require regulatory HBV sequences to function on HBsAg production(A) Schematics of the HBV sequences cloned in plasmid pcDNA3.1 tested for HBsAg production. The nucleotide number of the HBV genome is shown at the flanks of the DNA sequence. The green arrow represents the human cytomegalovirus promoter. The green box labeled with “An” represents the polyadenylation sequence. Both the promoter and polyadenylation sequence are present in pcDNA3.1. Boxes represent the coding sequence for HBV genes. The two red ovals denote HBV enhancer sequences. (B) Relative amounts of the HBsAg produced in HEK 293 cells transfected with STOPS, siRNAs, or ALG-20002. HBsAg levels were reproducibly increased by the knockdown of GRP78 in cells transfected with pPol_SAgtercDNA3.1 and pSAg-PsicDNA3.1. The percentages of HBsAg expressed from these plasmids relative to the mock-treated control (pCDNA3.1) are shown above the blue bars. Each error bar represents one standard deviation of uncertainty.
Fig 3: STOPS reduction of HBsAg involves ubiquitination of the HBsAg and proteasome degradation(A) HBsAg has post-translational modifications. The HBsAg was from HepG2.2.15 cells treated with 8 nM ALG-10000 or 10 nM siRPLP2 for 76 h. The most prominent band is the molecular mass expected of the large HBsAg. Additional higher-molecular-weight bands may be ubiquitinated forms of the HBsAg. (B) Intracellular HBsAg in HepG2.2.15 cells treated with ALG-10000 increased the amount of ubiquitin. The ubiquitinated HBsAg assayed was from cells treated for 76 h with 8 nM ALG-10000. (C) Knockdown of GRP78, RPLP1, and RPLP2 can increase HBsAg ubiquitination. All siRNAs were transfected into HepG2.2.15 cells at a final concentration of 10 nM. The amount of ubiquitinated HBsAg was normalized to the amount of HBsAg present in the cell lysate. (D) Proteasome inhibitors can partially reverse ALG-10000-mediated reduction of HBsAg and RPLP1 levels. The images of the western blot show the large HBsAg in HepG2.2.15 transfected with 10 nM ALG-10000 and treated with the proteasome inhibitor bortezomib (25 nM), or oprozomib (100 nM), or DMSO, the vehicle used to solubilize the proteasome inhibitors. The cells were treated for 24 h before their lysis for western blot analysis. The LC is the protein GAPDH. siRPLP1 was transfected at 10 nM and the cells treated with bortezomib are as described in (A). (E) The amount of ubiquitinated HBsAg present in HepG2.2.15 cells after treatment with ALG-10000 or knockdown of GRP78, RPLP1, and RPLP2 is increased by treatment with the proteasome inhibitor bortezomib. ∗p < 0.05, ∗∗p < 0.01. In the graphs in panels D, C, and E, each error bar represents one standard deviation of uncertainty.
Fig 4: Five host proteins that bind ALG-10000 affect extracellular HBsAg levels(A) Partial list of proteins that bind ALG-10000 affinity column. The proteins listed are identified with high confidence. (B) Dose responses for the inhibition of extracellular HBsAg levels and effects on cell viability by siRNA knockdowns of the host factors. Each error bar represents one standard deviation of uncertainty. (C) Recombinant proteins of RPLP1, RPLP2, GRP78, and HNRNPA2B1 can bind ALG-10000. The image is of a silver-stained polyacrylamide gel. The lane labeled “Proteins” contains the input proteins present in the analysis. The lane labeled “Tris” shows the amounts of the proteins pulled down by the affinity resin that lacks ALG-10000. (D) ALG-10000 can decrease the abundance of three of the cellular proteins that interact with ALG-10000. The image is from a western blot analysis of lysates from HepG2.2.15 cells that were transfected with 8 nM ALG-10000 and harvested 76 h after transfection.
Fig 5: RPLP2 affects HIF-1α by regulating the PI3K/AKT pathway.A KEGG pathway analysis of differentially expressed genes when RPLP2 was knocked down in Hep3B and Huh7 cells. B Western blot analysis showed a decrease in the protein levels of p-PI3K and p-AKT after RPLP2 knockdown and the quantitative analysis (n = 3, mean ± SD) is shown in (C). D After treatment of Hep3B cells with the AKT activator SC79 (10 µM) for 24 h, Western blotting was used to detect the expression of HIF-1α and p-AKT, and quantitative analysis (n = 3, mean ± SD) was performed in (E). After treatment with SC79, the expression of HIF-1α and p-AKT was rescued. F Western blotting was used to determine the effect of SC79 on HIF-1α levels in the cytoplasm and nucleus, and the quantitative analysis (n = 3, mean ± SD) was performed in (G). SC79 restored HIF-1α levels in the cytoplasm and nucleus. H Immunostaining of Hep3B cells with antibodies against HIF-1α (red). Knocking down RPLP2 led to a decrease in the nuclear expression level of HIF-1α, which was abrogated by SC79. Scale bar, 25 μm. *P < 0.05 versus corresponding control; ns means nonsignificant.
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