Fig 2: STAU1 indirectly binds to the core promoter via TARDBP(A) Operating principle of the tripartite NanoLuc system.(B) The tripartite Nanoluc system can be used to detect the interaction between HBx and DDB1.(C and D) The interactions between STAU1 and TARDBP or TARDBP mutants were assayed using the tripartite NanoLuc system. The data in (B) and (D) are represented as the mean ± SD of four independent experiments. Significance was tested with an unpaired t test. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = non-significant.(E) The interaction between STAU1 and TARDBP was assayed by co-IP.(F) Expression and purification of recombinant TARDBP protein from E. coli.(G) Detection of the interaction between STAU1, TARDBP, and core promoter by EMSA.(H and I) Knockdown efficiency of si-TARDBP was evaluated by qPCR and Western blot.(J) The si-Control (Lane1 and 3) or si-TARDBP (Lane2 and 4) were transfected into HepG2 cells. pCH9-STAU1 or the vector were co-transfected with pCore-Rluc into these cells after 48 h. After another 48 h, the activity of the core promoter was examined by renilla luciferase assays. The data in (I) and (J) are presented as the mean ± SD of three independent experiments and analyzed with an unpaired t-test. **p < 0.01, ***p < 0.001.(K and L) pCH9-STAU1 or vector were co-transfected with HBV1.3 into TARDBP-depleted HepG2 cells and the intracellular HBV DNA and HBV RNA were detected by Southern and Northern blots.

Fig 3: STAU1 inhibits the degradation of HBx in a ubiquitylation-independent manner(A–H) Plasmid pCH9-STAU1 and plasmid 3HA-HBx were co-transfected into HepG2 cells (A) and Huh7 cells (C). After 48 h, cells were treated with DMSO or MG132 (10 µM of final concentration) for 6 h, and then lysed for Western blot analysis. The si-STAU1 or si-control and plasmid 3HA-HBx were co-transfected into HepG2 cells (B) and Huh7 cells (D). After being treated with DMSO or MG132 for 6 h, cells were lysed for Western blot analysis. Plasmids pCH9-STAU1, pUBB and 3HA-HBx were co-transfected into HepG2 cells (E) and Huh7 cells (F). The si-STAU1, pUBB, and 3HA-HBx were co-transfected into HepG2 cells (G) and Huh7 cells (H). These cells were treated with MG132 for 6 h, and then lysed for IP 48 h post-infection.

Fig 4: STAU1 enhances HBV replication in cultured cells(A and B) Validation of STAU1 overexpression in HepG2 cells. The data are presented as mean ± SD. ∗∗∗p < 0.001.(C–E) STAU1 overexpression increased HBV replication in HepG2 cells. Different amounts of pCH9-STAU1 (0, 0.2, 0.5, and 1 μg plus vector) and HBV1.3 were co-transfected into HepG2 cells. The intracellular HBV DNA, HBV RNA, and secreted HBsAg and HBeAg were detected 5 days post-transfection.(F–H) STAU1 overexpression enhanced HBV replication in Huh7 cells.(I and J) Knockdown efficiency of si-STAU1 evaluated by RT-qPCR and Western blot. The data are presented as mean ± SD. ∗∗∗∗p < 0.0001.(K–P) STAU1 knockdown suppressed HBV replication in HepG2 and Huh7 cells. Different amounts of si-STAU1 (0, 20, 50, and 100 pmol plus control siRNA) were co-transfected with HBV1.3 into HepG2 and Huh7 cells. The intracellular HBV DNA, HBV RNA, and secreted HBsAg and HBeAg were assayed.(Q–S) STAU1 increased HBV replication in the HepG2-NTCP cell model. Lentiviruses expressing STAU1 and shRNA against STAU1 and control lentivirus (Control-OE and shControl) were used to infect HepG2-NTCP cells, respectively. After blasticidin selection for one week, the surviving cells (pooled) were expanded for the infection experiments. After HBV infection, the cells were maintained for another 5 days and then intracellular HBV DNA, HBV RNA, and secreted HBsAg and HBeAg were assayed by Southern blot, Northern blot, and ELISA, respectively.(T) STAU1 did not affect cccDNA level in the HepG2-NTCP cell model. cccDNA and total RNA were extracted from the STAU1-OE, shSTAU1, and control cells that were infected with HBV and analyzed by qPCR. The data are presented as mean ± SD and analyzed with an unpaired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 5: STAU1 interacts with HBx(A) Screening of HBV proteins interacting with STAU1 by the tripartite Nanoluc system.(B) Detection of the interaction between HBx and STAU1 by co-IP.(C) Analysis of the interactions between STAU1 mutants and HBx by co-IP.(D) Co-IP assays for the interactions between HBx mutants and STAU1.(E) Characterization of STAU1 domains required for its interaction with HBx by the tripartite Nanoluc system.(F) Characterization of HBx sequences required for its interaction with STAU1 by the tripartite Nanoluc system. The data in (E) and (F) are shown as the mean ± SD from four independent experiments. Significance was tested with an unpaired t test. ***p < 0.001, ****p < 0.0001.(G–J) The effects of STAU1 overexpression (G and I) and knockdown (H and J) on the level of HBx protein and mRNA were analyzed. The data in (I and J) are resented as the mean ± SD of three independent experiments.