Fig 2: MCPIP1 reduces HBV RNA. (A) Huh7 cells were transfected with pgRNA reporter (pCMV1.2xHBV/NL), helper plasmid (pcDNA-CP), and pCAG-SEAP, together with GFP or GFP-MCPIP expression vectors. Cells were harvested 3 days after transfection. The total lysates were immunoblotted with anti-GFP or β-actin antibody. Nanoluc (NL) activity was normalized with SEAP activity, and the value of GFP (mock) was defined as 100%. (B,C) C57BL/6 mice were intravenously administered the pgRNA reporter, the helper plasmid pcDNA-CPtds, pCAG-SEAP, and FLAG-MCPIP1 or mock vector (n = 8 each). Two days after injection, lysates from the livers were subjected to Western blotting analysis (B) and luciferase assay (C). NL activity is indicated after normalization by SEAP activity. (D–I) Parental or MCPIP1 knockout Hep38.7-Tet cells were cultured in the absence of tetracycline for 5 days (E). (D) Validation of MCPIP1 ablation by Western blotting analysis. (F) RT-qPCR analysis to determine HBV RNA level, normalized by HPRT. The result is indicated as the ratio to the parental cells. (G) Supernatant HBV DNA qPCR analysis. The result is indicated by the absolute copy numbers. (H, I) The HBsAg (H) and HBeAg (I) levels in the supernatant were measured by chemiluminescent enzyme immunoassay. Expressed as international units mIU/ml and Cut-Off-Index (C.O.I.), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: IL-1β adopts MCPIP1 for viral RNA downregulation. (A) IL-1β mRNA level in CHB (n = 122) and normal liver (n = 6, GSE83148). (B) IL-1β mRNA level in human liver-chimeric mice with 8 weeks after HBV infection (n = 9) and controls (n = 6, GSE52752). (C) Correlation between the mRNA levels of MCPIP1 and IL-1β in CHB patients. (D) Huh7 cells were cotransfected with pCMV1.2xHBV/NL, pCAG-SEAP, and pcDNA-CP, and cultivated for 24 h in the absence (PBS) or presence of IL-1β (100 and 200 ng/ml). The total lysates were subjected to luciferase assay and Western blotting. (E, F) Parental or MCPIP1 knockout Hep38.7-Tet cells were treated with or without 100 ng/ml IL-1β in the absence of tetracycline for 5 days. (E) RT-qPCR analysis to quantify HBV RNA level, normalized by HPRT. The result is indicated by the ratio to the untreated parental cells. (F) qPCR analysis to quantify HBV DNA in the culture supernatant. The result is presented as absolute copy numbers. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig 4: Epsilon structure of HBV RNA is required for MCPIP1-mediated reduction. (A) 293FT cells were transfected with pCMV1.2xHBV/NL, whose 3′- and/or 5′- epsilon structures are intact or deficient, along with the pCAG-SEAP, pcDNA-CP, and pGFP-MCPIP1. (B,C) 3 days after transfection, cells were harvested and subjected to Western blotting (B) and luciferase assay (C). In (C), NL activity of the cells transfected with pCMV1.2xHBV/NL and the mock plasmid was normalized to 100%. (D,E) Huh7 cells were cotransfected with pCMV1.2xHBV/NL, pCAG-SEAP, pcDNA-CP, and FLAG-MCPIP1 (or the mock plasmid, pFLAG-GFP). Transfected cells were further cultured for 3 days. Cell lysates were subjected to RNA immunoprecipitation assay using the FLAG M2 affinity gel. Crude extracts (input) and IP fractions were analyzed by Western blotting (D). The RNA (before and after immunoprecipitation) was subsequently subjected to RT-qRCR for quantitative evaluation of HBV RNA and HPRT mRNA. Relative enrichment of RNA in mock transfectants is defined as 1 (E). *P < 0.05, **P < 0.01, ***P < 0.001.