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. 2017 Mar 29;91(8):e01824-16. doi: 10.1128/JVI.01824-16

FIG 5.

FIG 5

MMP-9 facilitates HBV replication by repressing IFN-α action. (A and B) HepG2 cells were cotransfected with pHBV1.3 and pCMV-Tag2B-MMP-9 for 12 h and then treated with rhIFN-α for 36 h. (A) HBeAg and HBsAg in the supernatants were assayed by an ELISA. (B) HBV RNAs were detected by Northern blotting, and 18S and 28S rRNAs were used as loading controls. (C and D) HepG2 cells were cotransfected with pHBV1.3 and pCMV-Tag2B-MMP-9 for 12 h and then treated with JAK inhibitor I for 36 h. (C) HBeAg and HBsAg in the supernatants were assayed by an ELISA. (D) HBV RNAs were detected by Northern blot analyses. (E and F) HepG2 cells were cotransfected with pHBV1.3, pCMV-Tag2B-MMP-9, and pCAGGS-IFNAR1 for 48 h. (E) HBeAg and HBsAg in the supernatants were assayed by an ELISA. (F) HBV RNA levels were determined by Northern blotting. (G) HepG2 cells were transfected with sh-IFNAR1-1, and the protein level of IFNAR1 was determined by Western blotting. (H and I) HepG2 cells were cotransfected with sh-IFNAR1, pHBV1.3, and pCMV-Tag2B-MMP-9 for 48 h. (H) HBeAg and HBsAg in the supernatants were assayed by an ELISA. (I) HBV RNAs were detected by Northern blot analyses. (J to L) HepG2-NTCP cells were transfected with the vector, sh-IFNAR1-1, or sh-IFNAR1-2. Twenty-four hours later, cells were infected with HBV at 1,000 GEq. At 6 days postinfection, the mRNA level of IFNAR1 (J) was measured by qPCR, the HBeAg level in the supernatants of infected cells was determined by an ELISA (K), and the mRNA level of HBV 3.5-kb RNA was measured by qPCR (L). (M and N) HepG2-NTCP cells were transfected with the vector or pCMV-Tag2B-MMP-9 and sh-Ctrl or sh-IFNAR1-1. Twenty-four hours later, cells were infected with HBV at 1,000 GEq. At 6 days postinfection, HBeAg levels in the supernatants of infected cells were determined by an ELISA (M), and the levels of HBV 3.5-kb RNA were measured by qPCR (N). (O) HepG2-NTCP cells were transfected with the vector or pCMV-Tag2B-MMP-9 and sh-Ctrl, sh-IFNAR1-1, or sh-IFNAR1-2. Twenty-four hours later, cells were infected with HBV at 1,000 GEq. The expression of HBcAg in the cells was analyzed by immunostaining at 8 days postinfection. Results show means ± standard deviations (n = 3). *, P < 0.05.