武汉大学吴建国教授研究组发现乙肝病毒慢性感染新机制

rentianyixu

乙型肝炎病毒（HBV）感染可引起急性乙型肝炎，急性感染者有可能通过天然免疫系统清除病毒，如果免疫反应未能清除病毒，就会发生持续性感染、导致慢性乙型肝炎、肝硬化和肝细胞癌。全世界约有20亿人感染过HBV，其中3.6亿为慢性感染者，每年约有60万人死于慢性乙肝引发的肝硬化和肝细胞癌。干扰素是人体天然免疫反应的重要组成部分，具有广谱抗病毒效应。干扰素并不直接杀伤或抑制病毒，主要是通过细胞表面受体作用使细胞产生抗病毒蛋白，从而抑制病毒复制；同时还可增强自然杀伤细胞、巨嗜细胞和T淋巴细胞的活力，从而起到免疫调节作用，增强抗病毒能力。研究表明HBV感染只能短暂和微弱诱导干扰素产生，而且还能抑制干扰素效应，这可能是HBV慢性感染的分子基础。但是，HBV是如何逃避宿主天然免疫应答、导致慢性感染与肝细胞癌的分子机制在很大程度上还不明确。最新一期的病毒学权威性杂志Journal of Virology上发表了武汉大学病毒学国家重点实验室研究人员发现乙肝病毒慢性感染新机制的文章“Matrix metalloproteinase 9 facilitates hepatitis B virus replication through binding with type I interferon (IFN) receptor 1 to repress IFN/JAK/STAT signaling”，论文通讯作者为吴建国教授和刘映乐副教授。该研究发现，在HBV感染患者的外周血单核细胞中，基质金属蛋白酶9（MMP-9）的表达被显著上调；在巨噬细胞和外周血单核细胞中，HBV感染能激活MMP-9表达。MMP-9主要是在细胞外基质分解，炎症反应，肿瘤侵袭、转移和血管生成中起重要作用，但在病毒感染和复制中的作用则少有研究。该研究进一步证明，MMP-9通过抑制干扰素通路（IFN/IAK/STAT）、干扰素活性、STAT1/2磷酸化、干扰素激活基因（ISG）表达等来促进HBV复制。详细研究证明，MMP-9通过与I型干扰素受体直接结合，促进I型干扰素受体磷酸化、泛素化、亚细胞定位及蛋白降解，从而阻碍干扰素与其受体的相互作用。 因此，该研究揭示了一种关于HBV复制与MMP-9生产之间的新的正反馈调节机制。一方面，HBV在感染者的淋巴细胞中激活MMP-9表达；另一方面，MMP-9 通过抑制IFN/IAK/STAT通路、干扰素活性以及干扰素受体功能等促进HBV 复制。因此，HBV可能是通过调控MMP-9功能来逃避宿主免疫应答、维持慢性感染。

Figure 8. MMP-9 promotes phosphorylation, ubiquitination, degradation and sub-cellular distribution of IFNAR1

(A and B) HepG2 cells were transfected with pCMV-Tag2B-MMP-9 for 48 h. Cells were harvested. IFNAR1 mRNAs were analyzed by qPCR (A) and IFNAR1 and β-actin proteins were detected by Western blots (B). (C) HepG2 cells were treated with rhMMP-9 (50 ng/ml) for different times. Cells were harvested and IFNAR1 and β-actin were detected by Western blots. (D) HepG2 cells were treated with rhMMP-9 (50 ng/ml) for 12 h and treated with CHX (50 μg/ml) for indicated times. Cells were harvested and IFNAR1 and β-actin were detected by Western blots. (E) HEK293T cells were co-transfected with pCAGGS-IFNAR1 and pCMV-Tag2B-MMP-9 for 48 h and treated with CHX (50 μg/ml) for the indicated times. The proteins were analyzed by Western blot using corresponding antibodies. (F) HepG2 cells were transfected with pCMV-Tag2B-MMP-9 for 42 h and treated with proteasome inhibitors (MG132 and lactacystin) and lysosome inhibitor (bafilomycin A1) for 6 h. Cells were harvested and IFNAR1, MMP-9, and β-actin were detected by Western blot analyses. (G) HepG2 cells were transfected with pCMV-Tag2B-MMP-9 for 42 h, treated with MG132 (20 μM) for 2 h and with CHX (50 μg/ml) for 4 h. Cells were harvested and IFNAR1 and β-actin were detected by Western blot analyses. (H) HEK293T cells were co-transfected with pCDNA3.1-HA-Ub, pCDNA3.1-3×Flag-IFNAR1 and pcDNA3.1-Myc or pcDNA3.1-Myc-MMP-9 and treated with MG-132 (20 μM) for 9 h. Cell lysates were denatured, and subjected to immunoprecipitation (IP) with anti-Flag. The immunoprecipitates and whole cell lysates (WCL) were analyzed by Western blot with the indicated antibodies. (I) HepG2 cells were transfected with pCMV-Tag2B or pCMV-Tag2B-MMP-9 for 48 h. TYK2 and β-actin proteins expressed in the treated cells were determined by Western blot analyses using corresponding antibodies. (J) HepG2 cells were transfected with pCMV-Tag2B-MMP-9-wt or pCMV-Tag2B-MMP-9-mut for 42 h and treated with MG132 (20 μM) for 6 h. Cells were harvested and IFNAR1 and β-actin were detected by Western blot analyses. (K) HepG2 cells were transfected with pCMV-Tag2B-MMP-9 for 42 h and treated with signaling component inhibitors, SB203580 (p38 MAPK inhibitor), U0126 (ERK1/2 inhibitor), PD98059 (MEK inhibitor), for 6 h. Cells were harvested and IFNAR1, MMP-9, and β-actin proteins were detected by Western blot analyses. (L and M) HEK293T cells were co-transfected with pCAGGS-IFNAR1 (L) or pCAGGS-IFNAR1-S532A (M) and pCMV-Tag2B-MMP-9 for 33 h, treated with rhIFN-α or TG (1 μM) for 1 h, and with CHX (50 μg/ml) for the indicated times. IFNAR1-HA, IFNAR1-S532A-HA, GFP, and β-actin proteins were determined by Western blot. (N and O) HepG2 cells were transfected with pCMV-Tag2B-MMP-9 for 48 h and then harvested. IFNAR1 protein (N) and IFNAR2 protein (O) were analyzed by flow cytometry. (P) HepG2 cells were co-transfected with pCAGGS-IFNAR1 and pcDNA3.1-Myc-MMP-9 for 24 h. Cells were immunostained with anti-Myc and anti-HA antibodies and the nucleus was stained by DAPI, and analyzed by confocal microscopy. Results show means ± SD, ns, non-significant.

Figure 10. MMP-9 facilitates HBV replication by repressing the IFN/JAK/STAT signaling and IFNAR1 function

We propose a novel positive feedback regulation loop between HBV replication and MMP-9 production. Initially, HBV activates MMP-9 in infected patients and in HBV-stimulated leukocytes. Subsequently, MMP-9 facilitates HBV replication through repressing the IFN/JAK/STAT signaling, interacting with IFNAR1 to block its binding with IFN-α, and promoting the phosphorylation, ubiquitination, sub-cellular distribution, and degradation of IFNAR1.