Matrix Metalloproteinase 9 Facilitates Hepatitis B Virus Replication Through Repress Type Ⅰ Interferon Signaling

Around the world,2 billion people have been infected with HBV,among them,360 million are chronic infection,about 600,000 people died of liver disease caused by chronic hepatitis B each year.Liver cancer is the fifth most common cancer in men,accounting for 7.9%,and the seventh most common cancer in women,accounting for 6.5%.Hepatocellular carcinoma(HCC)accounts for the majority of liver cancer,and 80%of hepatocellular carcinoma is caused by HBV or HCV infection,of which HBV accounted for the majority.China is one of the regions with the highest hepatitis B virus infection rate.About 120 million people are carriers of hepatitis B virus,of which 20 million people suffer from chronic hepatitis B,and about 300,000 people die each year from HBV infection in China.HBV infection leads to various liver diseases,including acute hepatitis,chronic hepatitis,cirrhosis and hepatocellular carcinoma.The symptoms and consequences of HBV infection are associated with the age and immunity of the infected person.95%of adults infected with HBV will recover spontaneously,and 90%of the infants and 30%of the 1-5 year old children infected with HBV develop chronic hepatitis B.Once the chronic hepatitis B virus infection is established,the virus is difficult to be completely eliminated.Therefore,it is crucial to understand the mechanisms by which HBV establish and maintain chronic infection.Matrix metalloproteinase 9(MMP-9)is a protein that plays important roles in angiogenesis and tumor migration.The expression of MMP-9 is activated by HBV and HBx protein in hepatocytes.MMP-9 is mainly expressed by leukocytes in humans.We found that MMP-9 expression in PBMCs of patients with chronic hepatitis B was significantly higher than that of healthy individuals.Moreover,in PBMCs and monocytes differentiated macrophages,MMP-9 is activated by HBV,which is independent of the replication of HBV.MMP-9 plays important roles in development,angiogenesis,apoptosis,inflammatory response and cancer,and its function in virus infection and replication is rarely reported.In overexpression and interference experiments and HepG2-NTCP infection system,we analyzed the expression of HBV surface antigen and e antigen by ELISA,the expression of HBV 3.5 kb,2.4 kb and 2.1 kb RNA by Northern blot and the levels of intracellular and extracellular nuclear capsid-associated HBV DNA by qPCR,the results showed that MMP-9 promoted HBV replication.And this effect was independent of the protease activity of MMP-9.In the further investigations,we found that HBV infection and replication in HepG2-NTCP cells activated the phosphorylation of IRF3,the expression of IFN-βand interferon stimulated genes.Moreover,exogenous IFN-a inhibited the replication of HBV,and MMP-9 antagonized the inhibitory effect of IFN-a on HBV.Using JAK1 inhibitors and IFNAR1-specific interfering RNA,we confimed that MMP-9 promotes HBV replication by inhibiting IFN/JAK/STAT pathway.By determining the phosphorylation of JAK1,STAT1 and STAT2 proteins and the expression of PKR,OAS1 and Mx1 induced IFN-a,we found that MMP-9 inhibits the type I interferon activated signaling and effector genes.MMP-9 is secreted into the extracellular matrix and interacts with the receptor and membrane proteins on the cell membrane through its carboxyterminal hemopexin domain.By imunoprecipitation experiments,we demonstrated that MMP-9 interacts with IFNAR1 on the cell membrane.By truncating these two proteins and following immunoprecipitation experiments,we found that MMP-9 interacts with the extracellular domain of IFNAR1 through its hemopexin domain.Moreover,this interaction prevents the binding of IFN-a and IFNAR1.The expression of IFNAR1 can be degraded by ligand-dependent and ligand-independent pathways.We found that MMP-9 had no effect on the expression of IFNAR1 mRNA but reduced the protein level.Further study found that MMP-9 can promote the phosphorylation,ubiquitination,subcellular localization changes and degradation of IFNAR1.This degradation does not depend on its protease activity,and is independent of ERK and p38 signaling pathways,the exact mechanism remains unclear.Finally,we found that MMP-9 also promotes the replication of VSV by inhibiting the function of IFN-a.In conclusion,we identified a novel positive-feedback regulation loop between HBV replication and MMP-9 production.On one hand,HBV activates MMP-9 in infected patients and leukocytes.On the other hand,MMP-9 facilitates HBV replication through repressing IFN/JAK/STAT signaling,IFNAR1 function,and IFN-a action.Therefore,HBV may take the advantage of MMP-9 function to establish or maintain chronic infection.