Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model

AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS:The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP),which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence.Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy.Cellular localization and protein levels were examined by Western blotting.RESULTS:HCV NS3/4A protease cleaved eYFP-MAVSfrom mitochondria to block the activation of interferon (IFN)-β promoter,thus resulting in downregulation of SEAP activity.The decrease in SEAP activity was proportional to the dose of active NS3/4A protease.Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A.CONCLUSION:Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors.This system will constitute a new tool to allow the efficient screening of HCV inhibitors.

TAX1BP1 protects against myocardial infarction-associated cardiac anomalies through inhibition of inflammasomes in a RNF34/MAVS/NLRP3-dependent manner

Acute myocardial infarction(MI),one of the most common cardiovascular emergencies,is a leading cause of morbidity and mortality.Ample evidence has revealed an essential role for inflammasome activation and autophagy in the pathogenesis of acute MI.Tax1-binding protein 1(TAX1BP1),an adaptor molecule involved in termination of proinflammatory signaling,serves as an important selective autophagy adaptor,but its role in cardiac ischemia remains elusive.This study examined the role of TAX1BP1 in myocardial ischemic stress and the underlying mechanisms involved.Levels of TAX1BP1 were significantly downregulated in heart tissues of patients with ischemic heart disease and in a left anterior descending(LAD)ligation-induced model of acute MI.Adenovirus carrying TAX1BP1 was delivered into the myocardium.The acute MI induced procedure elicited an infarct and cardiac dysfunction,the effect of which was mitigated by TAX1BP1 overexpression with little effect from viral vector alone.TAX1BP1 nullified acute MI-induced activation of the NLRP3 inflammasome and associated mitochondrial dysfunction.TAX1BP1 overexpression suppressed NLRP3 mitochondrial localization by inhibiting the interaction of NLRP3 with mitochondrial antiviral signaling protein(MAVS).Further investigation revealed that ring finger protein 34(RNF34)was recruited to interact with TAX1BP1 thereby facilitating autophagic degradation of MAVS through K27-linked polyubiquitination of MAVS.Knockdown of RNF34 using siRNA nullified TAX1BP1 yielded protection against hypoxia-induced MAVS mitochondrial accumulation,NLRP3 inflammasome activation and associated loss of mitochondrial membrane potential.Taken together,our results favor a cardioprotective role for TAX1BP1 in acute MI through repression of inflammasome activation in a RNF34/MAVS-dependent manner.