The Study On The Function And Molecular Mechanism Of Fatty Acid Translocase CD36 In HBV Replication

Objective: Hepatitis B virus(HBV)is designated a “metabolovirus”due to the intimate connection between the virus and host metabolism.The nutrition state of the host plays a relevant role in the disease severity of HBV infection.The dysregulated lipid profile and altered lipid-related genes are likely due to metabolic hijacking by HBV in favor of its viral replication.A study demonstrated that fatty acid synthase(FAS)was upregulated in HBV-infected cells and the reduction of FAS by siRNA would suppress viral replication.Cluster of differentiation 36(CD36),also named fatty acid translocase(FAT),is known to facilitate long-chain fatty acid uptake and contribute to the development of some metabolic diseases.Recent studies revealed that fatty acid translocase CD36 is involved in the replication,assembly,storage,and secretion of certain viruses,such as hepatitis C virus(HCV)and human immunodeficiency virus(HIV).Our previous study found that the expression of CD36 was increased in the HBV-replicating cell line,suggesting that CD36 might play a role in HBV replication.In this study,we further explored the relationship andmolecular mechanism between CD36 and HBV replication.Methods: Both HBV-replicating cell lines HepG2.2.15 and HepAD38 were infected with recombinant lentivirus expressing CD36 cDNA or shRNA sequences to obtain stable overexpression or knock-down CD36 cell line models,respectively.The acute HBV infection model was established in C57BL/6 wild type and CD36 knockout mice via hydrodynamic injection.HBV DNA replicative intermediates were extracted and measured by real-time PCR and southern blot.An RNA sequencing(RNA-seq)approach was utilized to analyze the differential transcriptomic profiles in CD36 overexpression and the vector control of HepG2.2.15 cells.Cytosolic calcium was detected by a fluorescent Ca2+indicator Fluo-4/AM using flow cytometry.Chemical calcium reagents were utilized to assess the effects of calcium signals on CD36-induced HBV replication.Results: CD36 overexpression increased HBV replication,and CD36 knockdown inhibited HBV replication in vitro.Similarly,in vivo,the levels of HBV DNA in the serum and liver were remarkably decreased in CD36 knockout mice.RNA-seq found some of the differentially expressed genes were involved in ion homeostasis,especially calcium ion homeostasis.CD36 overexpression elevated the cytosolic calcium level,and CD36 knockdown decreased the cytosolic calcium level.Calcium chelator BAPTA-AM could override the HBV replication increased by CD36 overexpression,and the calcium activator thapsigargin could improve the HBV replication reduced by CD36 knockdown.We further found that CD36 overexpression activated Src kinase,which plays an important role in the regulation of the SOC channel and calcium influx.An inhibitor of Src kinase(SU6656)significantly reduced the CD36-induced HBV replication.Conclusions: In general,we have characterized new aspects of fatty acid translocase CD36 biology in HBV replication by showing that CD36 overexpression and knockdown are positive and negative regulators of HBV replication via cytosolic calcium and the Src kinase pathway,respectively.Given the relationship between CD36 and HBV replication,we hypothesize that for those HBV-infected patients with a higher expression level of CD36,such as NAFLD and MS patients,targeting CD36 may serve as an adjunctive therapeutic means for conventional anti-viral drugs.