Exploration Of Effect And Mechanism Of Single Amino Acid Substitution Of Cpam Binding Site On The Characteristics Of Viral Biology

Background and Aims:Hepatitis B virus(HBV)infection is a leading course of chronic Hepatitis.Without standard treatment,there is a risk of development of liver cirrhosis,liver failure and other end-stage liver diseases,and also the hepatocellular carcinoma(HCC).Current antiviral drugs mainly include two types:nucleos(t)ide analogues(NAs)and interferon drugs.NAs competitively binds to polymerase and inreversibly incorporate into the newly synthesized DNA chain,leading to HBV DNA replication inhibition.Whereas interferon have indirect antiviral effect via regulating host factors in infected cells or exert comprehensive antiviral effect via regulating host immune response.While neither of them effectively eliminates the "source" of the persistent infection by HBV,that is,the covalently closed circular DNA(cccDNA)that exists in the nuclei of infected liver cells.Scientists generally believe that the effective cure of chronic HBV infection requires combination of application of drugs that target multiple nodes in the HBV life cycle.Currently,a variety of antiviral drugs that inhibit various steps of HBV replication are in the preclinical or clinical development stages.Core protein allosteric modulator(CpAM)specificly binds to Cp dimer dimer interface to accelerate capsid assembly,leading to the formation of aberrant non-capsid polymers(type Ⅰ CpAM)or "normal" empty capsid structures(type Ⅱ CpAM),thereby inhibiting the packaging of pregenomic RNA(pgRNA)/pol protein complexes and the following HBV DNA synthesis.Additionally,CpAM binds to and disassemble the assembled capsid and "mature" nucleocapsid that harbors double stranded DNA and leads to inhibition of cccDNA formation.Because CpAM target multiple stages of virus replication cycle,CpAMs showed good potential of clinical application.At present,many of CpAM have entered phase Ⅱ clinical trials.Based on systematic analysis of site-directed mutagenesis for the hydrophobic pocket of HBV Cp dimers interface,we previously identified several amino acid residues important for capsid assembly,pgRNA/pol cpmplex encapsidation and the CpAM antiviral effect.Meanwhile,some mutation confers strong resistance of multiple CpAM antiviral effects.Based on these,this project further studied the interaction between CpAM and the virus,deeply explored the impact of CpAM-resistant mutations on the entire replication cycle of HBV to enrich the antiviral mechanism of CpAM,and also to reveale the biological characteristics of mutated HBV.The understanding of the antiviral mechanism of this class of antiviral drugs will promote the investigation of HBV resistance mutation strategies,which may contribute to the functional cure of HBV.Materials and Methods:HepG2 cells were transiently transfected with pHBV1.3 or pCMV-HBc plasmid carrying CpAM-resistant mutation on P25,T33 and 1105.The effect of CpAM-resistant mutation on the capsid assembly was measured by sucrose density ultracentrifugation,particle gel assay.the effect on the disassembly or uncoat ing of mature capsid or nucleocapsid were evaluated by combination of Southern blot and endogeneous polymerase reaction assay.The effect on the pgRNA encapsidation and the DNA replication were measured by Northern blot and Southern blot.The intact virion particle secretion was measured by anti-preS2 and anti-S antibody combined immune precipitation and the following real-time PCR.The C3ANTCP and the primary human hepatocytes(PHHs)HBV infection cell model were utilized by exploring the effect of mutation on the progeny virus infectivity and the cccDNA formation.In vitro phosphatase treatment,phos-tag gel derived SDS-PAGE,Western blot and ELISA assay were enrolled to explore the biosynthesis of precore,the phosphorylation of p22 and the secretion of HBeAg.The immunofluorescence(IF)and the cell fractionation assay were utilized to investigate the subcellular localization of p22.The virological function of p22 was measured by Northern blot and Southern blot etc.Results:1.Single amino acid substitutions at the Cp dimer-dimer interface(P25,T33 and 1105)not only resistance on CpAM inhibition of pgRNA encapsidation and the DNA replication,but also resistance on structural alteration of assembled capsids,"mature"nucleocapsid disassembly and the cccDNA formation inhibition.2.Although DNA-containing capsids and the empty capsid assembly are not affected by these CpAM-resistant mutations,several mutations(P25S,T33N,T33Q,I105Y and I105W)showed significant inhibition of pgRNA encapsidation.3.Except for P25A and I105F,others mutation significantly inhibited virion particle secretion and the infectivity of progeny virus.4.Mutations in the context of precore also inhibited HBeAg production.Biochemical analysis indicates mutations on P25,T33 and 1105 inhibited intracellular p17 but not p22,leading to the HBeAg oroduction inhibition.5.Incellular p22 exist in both phosphorylated and unphosphoiylated forms,while unphosphorylated p22 located in the membranes associated vesicles and is the precursor of HBeAg,the phosphorylated p22 located in the cytoplasm and nuclei to disrupt capsid assembly and the DNA replication.6.GLS4 inhibit the biosynthesis of HBeAg by directly exhibit effect on intrecellular p17 but not p22,and the CpAM-resistant Cp mutation also resisted on GLS4 inhibition of HBeAg production.Conclusion:1.Although the amino acid residues at the Cp dimer-dimer interface are not located at the surface of cpasid,they are critical to multiple stages of the virus lif cycle,including the pgRNA/pol encapsidation,the secretion of the intact virion particle,the infectivity of progeny virus and the production of HBeAg.2.The CpAM binds to similar amino acid residues in the HAP pocket of the Cp and the reduced p17 dimer dimer interface to disrupt the pgRNA/pol encapsidation,the mature capsid or nucleocapsid disassembly,the cccDNA formation and most interestly,the HBeAg biosynthesis,leading to CpAM exerts multiple antiviral effect on HBV replication.