The Screen Of HBV Variants With Core Internal Deletion And Its Regulation Mechanisms On Unfolded Protein Response

Background:HBV genome is partially double-stranded circular DNA molecule of about 3.2kb in length.HBV is highly variable within its genome,including point mutations,deletions mutation and frameshift mutations.Deletion mutation may occur in the different coding frame within genome.The core internal deletion(CID)mutations in the genome of HBV have also been reported within the core gene.HBV CID may alter the biological properties and pathogenic properties of HBV.Thus,it is has important for further understand of HBV pathogenesis by investigating the prevalence of HBV CID mutants from chronically HBV-infected adults in Jiangxi province and analyzing the basic biological properties of discovered HBV CID variants.Objective:This study was aimed to investigate the prevalence of the core internal deletion(CID)mutations in the genome of hepatitis B virus(HBV)from chronically HBV-infected adults in Jiangxi province and to elucidate the molecular characteristics of CID and the relationship with the occurrence and development of liver disease.The construction of HBV CID eukaryotic expression vector aimed to observe its expression characteristics in transfected cells and to realize the regulation of HBV CID mutants on unfolded protein response.Methods:1.A total of 189 serum samples with HBs Ag-positivity were collected in Department of Clinical Laboratory of the Second Affiliated Hospital of Nanchang University for investigation of HBV CID variants.2.PCR was used to amplify C region of HBV genome from HBV DNA extracted from the serum.PCR products were purified and sequencing directly.3.The sequences of nucleotide and amino acid of PCR products were aligned with the reference sequence using software Clustal X and Bioedit to analyze the deleted regions and the prevalence of the core internal deletion(CID)mutations.4.The large amount PCR was performed on the serum with HBV CID mutant to amplify C region.The complete C gene and deleted C gene were,respectively,purified and recombined with plasmid p Bud CE4.1 to make recombinant plasmid p Bud CE4.1-HBV-C and p Bud CE4.1-HBV-CID.Digestion of Hind? and Xba?and DNA sequence were used to identify the both recombinant plasmids5.ELISA was applied to detect HBe Ag in supernatant and lysates of Hep G2 cells transfected by p Bud CE4.1-HBV-C,p Bud CE4.1-HBV-CID and p Bud CE4.16.The total RNA was extracted from Hep G2 cells transfected the p Bud CE4.1-HBV-C,p Bud CE4.1-HBV-CID and p Bud CE4.1 at 24 h,48 h and 72 h after transfection.The m RNA expression level of ATF4,ATF6,XBP1 and GRP78 involved in the unfolded protein response were detected by RT-PCR.Result:1.The average age of 189 HBV patients with HBs Ag-positivity were 48± 6.51 years old.102 samples(53.97%)were collected from male and 87 samples(46.03%)from female.2.The results of PCR showed that seven samples had two PCR bands containing a DNA band with expected size of 840 bp and a smaller band of about 700 bp.The band of 840 bp contained enhancer II,core promoter,precore and Core gene.The smaller band of 700 bp carried the internal deletion mutation within the C gene region.3.The Alignment of CID gene and complete C region gene showed the deleted regions of nt2152-2265,nt2132-2239,nt2163-2306,nt2179-2283,nt2131-2247,nt2151-2252 and nt2180-2203 / nt2255-2332 were found in the seven discovered CID mutants respectively.4.The deletion of aa84-121,aa78-113,aa89-136,aa94-128,aa78-116,aa84-117 and aa94-101/aa119-144 within core proteins resulted in the loss of the immune epitopes of b1/b2/c2/t3,b1/b2/c2/t3,b1/b2/b3/c2/t3,b2/c2/t3,b1/b2/c2/t3,b1/b2/c2/t3 and b3/c2/t3 of seven HBV CID variants,respectively.5.The double enzyme-digestion and sequencing confirmed that both eukaryotic expression vector p Bud CE4.1-HBV-C and p Bud CE4.1-HBV-CID were constructed successfully,carrying the interest bands of 840 bp and 700 bp,respectively.6.HBe Ag expression levels in supernatant and lysates of Hep G2 cells transfected by p Bud CE4.1-HBV-C were higher significantly than ones in groups of p Bud CE4.1-HBV-CID and p Bud CE4.1(P<0.05).HBe Ag was only detected in the lysates of and no detectable in the supernatants of Hep G2 cells transfected with p Bud CE4.1-HBV-CID.7.The results of RT-PCR revealed the m RNA expression level of ATF4,ATF6,XBP1-s and GRP78 in Hep G2 cells transfected with p Bud CE4.1-HBV-C for 24 h,48 h and 72 h were significantly higher than ones of transfected groups with p Bud CE4.1-HBV-CID and p Bud CE4.1(P<0.05).Conclusions:1.The prevalence of HBV CID variants were found in the chronic HBV carriers in Jiangxi region.The prevalent rate of HBV CID variants was about 3.7% by the primary investigation.2.The deletion mutations occurred within HBV CID variants were all within open reading frame and encompass the regions of nt2131-2306.3.The deletion of aa78-136 within HBV CID variants may lead to the loss of some immune dominant epitopes and immune escape,serving as one important mechanism for chronic and persistent hepatitis B.4.The deleted regions of aa78-136 within HBV CID variants may not abrogate the domain of core protein required for the assembly of HBV capsid protein.5.The conformation of the expressed products of HBV CID gene may be different from HBe Ag expressed by the complete Core gene,explaining the mechanism for the secretion disability of the expressed products of HBV CID gene.6.HBV CID variants may activate the high expression of the proteins involved in the unfolded protein response,may contributing to one mechanism of complexity in HBV pathogenesis.