Construction Of Recombinant Hepatitis B Virus Vector Expressing APOBEC3C And Its Antiviral Effects In Vitro

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis in humans.Treatment has largely been unsuccessful, and there is clearly an urgent need to identify newand better therapeutic agents. Natsoulis and Boeke (1991) developed a new anti-viral strategy,referred as the capsid targeted viral inactivation (CTVI). CTVI is conceptually a powerfulantiviral strategy that exploits viral structural proteins or virion-associated protein as carriersthat target a destructive enzyme specifically into progeny virions to achieve the purpose ofinhibiting virus replication.Beterams and Nassal (2001) applied CTVI to the treatment of HBV. Calcium-dependentstaphylococcal nuclease (SN) was fused to the C-terminus of HBV capsid protein to yield achimeric protein, Core-SN. When the fusion protein was co-transfected into human hepatomacells with HBV in a ratio of1:10(<1Core-SN protein per10wild-type core proteins),>95%reduction in enveloped particles was reported. However, SN requires particles to reach a Ca2+rich environment. Intracellular calcium concentration is too low for activation of Core-SN.Core-SN can only be activated when the virus is released into the extracellular medium andstarts to degrade HBV DNA. This can avoid the effects DNase on DNA inside cells. But thiscannot inhibit HBV DNA inside the cells gettingt more cDNA in nucleus. Therefore it isnecessary to use directly the protein that has destroyed HBV DNA inside the cells.Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide (APOBEC) proteinscatalyse the hydrolytic deamination of cytosine, where cytosine (C) is converted to uracil (U)by the addition of water and the removal of an amine group. Thomas et al (2007) reported thedramatic effect of APOBEC3C (A3C) on HBV. The HBV core protein and A3C were verystable and can induced hypermutation in the genome of HBV, where guanine (G) is convertedto adenine (A). This showed that HBV were susceptible to the editing effect of A3C, andindicated that A3C can elicit an anti-HBV host response. But A3C cannot enter into thenucleus.In present study, we first observed the anti-HBV effects of the replicating-defective HBV vector plasmid expressing A3C (pCH-LJ3-A3C). Then, we tried to apply viral capsid-orientedCTVI to construct the nucleocapsid protein and A3C fusion protein (pCH-Core-A3C) andthen made a detailed analysis of the inhibitory effect of A3C on hepatitis B virus replicationin cell culture. The main methods are as follows: The levels of HBsAg and HBeAg from inthe culture supernatant were measured by ECLIA; viral DNA were extracted from the culturesupernatant and cytoplasmic lysates and visualized by Southern blotting analysis; HBV DNAextracted from cell lysates were analyzed by3D-PCR; the subcellular localization of thefusion protein in the cell was analyzed by cellular immunochemical method; pCH-LJ3-A3Cwas co-transfected with the helper construct pCH-3142that devoid of the encapsidation signalε, the viral DNA were extracted and visualized by Southern blotting analysis.The main results of our study were listed as follows:Part I: Construction of replicating-defective HBV vector plasmid expressing A3C andantiviral effects. A3C gene was amplified by PCR and was inserted into pCH-LJ3-hrGFPafter digestion by XhoI and BSP1407I, replaced the hrGFP gene of the pCH-LJ3-hrGFP toconstruct pCH-LJ3-A3C. The sequence was confirmed by DNA sequencing. Plasmid pCH-LJ3-A3C was cotransfected with wild-type HBV expressing plasmid (pCH-3093) into HepG2Cells. Firstly, the levels of HBsAg and HBeAg from in the culture supernatant were measuredby ECLIA. The results showed that pCH-LJ3-A3C had no effect on the expression of HBsAg(104.4％±2.25%) and HBeAg (98.65%±0.85%), respecterly. Secondly, as to elucidateantivirus function mediated by pCH-LJ3-A3C, pCH-LJ3-A3C was cotransfected into HepG2cells together with a construct harboring the wild-type HBV genome. Viral DNA wasextracted from the culture supernatant and cytoplasmic lysates and visualized by Southernblotting analysis. The results showed that accumulation of HBV replicative intermediates incytoplasmic lysates were reduced by30%with pCH-LJ3-A3C; release of viral particles intothe culture supernatant was reduced by39%with pCH-LJ3-A3C. Thirdly, as to investigatereplication and encapsidation capabilities of pCH-LJ3-A3C, pCH-LJ3-A3C was cotransfectedwith the helper construct (pCH-3142). The results showed that with aid of pCH-3142, thecapabilities of replication and encapsidation could be rescued. The progeny "therapeutic"HBV particles with A3C can be generated to secrete into the extracellular supernatant. Endlly,to determine the editing function of HBV DNA in viral nucleocapsids mediated by A3Cprotein, HBV DNA extracted from cell lysate was analyzed by3D-PCR. The PCR products were ligated into the pGEM-T cloning vector and transformed into TOP10competent E. colicells. The transformed cells were stored overnight, and this suspension was applied directlyfor DNA sequencing. Fifty individual clones were selected for HBV DNA sequence analysis.36clones displayed G-to-A mutations, while total number of G-to-A mutations was982.Part II: Construction of recombinant HBV vector plasmid expressing Core-A3C fusionprotein and its antiviral effects. A3C gene was amplified by PCR and was inserted into pdssXafter digestion by BstEII and Acor13HI to construct plasmid pCH-Core-A3C.The sequencewas confirmed by DNA sequencing. Plasmid pCH-Core-A3C was cotransfected withwild-type HBV expressing plasmid (pCH-3093) into HepG2Cells. Firstly, the levels ofHBsAg and HBeAg from in the culture supernatant were measured by ECLIA. The resultsshowed that pCH-Core-A3C had no effect on the expression of HBsAg (97.82%±1.37%) andHBeAg (97.16%±0.86%), respectively. Secondly, as to elucidate antivirus function ofpCH-Core-A3C, pCH-Core-A3C was cotransfected into HepG2cells together with aconstruct harboring the wild-type HBV genome. Viral DNA was extracted from the culturesupernatant and cytoplasmic lysates and visualized by Southern blotting analysis. The resultsshowed that accumulation of HBV replicative intermdiates in cytoplasmic lysates werereduced by84%with pCH-Core-A3C; release of viral particles into the culture supernatantwas reduced by91%with pCH-Core-A3C. Thirdly, to investigate the editing function ofHBV DNA in viral nucleocapsids mediated by pCH-Core-A3C, HBV DNA extracted fromcell lysate was analyzed by3D-PCR. The PCR products were ligated into the pGEM-Tcloning vector and transformed into TOP10competent E. coli cells. The transformed cellswere stored overnight, and this suspension was applied directly for DNA sequencing. Fiftyindividual clones were selected for HBV DNA sequence analysis.48clones displayingG-to-A mutations, while total number of G-to-A mutations was2416. Finally, the subcellularlocalization of the fusion protein in the cell was analyzed by cellular immunochemicalmethods, the results showed that the Core-A3C fusion protein was mainly localized in thecytoplasm.From these results, we drawed conclusions as follows:1．Recombinant HBV vector expressing Core-A3C fusion protein plasmid pCH-Core-A3C was successfully constructed. Replicating-defective HBV vector expressing A3Cplasmid pCH-LJ3-A3C was successfully constructed. 2．The levels of HBsAg and HBeAg from in the culture supernatant were measured byECLIA. The results showed that pCH-LJ3-A3C and pCH-Core-A3C had no effect on theexpression of HBsAg and HBeAg.3．The subcellular localization of the fusion protein in the cell was demonstrated bycellular immunochemical methods: the Core-A3C fusion protein was localized in thecytoplasm.4．The capabilities of replication and encapsidation in these replicating-defective HBVvector expressing A3C plasmid pCH-LJ3-A3C could be rescued with aid of the homologoushelper construct pCH-3142that devoid of encapsidation signal ε.5．Accumulation of HBV replicative intermdiates in cytoplasmic lysates and release ofviral particles into the culture supernatant were reduced by pCH-LJ3-A3C andpCH-Core-A3C, respectively. Compared with pCH-LJ3-A3C, the pCH-Core-A3C plasmidencoding Core-A3C chimerical protein substantially inhibited HBV production intracellularlyand extracellularly.6．HBV DNA extracted from cell lysate was analyzed by3D-PCR, fifty individualclones were selected for HBV DNA sequence analysis. Compared with pCH-LJ3-A3C, theediting function of pCH-Core-A3C on HBV genomes (total number of G-to-A mutations) wasmore potent.In conclusion, we constructed a new fusion protein with strong antiviral action. Thefusion protein can be assembled into HBV capsids with viral proteins. The editing function ofpCH-Core-A3C on HBV genomes was more potent. With aid of pCH-3142, the progeny"therapeutic" HBV particles with A3C can be generated and secreted into the extracellularmedium. The new A3C fusion protein could be regarded as an excellent candidate forfulfilling gene therapy strategies against HBV. It is expected to achieve "One-time treatment"for long-lasting anti-HBV effect in vivo.