Construciton Of Replication-competent Infectious Hepatitis B Virus Vectors Carrying Substantially Sized Transgenes

Background and objectiveViral vectors are engineered virus variants able to deliver nonviral genetic informationinto cells, usually by the same routes as the parental viruses. For several virus families,replication-competent vectors carrying reporter genes have become invaluable tools foreasy and quantitative monitoring of replication and infection, and thus also for identifyingantivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNAvirus causing B-type hepatitis, such vectors are not available because insertions into its tiny3.2kb genome almost inevitably affect essential replication elements. HBV replicates byreverse transcription of the pregenomic (pg) RNA which is also required as bicistronicmRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open readingframes (ORFs) overlap by some150basepairs. Translation of the downstream Pol ORFdoes not involve a conventional internal ribosome entry site (IRES). We reasoned thatduplicating the overlap region and providing artificial IRES control for translation of bothPol and an in-between inserted transgene might yield a functional tricistronic pgRNA,without interfering with envelope protein expression. As IRESs we used a22nucleotideelement termed Rbm3IRES to minimize genome size increase. Analogous plasmids forcomplete HBV genomes carrying399bp and720bp transgenes for blasticidin resistance(BsdR) and humanized Renilla green fluorescent protein (hrGFP) were constructed. Theyboth produced core and envelope proteins like wild-type HBV and remained replication.Both vectors, however, formed enveloped virions which were infectious forHBV-susceptible HepaRG cells. The new HBV vectors should become highly useful toolsto better understand, and combat this important pathogen.MethodThe study was followed as three parts. Part one: The activity of22-nt Rbm3IRES on the HBV pgRNA1. The first series of model plasmids were driven by CMV promoter and encodedEGFP as marker for visual inspection. I: pCH-EMCV IRES-EGFP; II: pCH-22ntIRES-EGFP; III: pCH-BsdR-22nt IRES-EGFP; IV: pCH-pATG-EGFP. The EGFPexpression was observed after HepG2or Huh7cells were transfected by the four plasmidsfor72h. HBeAg secretion was measured by ELISA in I, II and III group.2. The second series of model plasmids were driven by endogeous HBV core promoterand encoded Renilla luciferase (RLuc) for expression quantification. I: pHBV-EMCVIRES-RLuc; II: pHBV-22nt IRES-RLuc; III: pHBV-BsdR-22nt IRES-RLuc; IV:pHBV-pATG-RLuc. The Rluc constructs were cotransfected with the firely luciferasevector pGL3-Control and relative RLuc activity was monitored using the dual luciferaseluminometric assay.Part two: Construciton, Expression and Replication competence of Replication-competentHBV vectorpCH-BsdR and pCH-hrGFP were constructed. HepG2and Huh7cells were transfectedby the two plasmids and wild type plasmid pCH3093separately. Expression of hrGFP wasobserved by fluorescence microscope. Expression of functional BsdR was subsequentlydemonstrated by formation of stable Bsd resistant cell clones. HBV RNA was determinedby Northern blot. Envelope protein, core protein and assembled capsids were shown byWestern blot and Native western blot respectively. HBsAg and HBeAg secrection weredetermined by ELISA. Functional Pol molecule was detected by endogenous polymerasereaction (EPR). Replicative DNA intermediates were analyzed by Southern blot, and theamounts of viral genomes in the culture supernatants were determined by quantitative PCR.Viral particles were separated by CsCl density gradient centrifugation.Part three: Infection of Replication-competent HBV vectorHepG2cells were transfected by pCH-3093, pCH-BsdR and pCH-hrGFP respectively.Viral particles were collected from the culture supernatants by PEG8000precipitation.HepRG cells were infected by the enveloped virions for8days. HBV RNA wasdeterminded by Northern blot. HBsAg and HBeAg secrection were measured by ELISA.For blocking infection, the viral particles were preincubated with HBIG for1h beforeinfection. ResultsPart one: The activity of22-nt Rbm3IRES on the HBV pgRNA1. From the result of HBeAg ELISA, there were no significant difference amongconstruct I, II and III. EGFP fluorescence from the Rbm3IRES was stronger in HepG2cells than that from the corresponding EMCV IRES construct (II vs. I). EGFP fluorescencefrom the tricistronic construct (III) was substantially reduced yet remained easily detectable,clearly exceeding the signals from construct IV.2. Both HepG2and Huh7cells gave very similar results. Relative RLuc activity fromthe Rbm3IRES was more than2-fold higher than from the EMCV IRES (II vs. I, P＜0.01).RLuc activity from the tricistronic construct was reduced significantly compared withthe bicistronic EMCV IRES construct (III vs. II, P＜0.01), and it clearly exceeded that fromthe construct having the RLuc gene fused to the authentic Pol start (III vs. IV, P＜0.05).Part two: Construciton, Expression and Replication competence of Replication-competentHBV vectorpCH-BsdR and pCH-hrGFP were constructed. After transfected into HepG2or Huh7cells, high level hrGFP expression could be seen. Stable Bsd resistant cell clones could beformed after screened by Bsd. The recombinant pgRNA carrying transgenes could bemeasured by Northern blot. The two vectors produced core and envelope proteins likewild-type HBV. There were no significant differences in HBsAg and HBeAg secretionbetween groups. The data demonstrated that functional Pol was translated from the BsdRvector. While the hrGFP vector replicated poorly, the BsdR vector generated around40%asmuch replicative DNA as wild-type HBV. Both vectors formed enveloped virions.Part three: Infection of Replication-competent HBV vectorEnveloped virions formed by both vectors were infectious for HBV-susceptibleHepaRG cells. All inocula led to detectable presence of genomic and subgenomic viralRNAs8days post-inoculation. Infection by and active viral replication of the BsdR vectorparticles were supported by a wild-type HBV-like net increase of secreted HBsAg andHBeAg. GFP expression in hrGFP-HBV vector inoculated cells indicates successfulinfection. The test of preincubation with neutralizing hepatitis B immuno-globulin (HBIG)demonstrated that the infection was indeed mediated by the viral envelope proteins, as inwild-type HBV infection. Conclusion1. Wether driven by CMV or the authentic HBV core promoter, the Rbm3IRESdirected higher levels of reporter gene expression in human hepatoma cells than the EMCVIRES from bicistronic constructs. Importantly, substantial expression of the mostdownstream cistron was also achieved from the tricistronic constructs mimicking pgRNAwith a transgene inserted between core and Pol ORF.2. The C and P protein were expressed separately. Two22-nt IRES were used toexpress transgenes and P protein separately, which could maintain integrity and replicationcompetence, decrease the size of gene expansion, and realize the vector function.3. Both vectors formed enveloped virions which were infectious for HBV-susceptibleHepaRG cells. It demonstrated that the replication-competent HBV vector was infectious asthe wild-type HBV.4. Because numerous reporter and effector genes with sizes of around500bp or lessare available, the new HBV vectors should become highly useful tools to better understand,and combat this important pathogen.