![]() The HBV genome contains four open reading frames (ORFs): PreC/C (encoding precore protein giving rise to the hepatitis B e antigen and core protein), pS1/2 and S (encoding preS1, preS2, and S domains of the envelope proteins, respectively), X (encoding hepatitis B x antigen), and P (encoding viral polymerase, Pol). V01460.1), including the 1.056 HBV genome, in which the CMV promoter replaces the primitive HBV core promoter to start pregenomic RNA (pgRNA) transcription. Altogether, we describe a helpful tool for intracellular HBV replication and anti-viral drug screening studies.įigure 1 Genomic organization of wild-type hepatitis B virus vector pCH-3093 and replication-competent hepatitis B virus vectors.Ī: The parental plasmid pCH-3093 is based on hepatitis B virus (HBV) genotype D, subtype ayw (GenBank accession No. Interestingly, these recombinant HBV particles are significantly infectious for HepaRG lines. To detect the infectivity of recombinant HBV particles, we employed the available HBV infectable cell line HepaRG as the HBV infection model. Now, we report the new cell lines, which have the same background as HepG2.117 cells and produce high titer secNluc recombinant HBV particles. Previously, we established the tTA-expressing HepG2 TetOFF cell line HepG2.TA2-7 via a stably transfecting replication-competent HBV vector, and the HBV replication cell line HepG2.117 was successfully obtained, in which HBV particle production efficiency was ten times greater than HepG2.2.15 cell lines. As stable HBV-transfected human hepatoma cells, HepG2.2.15 cell lines are widely used in HBV molecular biology research, but HBV particle production efficiency is low. Transient transfection is an available method to establish suitable cell models of HBV infection and replication however, transfection efficiency varies. The results showed that the replication-competent HBV vector carrying secNluc reporter gene was able to replicate however, replication efficiency was decreased compared to the wild-type vector. In our study, we employed secNLuc reporter gene as foreign genetic material inserted into the replication-competent HBV vector to achieve a high level of expression of HBV particles carrying secNluc (597 bp) reporter gene, which is slightly larger than the BsdR gene (400 bp). The secreted luciferase (secNLuc) reporter gene (597 bp) can express luciferase protein that is secreted in culture supernatant and is beneficial for monitoring the transcriptional activation of the target gene, which is an extraordinarily helpful tool for molecular biology research. Data from several preliminary studies indicated that some medium-sized transgenes approximately 500 bp are compatible with replication competence. The latter could generate visible hrGFP expression by fluorescence microscopy, which is convenient for assessing the replication and expression of HBV however, replication efficiency is poor and cannot be quantified.įor this reason, we need to search for a new reporter gene that is convenient and quantifiable for further research. The replication efficiency of the former is higher but it is tedious to use in that the expression of functional BsdR needs to be detected by blasticidin, which generates stable Bsd-resistant cell clones upon transfection of pCH-BsdR into cells. Consequently, we have successfully constructed the replication-competent HBV vectors pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp). Hence, we uncoupled the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence, allowing it to carry nearly 400 bp (up to 720 bp) of foreign genetic information. Due to the characteristics of the HBV genome and replication strategy, interrupting any part of the genome by inserting a foreign sequence will inhibit HBV replication. ![]() In the preliminary research, we have successfully constructed replication-competent HBV vectors carrying foreign genetic material. Unfortunately, due to species restriction and tissue tropism of HBV, the development of models is limited. For this reason, it is absolutely necessary to establish suitable models of HBV infection and replication in vitro and in vivo. Hence, exploring the mechanisms of HBV replication and developing new drugs are imperative. The former is only partially effective, and the latter is essential to long-term medication and susceptible to resistance. Currently, antiviral therapy is critical for chronic infection with HBV, which includes type I interferon and nucleos(t)ide analogs. There are many patients with hepatitis B virus (HBV) infection all over the world who carry a high risk of liver fibrosis, cirrhosis, and hepatocellular carcinoma.
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