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Yoo, Junhee; Rho, Jinkyung; Lee, Dong Hoon; Shin, Suho; Jung, Guhung

doi:10.1023/a:1015320314201

Cited by 11 publications

(6 citation statements)

References 24 publications

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“…Rather than adding extra information, the commonly used approach was therefore to replace authentic viral sequences, e.g. the S ORF [18], [19], [20], [21] or the C ORF [22] with the transgene of interest. Some of these studies have indeed documented liver-specific gene transfer by such vectors; our own recent work, employing a chimeric replication-defective adenovirus-HBV vector carrying a truncated matrix-metalloproteinase-8 (MMP-8) has shown substantial therapeutic benefit in a rat model of liver cirrhosis [23].…”

Section: Introductionmentioning

confidence: 99%

Replication-Competent Infectious Hepatitis B Virus Vectors Carrying Substantially Sized Transgenes by Redesigned Viral Polymerase Translation

Wang

Cheng

et al. 2013

PLoS ONE

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Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better understand, and combat, this important pathogen.

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Section: Introductionmentioning

confidence: 99%

Replication-Competent Infectious Hepatitis B Virus Vectors Carrying Substantially Sized Transgenes by Redesigned Viral Polymerase Translation

Wang

Cheng

et al. 2013

PLoS ONE

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“…[ 1 , 3 ], the only option other than preS/polymerase spacer region was the central part of preC/C ORF that does not overlap with polymerase ORF ( Figure 1 ). Previously, this region has been targeted in recombinant HBV studies with limited inconclusive results [ 16 , 17 ]. We deleted 298 bp of this region (nucleotides 2009–2306) in 5c3c and inserted a short synthetic linker containing both a stop codon to terminate core ORF prematurely and restriction enzyme recognition sites to ease subsequent insertions.…”

Section: Resultsmentioning

confidence: 99%

Re-Designed Recombinant Hepatitis B Virus Vectors Enable Efficient Delivery of Versatile Cargo Genes to Hepatocytes with Improved Safety

Bai

Cui

Chen

et al. 2016

Viruses

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Hepatitis B virus (HBV) takes humans as its sole natural host, and productive infection in vivo is restricted exclusively to hepatocytes in the liver. Consequently, HBV-derived viral vectors are attractive candidates for liver-targeting gene therapies. Previously, we developed a novel recombinant HBV vector, designated 5c3c, from a highly replicative clinical isolate. 5c3c was demonstrated to be capable of efficiently delivering protein or RNA expression into infected primary tupaia hepatocytes (PTH), but the design of 5c3c imposes stringent restrictions on inserted sequences, which have limited its wider adoption. In this work, we addressed issues with 5c3c by re-designing the insertion strategy. The resultant vector, designated 5dCG, was more replicative than parental 5c3c, imposed no specific restrictions on inserted sequences, and allowed insertion of a variety of cargo genes without significant loss of replication efficiency. 5dCG-based recombinant HBV effectively delivered protein and RNA expression into infected PTH. Furthermore, due to the loss of functional core ORF, 5dCG vectors depend on co-infecting wild type HBV for replication and efficient expression of cargo genes. Development of the improved 5dCG vector makes wider applications of recombinant HBV possible, while dependence on co-infecting wild type HBV results in improved safety for certain in vivo applications.

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“…Replication-competent virus vectors carrying foreign reporter genes have become helpful tools in virus molecular biology research and anti-viral drug screening in various diseases, such as infections with HCV[19,20], human immunodeficiency virus (HIV)[21,22], and influenza virus[23,24]. Various replication-defective HBV vectors were constructed by substitution of the S gene[25-27] or the Core gene[28] with the gene of interest. We reported the construction of double shRNA expression in HBV Core and S regions[29].…”

Section: Discussionmentioning

confidence: 99%

Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines

Ruan¹,

Ping²,

Sun³

et al. 2019

WJG

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BACKGROUNDPreviously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling 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. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research.AIMTo establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODSWe utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTSThe replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity.CONCLUSIONOur research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.

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Untitled

Cited by 11 publications

References 24 publications

Replication-Competent Infectious Hepatitis B Virus Vectors Carrying Substantially Sized Transgenes by Redesigned Viral Polymerase Translation

Replication-Competent Infectious Hepatitis B Virus Vectors Carrying Substantially Sized Transgenes by Redesigned Viral Polymerase Translation

Re-Designed Recombinant Hepatitis B Virus Vectors Enable Efficient Delivery of Versatile Cargo Genes to Hepatocytes with Improved Safety

Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines

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