Replication of Hepatitis B Virus Which Carries Foreign DNA in Vitro

Hanafusa, Tadashi; Yumoto, Yasuhiro; Hada, Hajime; Shinji, Toshiyuki; Koide, Naoki; Tsuji, Takao

doi:10.1006/bbrc.1999.1224

Cited by 5 publications

(3 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Except that the 276 bp small insertion almost completely abolished generation of recombinant HBV, infectivity of its particles remained uncertain and was most probably lost because the expression of preS1 might be missed. Hanafusa[31] showed that the HBV could carry 63 bp of extra DNA by destroying the DR2 region, which should be a critical cis element of HBV replication. The only defined data is that a little of HBV DNA was observed by Southern blot in the cell line HuH-7 but not HepG2.…”

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

View full text Add to dashboard Cite

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.

show abstract

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

View full text Add to dashboard Cite

show abstract

“…Replication efficiency was an issue with a majority of previously reported recombinant HBV designs [ 5 , 7 , 8 , 20 ]. Our previously described vector 5c3c was based on a highly replicative clinical isolate 6898, which harbors an in-frame deletion in preS/polymerase spacer region [ 9 ].…”

Section: Discussionmentioning

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

View full text Add to dashboard Cite

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.

show abstract

“…HBV is the only DNA virus that targets the liver where it efficiently infects hepatocytes. This ability permits the development of an HBV-based vector for liver-directed gene transfer with foreign DNA inserted into the virus genome (33,34). However, this viral vector is not suitable for the delivery of substances other than DNA.…”

Section: Engineered Bio-nanocapsule Can Deliver Substances To Target mentioning

confidence: 99%

Engineered bio-nanocapsules, the selective vector for drug delivery system

Fukuda

Tuoya³

et al. 2006

TBMB

View full text Add to dashboard Cite

SummaryThe bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.

show abstract

Replication of Hepatitis B Virus Which Carries Foreign DNA in Vitro

Cited by 5 publications

References 15 publications

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

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

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

Engineered bio-nanocapsules, the selective vector for drug delivery system

Contact Info

Product

Resources

About