Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector

Lucifora, Julie; Durantel, David; Belloni, Laura; Barraud, Luc; Villet, Stéphanie; Vincent, Isabelle; Margeridon-Thermet, Severine; Hantz, Olivier; Kay, Alan; Levrero, Massimo; Zoulim, Fabien

doi:10.1099/vir.0.83659-0

Cited by 41 publications

(43 citation statements)

References 35 publications

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“…In infected cells, only cccDNA will be efficiently amplified and not integrated HBV sequences. We have used RCA to specifically amplify cccDNA from cells transducted with an HBV/baculovirus vector (15). This ability to directly probe the cccDNA pool will be important for the study of HBV variants, especially the emergence of drug-resistant mutants.…”

Section: Discussionmentioning

confidence: 99%

Rolling Circle Amplification, a Powerful Tool for Genetic and Functional Studies of Complete Hepatitis B Virus Genomes from Low-Level Infections and for Directly Probing Covalently Closed Circular DNA

Margeridon

Carrouée-Durantel²,

Chemin

et al. 2008

Antimicrob Agents Chemother

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Complete characterization of the biological properties of hepatitis B virus (HBV) variants requires the generation of full-length genomes. The aim of this study was to develop new tools for the efficient full-length genome amplification of virus from samples with low viral loads. Rolling circle amplification (RCA) was used to amplify full-length HBV genomes from both sera and liver biopsy samples from chronic HBV carriers. Serum-derived relaxed circular HBV DNA could be amplified only after completion and ligation of plus-strand DNA. Covalently closed circular DNA (cccDNA) from liver biopsies could be amplified directly from as few as 13 copies, using RCA, followed by a full-length HBV PCR. Three serial liver biopsy samples were obtained from a lamivudine-resistant patient who cleared detectable serum HBV after adefovir dipivoxil was added to the lamivudine therapy and then seroconverted to anti-HBs. Only the genomes from the last biopsy specimen obtained after the emergence of lamivudine resistance contained the lamivudine resistance-associated mutations rtL180M and rtM204V ("rt" indicates reverse transcriptase domain). Defective genomes were also found in this biopsy sample. Genomes cloned from the liver biopsy specimens were transfected into HuH7 cells to study their replication competence and their susceptibility to lamivudine. RCA is a powerful tool for amplifying full-length HBV genomes and will be especially useful for the study of occult or inactive HBV infections and patients undergoing antiviral treatment. It can also be used to probe HBV cccDNA, the crucial intermediate in viral persistence and the archive of resistance mutations. Chronic hepatitis B virus (HBV) infection is widespread,with an estimated 370 million carriers worldwide (1). Despite constraints imposed by a complex genetic architecture involving overlapping genes, the virus is highly variable, with eight known genotypes designated A to H (20).The complete molecular and biological characterization of HBV variants requires the isolation of full-length genomes. This has been done classically by amplifying overlapping fragments from serum-derived relaxed circular genomes (RC-DNA) (4, 21). However, the discontinuous structure of RC-DNA means that the amplification of at least one of the fragments can be problematical. Increasing the number of PCRs ineluctably augments the probability of incorporating PCR errors, and further steps are necessary to construct a full-length HBV genome. Günther et al. have developed an elegant method for amplifying full-length HBV genomes in a one-step PCR (7). However, this method requires serum with viral loads of Ͼ10 4 copies/ml of HBV DNA. In typical chronic HBV infections, viral loads usually range from 10 6 to 10 10 copies/ml. However, for inactive carriers (individuals who are hepatitis B surface antigen positive [HBsAg ϩ ]) with minimal virus replication and for occult HBV-infected patients (HBsAg Ϫ ), serum HBV DNA levels are usually very much lower. Consequently, few occult HBV infections have been studi...

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Section: Discussionmentioning

confidence: 99%

Rolling Circle Amplification, a Powerful Tool for Genetic and Functional Studies of Complete Hepatitis B Virus Genomes from Low-Level Infections and for Directly Probing Covalently Closed Circular DNA

Margeridon

Carrouée-Durantel²,

Chemin

et al. 2008

Antimicrob Agents Chemother

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“…The in vitro resistance phenotype detection assay used by Curtis et al is based on cytomegalovirus (CMV)-driven expression of the HBV genome and thus produces a large excess of viral pregenomes, a parameter used as a readout in their assay. Thus, CMV-driven in vitro systems seem less sensitive than those involving viral replicons driven by authentic promoters and thus seem inappropriate for measuring the relative extents of susceptibility of RT mutant genomes to nucleoside or nucleotide analogues (10,12,16). Nevertheless, the patients reported by Curtis and colleagues were susceptible to ADF therapy in vivo despite the rtI233V mutation.…”

mentioning

confidence: 99%

Selection and Counterselection of the rtI233V Adefovir Resistance Mutation during Antiviral Therapy

et al. 2010

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Recently, we reported on three patients with chronic hepatitis B virus (HBV) infection for whom adefovir (ADF) therapy virologically failed, most likely due to a preexisting rtI233V HBV polymerase mutation. Here, we describe two further patients with chronic HBV infection who were found to develop the rtI233V mutation after initiation of ADF therapy. These patients represent the first cases known so far in which the rtI233V ADF resistance mutation evolved under persistent HBV replication during HBV therapy with ADF. Interestingly, one of the previously described patients, who was initially successfully switched from ADF to tenofovir (TDF) and became virologically suppressed subsequently, experienced a moderate but remarkable rebound of HBV viremia after switching from TDF to entecavir, due to the emergence of renal toxicity. Thus, we provide evidence for the selection and counterselection of the rtI233V ADF resistance mutation during antiviral therapy.

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“…After transduction, AcMNPV neither replicates nor integrates into the genome of mammalian cells, making it in combination with its simple production and no pre-existing immunity to an efficient vector system for various applications. AcMNVPare extensively used for protein production [41,42], virus production [43,44], vaccine development [32,45] vaccine production [46] and cancer therapy [47]. However, although BV is replication-deficient in mammalian cells, several viral genes are expressed and lead to robust immune responses in vivo [48,49].…”

Section: Viral Vectors For Talen Deliverymentioning

confidence: 99%

Progress and Problems with Viral Vectors for Delivery of Talens

Bergmann¹

2014

J Mol Genet Med

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Transcription Activator-Like Effector Nucleases (TALENS) and Their Mode of ActionFor sequence-specific genome engineering and its biotechnological and medical applications various systems were tested. Most recent tools include designer nucleases and the bacteria derived clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system using short RNA to induce precise cleavage at endogenous genomic loci [1,2]. Designer nucleases are mainly represented by zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [3][4][5][6][7], combining features of a customized DNA binding motif for sequence-specific DNA binding domain and a nuclease for introduction of doubled-strand DNA (dsDNA) breaks at the target site.TALENs are a promising new class of designer nucleases that can be specifically designed to bind DNA sequences of interest and to introduce dsDNA breaks. They are chimeric proteins consisting of a N-terminal nuclear localization signal, a central DNA binding domain and a C-terminal FokI nuclease domain. The DNA binding domain originates from transcription activator like effectors (TALEs) of the bacterial plant pathogen Xanthomonas, used by this bacterium to alter the expression of several host genes [8]. Its main characteristic is a central repeat region consisting of a variable number of incomplete tandem repeats that are usually comprised of 33-35 amino acids (aa) that are identical except for the hypervariable repeat-variable diresidue (RVD) at a positions 12 and 13 [6]. With some degree of degeneracy, the RVD of each repeat is specific for binding a corresponding nucleotide in their contiguous target DNA sequence [6,8]. The C-terminal FokI domain provides non-specific endonuclease activity and similar to ZFNs, binding of the TALE domains upstream and downstream of the respective DNA target and subsequent dimerization at the FokI nuclease domain lead to dsDNA breaks. It is of note that the spacer between the TALEN DNA binding sites needs to be considered when designing novel TALENs [6,7]. After binding, dsDNA breaks are introduced which can than activate cellular pathways either leading to homologous recombination in the presence of the respective homologous donor AbstractIt has long been envisaged that gene disruption or gene correction in affected target cells can be efficiently conducted in vitro and in vivo and over the recent years several tools for achieving this goal were developed. Designer nucleases such as zinc finger nucleases (ZFNs) were extensively explored and more recently transcription activator-like effector nucleases (TALENs) were introduced for sequence-specific genome engineering in the mammalian genome. ZFNs and TALENs are fusion proteins containing a customized DNA-binding motif for sequence-specific DNA binding linked to a nuclease for introduction of double-stranded DNA breaks. Both systems were explored in mammalian cells using non-viral and viral delivery methods. Herein, we will provide a state-ofthe-art overview of available virus-base...

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Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector

Cited by 41 publications

References 35 publications

Rolling Circle Amplification, a Powerful Tool for Genetic and Functional Studies of Complete Hepatitis B Virus Genomes from Low-Level Infections and for Directly Probing Covalently Closed Circular DNA

Rolling Circle Amplification, a Powerful Tool for Genetic and Functional Studies of Complete Hepatitis B Virus Genomes from Low-Level Infections and for Directly Probing Covalently Closed Circular DNA

Selection and Counterselection of the rtI233V Adefovir Resistance Mutation during Antiviral Therapy

Progress and Problems with Viral Vectors for Delivery of Talens

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