Abstract
Hepatitis B virus (HBV) causes acute and chronic hepatitis B (CHB). Due to its error-prone replication via reverse transcription, HBV can rapidly evolve variants that escape vaccination and/or become resistant to CHB treatment with nucleoside/nucleotide analogs (NAs). This is particularly problematic for the first generation NAs lamivudine and adefovir. Though now superseded by more potent NAs, both are still widely used. Furthermore, resistance against the older NAs can contribute to cross-resistance against more advanced NAs. For lack of feasible HBV infection systems, the biology of such variants is not well understood. From the recent discovery of Na+-taurocholate cotransporting polypeptide (NTCP) as an HBV receptor new in vitro infection systems are emerging, yet access to the required large amounts of virions, in particular variants, remains a limiting factor. Stably HBV producing cell lines address both issues by allowing to study intracellular viral replication and as a permanent source of defined virions. Accordingly, we generated a panel of new tetracycline regulated TetOFF HepG2 hepatoma cell lines which produce six lamivudine and adefovir resistance-associated and two vaccine escape variants of HBV as well as the model virus woolly monkey HBV (WMHBV). The cell line-borne viruses reproduced the expected NA resistance profiles and all were equally sensitive against a non-NA drug. The new cell lines should be valuable to investigate under standardized conditions HBV resistance and cross-resistance. With titers of secreted virions reaching >3x107 viral genome equivalents per ml they should also facilitate exploitation of the new in vitro infection systems.
Highlights
Hepatitis B virus (HBV) is the type member of the hepdnaviridae, small hepatotropic DNAcontaining viruses that replicate through reverse transcription [1,2]
All constructs were based on vector pTRE-HBVT [44] in which authentic wild-type HBV pgRNA of genotype D (Genbank accession no.: J02203) is transcribed from the TRE promoter; a hygromycin resistance gene serves as selection marker
Better understanding the biology of these variant viruses is mandatory for knowledgebased management of the disease as well as for the development of further improved drugs
Summary
Hepatitis B virus (HBV) is the type member of the hepdnaviridae, small hepatotropic DNAcontaining viruses that replicate through reverse transcription [1,2]. ADV and TDF are acyclic nucleotide analogs, applied as more cell-permeable dipivoxil and disoproxil prodrugs. All these NAs target the viral polymerase and reduce reverse transcription of the viral pregenomic (pg) RNA into new relaxed-circular (RC) DNA genomes [2]. Sustained virus elimination is rarely achieved with any of the NAs [9], implying a need for life-long treatment [10] This aggravates the problem of cross-resistance [11], as illustrated by the massively enhanced emergence of ETV resistant variants in LAM experienced versus naive patients (51% vs 1.2% after 5 years; [8])
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