Suppression of hepatitis B virus antigen production and replication by wild-type HBV dependently replicating HBV shRNA vectors in vitro and in vivo

Li, Baosheng; Sun, Shili; Li, Min‐ran; Cheng, Xin; Li, Haijun; Kang, Fubiao; Kang, Jiwen; Dörnbrack, Katharina; Nassal, Michael; Sun, Dongdong

doi:10.1016/j.antiviral.2016.08.007

Cited by 9 publications

(6 citation statements)

References 57 publications

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“…To enrich capsids, lysates were sedimented through a 10% - 60% (w/v) Nycodenz step gradient for 2 h at 4°C in a TST 41.14 rotor at 41,000 rpm (302,500 g) and harvested in 14 fractions. Detection of HBV DNA in capsids and by Southern blotting was performed using 32 P labeled HBV DNA probes as described [84, 111]. Extracellular particles in the culture supernatants of HepG2.117 and H4-15 cells were enriched by precipitation with PEG8000 [84, 112].…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

et al. 2018

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Hepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target.

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Section: Methodsmentioning

confidence: 99%

Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

et al. 2018

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“…Inhibition of HBV protein expression (e.g., using shRNA-based knock-down) induces a recovery of an anti-viral adaptive immune response in the immune-competent mouse models [117,118,119]. Integrated forms are known to express both wild-type and mutant versions of the HBsAg.…”

Section: Unanswered Questionsmentioning

confidence: 99%

HBV DNA Integration: Molecular Mechanisms and Clinical Implications

Tu¹,

Budzinska²,

Shackel³

et al. 2017

Viruses

324

302

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Chronic infection with the Hepatitis B Virus (HBV) is a major cause of liver-related morbidity and mortality. One peculiar observation in cells infected with HBV (or with closely‑related animal hepadnaviruses) is the presence of viral DNA integration in the host cell genome, despite this form being a replicative dead-end for the virus. The frequent finding of somatic integration of viral DNA suggests an evolutionary benefit for the virus; however, the mechanism of integration, its functions, and the clinical implications remain unknown. Here we review the current body of knowledge of HBV DNA integration, with particular focus on the molecular mechanisms and its clinical implications (including the possible consequences of replication-independent antigen expression and its possible role in hepatocellular carcinoma). HBV DNA integration is likely to influence HBV replication, persistence, and pathogenesis, and so deserves greater attention in future studies.

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“…Extending from the latter points, the clinical consequences of decoupling HBsAg and HBx expression from cccDNA persistence are of particular interest. HBsAg has known immunosuppressive functions [ 93 , 94 , 95 ] and its expression can be maintained in clonally expanded hepatocytes containing HBV DNA integrations in the face of antiviral responses targeting productively-infected cells. Thus, expression from integrated HBV DNA may play an important role in HBV persistence in the HBeAg-negative phase.…”

Section: Open Questions Regarding Hepatitis B Virus Integrationmentioning

confidence: 99%

Cellular Genomic Sites of Hepatitis B Virus DNA Integration

Budzinska

Shackel

Urban

et al. 2018

Genes

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Infection with the Hepatitis B Virus (HBV) is one of the strongest risk-factors for liver cancer (hepatocellular carcinoma, HCC). One of the reported drivers of HCC is the integration of HBV DNA into the host cell genome, which may induce pro-carcinogenic pathways. These reported pathways include: induction of chromosomal instability; generation of insertional mutagenesis in key cancer-associated genes; transcription of downstream cancer-associated cellular genes; and/or formation of a persistent source of viral protein expression (particularly HBV surface and X proteins). The contribution of each of these specific mechanisms towards carcinogenesis is currently unclear. Here, we review the current knowledge of specific sites of HBV DNA integration into the host genome, which sheds light on these mechanisms. We give an overview of previously-used methods to detect HBV DNA integration and the enrichment of integration events in specific functional and structural cellular genomic sites. Finally, we posit a theoretical model of HBV DNA integration during disease progression and highlight open questions in the field.

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Suppression of hepatitis B virus antigen production and replication by wild-type HBV dependently replicating HBV shRNA vectors in vitro and in vivo

Cited by 9 publications

References 57 publications

Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

HBV DNA Integration: Molecular Mechanisms and Clinical Implications

Cellular Genomic Sites of Hepatitis B Virus DNA Integration

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