Evidence for Multiple Distinct Interactions between Hepatitis B Virus P Protein and Its Cognate RNA Encapsidation Signal during Initiation of Reverse Transcription

Feng, Hui; Chen, Ping; Zhao, Fei; Nassal, Michael; Hu, Kanghong

doi:10.1371/journal.pone.0072798

Cited by 10 publications

(8 citation statements)

References 55 publications

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“…In HBV, recognition of the apical pseudo‐triloop of the ε motif by the P protein is essential for encapsidation . While the internal bulge, but not the apical loop of ε is required for P protein–ε interaction, the protein priming reaction is dependent on apical loop, internal bulge and the short distance between cap and the ε element, similar to the requirements for RNA packaging . Even though the overall structure of the ε element is conserved in Hepadnaviridae, the human ε displayed relatively stronger primer loop and apical stem‐loop stability than its avian counterparts .…”

Section: Discussionmentioning

confidence: 99%

The epsilon motif of hepatitis B virus RNA exhibits a potassium‐dependent ribonucleolytic activity

Chakraborty

Ghosh

2017

The FEBS Journal

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Fourteen different classes of ribozymes are known that catalyse a range of diverse chemical reactions. We report here a novel potassium-dependent nucleolytic activity present in hepatitis B virus (HBV) RNA. A short RNA region (53 nt) with enzymatic properties released itself from the viral sequence by cis cleavages and could subsequently act in trans. The released region encompassed the epsilon motif present in the HBV RNA. The 3 0 end of the liberated fragment was within the noncanonical polyadenylation signal (UAUAAA) of the viral RNA while cleavages at about 53 nt upstream sites released the fragment. Mutations of the primary scissile sites or annealing these sites with the antisense oligodeoxyribonucleotides blocked the release of this short fragment and annulled subsequent trans cleavage activity. An exogenously synthesized short transcript of only this 53 nt was active as a sequence-independent trans-acting nuclease and cleaved after pyrimidines in viral or other substrate transcripts under physiological potassium ion concentrations. Formation of a G-quadruplex within this region was suggested by circular dichroism and nondenaturing polyacrylamide gel analyses. Our results reveal a unique natural example of a trans-acting ribonuclease that cleaves at multiple sites in a sequence-independent fashion. The presence of this novel activity implores a dynamic structural behaviour in the e region and raises new questions about HBV gene regulation.

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Section: Discussionmentioning

confidence: 99%

The epsilon motif of hepatitis B virus RNA exhibits a potassium‐dependent ribonucleolytic activity

Chakraborty

Ghosh

2017

The FEBS Journal

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“…Intracellular viral core DNA was isolated using the method described previously [Feng et al, ]. Briefly, cells were lysed in either 1 ml (HepG2.2.15, 10 7 cells per 10 cm plate,) or 0.6 ml (HuH‐7, 3×10 6 cells per 6 cm plate) lysis buffer (50 mM Tris‐HCl pH 7.5, 140 mM NaCl, 0.5% NP‐40).…”

Section: Methodsmentioning

confidence: 99%

Sodium selenite suppresses hepatitis B virus transcription and replication in human hepatoma cell lines

Cheng

Zhi

Guo

et al. 2015

Journal of Medical Virology

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Hepatitis B virus (HBV) infection is one of the most serious and prevalent health problems worldwide. Current anti-HBV medications have a number of drawbacks, such as adverse effects and drug resistance; thus, novel potential anti-HBV reagents are needed. Selenium (Se) has been shown to be involved in both human immunodeficiency virus and hepatitis C virus infections, but its role in HBV infection remains unclear. To address this, sodium selenite (Na2SeO3 ) was applied to three HBV cell models: HepG2.2.15 cells, and HuH-7 cells transfected with either 1.1 or 1.3× HBV plasmids. Cytotoxicity of Na2SeO3 was examined by Cell Counting Kit-8. Levels of viral antigen expression, transcripts, and encapsidated viral DNA were measured by enzyme-linked immunosorbent assay, northern blot, and Southern blot, respectively. There was no obvious cytotoxicity in either HepG2.2.15 or HuH-7 cells with <2.5 µM Na2SeO3 . Below this concentration, Na2SeO3 suppressed HBsAg and HBeAg production, HBV transcript level, and amount of genomic DNA in all three tested models, and suppression level was enhanced in line with increases in Na2 SeO3 concentration or treatment time. Moreover, the inhibitory effect of Na2SeO3 on HBV replication can be further enhanced by combined treatment with lamivudine, entecavir, or adefovir. Thus, the present study clearly proves that Na2SeO3 suppresses HBV protein expression, transcription, and genome replication in hepatoma cell models in a dose- and time-dependent manner.

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“…Otherwise, a specific architecture of the bulge region itself appears also crucial in HBV 13 , 14 , 24 , 40 , 41 . The classical sequence in orthohepadnaviruses is similar to that in avihepadnaviruses (CUGUGC vs. CUGUUGU) and also here the central GUG motif appears most important 24 , 34 . However, any deeper mechanistic understanding will require an HBV in vitro priming system, ideally comprising just P protein and ε RNA; obviously, this could also serve to screen for new antivirals that interfere with protein-priming as a highly virus-specific target.…”

Section: Discussionmentioning

confidence: 52%

Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming

Gajer

Dörnbrack

Rösler

et al. 2017

Sci Rep

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Hepadnaviruses, including human hepatitis B virus (HBV), replicate their tiny DNA genomes by protein-primed reverse transcription of a pregenomic (pg) RNA. Replication initiation as well as pgRNA encapsidation depend on the interaction of the viral polymerase, P protein, with the ε RNA element, featuring a lower and an upper stem, a central bulge, and an apical loop. The bulge, somehow assisted by the loop, acts as template for a P protein-linked DNA oligo that primes full-length minus-strand DNA synthesis. Phylogenetic conservation and earlier mutational studies suggested the highly based-paired ε structure as crucial for productive interaction with P protein. Using the tractable duck HBV (DHBV) model we here interrogated the entire apical DHBV ε (Dε) half for sequence- and structure-dependent determinants of in vitro priming activity, replication, and, in part, in vivo infectivity. This revealed single-strandedness of the bulge, a following G residue plus the loop subsequence GUUGU as the few key determinants for priming and initiation site selection; unexpectedly, they functioned independently of a specific structure context. These data provide new mechanistic insights into avihepadnaviral replication initiation, and they imply a new concept towards a feasible in vitro priming system for human HBV.

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Evidence for Multiple Distinct Interactions between Hepatitis B Virus P Protein and Its Cognate RNA Encapsidation Signal during Initiation of Reverse Transcription

Cited by 10 publications

References 55 publications

The epsilon motif of hepatitis B virus RNA exhibits a potassium‐dependent ribonucleolytic activity

The epsilon motif of hepatitis B virus RNA exhibits a potassium‐dependent ribonucleolytic activity

Sodium selenite suppresses hepatitis B virus transcription and replication in human hepatoma cell lines

Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming

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