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

Gajer, M.; Dörnbrack, Katharina; Rösler, Christine; Schmid, Bernadette; Beck, Jürgen; Nassal, Michael

doi:10.1038/s41598-017-07657-z

Cited by 5 publications

(17 citation statements)

References 43 publications

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“…Intriguingly, cryptic elements inside the HBV transcripts fulfill some of the tasks. These include the pgRNA packaging signal [ 38 , 39 ], the PRE regulating the nuclear export of the HBV transcripts [ 40 , 41 , 42 ], the RNA region involved in destabilizing HBV RNA in response to cytokine treatments [ 43 , 44 ] and the ERE described here. Along with the minimization of the HBV genome and the number of ORFs, non-coding RNAs became an effective alternative.…”

Section: Discussionmentioning

confidence: 99%

RNR-R2 Upregulation by a Short Non-Coding Viral Transcript

et al. 2021

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DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells.

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Section: Discussionmentioning

confidence: 99%

RNR-R2 Upregulation by a Short Non-Coding Viral Transcript

et al. 2021

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“…The HBV transcripts are made of a number of functional entities embedded inside the RNA sequence. These include the encapsidation signal necessary for pgRNA packaging [29,30], the PRE regulating the nuclear export of the HBV transcripts [2,3,31], the RNA region involved in destabilizing HBV RNA in response to cytokines treatments [32,33] and the ERE described here. It is therefore possible that with the minimization of the HBV genome, some of the essential activities have become adapted to be executed by the viral RNA and therefore the emergence of multitasking HBV RNA is an important evolutionary step in establishing the hepadnavirus family.…”

Section: Discussionmentioning

confidence: 99%

A short HBV RNA region induces RNR-R2 expression in non-cycling cells and in primary human hepatocytes

Ricardo-Lax

Broennimann

Adler

et al. 2018

Preprint

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25 Hepatitis B virus infects non-dividing cells in which dNTPs are scarce. HBV replication 26 requires dNTPs. To cope with this constraint the virus induces the DNA damage response 27 (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR 28 holoenzyme, the key regulator of dNTP levels. Previously we reported that the HBx open 29 reading frame (ORF) triggers this pathway. Unexpectedly however, we report here that 30 the production of HBx protein is not essential. We found that a small region of 125 bases 31 within the HBx transcript is sufficient to induce RNR-R2 expression in growth arrested 32 HepG2 cells and in primary human hepatocytes (PHH). The observed HBx embedded 33 regulatory element is named ERE. We demonstrate that ERE is functional as a positive 34 strand RNA polymerase-II transcript. Remarkably, ERE is sufficient to induce the Chk1-35 E2F1-RNR-R2 DDR pathway, previously reported to be activated by HBV. Furthermore, 36 we found that ERE activates ATR but not ATM in eliciting this DDR pathway in 37 upregulating RNR-R2. These findings demonstrate the multitasking role of HBV 38 transcripts in mediating virus-host cell interaction, a mechanism that explains how such a 39 small genome effectively serves such a pervasive virus. 40 41 Author summary 42 The hepatitis B virus (HBV) infects the human liver and over 250 million people 43 worldwide are chronically infected with HBV and at risk for cirrhosis and liver cancer.44 HBV has a very small DNA genome with only four genes, much fewer than other 45 viruses. For propagation the virus consumes dNTPs, the building blocks of DNA, in 46 much higher amounts than the infected cells provide. To cope with this constraint, the 3 47 virus manipulates the cells to increase the production of dNTPs. We found that the virus 48 activates the cellular response to DNA damage upon which the cells increase the 49 production of dNTPs, but instead of repairing cellular DNA, the virus uses them for 50 production of its own DNA. Usually viruses manipulate host cells with one or more of 51 their unique proteins, however the small HBV genome cannot afford having such a 52 unique gene and protein. Instead, we found that here the virus relies on RNA to 53 manipulate the host cells. Our findings highlight the unprecedented principle of a 54 multitasking viral RNA that is not only designed to be translated into proteins but also 55 harbors an independent role in activating the cellular DNA damage response. 56 57 Introduction 58 Hepatitis B virus (HBV) is a non-cytopathic enveloped virus containing a small circular 59 partially double-stranded DNA genome. Upon entering the cell the genome is converted 60 into a covalently closed circular DNA (cccDNA), the viral transcription template [1]. The 61 HBV genome harbors enhancers and promoters regulating the transcription of a number 62 of positive strand transcripts. The generated RNA species are nuclear exported by a 63 unique mechanism that is not entirely understood but requires a RNA region shared by all 64 t...

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“…Strikingly, in avihepadnaviruses, the same two motifs are strictly conserved (Fig. 2B) and functionally essential (10,12,23), revealing an unexpectedly high similarity between the two divergent virus families. The still higher sequence similarity between avian HBVe and NDVe than between avian and mammalian HBVe (Fig.…”

Section: Nackednaviral P Protein Initiates Reverse Transcription By Proteinmentioning

confidence: 92%

Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription

Beck

Seitz

Lauber

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Replication is initiated by a unique protein-priming mechanism whereby a hydroxy amino acid side chain of the terminal protein (TP) domain of the viral polymerase (P) is extended into a short DNA oligonucleotide, which subsequently serves as primer for first-strand synthesis. A key component in the priming of reverse transcription is the viral RNA element epsilon, which contains the replication origin and serves as a template for DNA primer synthesis. Here, we show that recently discovered non-enveloped fish viruses, termed nackednaviruses [C. Lauber et al., Cell Host Microbe 22, 387–399 (2017)], employ a fundamentally similar replication mechanism despite their huge phylogenetic distance and major differences in genome organization and viral lifestyle. In vitro cross-priming studies revealed that few strategic nucleotide substitutions in epsilon enable site-specific protein priming by heterologous P proteins, demonstrating that epsilon is functionally conserved since the two virus families diverged more than 400 Mya. In addition, other cis elements crucial for the hepadnavirus-typical replication of pregenomic RNA into relaxed circular double-stranded DNA were identified at conserved positions in the nackednavirus genomes. Hence, the replication mode of both hepadnaviruses and nackednaviruses was already established in their Paleozoic common ancestor, making it a truly ancient and evolutionary robust principle of genome replication that is more widespread than previously thought.

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

Cited by 5 publications

References 43 publications

RNR-R2 Upregulation by a Short Non-Coding Viral Transcript

RNR-R2 Upregulation by a Short Non-Coding Viral Transcript

A short HBV RNA region induces RNR-R2 expression in non-cycling cells and in primary human hepatocytes

Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription

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