Abstract
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.
Highlights
Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate
Unlike the host transfer RNA priming employed by retroviruses, hepadnaviral reverse transcription is initiated by de novo synthesis of a short DNA primer that is copied from e and, upon transfer to a complementary 3′-proximal site, is elongated into full-length minus-strand DNA [(−)DNA] [9, 10, 13, 14] (SI Appendix, Fig. S1A)
Our data imply that the Hepatitis B virus (HBV)-like reverse transcription mechanism was already well established in the last common ancestor (LCA) of hepadna- and nackednaviruses and has been preserved for more than
Summary
Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Unlike the host transfer RNA priming employed by retroviruses, hepadnaviral reverse transcription is initiated by de novo synthesis of a short DNA primer that is copied from e and, upon transfer to a complementary 3′-proximal site, is elongated into full-length minus-strand DNA [(−)DNA] [9, 10, 13, 14] (SI Appendix, Fig. S1A) Peculiar to this initiation process is the covalent attachment of the first nucleotide (nt) to P itself, mediated by an auto-nucleotidylation mechanism that, beyond the cognate eRNA, requires the DNA polymerase activity of RT and a specific tyrosine residue of TP as an acceptor [15,16,17]. The rcDNA is converted into covalently closed circular DNA (cccDNA) that, as an episomal minichromosome, serves as a template for viral transcripts and establishes viral persistence [6]
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