A High Level of Mutation Tolerance in the Multifunctional Sequence Encoding the RNA Encapsidation Signal of an Avian Hepatitis B Virus and Slow Evolution Rate Revealed by In Vivo Infection

Abstract

In all hepadnaviruses, protein-primed reverse transcription of the pregenomic RNA (pgRNA) is initiated by binding of the viral polymerase, P protein, to the RNA element. Universally, consists of a lower stem and an upper stem, separated by a bulge, and an apical loop. Complex formation triggers pgRNA encapsidation and the -templated synthesis of a DNA oligonucleotide (priming) that serves to generate minus-strand DNA. In vitro systems for duck hepatitis B virus (DHBV) yielded important insights into the priming mechanism, yet their relevance in infection is largely unexplored. Moreover, additional functions encoded in the DHBV (D) sequence could affect in vivo fitness. We therefore assessed the in vivo performances of five recombinant DHBVs bearing multiple mutations in the upper D stem. Three variants with only modestly reduced in vitro replication competence established chronic infection in ducks. From one variant but not another, three adapted new variants emerged upon passaging, as demonstrated by increased relative fitness in coinfections with wild-type DHBV. All three showed enhanced priming and replication competence in vitro, and in one, DHBV e antigen (DHBeAg) production was restored. Pronounced impacts on other D functions were not detected; however, gradual, synergistic contributions to overall performance are suggested by the fact of none of the variants reaching the in vivo fitness of wild-type virus. These data shed more light on the P-D interaction, define important criteria for the design of future in vivo evolution experiments, and suggest that the upper D stem sequences provided an evolutionary playground for DHBV to optimize in vivo fitness.Hepadnaviruses are small enveloped hepatotropic DNA viruses that replicate through protein-primed reverse transcription (for reviews, see references 8 and 38). Human hepatitis B virus (HBV) and duck HBV (DHBV) are the respective prototypes of the mammalian and avian HBVs, which share a similar genome organization and replication strategy (Fig. 1A). Their ϳ3-kb genomes encode a single core protein, three or two (avian viruses) envelope proteins, an unusual reverse transcriptase (P protein), and, only in the mammalian viruses, the poorly understood X protein. Virions contain the genome as a relaxed circular DNA (rcDNA), which upon infection is converted into nuclear covalently closed circular DNA (cccDNA), the template for all viral transcripts. In addition to the subgenomic RNAs (sgRNAs), two types of greater-than-genomelength RNAs are produced. The precore RNAs serve for translation of the precursors of the secretory hepatitis B virus e antigens (HBeAg and DHBeAg, respectively), which may modulate the host's immune response but are nonessential. The pregenomic RNAs (pgRNAs), by contrast, are vital as mRNAs for core and P protein and by constituting the obligate template for new DNA genomes.Reverse transcription begins with specific packaging of pgRNA into newly forming capsids, triggered by binding of P protein to the 5Ј-proximal ε (Dε for DHBV) stem-loop (...

Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase

“…The regions around the bulge (which serves as the template) and the apical loop are particularly important for P protein binding (7,40,41), and once the RNA is bound, the unfolding of the upper stem is critical for its template function (9). On the P protein, one RNA binding site appears to comprise the conserved T3 motif in the C-terminal TP region (2,45) and the so-called RT-1 motif (2) in the N-terminal RT domain part (residues 381 to 416), far apart in primary sequence from box E. Most notable in this light is the enhanced D⑀ RNA binding yet reduced priming activity observed for the L559A variant (Fig.…”

“…Beyond providing a feasible in vivo infection system (41,42), DHBV is the only hepadnavirus for which replication initiation has successfully been reconstituted in vitro. The DHBV P protein in vitro translated in rabbit reticulocyte lysate (RRL) (51) or expressed as a fusion protein in Escherichia coli and supplemented with RRL or purified chaperones (Hsc70 and Hsp40, with further stimulation by Hsp90 and Hop [46]) displays authentic protein-priming activity when supplied with the cognate DHBV ⑀ (D⑀) RNA and deoxynucleoside triphosphates (dNTPs), as manifested by the covalent labeling of the protein if ␣-32 P-labeled dNTPs are used (8,25,46).…”

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

“…The ten HBV ε mutants from our study which showed no substantial defect in DNA accumulation indicate that the authentic ε sequence is not a prerequisite for productive interactions with P protein; notably, tolerated mutations were found at all investigated positions, including the structured apical loop [38], [39]. Hence natural sequence conservation may be driven by minor differences in replication efficiency that come to bear only upon continuous genome propagation in vivo but not in single round transfection studies [33]. Candidate factors beyond the immediate P - ε interaction might be the overlapping preC ORF and its translation products, or cis-elements that act after the initial priming step.…”

“…stop codons in the overlapping preC ORF which prevent precore translation and HBeAg formation, are almost invariably accompanied by additional compensatory mutations that restore the genuine stem-loop architecture. The HBV ε sequence is also largely conserved in the other mammalian hepadnaviruses; in contrast, especially the upper stem is much more variable between avian hepadnaviruses [32], and DHBVs with various mutations in the upper ε stem are viable even in ducks [33]. Hence a reason for the strict conservation of ε amongst the mammalian viruses is not obvious.…”

“…Collectively, the DHBV data suggest that the P - ε interaction during priming is a dynamic multi-step process in which the initial RNA binding is followed by conformational changes in both protein [23], [24], [25] and RNA [26]; these alterations are crucial to reach the priming-active state and require assistance by cellular chaperones [27], [28], [25], [22], although chaperone-dependence can be circumvented by employing truncated "miniP" proteins which lack the RH domain, the spacer and nonessential parts of the TP and RT domains but remain priming-competent [29], [30]. Most important within the DHBV ε RNA for achieving a priming-active complex are the central region comprising the bulge [31] and the apical loop, whereas in the upper stem various nt exchanges are tolerated [32], even in vivo [33]; in fact, part of the right half of the upper stem becomes expelled in priming-active P - ε complexes [26].…”