SELEX-derived aptamers of the duck hepatitis B virus RNA encapsidation signal distinguish critical and non-critical residues for productive initiation of reverse transcription

Abstract

Protein-primed replication of hepatitis B viruses (HBVs) is initiated by the chaperone dependent binding of the reverse transcriptase (P protein) to the bulged epsilon stem-loop on the pregenomic RNA, and the epsilon-templated synthesis of the 5' terminal nucleotides of the first DNA strand. How P protein recognizes the initiation site is poorly understood. In mammalian HBVs and in duck HBV (DHBV) the entire stem-loop is extensively base paired; in other avian HBVs the upper stem regions have a low base pairing potential. Initiation can be reconstituted with in vitro translated DHBV, but not HBV, P protein and DHBV epsilon (Depsilon) RNA. Employing the SELEX method on a constrained library of Depsilon upper stem variants, we obtained a series of well-binding aptamers. Most contained C-rich consensus motifs with very low base pairing potential; some supported initiation, others did not. Consensus-based secondary mutants allowed to pin down this functional difference to the residues flanking the conserved loop, and an unpaired U. In vitro active consensus sequences also supported virus replication. Hence, most of the upper stem acts as a spacer, which, if not base paired, warrants accessibility of relevant anchor residues. This suggests that the base paired Depsilon represents an exceptional rather than a prototypic avian HBV epsilon signal, and it offers an explanation as to why attempts to in vitro reconstitute initiation with human HBV have thus far failed.

Hepatitis B virus replication

“…Surprisingly, the corresponding ε signal (Hε) from the related heron HBV (HHBV) has much reduced basepairing in the upper stem region ( Figure 4C), yet it functionally interacts with DHBV P protein in vitro whereas HBV ε does not [70] . Selection, from a library of RNAs with partially randomized upper stems for individuals able to bind to in vitro translated DHBV P protein (see Cell-free reconstitution of hepadnaviral replication initiation) revealed the absence of base-pairing in the upper stem region as a common theme ( Figure 4D) [71] . Some of the selected P-binding RNAs supported in vitro priming while others did not, confirming that a productive interaction, leading to DNA synthesis, requires more than mere physical binding (see below).…”

“…Some of the selected P-binding RNAs supported in vitro priming while others did not, confirming that a productive interaction, leading to DNA synthesis, requires more than mere physical binding (see below). Hence for avihepadnaviral ε signals an open upper stem structure is beneficial for both physical and productive binding to P; in fact, deliberate Dε stabilization strongly reduces P binding [71] . This is one line of evidence that structural reshaping of the upper stem is a crucial event for initiation of DNA synthesis.…”

“…The overall 2D structure is similar to that of HBV ε, including a largely base-paired upper stem [69] , as confirmed by preliminary NMR data. The boxed positions were randomized in a SELEX approach, and P binding individuals were selected from the corresponding RNA library [71] ; C: HHBV ε (Hε). Hε shows substantial sequence variation to D: (encircled nt), leading to a largely open upper stem structure; D: Generalized secondary structure of an avihepadnaviral ε signal.…”

“…Hε shows substantial sequence variation to D: (encircled nt), leading to a largely open upper stem structure; D: Generalized secondary structure of an avihepadnaviral ε signal. The scheme summarizes major determinants for productive interaction with P protein [71] . Grey background indicates nt positions that are probably in contact with protein.…”

“…Mutants in the upper stem (see Figure 4B) which favor stable non-bulged structures do not bind to P [70] . Similarly critical is the sequence and structure at the junction between the lower stem and the bulge: Mutation of the unpaired U2604 opposite the bulge to G substantially reduced binding (and nearly abolished priming) [71] . At the tip base-pair of the lower stem, base identity of G2605, but not base-pairing itself, is important; in addition, most ribose residues in Dε could be replaced by deoxyribose residues, except in the two top base pairs of the lower stem and in the bulge residue templating the first nt of (-)-DNA [107] .…”