Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals

Abstract

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5′-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P–epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35°C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL.

1H, 13C and 15N NMR assignments of Duck HBV apical stem loop of the epsilon encapsidation signal

“…The RNA sequence was prepared as previously described (Girard et al 2007). NMR samples were prepared in buffer (10 mM Na-Phosphate, pH 6.7, 0.1 mM EDTA) giving a 1.2 mM non-labelled sample, a 1.0 mM 13 …”

“…00 -U-labelled sample and a 0.7 mM fully 13 C/ 15 N labelled sample (Girard et al 2007 Spectra were processed using NMRpipe (Delaglio et al 1995) and resonance assignment was performed with Sparky software (Kneller and Kuntz 1993).…”

“…The sequential walks are interrupted at residue U25 due to dynamic movement of this residue. Sugar puckering modes were derived from observable/non observable H1 0 -H2 0 coherences in the COSY spectrum (Girard et al 2007). Spectra recorded at 15°C were used to verify the initial assignments as well as supplement the 13 C and 15 N chemical shifts and sugar-base correlation via 2D HSQC, 3D HCN, 3D HCNCH ( Fig.…”

“…Subsequently, a 4nt DNA primer is synthesized using the e-primer loop as template. The resulting complex translocates to a 3 0 -proximal e RNA element of the pgRNA, where full-length DNA synthesis is started using the 4-nt DNA as primer (Flodell et al 2006;Girard et al 2007;Petzold et al 2007). Although the structural basis and sequence requirements for RT-e binding and priming are emerging, several questions remain and a full understanding of the molecular basis for the specific interactions between P and e awaits high-resolution structural and thermodynamic data.…”

“…HBV is a member of the Hepadnaviridae family consisting of the hepatotropic DNA viruses, which also includes related animal viruses such as duck HBV and heron HBV. The genome of HBV is a small (3.2 kb), relaxed circular, partially double stranded DNA genome which replicates by reverse transcription of an RNA intermediate, the pregenomic RNA (Beck and Nassal 2007;Girard et al 2007). The pregenomic RNA is transported into the cytoplasm and encapsidated into immature core particles together with HBV reverse transcriptase (RT).…”