Solution structure of the apical stem–loop of the human hepatitis B virus encapsidation signal

Abstract

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5′ end of the RNA pregenome. Epsilon contains an apical stem–loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem–loop based on NOE, RDC and 1H chemical shift NMR data. The 1H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5′-CUGUGC-3′ folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

Review NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings

“…Interhelical bend angles are also shown. The examples include the TBR, 84 domain II of HCV IRES (IIa), 119 SLD from the 5 0 UTR of enterovirus and rhinoviruses genomes (SLD), 120 a stem-loop at the 5 0 end of the HBV, 121 , stem-loop derived from helix-35 of E. coli 23S ribosomal RNA (Helix 35), 108 Tetrahymena thermophila SL-IV RNA in two variant constructs (SL-IV) 122 and (SL-IV-Fragment), 123 the CR4-CR5 domain in the vertebrate telomerase (hTR J6), 124 HIV-1 TAR RNA, 86 the frame-shift inducing element in HIV-1 (Frameshift), 125 RNase P P4, 126 HIV SL1m, 127 and the iron-responsive element (IRE). 87 NMR Studies of RNA Dynamics by RDCs 397 ble secondary structural rearrangement occurring at ls-ms timescales involving the internal loop and base-pairs in the upper stem.…”

Structural and thermodynamic signatures that define pseudotriloop RNA hairpins

“…Pseudotriloop (PTL) hairpins (Haasnoot et al 2002) or lonepair triloop hairpins (Lee et al 2003) are common, albeit rarely studied, structures in RNA (Addess et al 1997;Flodell et al 2002Flodell et al , 2006Kulinski et al 2003;Welting et al 2008). A PTL is proposed to form by creating a cross-loop base pair in an RNA hexaloop such that a triloop is created and the 3 ′ sixth loop base is bulged out (Fig.…”

“…RNA loop structures with the potential to form PTLs are found in a variety of sequences, e.g., BMV (Haasnoot et al 2000(Haasnoot et al , 2002, domain IIId of the internal ribosomal entry site (IRES) of hepatitis C virus (Klinck et al 2000;Lukavsky et al 2000Lukavsky et al , 2003, RNAse MRP P3 domain (Welting et al 2008), HIV-1 TAR (Critchley et al 1993;Kulinski et al 2003), the apical loop of hepatitis B virus (HBV) ɛ domain (Flodell et al 2002(Flodell et al , 2006, and iron responsive element (IRE) (Sierzputowska et al 1995;Liang and Hall 1996;Hall and Williams 2004). The BMV, HBV, HCV, and IRE PTL structures have been established experimentally (Addess et al 1997;Lukavsky et al 2000;Flodell et al 2002Flodell et al , 2006Skov et al 2012).…”

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

“…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. The importance of high-resolution structural data is underlined by the NMR studies of the human HBV e apical stem-loop, which showed that its conserved apical loop folds into a pseudotriloop, whereas secondary structure programs predicted a hexaloop (Flodell et al 2002(Flodell et al , 2006. A functional in-vitro RT-e replication system exists for Duck, but not for human HBV.…”

“…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.…”