Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element

Abstract

Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel.

Keywords: Covariance model; RNA secondary structure; hepadnavirus; stem-loop α; stem-loop β.

Figure 1.

Map of the human HBV genome highlighting the multiple overlapping HBV ORFs and transcripts (RefSeq: NC_003977.1; GenBank:X04615.1). The inner ring represents the main HBV ORFs whereas the outer ring represents the main HBV transcripts. Most of the elements of the PRE (light gray shading) are present in all the main HBV transcripts. At least 2 of the 3 conserved RNA stem-loops (dark gray shadings), SLα, SLβ, and ɛ are present in all the main HBV transcripts.

Figure 2.

Functional elements and RNA secondary structures within the PRE. (A) A Map of the known functional elements and RNA secondary structures of HBV PRE. HBV (RefSeq: NC_003977.1; GenBank: X04615.1) PRE contains 2 major nuclear export elements, recently named SEP1 (nucleotides 1590-1705) and SEP2 (nucleotides 1239-1442). SEP1 contains the binding site for ZC3H18, a cellular factor for PRE-mediated nuclear export. SEP2 contains previously described SRE-1 (nucleotides 1252-1348), La binding site (nucleotides 1275-1291), SLα (nucleotides 1292-1321) and SLβ (nucleotides 1411-1433). The PRE intronic splicing silencer (PRE-ISS) region encompasses PRE III (nucleotides 1458-1584). The PRE III is a binding site for GAPDH and an alternative splicing regulator, polypyrimidine tract binding protein (PTB). PTB is also a cellular factor for a PRE-mediated nuclear export activity. The secondary structures of SLα and SLβ were inferred from NMR data (PDB: 2JYM) and/or previous mutation analyses. (B) Plot of conservation for an aligned, annotated, representative set of human HBV genotypes A-H (n = 32) and woodchuck hepadnavirus (n = 9) sequences. The p-value plot was produced by CDS-plotcon, in which the probability of the conservation in a 21 nucleotide running-mean is as great or greater than what is expected from a null model using a default "non-coding" setting. The gray boxes indicate the nucleotide positions for SLα and SLβ. (C) Plot of the frequencies of conserved paired sites (predicted RNA secondary structures) that was done using pairwise comparisons on the same set of aligned sequences (n = 41). The analysis was done using StructureDist in the SSE package, in which the maximum value of each frequency is equal to 1.