The Hepatitis B Virus Pregenome: Prediction of RNA Structure and Implications for the Emergence of Deletions

The terminally redundant pregenomic RNA of human hepatitis B virus (HBV) comprises some 3,330 nucleotides and is a replicative intermediate in the production of the circular DNA genome. Deletions are known to arise in the HBV genome during the course of chronic infection and are sometimes associated with interferon therapy. These deletions are limited to small parts of the genome such as the 357-nucleotide pre-S1 region. Long RNA molecules such as the HBV pregenome have considerable structural flexibility and will undergo secondary structure shifts between energetically favourable states in a continuous and semi-random fashion. Since prediction of structure elements that are highly conserved in different forms of one RNA molecule is now feasible by computer modelling, we have analysed the whole HBV pregenome by two different RNA structure prediction algorithms and by new methods that exploit these algorithms. Significantly, the ends of pregenomic RNA were predicted to undergo both short-range and long-range interactions, which has relevance to our knowledge of the virus replicative cycle. By incorporating phylogenetic information relating to the 6 recognised genotypes of HBV, it was possible to highlight short secondary structures that may be common to all HBV strains. For example, although the pre-S1 region was predicted to undergo local folding of a loosely defined nature, most observed pre-S1 deletions mapped to all or part of an arm carrying a better-defined structure. The loss of such sequences may be mechanistically attributable to polymerase skipping during reverse transcription, and the possible advantages of such deletions are considered.