THE ENCAPSIDATION SIGNAL ON THE HEPATITIS-B VIRUS-RNA PREGENOME FORMSA STEM-LOOP STRUCTURE THAT IS CRITICAL FOR ITS FUNCTION

Hepatitis B virus (HBV) is the type member of the hepadnaviridae, smal l enveloped DNA viruses that replicate through reverse transcription o f an RNA intermediate, the pregenome. This reaction occurs usually ins ide the viral nucleocapsid, the assembly of which requires specific in teractions between multiple copies of the core protein, the viral repl ication enzyme (P protein) and the RNA pregenome which also serves as mRNA for both proteins. Deletion studies have established that specifi c packaging of the RNA is mediated by a short cis-acting sequence, the encapsidation signal epsilon. Using nuclease sensitivity experiments we provide experimental evidence that part of this sequence can adopt a stem-loop structure that is interrupted by a bulge and a single unpa ired U residue. The structural consequences of deletions of the unpair ed regions and changes in their primary sequences were investigated in vitro, and their influence on the function of the epsilon-signal was tested in animal cells by monitoring encapsidation of RNAs carrying th e mutant epsilon-sequences in front of a 2.7 kb foreign RNA fragment, or within the context of a complete HBV genome. The data indicate that the entire stem-loop structure containing the bulge and the loop is c ritical for encapsidation competence. While gross alterations in the p rimary sequences of the unpaired regions interfere with encapsidation, data obtained with additional mutants suggest that the bulge region i s more tolerant to sequence changes than the loop.