The encapsidation signal on the hepatitis B virus RNA pregenome forms a stem-loop structure that is critical for its function.
Hepatitis B virus (HBV) is the type member of the hepadnaviridae, small enveloped DNA viruses that replicate through reverse transcription of an RNA intermediate, the pregenome. This reaction occurs usually inside the viral nucleocapsid, the assembly of which requires specific interactions between multiple copies of the core protein, the viral replication enzyme (P protein) and the RNA pregenome which also serves as mRNA for both proteins. Deletion studies have established that specific 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 unpaired U residue. The structural consequences of deletions of the unpaired 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 the 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 critical for encapsidation competence. While gross alterations in the primary sequences of the unpaired regions interfere with encapsidation, data obtained with additional mutants suggest that the bulge region is more tolerant to sequence changes than the loop.
Knaus
T
T
Zentrum für Molekulare Biologie, Universität Heidelberg, Germany.
Nassal
M
M
D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000378:metabolism*; D003001:Cloning, Molecular; D003198:Computer Simulation; D015684:Gene Products, pol / Q000378:metabolism; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011233:Precipitin Tests; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase