Distinct requirements for primary sequence in the 5' - and 3'-part of a bulge in the hepatitis B virus RNA encapsidation signal revealed by a combined in vivo selection/in vitro amplification system

摘要

Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription of a terminally redundant RNA, the pregenome. Specific packaging of this transcript into viral capsids .is mediated by interaction of the reverse transcriptase, P protein, with the 5'-proximal encapsidation signal e. e-function is correlated with the formation of a hairpin structure containing a bulge and a loop, each consisting of 6 nt To analyse the importance of primary sequence in these regions, we have combinedselection of encapsidation competent individuals from pools of randomized epsilon-sequences in transfected cells with in vitro amplification, thus bypassing the current experimental limitations of the HBV system. While no alterations of the authentic loop sequence were detectable, many different sequences were tolerated in the 3'-part of the bulge. However, at the two 5'-proximal bulge positions the wt sequence was strongly selected for, indicating that for RNA packaging close contacts between proteinand the 5'- but not the 3'-part of the bulge are important. Such 8 bipartite organisation provides a structural basis for the recently demonstrated special role of the 3'-part of the bulge as template for the first nucleotides of DNA in HBV reverse transcription.