DISTINCT REQUIREMENTS FOR PRIMARY SEQUENCE IN THE 5'-PART AND 3'-PARTOF A BULGE IN THE HEPATITIS-B VIRUS-RNA ENCAPSIDATION SIGNAL REVEALEDBY A COMBINED IN-VIVO SELECTION IN-VITRO AMPLIFICATION SYSTEM

Hepatitis B virus (HBV) is a small DNA virus that replicates by revers e transcription of a terminally redundant RNA, the pregenome. Specific packaging of this transcript into viral capsids is mediated by intera ction of the reverse transcriptase, P protein, with the 5'-proximal en capsidation signal epsilon. epsilon-function is correlated with the fo rmation of a hairpin structure containing a bulge and a loop, each con sisting of 6 nt, To analyse the importance of primary sequence in thes e regions, we have combined selection of encapsidation competent indiv iduals from poets 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 l oop sequence were detectable, many different sequences were tolerated in the 3'-part of the bulge, However, at the two 5'-proximal bulge pos itions the wt sequence was strongly selected for, indicating that for RNA packaging close contacts between protein and the 5'- but not the 3 '-part of the bulge are important. Such a bipartite organisation provi des a structural basis for the recently demonstrated special role of t he 3'-part of the bulge as template for the first nucleotides of (-)-s trand DNA in HBV reverse transcription.