
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 combinedin vivoselection/in vitroamplification system
Author(s) -
Andrea Rieger,
Michael Nassal
Publication year - 1995
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/23.19.3909
Subject(s) - biology , sequence (biology) , rna , hepatitis b virus , virology , viral evolution , virus , genetics , evolutionary biology , computational biology , gene
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 epsilon, epsilon-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 combined selection 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 protein and the 5'- but not the 3'-part of the bulge are important. Such a 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 (-)-strand DNA in HBV reverse transcription.