EFFICIENT HAMMERHEAD RIBOZYME-MEDIATED CLEAVAGE OF THE STRUCTURED HEPATITIS-B VIRUS ENCAPSIDATION SIGNAL IN-VITRO AND IN CELL-EXTRACTS, BUTNOT IN INTACT-CELLS

Hepatitis B virus (HBV), the causative agent of B-type hepatitis in ma n, is a small enveloped DNA virus that replicates through reverse tran scription of an RNA intermediate, the terminally redundant RNA pregeno me, An essential highly conserved cis-element present twice on this RN A is the encapsidation signal epsilon, a stem-loop structure that is c ritical for pregenome packaging and reverse transcription, epsilon is hence an attractive target for antiviral therapy, Its structure, howev er, is a potential obstacle to antivirals whose action depends on hybr idization, e.g. ribozymes, Here we demonstrate effective in vitro clea vage inside epsilon by hammerhead ribozymes containing flanking sequen ces complementary to an adjacent less structured region, Upon co-trans fection with a HBV expression construct corresponding ribozymes embedd ed in a U6 snRNA context led to a significant, though modest, reductio n in the steady-state level of HBV pregenomes, Inactive ribozyme mutan ts revealed that antisense effects contributed substantially to this r eduction, however, efficient epsilon cleavage by the intracellularly e xpressed ribozymes was observed in Mg2+-supplemented cell lysates, Art ificial HBV pregenomes carrying the ribozymes in cis and model RNAs la cking all HBV sequences except epsilon exhibited essentially the same behaviour, Hence, neither the absence of co-localization of ribozyme a nd target nor a viral component, but rather a cellular factor(s), is r esponsible for the strikingly different ribozyme activities inside cel ls and in cellular extracts.