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Nucleic Acids Research, 1995, Vol. 23, No. 24 4954-4962
© 1995

Articles

Efficient hammerhead ribozyme-mediated cleavage of the structured heapatitis B virus encapsidation signal in vitro and in cell extracts, but not in intact cells

Jürgen Beck and Michael Nassal*

Zenturm fuür Molekulare Biologie, Universitaät Heidelberg Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany

*To whom correspondence should be addressed

Received October 9, 1995. Accepted November 8, 1995.

Hepatitis B virus (HBV), the causative agent of B-type hepatitis in man, is a small enveloped DNA virus that replicates through reverse transcription of an RNA intermediate, the terminally redundant RNA pregenome. An essential highly conserved cls-element present twice on this RNA is the encapsidation signal {varepsilon}, a stem-loop structure that is critical for pregenome packaging and reverse transcription, {varepsilon} is hence an attractive target for antiviral therapy. Its structure, however, is a potential obstacle to antivirals whose action depends on hybridization, e.g. ribozymes. Here we demonstrate effective In vitro cleavage inside e by hammerhead ribozymes containing flanking sequences complementary to an adjacent less structured region. Upon cc-transfection with a HBV expression construct corresponding ribozymes embedded in a U6 snRNA context led to a significant, though modest, reduction in the steady-state level of HBV pregenomes. Inactive ribozyme mutants revealed that antisense effects contributed substantially to this reduction, however, efficient {varepsilon} cleavage by the intracellulariy expressed ribozymes was observed in Mg2+-supplemented cell lysates. Artificial HBV pregenomes carrying the ribozymes in els and model RNAs lacking all HBV sequences except {varepsilon} exhibited essentially the same behaviour. Hence, neither the absence of co-localization of ribozyme and target nor a viral component, but rather a cellular factors), is responsible for the strikingly different ribozyme activities inside cells and in cellular extracts.


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