INCORPORATION OF THE CATALYTIC DOMAIN OF A HAMMERHEAD RIBOZYME INTO ANTISENSE RNA ENHANCES ITS INHIBITORY EFFECT ON THE REPLICATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. + 222 to + 634). The resulting catalytic antisense RNA was shown to cleave i ts target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into hum an SW480 cells together with infectious complete proviral HIV-1 DNA, f ollowed by analysis of HIV-1 replication. The presence of the catalyti cally active domain resulted in 4 to 7 fold stronger inhibition of HIV -1 replication as compared to the parental antisense RNA and the inact ive mutant. Kinetic and structural studies performed in vitro indicate d that the ability for double strand formation was not changed in cata lytic antisense RNA versus parental antisense RNA. Together, these dat a suggest that the ability to cleave target RNA is a crucial prerequis ite for the observed increase of inhibition of the replication of HIV- 1.