RIBOZYME CLEAVES REX TAX MESSENGER-RNA AND INHIBITS BOVINE LEUKEMIA-VIRUS EXPRESSION

Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long ter minal repeat to promote viral transcription and may be involved in tum origenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammerhead catalytic motif flanked by antise nse sequences that hybridize with the complementary rex/tax mRNA. To e valuate cleavage in a cell-free system, we transcribed portions of rex /tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves >80% of the target RNA. Synthetic DNA enco ding this ribozyme was cloned into the expression vector pRc/RSV and t ransfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells exp ressing the ribozyme, viral expression was markedly inhibited. Express ion of the BLV core protein p24 was inhibited by 61%, and reverse tran scriptase activity in supernatant was inhibited by 92%. Ribozyme inhib ition of BLV expression suggests that cattle expressing these sequence s may be able to control BLV replication.