Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. 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 antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate 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 encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication.Bovine leukemia virus (BLV), a retrovirus structurally similar to human T-cell leukemia viruses I and II (HTLV-I and -II), causes persistent lymphocytosis and B-lymphocyte lymphoma in cattle and sheep (1). After initial infection, BLV expresses a doubly spliced transcript encoding the regulatory proteins Rex and Tax (2). Tax trans-activates the viral long terminal repeat and also cellular promoters, including c-fos and somatostatin (3). Cotransfection experiments have shown that Tax is necessary for viral expression in vitro (4). Rex regulates the transition from early expression of the doubly spliced transcript encoding regulatory proteins to the later expression of singly spliced or unspliced transcripts that express the env gene or the gag and pol genes (5). Recently, the 3' region of HTLV-I and BLV has been shown to encode RNA with alternative splice patterns that may express other regulatory proteins (6-8).Because of the critical role of regulatory proteins such as Tax and Rex in the HTLV/BLV group of viruses, we hypothesized that the rex/tax mRNA would be a rational target for ribozyme-mediated inhibition. The hammerhead motif, first identified in plant RNA pathogens (9, 10), cleaves the phosphodiester bond downstream of a GUC triplet (9,10). By flanking the hammerhead motif with antisense sequences, Haseloff and Gerlach (11) demonstrated cleavage of specific target RNAs. In this study, we demonstrate a ribozyme that cleaves rex/tax mRNA and, when transfected into BLV-infected cells, markedly inhibits viral expression.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact...
