USE OF THE BOVINE LEUKEMIA-VIRUS LTR U3 PROMOTER FOR EXPRESSING ANTISENSE ANTIVIRAL RNAS AND COMPETITIVE-INHIBITION OF VIRAL-INFECTION IN CELL-CULTURE

Use of viral inducible promoters which can be activated by virus-speci fic transactivator proteins to drive expression of antisense (as)RNA g enes appears to be an attractive approach to inhibit virus infections in vivo. To this end, we have constructed an asRNA gene expressed from the bovine leukaemia virus (BLV) U3 promoter that is complementary to the R-U5 region of the BLV genome, This is the region that is most su sceptible to inhibition by asRNA, With plasmid pLU, which expresses th e asRNA gene under the control of the BLV U3 promoter, 75% inhibition of virus replication was attained in CC81 cells (the molar ratio of pL U DNA over BLV proviral DNA in the transfection mixture was 5:1). Plas mid pLT, which contains only the BLV U3 promoter without any asRNA-cod ing region, also efficiently (up to 60%) inhibited virus replication w hen cotransfected with BLV proviral DNA at a ratio of 20:1. It was sug gested that competition between functional and 'empty' viral promoters for the viral transactivator protein p38(tax) could account for this inhibition. An immunoblotting assay showed that in the presence of nuc lear extracts from CC81 cells exogenous BLV p38(tax) specifically asso ciates with its responsive sequence located in the BLV U3 promoter, Mo reover, the additional expression of p38(tax) in CCS 1 cells abolishes the inhibitory effect of the empty viral promoter, These observations suggest a new mechanism of BLV inhibition caused, most probably, by s equestering of the viral transactivator protein.