Inhibition of SV40 replicon function by engineered antisense RNA transcribed by RNA polymerase III

Abstract

Promoters recognized by RNA polymerase III were used to direct synthesis of RNAs of opposite polarity to the 5' end of the mRNA for the large T-antigen of SV40. A construct was made utilizing the adenovirus (human type II) VA1 gene promoter linked to 163 bp of SV40 DNA sequences cloned in antisense orientation relative to the promoter. The SV40 sequence corresponds to the 5' end of the large T-antigen gene. In addition to the antisense constructs control plasmids were utilized which either lacked both promoter and SV40 elements, lacked RNA polymerase III promoter elements but contained SV40 sequences or contained the VA1 gene promoter fused to SV40 sequences in the sense orientation. The function of the various gene fusions was demonstrated in an in vitro transcription system and in vivo by S1 nuclease 5' end mapping following transfection into COS1 cells. Cotransfection of COS1 cells with the 'antisense' gene and a plasmid containing an SV40 origin of replication resulted in a substantial transient inhibition of SV40-replicon function when compared to control determinations (50% to nearly complete inhibition of large T-antigen dependent DNA replication for 18-36 h). These results show that an antisense RNA generated by RNA polymerase III can effectively block expression of a chromosomally located gene.