Summary auto-generated
Researchers developed an antisense RNA (asRNA) system driven by the bovine leukemia virus (BLV) U3 promoter to inhibit BLV replication in cultured feline CC81 cells. The BLV U3 promoter is normally activated only by the viral transactivator protein p38tax, making it an inducible system that activates only during viral infection. An asRNA targeting the R-U5 region of the BLV genome was constructed under this promoter's control. Transfection of plasmid pLU (containing the asRNA gene) with BLV proviral DNA achieved 75% inhibition of viral replication at a 5:1 molar ratio. Unexpectedly, a plasmid containing only the empty BLV U3 promoter (pLT) also inhibited replication up to 60% at high molar ratios, suggesting competition for the limiting p38tax protein rather than asRNA action. Immunoblotting confirmed that p38tax physically associates with its responsive sequences in the viral promoter. Adding extra p38tax protein abolished the inhibitory effect of the empty promoter, confirming that sequestering this transactivator was the mechanism of inhibition. These results suggest a novel antiviral strategy based on competitive binding of viral transactivators to exogenous DNA sequences.
Key findings
- An asRNA construct driven by the BLV U3 promoter inhibited viral replication 75% at a 5:1 plasmid-to-proviral DNA ratio in cultured cells
- An empty BLV U3 promoter (without asRNA-coding sequences) also inhibited BLV replication by up to 60%, indicating a mechanism independent of asRNA
- Competition for the limiting viral transactivator protein p38tax appears to be the primary inhibition mechanism, as overexpression of p38tax abolished the empty promoter's inhibitory effect
- p38tax protein physically associates with its CRE/ATF responsive sequences in the BLV U3 promoter, demonstrated by indirect DNA-binding assays
- The BLV U3 promoter is an inducible system useful for antiviral strategies because it is activated only during viral infection by viral transactivator proteins
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Abstract
Use of viral inducible promoters which can be activated by virus- specific transactivator proteins to drive expression of antisense (as)RNA genes 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 susceptible to inhibition by asRNA. With plasmid pLU, which expresses the asRNA gene under the control of the BLV U3 promoter, 75% inhibition of virus replication was attained in CC81 cells (the molar ratio of pLU DNA over BLV proviral DNA in the transfection mixture was 5:1). Plasmid pLT, which contains only the BLV U3 promoter without any asRNA-coding region, also efficiently (up to 60%) inhibited virus replication when cotransfected with BLV proviral DNA at a ratio of 20:1. It was suggested that competition between functional and 'empty' viral promoters for the viral transactivator protein p38tax could account for this inhibition. An immunoblotting assay showed that in the presence of nuclear extracts from CC81 cells exogenous BLV p38tax specifically associates with its responsive sequence located in the BLV U3 promoter. Moreover, the additional expression of p38tax in CC81 cells abolishes the inhibitory effect of the empty viral promoter. These observations suggest a new mechanism of BLV inhibition caused, most probably, by sequestering of the viral transactivator protein.