Inhibition of 2-5A synthetase expression by antisense RNA interferes with interferon-mediated antiviral and antiproliferative effects and induces anchorage-independent cell growth.

It has been shown previously that the IFN-induced enzyme 2-5A synthetase is sufficient to induce antiviral and antiproliferative effects in transfected cells expressing the protein. In this study, the possibility that this enzyme is also essential in generating these biological activities was investigated. For this purpose, a plasmid, pMSas-NEO, was constructed. This plasmid carries an active neomycin-resistant gene. In addition, it contains a metallothionein promoter fused to an inverted 180-bp fragment derived from the 5' end of cDNA encoding the 43-kDa isoform of murine 2-5A synthetase. NIH/3T3 mouse fibroblasts were transfected with the plasmid, about 50 neomycin-resistant clones were isolated, and two, clone 11 and clone 22, were chosen for further studies. One clone transfected only with the neomycin-resistant gene, clone Neo, was used as a control. The results show that in the case of clone 11, the combined treatment of IFN and ZnCl2 reduced significantly the level of the IFN-induced 2-5A synthetase activity, the amount of the 40-, 43-, and 71-kDa 2-5A synthetase isoforms and the level of the 1.7-kb specific RNA transcript. An even stronger effect on these parameters was observed with clone 22 cells. No difference in PKR activity was evident under the same conditions with all three clones tested. Most important, the combined treatment of IFN and ZnCl2 reversed the IFN-mediated antiproliferative and antiviral activities, as determined by the kinetics of cell growth, thymidine incorporation, cloning efficiency, and infection with mengovirus. Strikingly, the growth of colonies in soft agar were observed in both clone 11 (small colonies) and clone 22 (large colonies) cells, particularly following treatment with ZnCl2. We conclude that 2-5A synthetase is an essential component in the IFN-induced biological activities and that interference with its function results in anchorage-independent growth of the transfected cells.