Mechanism of Antiviral Action of Interferon: Analysis Using 2-5A Synthetase Producing Cells

为了阐明2-5A合成酶的功能，我们构建了产生人2-5A合成酶的小鼠细胞。将人2-5A合成酶cDNA直接连接在SV 40启动子下游的表达质粒与neo基因共转染小鼠L929细胞。在所选择的G418抗性细胞中，获得了表达人2-5A合成酶cDNA的三个菌株，将其命名为LAS 0、LAS 3和LAS 4。用VSV或EMCV攻击时，病毒增殖比L929细胞低1/100 ~ 1/1000。VSV在LAS 3细胞中的生长与在L929细胞中的生长几乎相同，而EMCV在LAS 3细胞中的生长比在L929细胞中的低约1/1000。细胞倍增时间在LAS 0、LAS 3和LAS 4细胞中比在L929细胞中慢，且与2-5A合成酶水平相关。当血清饥饿的HeLaS 3细胞被FCS、PDGF、EGF或胰岛素刺激时，2 - 5A合成酶被诱导。抗IFN-γ单克隆抗体的加入抑制了2-5A合成酶基因的表达和2 -5A合成酶的产生，表明自分泌IFN-γ参与了2-5A合成酶的诱导。2-5A合成酶的诱导抑制抗IFN-抗体增强了第二个，但不是第一个循环的DNA合成。这些结果表明，在HeLa细胞中，IFN/2-5A合成酶系统在生长因子刺激后的细胞生长负调控机制中起作用。

To clarify the function of 2-5A synthetase, we constructed a mouse cell producing human 2-5A synthetase. The expression plasmid having human 2-5A synthetase cDNA ligated immediately down stream of SV40 promoter was cotransfected with neo gene into mouse L929 cells. Of G418 resistant cells selected, three strains expressing the human 2-5A synthetase cdna were obtained, which were designated as LAS0, LAS3 and LAS4. When challenged with VSV or EMCV, the virus multiplications were 1/100 - 1/1000 lower than L929 cells. On the other hand, the growth of VSV in LAS3 was almost the same as in L929 cells, but that of EMCV in LAS3 was about 1/1000 lowere than in L929 cells. Furthermore the doubling time of the cell growth became slower in LAS0, LAS3 and LAS4 cells than in L929 cells, and it was correlated with the level of 2-5A synthetase. Thus, 2-5A synthetase is responsible independently of other enzymes induced by IFN for the suppression of virus growth and cell growth.When serum-starved HeLaS3 cells were stimulated by the addition of FCS, PDGF, EGF or insulin, 2-5A synthetase was induced. Addition of anti-IFN- monoclonal antibody inhibited both the expression of 2-5A synthetase gene and the production of the enzyme, suggesting that autocrine IFN- was involved in 2-5A synthetase induction. Inhibition of 2-5A synthetase induction by anti-IFN- antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa cells after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.