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

为了明确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。将直接连接SV40启动子下游的人2-5A合成酶cDNA表达质粒与neo基因共转染小鼠L929细胞。从筛选的G418耐药细胞中获得3株表达人2-5A合成酶cdna的菌株，分别命名为LAS0、LAS3和LAS4。当用VSV或EMCV攻毒时，病毒增殖比L929细胞低1/100 - 1/1000。另一方面，VSV在LAS3中的生长与L929细胞中几乎相同，但EMCV在LAS3中的生长比L929细胞低约1/1000。LAS0、LAS3和LAS4细胞的细胞生长倍增时间较L929细胞慢，且与2-5A合成酶水平有关。因此，2-5A合成酶独立于IFN诱导的其他酶抑制病毒生长和细胞生长。当添加FCS、PDGF、EGF或胰岛素刺激血清饥饿的HeLaS3细胞时，可诱导2-5A合成酶。添加抗IFN-单克隆抗体可抑制2-5A合成酶基因的表达和2-5A合成酶的产生，提示自分泌IFN-参与了2-5A合成酶的诱导。抗ifn -抗体抑制2-5A合成酶诱导可促进第二周期DNA合成，但不能促进第一周期DNA合成。上述结果提示，在HeLa细胞受生长因子刺激后，IFN/2-5A合成酶系统在细胞生长负调控机制中发挥作用。

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.