Castelli J, Wood K A, Youle R J
Biochemistry Section, NINDS, NIH, Bethesda, MD 20892, USA.
Biomed Pharmacother. 1998;52(9):386-90. doi: 10.1016/s0753-3322(99)80006-7.
The 2-5A system is an established endogenous antiviral pathway. Interferon treatment of cells leads to an increase in basal, but latent, levels of 2-5A-dependent RNase (RNase L) and the family of 2'-5' oligoadenylate synthetases (OAS). Double-stranded RNA, thought to be derived from viral replication intermediates, activates OAS. Activated OAS converts ATP into unusual short 2'-5' linked oligoadenylates called 2-5A [ppp5'(A2'p5')2A]. The 2-5A binds to and activates RNase L which cleaves single stranded RNA with moderate specificity for sites 3' of UpUp and UpAp sequences, and thus leads to degradation of cellular rRNA. During apoptosis, generalized cellular RNA degradation, distinct from the differential expression of mRNA species that may regulate specific gene expression during apoptosis, has been observed. The mechanism of RNA breakdown during apoptosis has been commonly considered a non-specific event that reflects the generalized shut down of translation and homeostatic regulation during cell death. Due to the similar RNA degradation that occurs during both apoptosis and viral infection we investigated the potential role of RNase L in apoptosis. To investigate whether RNase L activity could lead to apoptosis, NIH3T3 cells were transfected with a lac-inducible vector containing the human RNase L gene. Treatment of these cells with isopropylthiogalactoside (IPTG) caused loss of cell viability that was confirmed as an apoptotic cell death by morphological and biochemical criteria. Similarly, specific allosteric activation of endogenous RNase L by introduction of 2-5A directly into L929 cells also induced apoptosis. In L929 cells poly(I).poly(C) treatment in combination with interferon caused an increase in apoptosis whereas neither interferon or double stranded RNA alone altered cell viability. Therefore, increased expression or activation of RNase L causes apoptosis. Inhibition of RNase L, specifically with a dominant negative mutant, suppressed poly(I)Ypoly(C)-induced apoptosis in interferon-primed fibroblasts. Poliovirus, a picornovirus with a single-stranded RNA genome, causes apoptosis of HeLa cells. Expression of the dominant negative inhibitor of RNase L in HeLa prevented virus-induced apoptosis and maintained cell viability. Thus, reduction or inhibition of RNase L activity prevents apoptosis. Both apoptosis and the 2-5A system can provide defense against viral infection in multicellular organisms by preventing production and therefore spread of progeny virus. RNase L appears to function in both mechanisms, therefore, initiation of apoptosis may be one mechanism for the antiviral activity of the 2-5A system.
2-5A系统是一种既定的内源性抗病毒途径。用干扰素处理细胞会导致2-5A依赖性核糖核酸酶（RNase L）和2'-5'寡腺苷酸合成酶（OAS）家族的基础水平升高，但处于潜伏状态。双链RNA被认为源自病毒复制中间体，可激活OAS。被激活的OAS将ATP转化为称为2-5A [ppp5'(A2'p5')2A]的异常短的2'-5'连接的寡腺苷酸。2-5A与RNase L结合并激活RNase L，RNase L以中等特异性切割UpUp和UpAp序列3'位点的单链RNA，从而导致细胞rRNA降解。在细胞凋亡过程中，已观察到普遍的细胞RNA降解，这与可能在细胞凋亡过程中调节特定基因表达的mRNA种类的差异表达不同。细胞凋亡过程中RNA分解的机制通常被认为是一种非特异性事件，反映了细胞死亡期间翻译的普遍关闭和稳态调节。由于细胞凋亡和病毒感染期间都会发生类似的RNA降解，我们研究了RNase L在细胞凋亡中的潜在作用。为了研究RNase L活性是否会导致细胞凋亡，将含有人类RNase L基因的乳糖诱导型载体转染到NIH3T3细胞中。用异丙基硫代半乳糖苷（IPTG）处理这些细胞会导致细胞活力丧失，通过形态学和生化标准证实这是一种凋亡性细胞死亡。同样，通过直接将2-5A引入L929细胞中对内源性RNase L进行特异性变构激活也会诱导细胞凋亡。在L929细胞中，聚肌苷酸-聚胞苷酸（poly(I).poly(C)）处理与干扰素联合使用会导致细胞凋亡增加，而单独使用干扰素或双链RNA都不会改变细胞活力。因此，RNase L表达增加或激活会导致细胞凋亡。用显性负突变体特异性抑制RNase L可抑制聚肌苷酸-聚胞苷酸诱导的经干扰素预处理的成纤维细胞凋亡。脊髓灰质炎病毒是一种具有单链RNA基因组的小RNA病毒，可导致HeLa细胞凋亡。在HeLa细胞中表达RNase L的显性负抑制剂可预防病毒诱导的细胞凋亡并维持细胞活力。因此，降低或抑制RNase L活性可预防细胞凋亡。细胞凋亡和2-5A系统都可以通过阻止子代病毒的产生从而阻止其传播，为多细胞生物提供抗病毒感染的防御。因此，RNase L似乎在这两种机制中都起作用，细胞凋亡的启动可能是2-5A系统抗病毒活性的一种机制。
