Rice A P, Roberts W K, Kerr I M
J Virol. 1984 Apr;50(1):220-8. doi: 10.1128/JVI.50.1.220-228.1984.
We investigated the effects of interferon treatment on virus yield, protein synthesis, and the 2-5A system in vaccinia virus-infected HeLa, L929, and CV1 cells. Under the culture conditions used, vaccinia virus replication was relatively insensitive to the antiviral effects of interferon. In L929 and HeLa cells, interferon at 400 reference units (r.u.) per ml had little effect on viral protein synthesis, the virus-induced inhibition of host protein synthesis, or virus yield: 2,000 to 20,000 r.u./ml were required to inhibit these. Despite this, high levels (up to 5 microM) of 2-5A [ppp(A2'p)nA; n greater than or equal to 2] were found during vaccinia infection of all of these types of cells treated with 400 r.u. of interferon per ml, i.e., at interferon concentrations too low to inhibit significantly virus growth. High levels (up to 5 microM) were also found in non-interferon-treated HeLa cells (which have a high constitutive level of 2-5A synthetase) in which vaccinia virus replicates perfectly well. It can be concluded that high levels of 2-5A per se have no necessary antiviral effect on vaccinia virus in these systems. These results are in marked contrast to those obtained here and previously with encephalomyocarditis virus. For example, in HeLa cells less than 20 nM 2-5A accumulated, but virus replication was inhibited by 50 r.u. of interferon per ml (Silverman et al., Eur. J. Biochem. 124:131-138, 1982). The characteristic cleavage of rRNA by the 2-5A-dependent RNase was delayed relative to 2-5A accumulation in the vaccinia virus-infected cells. This delay was not the result of either defective 2-5A or of a stable virus-induced inhibition of the 2-5A-dependent RNase; 2-5A extracted from the cells had full biological activity when assayed by activation of the 2-5A-dependent RNase in cell extract, and the 2-5A-dependent RNase extracted from the vaccinia virus-infected cells was fully active in vitro. The basis for the delay remains to be determined. High levels of 2-5A were not observed when late (DNA synthesis-dependent) vaccinia transcription was inhibited by either cycloheximide or cytosine arabinoside. The only known activator of the 2-5A synthetase is double-stranded RNA. The presence of 2-5A therefore implies the natural occurrence of double-stranded structures in late viral RNA in intact vaccinia virus-infected cells.
我们研究了干扰素处理对痘苗病毒感染的HeLa、L929和CV1细胞中病毒产量、蛋白质合成及2-5A系统的影响。在所用的培养条件下，痘苗病毒复制对干扰素的抗病毒作用相对不敏感。在L929和HeLa细胞中，每毫升400参考单位（r.u.）的干扰素对病毒蛋白质合成、病毒诱导的宿主蛋白质合成抑制或病毒产量几乎没有影响：抑制这些需要每毫升2000至20000 r.u.。尽管如此，在用每毫升400 r.u.干扰素处理的所有这些类型的细胞进行痘苗感染期间，都发现了高水平（高达5 microM）的2-5A [ppp(A2'p)nA；n大于或等于2]，即干扰素浓度低至不足以显著抑制病毒生长。在未用干扰素处理的HeLa细胞（其2-5A合成酶的组成水平较高）中也发现了高水平（高达5 microM），在这些细胞中痘苗病毒复制良好。可以得出结论，在这些系统中，高水平的2-5A本身对痘苗病毒没有必然的抗病毒作用。这些结果与我们在此以及先前用脑心肌炎病毒获得的结果形成明显对比。例如，在HeLa细胞中积累的2-5A不到20 nM，但每毫升50 r.u.的干扰素就能抑制病毒复制（Silverman等人，《欧洲生物化学杂志》124:131 - 138，1982）。在痘苗病毒感染的细胞中，相对于2-5A的积累，2-5A依赖性核糖核酸酶对rRNA的特征性切割有所延迟。这种延迟既不是由于有缺陷的2-5A，也不是由于病毒诱导的对2-5A依赖性核糖核酸酶的稳定抑制所致；当通过细胞提取物中2-5A依赖性核糖核酸酶的激活来检测时，从细胞中提取的2-5A具有完全的生物学活性，并且从痘苗病毒感染的细胞中提取的2-5A依赖性核糖核酸酶在体外具有完全活性。延迟的原因尚待确定。当用环己酰亚胺或阿糖胞苷抑制晚期（依赖DNA合成的）痘苗转录时，未观察到高水平的2-5A。2-5A合成酶唯一已知的激活剂是双链RNA。因此，2-5A的存在意味着在完整的痘苗病毒感染细胞的晚期病毒RNA中天然存在双链结构。
