Sawicki D L, Kaariainen L, Lambek C, Gomatos P J
J Virol. 1978 Jan;25(1):19-27. doi: 10.1128/JVI.25.1.19-27.1978.
When cells infected with the Semliki Forest virus (SFV) mutant ts-4 were shifted to the nonpermissive temperature, synthesis of 26S RNA ceased, whereas synthesis of 42S RNA continued normally. These two single-stranded SFV RNAs are synthesized in two types of replicative intermediate (RI), 26S RNA in RI(b) and 42S RNA in RI(a). Cessation of 26S RNA synthesis after shift up in temperature was accompanied by loss of RI(b). When infected cells were shifted back down to 27 degrees C, 26S RNA synthesis resumed, coincident with the reappearance of RI(b). In both types of RI, the 42S minus-strand RNA is template for synthesis of plus-strand RNA. In pulse-chase experiments, we obtained RIs labeled only in their minus-strand RNA, and thus could follow the fate of RIs assembled at 27 degrees C when they were shifted to 39 degrees C. Our results show that, after shift up to 39 degrees C, there was a quantitative conversion of RIs in which 26S RNA had been synthesized to RIs in which 42S RNA was synthesized. This conversion of RI(b) to RI(a) was reversible, since RIs in which 26S RNA was synthesized reappeared when the infected cultures were shifted back down to 27 degrees C. We propose that, associated with RI(b), in which 26S RNA is synthesized, there is a virus-specific protein that functions to promote initiation of 26S RNA transcription at an internal site on the 42S minus-strand RNA and to block transcription on the minus strand in this region by the SFV RNA polymerase that had bound and was copying the minus-strand RNA from its 3' end. A ribonuclease-sensitive region would thus result in the sequence adjacent to the one that was complementary to 26S RNA. This virus-specific protein is not a component of the SFV RNA polymerase that continues to transcribe 42S RNA, and it is temperature sensitive in ts-4 mutant-infected cells. When this virus-specific protein is not present on RIs, the SFV polymerase transcribes the whole 42S minus-strand RNA and yields 42S plus-strand RNA.
当感染了辛德毕斯病毒（SFV）突变体ts - 4的细胞转移到非允许温度时，26S RNA的合成停止，而42S RNA的合成仍正常继续。这两种单链SFV RNA是在两种类型的复制中间体（RI）中合成的，26S RNA在RI(b)中合成，42S RNA在RI(a)中合成。温度升高后26S RNA合成的停止伴随着RI(b)的丢失。当感染的细胞转移回27℃时，26S RNA合成恢复，与RI(b)的重新出现同时发生。在两种类型的RI中，42S负链RNA是正链RNA合成的模板。在脉冲追踪实验中，我们获得了仅在其负链RNA上被标记的RI，因此可以追踪在27℃组装的RI转移到39℃时的命运。我们的结果表明，转移到39℃后，已合成26S RNA的RI发生了定量转化，变成了合成42S RNA的RI。这种RI(b)到RI(a)的转化是可逆的，因为当感染培养物转移回27℃时，合成26S RNA的RI重新出现。我们提出，与合成26S RNA的RI(b)相关联，存在一种病毒特异性蛋白，其作用是促进在42S负链RNA内部位点启动26S RNA转录，并阻止已结合并从其3'端复制负链RNA的SFV RNA聚合酶在该区域的负链上进行转录。因此，一个核糖核酸酶敏感区域将出现在与26S RNA互补的序列相邻处。这种病毒特异性蛋白不是继续转录42S RNA的SFV RNA聚合酶的组成部分，并且在ts - 4突变体感染的细胞中对温度敏感。当这种病毒特异性蛋白不存在于RI上时，SFV聚合酶转录整个42S负链RNA并产生42S正链RNA。
