Shapiro G I, Gurney T, Krug R M
J Virol. 1987 Mar;61(3):764-73. doi: 10.1128/JVI.61.3.764-773.1987.
Single-stranded M13 DNAs specific for various influenza virus genomic segments were used to analyze the synthesis of virus-specific RNAs in infected cells. The results show that influenza virus infection is divided into two distinct phases. During the early phase, the syntheses of specific virion RNAs, viral mRNAs, and viral proteins were coupled. Thus, the NS (nonstructural) virion RNA was preferentially synthesized early, leading to the preferential synthesis of NS1 viral mRNA and NS1 protein; in contrast, M (matrix) RNA synthesis was delayed, leading to the delayed synthesis of M1 viral mRNA and M1 protein. This phase lasted for 2.5 h in BHK-21 cells, the time at which the rate of synthesis of all the viral mRNAs was maximal. During the second phase, the synthesis of all the virion RNAs remained at or near maximum until at least 5.5 h postinfection, whereas the rate of synthesis of all the viral mRNAs declined dramatically. By 4.5 h, the rate of synthesis of all the viral mRNAs was 5% of the maximum rate. Viral mRNA and protein syntheses were also not coupled, as the synthesis of all the viral proteins continued at maximum levels, indicating that protein synthesis during this phase was directed principally by previously synthesized viral mRNAs. Short pulses (3 min) with [3H]uridine and nonaqueous fractionation of cells were used to show that influenza virion RNA synthesis occurred in the nucleus, demonstrating that all virus-specific RNA synthesis was nuclear. Virion RNAs, like viral mRNAs, were efficiently transported to the cytoplasm at both early and late times of infection. In contrast, the full-length transcripts of the virion RNAs, which are the templates for virion RNA synthesis, were sequestered in the nucleus. Thus, the template RNAs, which were synthesized only at early times, remained in the nucleus to direct virion RNA synthesis throughout infection. These results enabled us to present an overall scheme for the control of influenza virus gene expression.
使用针对各种流感病毒基因组片段的单链M13 DNA来分析感染细胞中病毒特异性RNA的合成。结果表明，流感病毒感染分为两个不同阶段。在早期阶段，特异性病毒粒子RNA、病毒mRNA和病毒蛋白的合成是偶联的。因此，NS（非结构）病毒粒子RNA在早期优先合成，导致NS1病毒mRNA和NS1蛋白的优先合成；相反，M（基质）RNA合成延迟，导致M1病毒mRNA和M1蛋白的合成延迟。此阶段在BHK - 21细胞中持续2.5小时，此时所有病毒mRNA的合成速率达到最大值。在第二阶段，所有病毒粒子RNA的合成在感染后至少5.5小时内保持在或接近最大值，而所有病毒mRNA的合成速率则急剧下降。到4.5小时时，所有病毒mRNA的合成速率为最大速率的5%。病毒mRNA和蛋白的合成也不再偶联，因为所有病毒蛋白的合成继续保持在最高水平，这表明此阶段的蛋白合成主要由先前合成的病毒mRNA指导。用[3H]尿苷进行短脉冲（3分钟）处理并对细胞进行非水相分级分离，结果表明流感病毒粒子RNA合成发生在细胞核中，这证明所有病毒特异性RNA合成均在细胞核中进行。病毒粒子RNA与病毒mRNA一样，在感染的早期和晚期都能有效地转运到细胞质中。相比之下，作为病毒粒子RNA合成模板的病毒粒子RNA全长转录本被隔离在细胞核中。因此，仅在早期合成的模板RNA保留在细胞核中，以在整个感染过程中指导病毒粒子RNA的合成。这些结果使我们能够提出一个控制流感病毒基因表达的总体方案。
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