Synthesis of template-sense, single-strand Flockhouse virus RNA in a cell-free replication system.
Synthesis of template-sense, single-strand Flockhouse virus RNA in a cell-free replication system.
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DOI：
10.1016/0042-6822(91)90153-3
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发表时间：
1991-07
期刊：
影响因子：
3.7
通讯作者：
Shi-Xuan Wu;P. Kaesberg
中科院分区：
文献类型：
--
作者：
Shi-Xuan Wu;P. Kaesberg
感染FHV（弗洛克豪斯病毒）的果蝇细胞的无细胞提取物具有合成FHV RNA的能力。当适当孵育这些提取物时，可以检测到ss和ds FHV RNA 1、-2和-3作为合成产物。脉冲追踪分析表明，掺入到ds RNA 1、-2和3中的放射性被追踪到ss RNA 1、-2和3中，表明这些ds RNA是FHV RNA复制的中间体。当用去污剂处理无细胞提取物以溶解细胞膜时，仅可检测到ds RNA 1、-2和-3作为合成产物。用核糖核酸酶处理以去除内源性模板的无细胞提取物，当这些RNA与脂质体一起作为模板提供时，具有复制FHV RNA 1和-2的能力。检测到as和ds RNA 1和-2都是合成产物。当RNA 3作为模板提供时，仅合成ds RNA 3。在不存在lipofectin或存在去污剂的情况下，仅检测到ds RNA作为合成产物，并且在与模板互补的链中发现所有掺入的放射性。我们的研究结果是一致的机制，其中第二链的合成，虽然不是互补链的合成，需要完整的膜。它们还表明ds RNA 3是合成ss RNA 3的中间体。
Cell-free extracts of Drosophila melanogaster cells infected with FHV (Flockhouse virus) have the capacity to synthesize FHV RNA. When such extracts are incubated appropriately, ss and ds FHV RNA 1, -2, and -3 can be detected as synthesis products. Pulse-chase analyses indicate that radioactivity, incorporated into ds RNA 1, -2, and -3 is chased into ss RNA 1, -2, and -3, suggesting that these ds RNAs are intermediates in FHV RNA replication. When the cell-free extracts are treated with detergents to solubilize cellular membranes, only ds RNA 1, -2, and -3 can be detected as synthesis products. The cell-free extracts, treated with a ribonuclease to remove endogenous templates, have the capacity to replicate FHV RNA 1 and -2 when these RNAs are provided as templates, together with lipofectin. Both as and ds RNA 1 and -2 are detected as synthesis products. When RNA3 is provided as a template, only ds RNA3 is synthesized. In the absence of lipofectin or in the presence of detergent, only ds RNAs are detected as synthesis products and all the incorporated radioactivity is found in the strand complementary to that of the template. Our results are consistent with a mechanism in which second-strand synthesis, although not complementary strand synthesis, requires intact membranes. They also suggest that ds RNA3 is an intermediate in the synthesis of ss RNA3.