Active complete in vitro replication of nodavirus RNA requires glycerophospholipid.
Active complete in vitro replication of nodavirus RNA requires glycerophospholipid.
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DOI：
10.1073/pnas.89.23.11136
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发表时间：
1992-12
影响因子：
11.1
通讯作者：
Shi-Xuan Wu;P. Ahlquist;Paul Kaesberg
中科院分区：
文献类型：
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作者：
Shi-Xuan Wu;P. Ahlquist;Paul Kaesberg
鸡窝病毒（FHV）是正链RNA病毒诺达病毒组的一员。在缺乏其他化合物的情况下，从感染FHV的细胞中提取的模板依赖性RNA依赖性RNA聚合酶合成添加的FHV RNA的互补（-）链拷贝，产生双链RNA产物。在加入甘油磷脂（GPL）后，该系统可重复地对添加的FHV RNA进行完整的高活性复制，产生以单链RNA形式为主的新合成（+）链RNA。这解释了先前观察到的脂效素（GPL和阳离子脂质的混合物）在系统中的作用。除了磷脂酸外，所有测试的中性和带负电荷的gpl都支持体外系统中FHV RNA的完整复制，从未感染和FHV感染细胞中提取的磷脂提取物也是如此。鞘磷脂（一种不是来源于甘油的膜磷脂）和胆固醇都不支持FHV RNA复制。从GPL衍生的化合物测试表明，活性GPL支持FHV（+）链RNA合成的能力取决于头基和酰基链的结构。磷酸化的头基团和二酰基甘油脂段都不能单独支持RNA复制。酰基链的长度和饱和度强烈影响GPL支持RNA复制的能力。本文还讨论了这种体外RNA复制系统的其他特性以及膜及其组分在FHV RNA复制中可能发挥的作用。
Flockhouse virus (FHV) is a member of the nodavirus group of positive-strand RNA viruses. In the absence of additional compounds, a template-dependent RNA-dependent RNA polymerase extracted from FHV-infected cells synthesizes complementary (-)-strand copies of added FHV RNA to yield a double-stranded RNA product. Upon addition of glycerophospholipid (GPL), this system reproducibly carries out complete highly active replication of added FHV RNA, producing newly synthesized (+)-strand RNA in predominantly single-stranded RNA form. This accounts for previously observed effects of Lipofectin (a mixture of GPL and cationic lipid) in the system. All tested neutral and negatively charged GPLs except phosphatidic acid support complete FHV RNA replication in this in vitro system, as do phospholipid extracts from uninfected and FHV-infected cells. Neither sphingomyelin, a membrane phospholipid that is not derived from glycerol, nor cholesterol supported FHV RNA replication. Testing of compounds derived from GPL shows that the ability of active GPL to support FHV (+)-strand RNA synthesis is dependent on the structures of both the head group and the acyl chains. Neither the phosphorylated head group nor the diacylglycerol lipid moiety alone supports RNA replication. The length and saturation of acyl chains strongly influence the ability of GPL to support RNA replication. Other characteristics of this in vitro RNA replication system and the possible role played by membranes and their components in FHV RNA replication are discussed.