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首页> 美国卫生研究院文献>Journal of Virology >Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.
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Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.

机译：蚜虫对牛细小病毒感染过程中复制型DNA产生的抑制作用。

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Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.
机译：由于细小病毒显然不具有DNA聚合酶活性，因此一种或多种宿主细胞DNA聚合酶必须负责复制单链DNA基因组。我们专注于确定哪种聚合酶，α，β或γ（分别为polα，polβ或polγ）负责牛细小病毒DNA复制的第一步：将单链DNA基因组转化为a父母复制形式（RF）。在这项研究中，我们使用了aphidicolin（一种特定的DNA pol alpha抑制剂）来测定体内亲本RF形成对pol alpha活性的需求。用牛细小病毒在有或没有蚜虫的情况下感染同步细胞培养物，并在琼脂糖凝胶上分离病毒复制的产物，并通过Southern印迹分析进行鉴定。我们发现，当整个感染过程中都存在20 microM蚜虫时，会完全抑制病毒DNA的合成。此外，病毒DNA的合成被少至1 microM的蚜虫抑制，而较低的浓度（0.1和0.01 microM）导致复制过程的部分抑制。使用32P标记的牛细小病毒作为输入病毒，我们将亲本RF与子RF和子代DNA合成区分开来。我们得出结论，牛细小病毒在体内复制过程中产生RF需要DNA pol alpha，而这一要求最有可能将牛细小病毒输入单链DNA转化为亲代RF。这些结果不排除在第一步中DNA pol gamma的可能作用，也不排除在复制周期的后期对pol alpha或pol gamma的作用。

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