Selection and Characterization of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Mutations
Selection and Characterization of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Mutations
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DOI：
10.1128/jvi.01507-12
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发表时间：
2012-12-01
影响因子：
5.4
通讯作者：
Krell, Peter J.
中科院分区：
文献类型：
--
作者：
Feng, Guozhong;Thumbi, David K.;Krell, Peter J.
加州签名多核多角体病毒（AcMNPV） DNA聚合酶（DNApol）是病毒DNA复制所必需的。AcMNPV突变体对阿菲迪克林（一种选择性的病毒DNA复制抑制剂）和阿巴卡韦（一种有效的核苷类似物，具有抑制逆转录酶的活性）具有抗性，通过在阿菲迪克林或阿巴卡韦浓度增加的情况下对亲本AcMNPV进行连续传代，选择了AcMNPV突变体。这些耐药突变体在保守区II和III内具有单点（C543R）（阿菲迪克林）或双点（C543R和S611T）（阿巴卡韦）突变。为了证实这些点突变在AcMNPV DNA聚合酶中的作用，首先产生了一个dnapol敲除病毒，并通过将dnapol野生型基因或含有单点或双点突变的基因转置到dnapol敲除bacmid的多面蛋白位点构建了几个修复病毒。单个C543R或双C543R/S611T突变显示出对阿菲迪克林和阿巴卡韦的抗性增加，即使在没有药物的情况下，与野生型修复病毒相比，病毒和病毒DNA复制水平降低。令人惊讶的是，dnapol突变修复病毒导致了闭塞衍生病毒的产生，这些病毒在环区和多面体内大多数是单个核衣壳，只有少数是多个核衣壳。因此，AcMNPV DNA聚合酶的这些点突变增加了耐药性，轻微损害了病毒和病毒DNA复制，并影响了闭塞源病毒的病毒形态发生。
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus.