In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication
In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication
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DOI：
10.1016/j.antiviral.2016.08.021
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发表时间：
2016-10-01
影响因子：
7.6
通讯作者：
Ferreira, Fernando
中科院分区：
文献类型：
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作者：
Freitas, Ferdinando B.;Frouco, Goncalo;Ferreira, Fernando
非洲猪瘟病毒(ASFV)是一种高度传染性和致死性的家猪疾病的病原体，在受影响的国家造成严重的社会经济影响。到目前为止，还没有疫苗或选择性抗病毒药物可用于预防或治疗非洲猪瘟(ASF)，强调需要对参与病毒DNA复制和转录的ASFV蛋白的作用进行更详细的研究。值得注意的是，ASFV编码一种功能性的II型拓扑异构酶(ASFV-TOPO II)，我们最近发现几种氟喹诺酮类药物(细菌DNA拓扑异构酶抑制剂)在体外完全抑制ASFV的复制。在此，我们报道了ASFV-Topo II基因在整个感染过程中都被活跃地转录，转录本最早在2HPI被检测到，并在16HPI左右达到最大浓度，此时病毒DNA合成、转录和翻译更活跃。SiRNA敲除实验表明，ASFV-Topo II在病毒DNA复制和基因表达中起关键作用，与对照组相比，转基因细胞的病毒转录本减少(最多减少89%)，细胞病变效应减少(-66%)。此外，只有在针对ASFV-Topo II的siRNA转染的细胞中，感染细胞的数量(75.5%)和病毒工厂数以及病毒产量(高达99.7%，2.5log)都显著减少。我们还证明，在感染后期短期暴露于恩诺沙星(从15 HPI到1HPI)会导致病毒基因组的片段化，而从感染早期(从2 HPI到16HPI)加入恩诺沙星则没有检测到病毒基因组，表明氟喹诺酮类药物是ASFV-TOPO II毒物。综上所述，我们的结果表明ASFV-Topo II酶在病毒基因组复制和转录过程中起着至关重要的作用，强调了这种酶可以成为治疗ASF的药物和疫苗开发的潜在靶点。(C)2016爱思唯尔B.V.保留所有权利。
African swine fever virus (ASFV) is the etiological agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic impact in affected countries. To date, neither a vaccine nor a selective anti-viral drug are available for prevention or treatment of African swine fever (ASF), emphasizing the need for more detailed studies at the role of ASFV proteins involved in viral DNA replication and transcription. Notably, ASFV encodes for a functional type II topoisomerase (ASFV-Topo II) and we recently showed that several fluoroquinolones (bacterial DNA topoisomerase inhibitors) fully abrogate ASFV replication in vitro. Here, we report that ASFV-Topo II gene is actively transcribed throughout infection, with transcripts being detected as early as 2 hpi and reaching a maximum peak concentration around 16 hpi, when viral DNA synthesis, transcription and translation are more active. siRNA knockdown experiments showed that ASFV-Topo II plays a critical role in viral DNA replication and gene expression, with transfected cells presenting lower viral transcripts (up to 89% decrease) and reduced cytopathic effect (-66%) when compared to the control group. Further, a significant decrease in the number of both infected cells (75.5%) and viral factories per cell and in virus yields (up to 99.7%, 2.5 log) was found only in cells transfected with siRNA targeting ASFV-Topo II. We also demonstrate that a short exposure to enrofloxacin during the late phase of infection (from 15 to 1 hpi) induces fragmentation of viral genomes, whereas no viral genomes were detected when enrofloxacin was added from the early phase of infection (from 2 to 16 hpi), suggesting that fluoroquinolones are ASFV-Topo II poisons. Altogether, our results demonstrate that ASFV-Topo II enzyme has an essential role during viral genome replication and transcription, emphasizing the idea that this enzyme can be a potential target for drug and vaccine development against ASF. (C) 2016 Elsevier B.V. All rights reserved.