The combined use of alphavirus replicons and pseudoinfectious particles for the discovery of antivirals derived from natural products.
The combined use of alphavirus replicons and pseudoinfectious particles for the discovery of antivirals derived from natural products.
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DOI：
10.1177/1087057114564868
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发表时间：
2015-06
影响因子：
--
通讯作者：
Miller DJ
中科院分区：
文献类型：
--
作者：
Delekta PC;Raveh A;Larsen MJ;Schultz PJ;Tamayo-Castillo G;Sherman DH;Miller DJ
甲型病毒是一类突出的再发病原体，因为它们的全球范围扩大，潜在的致命性，并可能用作生物武器。甲型病毒缺乏有效的治疗方法，突显了确定抗病毒药物的创新战略的必要性。使用非传染性自我复制RNA(称为复制子)的初级筛选已被用于识别潜在的甲型病毒抗病毒化合物。然而，只有病毒基因组复制的抑制剂将使用复制子来识别，这排除了病毒生命周期中的许多其他可用药步骤。为了解决这一局限性，我们开发了一种西部马脑炎病毒假感染颗粒系统，除了基因组复制外，它还复制了几个关键的病毒生命周期步骤。我们使用这个系统筛选了一个包含来自海洋微生物的∼26,000提取物的文库，并鉴定了多个产生潜在抗病毒活性化合物的细菌菌株。随后，我们并行使用假感染颗粒和复制子分析来反筛选候选提取物，并在生化分离和纯化过程中跟踪抗病毒活性，以区分病毒进入的抑制剂和基因组复制的抑制剂。这一新的方法导致了已知的甲型病毒进入抑制剂巴菲罗霉素的分离，从而验证了用于筛选和鉴定潜在抗病毒化合物的方法。
Alphaviruses are a prominent class of reemergent pathogens due to their globally expanding ranges, potential for lethality, and possible use as bioweapons. The absence of effective treatments for alphaviruses highlights the need for innovative strategies to identify antiviral agents. Primary screens that use noninfectious self-replicating RNAs, termed replicons, have been used to identify potential antiviral compounds for alphaviruses. Only inhibitors of viral genome replication, however, will be identified using replicons, which excludes many other druggable steps in the viral life cycle. To address this limitation, we developed a western equine encephalitis virus pseudoinfectious particle system that reproduces several crucial viral life cycle steps in addition to genome replication. We used this system to screen a library containing ∼26,000 extracts derived from marine microbes, and we identified multiple bacterial strains that produce compounds with potential antiviral activity. We subsequently used pseudoinfectious particle and replicon assays in parallel to counterscreen candidate extracts, and followed antiviral activity during biochemical fractionation and purification to differentiate between inhibitors of viral entry and genome replication. This novel process led to the isolation of a known alphavirus entry inhibitor, bafilomycin, thereby validating the approach for the screening and identification of potential antiviral compounds.
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