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A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms
Frontiers in Cellular and Infection Microbiology ( IF 5.7 ) Pub Date : 2020-11-25 , DOI: 10.3389/fcimb.2020.536150
Ulfert Rand 1 , Tobias Kubsch 1 , Bahram Kasmapour 1, 2 , Luka Cicin-Sain 1, 2, 3, 4
Affiliation  

Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To date, no vaccines are available and there are only few drugs for anti-HCMV therapy. Adverse effects and the continuous emergence of drug-resistance strains require the identification of new drug candidates in the near future. Identification and characterization of such compounds and biological factors requires sensitive and reliable detection techniques of HCMV infection, gene expression and spread. In this work, we present and validate a novel concept for multi-reporter herpesviruses, identified through iterative testing of minimally invasive mutations. We integrated up to three fluorescence reporter genes into replication-competent HCMV strains, generating reporter HCMVs that allow the visualization of replication cycle stages of HCMV, namely the immediate early (IE), early (E), and late (L) phase. Fluorescent proteins with clearly distinguishable emission spectra were linked by 2A peptides to essential viral genes, allowing bicistronic expression of the viral and the fluorescent protein without major effects on viral fitness. By using this triple color reporter HCMV, we monitored gene expression dynamics of the IE, E, and L genes by measuring the fluorescent signal of the viral gene-associated fluorophores within infected cell populations and at high temporal resolution. We demonstrate distinct inhibitory profiles of foscarnet, fomivirsen, phosphonoacetic acid, ganciclovir, and letermovir reflecting their mode-of-action. In conclusion, our data argues that this experimental approach allows the identification and characterization of new drug candidates in a single step.



中文翻译:

新型三荧光HCMV菌株揭示基因表达动力学和抗疱疹病毒药物机制。

人巨细胞病毒(HCMV)感染可能会在免疫功能低下的患者(如AIDS患者,移植受者和新生儿)中导致严重后果。迄今为止,尚无疫苗可用,抗HCMV治疗药物也很少。不良反应和耐药菌株的不断出现要求在不久的将来确定新的候选药物。此类化合物和生物学因素的鉴定和表征需要HCMV感染,基因表达和传播的灵敏可靠的检测技术。在这项工作中,我们提出并验证了一种针对多报告者疱疹病毒的新颖概念,该概念是通过对微创突变的迭代测试来确定的。我们将多达三个荧光报告基因整合到具有复制能力的HCMV菌株中,生成报告者HCMV,使HCMV复制循环阶段可视化,即早期(IE),早期(E)和晚期(L)。具有清晰可辨的发射光谱的荧光蛋白通过2A肽与必需的病毒基因相连,从而使病毒和荧光蛋白双顺反子表达而对病毒适应性没有重大影响。通过使用此三色报告器HCMV,我们通过测量受感染细胞群体中病毒基因相关的荧光团的荧光信号并以高时间分辨率监视了IE,E和L基因的基因表达动态。我们展示了膦甲酸,氟米韦森,膦乙酸,更昔洛韦和莱特莫韦的不同抑制特征,反映了它们的作用方式。结论,

更新日期:2021-01-08
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