Tillmanns Julia, Kicuntod Jintawee, Ehring Antonia, Elbasani Endrit, Borst Eva Maria, Obergfäll Debora, Müller Regina, Hahn Friedrich, Marschall Manfred
Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany.
Institute of Virology, Hannover Medical School (MHH), 30625 Hannover, Germany.
Pathogens. 2024 Jul 31;13(8):645. doi: 10.3390/pathogens13080645.
Human cytomegalovirus (HCMV) represents a highly medically important pathogen which has constantly been the subject of both molecular and clinical investigations. HCMV infections, especially those in high-risk patients, still raise many unanswered questions, so current investigations are focused on viral pathogenesis, vaccine development, and options for antiviral drug targeting. To this end, the use of suitable viral strains as well as recombinant reporter constructs in cultured cells and model systems has specific significance. We previously reported on the application of various herpesviruses that express green, red, or related fluorescent proteins, especially in the fields of virus-host interaction and antiviral research. Here, we characterized a recombinant version of the clinically relevant and cell type-adaptable HCMV strain TB40, which expresses firefly luciferase as a quantitative reporter of viral replication (TB40-FLuc). The data provide evidence for five main conclusions. First, HCMV TB40-FLuc is employable in multiple settings in primary human cells. Second, viral reporter signals are easily quantifiable, even at early time points within viral replication. Third, the FLuc reporter reflects the kinetics of viral intracellular replication, cascade-like viral IE-E-L protein production, and progeny release. Fourth, as relates to specific applications of the TB40-FLuc system, we demonstrated the reliability of quantitative antiviral compound determination in multi-well formats and its independence from fluorescence-based measurements in the case of autofluorescent inhibitors. Finally, we illustrated increased reporter sensitivity in comparison to other recombinant HCMVs. In essence, recombinant HCMV TB40-FLuc combines several molecular properties that are considered beneficial in studies on viral host tropism, replication efficiency, and antiviral drug assessment.
人巨细胞病毒（HCMV）是一种具有高度医学重要性的病原体，一直是分子研究和临床研究的对象。HCMV感染，尤其是高危患者的感染，仍然存在许多未解决的问题，因此目前的研究集中在病毒发病机制、疫苗开发以及抗病毒药物靶点选择方面。为此，在培养细胞和模型系统中使用合适的病毒株以及重组报告构建体具有特殊意义。我们之前报道了各种表达绿色、红色或相关荧光蛋白的疱疹病毒的应用，特别是在病毒-宿主相互作用和抗病毒研究领域。在这里，我们对临床相关且细胞类型适应性强的HCMV毒株TB40的重组版本进行了表征，该毒株表达萤火虫荧光素酶作为病毒复制的定量报告基因（TB40-FLuc）。这些数据为五个主要结论提供了证据。第一，HCMV TB40-FLuc可用于原代人细胞的多种环境。第二，即使在病毒复制的早期时间点，病毒报告信号也易于定量。第三，FLuc报告基因反映了病毒细胞内复制的动力学、级联式病毒IE-E-L蛋白的产生以及子代释放。第四，关于TB40-FLuc系统的具体应用，我们证明了在多孔板形式中定量抗病毒化合物测定的可靠性，以及在自发荧光抑制剂情况下其独立于基于荧光的测量。最后，我们说明了与其他重组HCMV相比，报告基因的敏感性有所提高。实质上，重组HCMV TB40-FLuc结合了几种在病毒宿主嗜性、复制效率和抗病毒药物评估研究中被认为有益的分子特性。
