Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

• Published in: Scientific reports Vol. 9; no. 1; p. 5397
• Main Authors: Sáez-Álvarez, Yanira, Arias, Armando, del Águila, Carmen, Agudo, Rubén
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2019; Nature Publishing Group
• Subjects: 38/44; 38/70; 38/71; 42/34; 45/77; 631/154/1435/2163; 631/326/596/1296; 82/29; 82/47; 82/80; 82/83; Animals; Antiviral agents; Antiviral Agents - isolation & purification; Antiviral Agents - pharmacology; DNA-directed RNA polymerase; Drug Discovery - methods; Drug screening; Fluorescence; Fluorescent indicators; Foot & mouth disease; Guillain-Barre syndrome; Guillain-Barre Syndrome - chemically induced; Guillain-Barre Syndrome - prevention & control; Hepatitis C; High-throughput screening; High-Throughput Screening Assays - methods; Humanities and Social Sciences; Humans; Microcephaly - prevention & control; Microcephaly - virology; Microencephaly; Monitoring methods; multidisciplinary; Neurological diseases; Nucleotides; Polymerization; Public health; RNA polymerase; RNA viruses; RNA-Dependent RNA Polymerase - genetics; RNA-Dependent RNA Polymerase - metabolism; RNA-directed RNA polymerase; Science; Science (multidisciplinary); Transcription; Vector-borne diseases; Virus Replication - drug effects; Virus Replication - genetics; Zika virus; Zika Virus - enzymology; Zika Virus - genetics; Zika Virus - physiology; Zika Virus Infection - prevention & control; Zika Virus Infection - virology
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-41998-1

Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fluorescent dye, which permits the specific quantification and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.