Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus

• Type of Material: Article
• Authors: Dandan Liu, University of Missouri; Philip Tedbury, Emory University; Shuiyun Lan, Emory University; Andrew D. Huber, University of Missouri; Maritza N. Puray-Chavez, University of Missouri; Juan Ji, University of Missouri; Eleftherios Michailidis, Rockefeller University; Mohsan Saeed, Rockefeller University; Tanyaradzwa P. Ndongwe, University of Missouri; Leda Bassit, Emory University; Raymond Schinazi, Emory University; Robert Ralston, University of Missouri; Charles M. Rice, Rockefeller University; Stefan Sarafianos, Emory University
• Language: English
• Date: 2019-11-01
• Publisher: MDPI
• Publication Version: Final Published Version
• Copyright Statement: © 2019 by the authors. Licensee MDPI, Basel, Switzerland. CC BY 4.0
• Final Published Version (URL): http://dx.doi.org/10.3390/v11111039
• Title of Journal or Parent Work: Viruses
• ISSN: 1999-4915
• Volume: 11
• Issue: 11
• Grant/Funding Information: This work was supported by the NIH (AI121315, AI120860, and GM103368 to S.G.S., AI099284 to C.M.R. and by the Emory University Center for AIDS Research 2P30-AI-050409 to R.F.S.).
• Keywords: IN-SITU HYBRIDIZATION; NS5A INHIBITOR; INFECTION; DACLATASVIR; hepatitis C virus; REPLICATION; RNA FISH; INTERFERON; PERIPHERAL-BLOOD; DENGUE VIRUS; Science & Technology; Virology; Life Sciences & Biomedicine; positive-sense; CELLS; negative-sense; POTENT; Zika virus; direct acting antivirals
• Research Categories: Chemistry, Biochemistry; Health Sciences, Pharmacology; Biology, Virology

Abstract

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)-and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.