Lighting up the Native Viral RNA Genome with a Fluorogenic Probe for the Live-Cell Visualization of Virus Infection

Abstract

RNA viruses represent a major global health threat, and the visualization of their RNA genome in infected cells is essential for virological research and clinical diagnosis. Due to the lack of chemical toolkits for the live-cell imaging of viral RNA genomes, especially native viral genomes without labeling and genetic modification, studies on native virus infection at the single-live-cell level are challenging. Herein, taking hepatitis C virus (HCV) as a representative RNA virus, we propose that the innate noncanonical G-quadruplex (G4) structure of viral RNA can serve as a specific imaging target and report a new benzothiazole-based G4-targeted fluorescence light-up probe, ThT-NE, for the direct visualization of the native RNA genome of HCV in living host cells. We demonstrate the use of the ThT-NE probe for several previously intractable applications, including the sensitive detection of individual virus-infected cells by small-molecule staining, real-time monitoring of the subcellular distribution of the viral RNA genome in live cells, and continuous live-cell tracking of the infection and propagation of clinically isolated native HCV. The fluorogenic-probe-based viral RNA light-up system opens up a promising chemical strategy for cutting-edge live-cell viral analysis, providing a potentially powerful tool for viral biology, medical diagnosis, and drug development.

G-Quadruplex-Based Fluorescent Turn-On Ligands and Aptamers: From Development to Applications

“…Similarly, ThT derivatives have been applied in viral RNA genome detection and monitoring. This was demonstrated very recently by Luo et al [107], who developed the ligand ThT–NE, with the excitation and emission wavelengths shown in Table 1 (ThT derivative). The ligand was a cell permeable and highly specific G4-based fluorescence turn-on probe for real-time imaging of native viral RNA in the hepatitis C virus (HCV).…”

“…The ligand was a cell permeable and highly specific G4-based fluorescence turn-on probe for real-time imaging of native viral RNA in the hepatitis C virus (HCV). This method was shown to allow subcellular monitoring and continuous live-cell monitoring of infected cells [107]. However, the limitation of this ligand class include the fact that only few were shown to penetrate the cells [107] and reach their desired target.…”

G-Quadruplexes Are Present in Human Coronaviruses Including SARS-CoV-2

“…SARS-CoV-2 G4 could be used to develop tools for SARS-CoV-2 studies. G4 has been applied to detect HCV (Luo et al, 2019) and HIV (Sontakke and Srivatsan, 2020). Likewise, G4 could be developed as a potential biosensor for SARS-CoV-2 detection.…”

Viral G-quadruplexes: New frontiers in virus pathogenesis and antiviral therapy

“…Moreover, the HCV replication machinery promotes formation of vesicle-like viral replication complexes that contain most of the newly synthesized viral RNA, generating a localized G4 enrichment that improves ThT-NE recognition. 52 If this strategy were applicable also to other pathogens, it would represent a striking turning point in pathological research and clinical diagnosis.…”