G-quadruplex-forming small RNA inhibits coronavirus and influenza A virus replication

Future pandemic threats may be caused by novel coronaviruses and influenza A viruses. Here we show that when directly added to a cell culture, guanosine 12mer (G12) and its phosphorothioate-linked derivatives (G12(S)), rapidly entered cytoplasm and suppressed the propagation of human coronaviruses and influenza A viruses to between 1/100 and nearly 1/1000 of normal virus infectivity without cellular toxicity and induction of innate immunity. Moreover, G12(S) alleviated the weight loss caused by coronavirus infection in mice. G12(S) might exhibit a stable G-tetrad with left-handed parallel-stranded G-quadruplex, and inhibit the replication process by impeding interaction between viral nucleoproteins and viral RNA in the cytoplasm. Unlike previous antiviral strategies that target the G-quadruplexes of the viral genome, we now show that excess exogenous G-quadruplex-forming small RNA displaces genomic RNA from ribonucleoprotein, effectively inhibiting viral replication. The approach has th..., QUMA-1 stain for the cells
A549 cells were cultured in 96-well plates (ViewPlate). After being washed with MEM, cells were cultured in the presence of 2 μM of each PS-RNA for 17 h. Cells were then washed with MEM and fixed with 4% paraformaldehyde in PBS for 15 min on ice. After washing three times with PBS, the cells were observed after 7 h or later by adding PBS containing 50 nM QUMA-1 and 1 μg/mL DAPI. Confocal imaging was performed using LSM900 (Zeiss) equipped with Airyscan 2 using Plan-Apochromat 63x/1.40 Oil DIC M27 (objective). Excitation and detection wavelengths were 353 nm laser and 400-564 nm for DAPI, 612 nm laser and 590-700 nm (AF610) for QUMA-1. Images of 8 slices (7 µm thick) were processed by Airyscan-2 were superimposed by ZEN (Zeiss). TIFF files (16 bit) were exported and intensity values of QUMA-1 fluorescence were quantitated using Fiji (ImageJ 2.14.0). To quantify PS-RNA specific QUMA-1 fluorescence, Image-J (Fiji) was used to set the threshold at approximately 40..., , # G-quadruplex-forming small RNA inhibits coronavirus and influenza A virus replication

[https://doi.org/10.5061/dryad.8931zcs1s](https://doi.org/10.5061/dryad.8931zcs1s)

## Principle Investigator Contact Information

Name: Shusuke Kuge

Institution: Division of Microbiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University.

e-mail: [[e-mail]](mailto:[e-mail])

**QUMA-1 stain for the cells**

A549 cells were cultured in 96-well plates (ViewPlate). After being washed with MEM, cells were cultured in the presence of 2 μM of each PS-RNA for 17 h. Cells were then washed with MEM and fixed with 4% paraformaldehyde in PBS for 15 min on ice. After washing three times with PBS, the cells were observed after 7 h or later by adding PBS containing 50 nM QUMA-1 and 1 μg/mL DAPI. Confocal imaging was performed using LSM900 (Zeiss) equipped with Airyscan 2 using Plan-Apochromat 63x/1.40 Oil DIC M27 (objective). Excitation and detection wa...