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 the potential to facilitate the creation of versatile middle-molecule antivirals featuring lipid nanoparticle-free delivery.

Methods
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 4000 to exclude autofluorescence (lower panel). The DAPI stained area of each image was quantified and divided by the number of cells to calculate the area per cell. The brightness value per cell was divided by this value to correct for the cell size between the images.