Novel ionophores active against La Crosse virus identified through rapid antiviral screening

Zachary J. Sandler; Michelle N. Vu; Vineet D. Menachery; Bryan C. Mounce

Huh7 cells to confluency in 96-well plates, to which we added 2 µM drug from the NIH NCI Importantly, any cytotoxic molecule would not stain with crystal violet; thus, stained cells indicate 120 antiviral molecules that are not cytotoxic at 2 µM. Crystal violet stain was subsequently 121 resuspended in 10% acetic acid and absorbance read at 595 nm. To control for inter-plate 122 variability, each plate contained an untreated and infected (low survival), untreated and uninfected 123 (high survival), and ribavirin-treated (400 µM) control (high survival). Absorbances of drug-treated 124 and infected wells ( Figure 1B ) were compared to untreated and uninfected controls by dividing 125 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.21.914929 doi: bioRxiv preprint their absorbance values ( Figure 1C ). This ratio highlighted several candidate antivirals, including 126 lagistase, lapachol, superacyl, and valinomycin. Interestingly, we identified several known 127 antivirals in our screen, including deoxyuridine and nelarabine (summarized in Table 1 ). Thus, 128 our assay identified molecules with novel activity against LACV, including some recognized 129 antivirals. 130 131 Valinomycin restricts LACV replication. We focused on valinomycin, as the molecule was a 132 prominent hit in our screen, had a known mechanism relevant to LACV infection, and was not 133 previously described to have antiviral activity against LACV. As an initial consideration of its 134 antiviral activity, we performed secondary screening on Huh7 cells. Cells were seeded to 135 confluency, treated with increasing doses of valinomycin, from 1 to 64 µM, and infected at MOI 136 0.1. At 48h, cells were fixed and stained with crystal violet, and stain was quantified by absorbance 137 reading. We observed that valinomycin exhibited antiviral activity at doses above 10 µM, as crystal 138 violet staining was stronger, suggesting more surviving cells (Figure 2A ). Doses as high as 64 µM 139 did not affect crystal violet stain, suggesting that cellular viability was not compromised. To 140 confirm this phenotype with titers, we treated cells with increasing doses of valinomycin 2h prior 141 to infection at MOI 0.1 and measured titers by plaque assay at 48 hpi. We observed that viral 142 titers were significantly decreased compared to untreated controls ( Figure 2B , dotted line) at 143 concentrations above 1 µM. In fact, viral titers were reduced over 100-fold at 10 µM. We calculated 144 an IC50 value of 1.4 µM. To confirm that cellular viability was not compromised, we used a 145 fluorescent assay to measure cellular ATP content after treatment with increasing doses of 146 valinomycin. We observed cellular toxicity at doses at and above 16 µM ( Figure 2C , CC50 value 147 of 14 µM), though no toxicity was observed either by cellular morphology or fluorescent ATP assay 148 below 10 µM. 149 150 As further confirmation of valinomycin's antiviral activity, we measured cell-associated viral 151 genomes. Huh7 cells were treated and infected as above and cell-associated RNA was collected 152 at 48 hpi. RNA was purified, reverse transcribed, and analyzed via qPCR for Small, Medium, and 153