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

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

Large genome segments, normalizing to cellular β-actin. Paralleling our titer data, valinomycin 154 treatment reduced viral genome content in a dose-dependent manner ( Figure 2D ). Viral genome 155 content was reduced upwards of 100-fold with 10 µM valinomycin treatment. To extend these 156 results to other cell types, we treated and infected Vero-E6 cells as above and determined viral 157 titers by plaque assay at 48 hpi. Again, we observed a significant reduction in viral titer with an 158 in multiple cell types, reducing both viral titers and cell-associated viral genomes in a dose-160 dependent manner. 161 162 Valinomycin is antiviral over multiple rounds of infection. Our initial assays were performed 163 at low MOI and viral titer measured at 48 hpi. To determine if valinomycin was antiviral over 164 several rounds of replication, we treated Huh7 cells with 2 µM valinomycin two hours prior to 165 infection at MOI 0.1 and subsequently collected samples to titer every 8h for 56h total. We found 166 that LACV titers were significantly reduced at all times after 8 hpi ( Figure 3A ); in fact, virus failed 167 to replicate above input virus titers (0h). To confirm that valinomycin was reducing virus 168 replication, we measured viral RNA genomes. To this end, we treated cells with increasing doses 169 of valinomycin, infected with LACV and collected cell-associated RNA in Trizol at 24 hpi. After 170 purifying and reverse-transcribing, we performed qPCR using primers specific to the small, 171 medium, and large genome segments. We observed that treatment with valinomycin significantly 172 reduced the number of viral genomes by >90% with treatment and that no individual genome 173 segment was affected more than another ( Figure 3B ). Together, these data suggest that 174 valinomycin blocks virus replication and reduces viral RNA accumulation. 175 176 Valinomycin does not reduce viral particle infectivity. Because we observe significant 177 reductions in LACV titers with valinomycin treatment, we hypothesized that valinomycin might be 178 directly affecting cellular processes to reduce virus infection. Nonetheless, valinomycin is a cyclic 179 peptide and could potentially directly inactivate viral particles, as seen previously 20 . To test 180 whether valinomycin directly reduced virus infectivity, we directly incubated LACV with 2 µM 181 valinomycin for 24h and directly titered the surviving virus at regular intervals. We found that 182 valinomycin did not significantly alter viral titer over the time examined ( Figure 4A ), suggesting 183 that valinomycin is not directly inactivating viral particles. We further examined the capacity of 184 valinomycin to inactivate particles by incubating with increasing doses, up to 10 µM valinomycin, 185 for 24h prior to directly titering. As in our timecourse, we observed no significant change in viral 186 titers at any dose ( Figure 4B ), again suggesting that valinomycin does not directly inactivate LACV 187 particles. As final confirmation of this phenotype, we measured viral RNA in viruses exposed to 188 increasing doses of valinomycin. We then compared the relative number of genomes to the titer 189 to calculate the genome-to-PFU ratio. We observed that this number did not change with 190 valinomycin treatment, suggesting no change in specific infectivity ( Figure 4C ). In sum, these data 191 suggest that valinomycin does not affect virus infectivity by directly acting on the virion. 192 193 . CC