Inhibition of arterivirus RNA synthesis by cyclophilin inhibitors is counteracted by mutations in replicase transmembrane subunits

Adriaan H. de Wilde; A. Linda Boomaars-van der Zanden; Anja W. M. de Jong; Montserrat Barcéna; Eric J. Snijder; Clara C. Posthuma

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/587261 doi: bioRxiv preprint 32 P-labeled reaction products were isolated and separated in a 1.5% denaturing agarose gel. Agarose 209 gels were dried and reaction products were visualized by phophorimaging using a 2013a). We now aimed to investigate the mechanism-of-action by which arterivirus infection is 232 inhibited in the presence of this cyclophilin inhibitor. To this end, wt EAV (strain Bucyrus; MOI 233 0.005) was serially passaged in Huh7 cells in the presence of increasing concentrations of CsA (4 to 234 20 µM). Three independent virus lineages were generated (named EAV CsA-1 to -3), which exhibited 235 clearly decreasing sensitivity to CsA ( Fig. 2A) . By passage 7 (P7), all three lineages replicated in the 236 presence of 20 µM CsA (data not shown), a dose that is approximately 10-fold higher than the EC50 237 value observed when using wtEAV ( passages in the absence of CsA did not alter the drug sensitivity of the wtEAV control (EC50 2.1 µM), 247 P7 virus of the CsA-treated EAV lineages CsA-1 to -3 had become five-fold less sensitive to the 248 compound ( Fig. 2B and Table 1 ). Of note, additional passaging (up to passage 9) did not decrease CsA 249 sensitivity any further. 250