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

both variants were engineered and tested. Compared to the viruses carrying singular mutations, mutant 287 rEAV nsp5 Y113H+A134V displayed a decreased sensitivity to CsA, which now approached that of the 288 EAV CsA-1 lineage ( Fig. 3A and Table 1 ). When the Q21R mutation was added, the CsA resistance 289 diminished slightly compared to the Y113H+A134V double mutant ( Fig. 3B and Table 1 ; EC50 of 9.1 290 vs. 7.2 µM). However, upon passaging and resequencing (data not shown), the double mutant 291 appeared to be genetically instable, which may also explain the mixed sequence found at nsp5 codon 292 21 in the lineage CsA-1 consensus sequence. For lineage CsA-2, combining the three mutations (in 5'-293 UTR, nsp5, and nsp10) into the same genome did not further improve CsA resistance compared to 294 rEAV nsp5 Y113H (Fig. 3C) . This lack of a synergistic effect indicates that the mutations in 5'-UTR and 295 nsp10 do not contribute substantially to the level of CsA resistance of CsA-2. In the case of EAV 296