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

To explore the development of ALV resistance in EAV, two independent passaging 394 experiments in Huh7 cells were performed (EAV ALV-1 and ALV-2) in the presence of increasing 395 ALV concentrations (from 4 µM in passage 1 to 75 µM in passage 10), similar to the approach used to 396 generate CsA-resistant viruses (Fig. 2) . By passage 10, a CPE-based assay revealed that, compared to 397 the wtEAV-P10, the sensitivity to ALV was reduced by 7.4-and 2.8-fold for EAV lineages ALV-1 398 and ALV-2, respectively ( Fig. 8A and Table 3 ). Interestingly, the P10 samples of EAV lineages ALV-399 1 and -2 were ~4-fold less sensitive to CsA as well (Fig. 8B) . Analysis of the ALV-1 and -2 P10 400 consensus sequences revealed that each population contained multiple mutations (Table 2) . These 401 included singular mutations in the 5'-UTR, in ORF1a (nsp1 region), and in the structural protein-402 coding region. Strikingly, the P10 viruses from both lineages also carried two substitutions in nsp5. 403