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

This study demonstrates that adaptive mutations in the transmembrane replicase subunit nsp5 can 425 reduce the sensitivity of EAV replication to CsA treatment. Independently, experiments using the CsA 426 analog ALV pointed in the same direction, in addition to implicating nsp2 in ALV sensitivity and 427 resistance. Substitutions in nsp5 and nsp2 enable EAV to tolerate CsA or ALV concentrations that 428 would normally block virus replication (Figs. 2, 9 and 10 ). The largest EC50 increase, about 5-fold, 429 was achieved when multiple substitutions in nsp5 (and nsp2, in the case of ALV) were combined 430 (Table 1 and Four substitutions in nsp5 were implicated in CsA resistance (L8S, Q21R, Y113H, and 465 A134V), of which only the L8 residue is conserved among arteriviruses (Fig. 1C) . Interestingly, this 466 residue is also part of a potential CypA binding motif (GPxL, as identified by (Piotukh et al., 2005) ), 467 constituting a potential interaction site between the two proteins. However, during infection, this L8S 468 mutation quickly reverted, and therefore the contribution of the L8S substitution to CsA resistance 469 remains unclear. All other three nsp5 mutations (Q21R, Y113H, and A134V) are present in predicted 470 transmembrane domains (Fig. 1B) , and therefore may not be directly involved in interactions of nsp5 471 with Cyps or other proteins. There is no experimental data available on the membrane topology of 472 nsp5, but upon in silico modeling these nsp5 mutations did not appreciably alter the predicted 473 conformation of the various transmembrane domains (data not shown). In addition, our EM analysis of 474 DMV formation upon rEAV nsp5 QYA infection did not reveal any ultrastructural differences, either in 475 the absence or presence of CsA (Fig 5) . Clearly, more detailed information on the 3D-structure of nsp5 476 All rights reserved. No reuse allowed without permission.