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

CsA concentrations, to induce resistance to the compound. Subsequently, adaptive mutations that 116 markedly decreased the sensitivity of EAV replication to CsA treatment were found to map to nsp5, 117 one of the transmembrane subunits of the arterivirus replicase. We could show that CsA treatment 118 interferes with EAV RNA synthesis, and that the CsA-resistance mutations in nsp5 render replication, 119 and in particular RNA synthesis, less sensitive to CsA treatment. Furthermore, we show that resistance 120 to the closely related Cyp inhibitor ALV requires an additional adaptive mutation in nsp2, a second 121 transmembrane subunit of the arterivirus replicase that is essential for both polyprotein processing and 122 RO formation. Our studies implicate both nsp2 and nsp5 in the mechanism underlying the sensitivity 123 of arterivirus replication to cyclophilin inhibitor treatment. Moreover, they suggest that the role of 124 cyclophilins as arterivirus host factors is specifically linked to viral RNA synthesis, possibly through 125 interactions with key players in RO formation. Membrane-associated EAV RTCs were isolated from BHK-21 cells and in vitro RNA synthesis assays 200 (IVRAs) were performed essentially as described previously (van Hemert et al., 2008) . In short, 201 approximately 5 × 10 7 EAV-infected BHK-21 cells (MOI 5) were harvested at 7.5 h p.i. and lysed 202 using a ball-bearing homogenizer (Isobiotek) with 16 µm clearance. Lysates were centrifuged for 10 203 min at 1000 x g to obtain a post nuclear supernatant (PNS). A standard IVRA contained PNS (the 204 equivalent of 6 x 10 4 infected cells) and a 5x concentrated CsA stock (final concentration 4 to 32 µM) 205 or RTC dilution buffer (control) and was performed in the presence of [α-32 P]CTP that was 206 incorporated in the newly synthesized EAV RNA. Assays were incubated at 30°C for 100 min and 207 terminated by the addition of 60 µl of 5% LiDS-LET-ProtK. After a 15-min incubation at 42°C, the 208 All rights reserved. No reuse allowed without permission.