Previously, the
cyclophilin inhibitors
cyclosporine (CsA) and
alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales In this study, we aimed to identify arterivirus
proteins involved in the mode of action of
cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus
RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced
drug sensitivity is associated with the emergence of adaptive mutations in nonstructural
protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase
polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in arterivirus
RNA synthesis. Particularly, in an in vitro assay, EAV
RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related
drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by
cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat
infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the
cyclophilin inhibitors
cyclosporine (CsA) and
alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural
proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by
cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future
drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.