FIG 1.

Positions of CsA resistance-associated mutations in the EAV 5′ UTR and ORF1ab. (A) Map of CsA resistance-associated mutations identified in the consensus sequence of replicates CsA-1, -2, and -3 after seven passages. Depicted are the 5′ UTR and ORF1ab, with replicase polyprotein 1ab amino acid numbers, cleavage sites, and nsp cleavage products indicated. The seven mutations identified in the sequences of the CsA-resistant viruses are indicated with black arrows, and the amino acid position/change in the respective nsp is indicated, as well as the replicate in which the mutation was identified (see also Table 2). nsp5 is highlighted in green. P1 and P2, papain-like proteinases in nsp1 and nsp2, respectively; TM, transmembrane domains; Mpro, main proteinase; NT, nidovirus RdRp-associated nucleotidyltransferase (NiRAN); RdRp, RNA-dependent RNA polymerase; Z, zinc-binding domain; Hel, helicase; U, endoribonuclease; RFS, ribosomal frameshift site. (B) Membrane topology of EAV nsp5 as predicted using the TMHMM topology prediction method within the Geneious software package. The position/change of the four nsp5 mutations identified in replicates CsA-1, -2, and -3 is indicated with a black arrow. (C) Multiple-sequence alignment of nsp5 from selected arteriviruses, performed by Clustal Omega (51). Fully conserved residues are indicated in gray, predicted transmembrane helices (TMHMM topology prediction from Geneious software package) are boxed, and adaptive mutations are marked with an asterisk. EAV, equine arteritis virus (GenBank accession number DQ846750); SHFV, simian hemorrhagic fever virus (AF180391); PRRSV-1, porcine reproductive and respiratory syndrome virus, European genotype (GU737264.2); PRRSV-2, porcine reproductive and respiratory syndrome virus, North American genotype (JX138233); LDV, lactate dehydrogenase-elevating virus (U15146).