Adaptive Mutations in Replicase Transmembrane Subunits Can Counteract Inhibition of Equine Arteritis Virus RNA Synthesis by Cyclophilin Inhibitors

Abstract

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.

Keywords: alisporivir; arterivirus; cyclosporine; drug resistance; host factors; nidovirus; nonstructural proteins; replication.

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).

FIG 2

Selection of CsA-resistant EAV mutants. (A) Passaging scheme for wild-type (wt) EAV (strain Bucyrus) in Huh7 cells (MOI, 0.005) to generate three independent CsA-resistant replicates (EAV CsA-1, CsA-2, and CsA-3). Passaging was performed in the presence of increasing CsA concentrations, ranging from 4 μM during passage 1 (P1) to 20 μM during P7. As a control, EAV^wt was passaged in the absence of CsA to assess the acquisition of mutations unrelated to CsA resistance. (B) After P7, EAV CsA-1 (red), CsA-2 (blue), and CsA-3 (green) were tested for CsA resistance, while including the original (P0, gray) and passaged (P7, black) EAV^wt as controls. Huh7 cells in 96-well plates were infected at an MOI of 0.05 in the presence of 0 to 50 μM CsA. Cells were incubated for 3 days and cell viability was monitored using a commercial assay. Furthermore, the cytotoxicity of CsA treatment only was monitored in parallel in mock-infected Huh7 cells (light gray). Graphs show the results (averages and SD) of a representative experiment performed in quadruplicate. All experiments were repeated at least twice. Huh7 cells (C) or BHK-21 cells (D) were infected with EAV CsA-1, -2, or -3 or with lineage EAV^wt P7 (MOI, 0.01). At 1 h p.i., the inoculum was replaced by medium containing either 0.02% DMSO (bars labeled “−”; solvent control) or 4 μM CsA (bars labeled “+”). Medium was collected at 32 h p.i., and EAV progeny titers were determined by plaque assay.

FIG 3

CsA resistance analysis of engineered rEAV mutants reflecting the consensus sequences of CsA-1, -2, and -3. Huh7 cells in 96-well plates were infected (MOI, 0.05) with rEAV^wt (control; black line) or mutants reflecting the consensus sequences of CsA-1, -2, and -3. Cells were incubated for 3 days in the presence of 0 to 50 μM CsA, and cell viability was monitored using a commercial assay. The cytotoxicity of CsA treatment only was monitored in parallel in mock-infected Huh7 cells (light gray line). Above each panel, the EC₅₀ value of CsA inhibition is indicated (see also Table 1). Graphs show the results (average and SD) of a representative experiment that was performed in quadruplicate. All experiments were repeated at least twice.

FIG 4

Virus yields of rEAV^wt and rEAV^QYA infection in the absence or presence of CsA. Shown are growth kinetics of rEAV^wt and rEAV^QYA in Huh7 cells (MOI, 3). Infections were performed in the absence or presence of 4 μM CsA. EAV yields at the indicated time points were determined by plaque assay (averages and SD are given [n = 3]).

FIG 5

Adaptive mutations in EAV nsp5 do not affect the morphology of virus-induced double membrane vesicles. Huh7 cells were infected with rEAV^wt (B and E) or rEAV^QYA (C and F) at an MOI of 5 or were mock infected (A and D). After an incubation of 11 h (untreated [A to C]) or 14 h (CsA treated [D to F]), cells were fixed and processed for transmission electron microscopy. DMV formation is very similar upon infection with rEAV^wt and rEAV^QYA, while CsA treatment induces dilated ER both in mock-infected and in infected cells. Scale bars: 2 μm (left images) and 500 nm (middle and right images).

FIG 6

rEAV^QYA does not replicate in the absence of CypA. (A) Virus yields of rEAV^wt and rEAV^QYA (MOI, 0.01) at 32 h after infection of parental Huh7 cells and Huh7 CypA^KO cells. EAV yields were determined by plaque assay (averages and SD [n = 3]).

FIG 7

rEAV^QYA RNA synthesis is impaired but less sensitive to CsA. (A and B) Viral RNA synthesis in BHK-21 cells infected with rEAV^wt or rEAV^QYA was metabolically labeled between 6.5 and 7.5 h p.i. using [³H]uridine. This was done in the presence or absence of 8 μM CsA and dactinomycin. Total intracellular RNA was isolated at 7.5 h p.i. (A) The total incorporation of ³H label was quantified by liquid scintillation counting. (B) ROs were semipurified from lysates of rEAV^wt- or rEAV^QYA-infected BHK-21 cells (MOI, 5) at 7.5 h p.i. and were used in an in vitro RNA synthesis assay (IVRA) in which [³²P]CTP was incorporated into viral RNA products. Reactions were performed in the presence of increasing concentrations of CsA (indicated above the lanes) and were terminated after 100 min. Labeled RNA products were isolated, separated in a denaturing formaldehyde agarose gel, and visualized by phosphorimaging. The positions of the genomic RNA (RNA1) and subgenomic RNAs (positions 2 to 7) are indicated on the left side of the gel. (C) Hybridization analysis of RNA synthesis in rEAV^wt- and rEAV^QYA-infected cells. Intracellular RNA was isolated at 7.5 h p.i. from rEAV^wt- and rEAV^QYA-infected BHK-21 cells and analyzed in a denaturing formaldehyde agarose gel. The EAV RNA was visualized by hybridization to a ³²P-labeled oligonucleotide probe (see Materials and Methods) complementary to the 3′ end of EAV genome and sg mRNAs. The positions of the genomic RNA (RNA1) and subgenomic mRNAs 2 to 7 are indicated on the left side of the gel. Subgenomic RNA abundance was measured by phosphorimaging-based quantification of RNA bands and is given relative to the abundance of RNA1, which was placed at 100%.

FIG 8

Passaging in the presence of ALV reduces EAV sensitivity toward both ALV and CsA. Virus was passaged at increasing ALV concentrations, ranging from 4 μM during passage 1 (P1) to 75 μM during P10. P10 harvests of EAV replicates ALV-1 and ALV-2 were tested for resistance to ALV (A) and CsA (B) treatment. Huh7 cells in 96-well plates were infected at an MOI of 0.05 in the presence of 0 to 50 μM ALV or CsA. Cells were incubated for 3 days, and cell viability was monitored using a commercial assay. In addition, the cytotoxicity of CsA treatment was monitored in parallel in mock-infected Huh7 cells. Graphs show the results (average and SD) of a representative experiment that was performed in quadruplicate. All experiments were repeated at least twice.

FIG 9

Analysis of ALV and CsA sensitivities of engineered rEAV ALV-resistant mutants. Huh7 cells in 96-well plates were infected with rEAV^wt (control) and rEAV ALV-resistant mutants (MOI, 0.05), grouped per ALV replicate, in the presence of 0 to 50 μM ALV (A and B) or CsA (C and D). Cells were incubated for 3 days and cell viability was monitored using a commercial assay. The cytotoxicity of ALV or CsA treatment only was monitored in parallel in mock-infected Huh7 cells (light gray line). Graphs show the results (average and SD) of a representative experiment that was performed in quadruplicate. All experiments were repeated at least twice.

FIG 10

EAV resistance to ALV requires an additional mutation in nsp2. (A and B) Huh7 cells in 96-well plates were infected with rEAV^wt (control) and the rEAV mutants rEAV-nsp2^H114R, rEAV^QYA, and rEAV^QYA-nsp2^H114R in the presence of 0 to 50 μM ALV (A) or CsA (B). Cells were incubated for 3 days and cell viability was monitored using a commercial assay. In addition, the cytotoxicity of ALV or CsA treatment only was monitored in parallel in mock-infected Huh7 cells (light gray line). Graphs show the results (average and SD) of a representative experiment that was performed in quadruplicate. All experiments were repeated at least twice.