ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells

Abstract

In addition to transporting ions, the multisubunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na(+),K(+)-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na(+),K(+)-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections.

Importance: Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na(+),K(+)-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na(+),K(+)-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

FIG 1

RNAi-mediated downregulation of ATP1A1 affects MHV, FIPV, and VSV but not IAV. (A) Effect of RNAi-mediated downregulation of ATP1A1 on MHV-ERLM, FIPV-RLuc, IAV-RLuc, and VSV-FLuc. Gene silencing was performed using individual transfection of three different siRNAs targeting ATP1A1 (ATP1A1-1 to ATP1A1-3) in HeLa cells expressing the appropriate virus receptors. Negative siRNA (neg siRNA) was included as a control. Cells were infected with luciferase-expressing viruses at an MOI of 0.1 for 7 h or overnight for IAV. Infection levels were determined by assaying the luciferase activity in cell lysates relative to lysates of infected cells that had been mock treated. Infection levels were corrected for cell number and viability as determined by the Wst-1 assay. Error bars represent standard errors of the means (n = 3 replicates of 3). (B) Confirmation of siRNA-mediated reduction in mRNA levels. mRNA levels at 72 h posttransfection were measured by qRT-PCR relative to mock-transfected cells. Expression levels were corrected for cell number and viability as determined by the Wst-1 assay. Error bars represent standard errors of the means (n = 3 replicates of 3). Dotted lines indicate the lower 95% confidence interval of negative siRNA controls (A) or mock treatment (B).

FIG 2

Knockdown of ATP1A1 affects MHV and VSV fusion. (A) Effects of siRNA-mediated gene silencing on viral binding, internalization, and fusion using replication-independent assays. Three different siRNAs against ATP1A1 (ATP1A1-1 to ATP1A1-3) were transfected individually into HeLa-(mCC1a-)ΔM15 cells. Negative siRNA (neg siRNA) was included as a control. At 72 h posttransfection MHV-αN was allowed to bind to the cells on ice at an MOI of 20 for 90 min. Unbound virus was washed off. For the binding assay, cells and viruses were subsequently lysed, and complementation of ΔM15 by αN was determined relative to mock-treated samples using Beta-Glo substrate and a luminometer. For internalization and fusion assays, the cells were warmed to 37°C, and virus was allowed to enter cells for 60 and 100 min, respectively. To assay internalization, cell surface-bound virus was removed using trypsin, and cells and viruses were subsequently lysed. Complementation of ΔM15 by αN was determined relative to mock-treated samples using Beta-Glo substrate and a luminometer. For the fusion assay, cells were preloaded with FDG by hypotonic shock before inoculation. Upon infection for 100 min, cells were collected and analyzed by FACS. Fusion was determined relative to the number of FIC-positive cells observed upon mock treatment of infected cells. Error bars represent standard errors of the means (n = 3 replicates of 3) for binding and internalization; n = 3 for fusion). (B) VSV fusion was determined as described in panel A using VSV-ΔG/Luc-Gα. Dotted lines indicate the lower 95% confidence intervals of the mock treatment.

FIG 3

Knockdown of ATP1A1 inhibits infection with MHV independent of the intracellular site of fusion or the receptor used. Gene silencing was performed as described in the legend to Fig. 1. (A) Cells were infected with luciferase-expressing MHV or MHV-S2′FCS at an MOI of 0.1 for 7 h. Infection levels were determined by assaying the luciferase activity in cell lysates relative to lysates of infected cells that had been mock treated. Infection levels were corrected for cell number and viability as determined by the Wst-1 assay. Error bars represent standard errors of the means (n = 3 replicates of 3). (B) Cells were infected with GFP-expressing MHV or MHV-SRec at an MOI of 0.5 for 8 h. Cells were collected, and virus replication and cell viability were analyzed by FACS relative to mock-treated samples. Negative siRNA and an siRNA targeting GFP were included as controls. Error bars represent standard errors of the means (n = 3).

FIG 4

Low levels of ouabain and bufalin affect entry of CoVs and VSV but not of IAV. (A) HeLa (MHV, FIPV [FIPV-H], VSV, and IAV), Huh7 (MERS-CoV), or FCWF (FIPV [FIPV-F]) cells were inoculated with the indicated viruses at an MOI of 0.1 for 2 h. Cells were treated with 50 nM ouabain from 30 min prior to (pre) or 2 h after (post) inoculation until 7 h (MHV and FIPV), 8 h (MERS-CoV), or 16 h (IAV) postinfection. Infection levels were determined by measuring the luciferase activity in cell lysates or by determining the number of infected cells (MERS-CoV) by immunocytochemistry relative to levels in mock-treated cells. Error bars represent standard errors of the means (n = 3 replicates of 3). (B) Effect of low doses of bufalin on MHV, FIPV, MERS-CoV, IAV, and VSV infection. Cells were infected and treated as described in panel A with 10 nM bufalin instead of ouabain. Error bars represent standard errors of the means (n = 3 replicates of 3).

FIG 5

Effect of ouabain on virus entry is linked to ATP1A1-encoded α1 subunit. HeLa cells were transfected with plasmids expressing either human- or murine-derived ATP1A1 (hATP1A1 and mATP1A1, respectively). Cells were treated with 50 nM ouabain from 30 min prior to (pre) or 2 h after (post) inoculation with luciferase-expressing MHV, IAV, or VSV at an MOI of 0.1 until 7 h (MHV and VSV) or 16 h (IAV) postinfection. Infection levels were determined by measuring the luciferase activity in cell lysates relative to that in lysates of mock-treated cells. Infection levels were corrected for cell number and viability as determined by the Wst-1 assay prior to infection. Error bars represent standard errors of the means (n = 3 replicates of 3).

FIG 6

Low levels of ouabain and bufalin prevent fusion of MHV and VSV. (A) Time-of-addition experiment using 50 nM ouabain. Luciferase-expressing MHV was bound to HeLa-mCC1a cells at an MOI of 0.5 for 90 min on ice. Unbound virus was washed off, and incubation continued at 37°C. At the indicated time points medium was replaced by warm medium containing 50 nM ouabain. Luciferase expression levels were determined relative to those of mock-treated cells. Error bars represent standard errors of the means (n = 3). (B) Binding, internalization, and fusion assays of MHV upon ouabain or bufalin treatment were performed as described in the legend to Fig. 2. HeLa-(mCC1a-)ΔM15 cells were pretreated with 50 nM ouabain or 10 nM bufalin. (C) Binding, internalization, and fusion assays of VSV were performed using VSV-ΔG/Luc-Gα as described for MHV in the legend to Fig. 2. In panels B and C, error bars represent standard errors of the means (n = 3 replicates of 3 for binding and internalization; n = 3 for fusion). (D) Effect of ouabain treatment on infection with MHV-SRec or MHV-S2′FCS. Cells were treated with ouabain as described in the legend to Fig. 5 and inoculated with luciferase-expressing MHV, MHV-S2′FCS, or MHV-SRec at an MOI of 0.1. As a control, cells were treated with U18666A. Infection levels were determined by measuring the luciferase expression levels in cells at 7 h postinfection relative to those in mock-treated cells. Error bars represent standard errors of the means (n = 3 replicates of 3).

FIG 7

Ouabain inhibits virus entry upstream of inhibitors of CME. (A) The inhibitory effect of ouabain is not observed when the compound is removed after virus inoculation. Cells were mock treated or treated with 50 nM ouabain (Ou) starting at 30 min prior to and during inoculation (the period 0 to 2 h) and/or after removal of the inoculum (the period 2 to 18 h). Cells were inoculated with luciferase-expressing MHV at an MOI of 0.1. (B) Virus infection is not inhibited when the fusion-inhibitory peptide HR2 is added after removal of ouabain. Cells were mock treated or treated with the indicated compound (ouabain [Ou] or HR2 peptide) starting at 30 min prior to and during inoculation (0 to 2 h) and/or after removal of the inoculum (2 to 18 h). (C) After the removal of ouabain, virus infection was inhibited by the addition of CME inhibitors. Cells were mock treated or treated with 50 nM ouabain (Ou) starting at 30 min prior to and during inoculation (0 to 2 h). After removal of the inoculum the medium was replaced by drug-containing medium (Dyngo, Dyngo-4A; CPZ, chlorpromazine; and BafA1, bafilomycin A1). In panels A to C, after overnight infection cells were lysed, and infection levels were determined by measuring the luciferase activity in cell lysates relative to that of control cells that were treated only with ouabain prior to and during inoculation (Ou, 0 to 2 h only; black bar). Error bars represent standard errors of the means (n = 3 replicates of 3).

FIG 8

Ouabain inhibits virus entry at an early stage. (A) MHV particles accumulate close to the cell surface in the presence of ouabain. Images show ouabain-treated cells inoculated with DyLight 488-labeled MHV by confocal microscopy. Cells were mock treated (upper two rows) or treated with 50 nM ouabain (lower two rows) throughout the experiment starting at 30 min prior to inoculation. MHV covalently labeled with DyLight 488 (MHV particles) was bound to cells at an MOI of 20 for 70 min on ice. Unbound virus was removed, and cell-bound virus was allowed to infect at 37°C for 90 min. Cells were fixed, stained with DAPI (nuclei) and phalloidin (actin), and analyzed by confocal microscopy. Single z-slices are shown. (B) The inhibitory effect of ouabain on VSV entry can be bypassed by low-pH shock-induced fusion. Cells were pretreated with 50 nM ouabain. VSV-FLuc virus was bound to the pretreated cells at an MOI of 0.3 in the presence of 50 nM ouabain on ice for 90 min. Unbound virus was removed, and cells were incubated for 2 h at 37°C in the presence of ouabain. At 2 hpi the inoculum was removed, and cells were incubated for 2 min with warm buffers at different pHs (7.2, 6.5, 5.5, and 5.0) that contained 50 nM ouabain. Incubation at 37°C in ouabain-containing medium was continued until 11 hpi. Infection levels were determined by measuring the luciferase expression levels in cell lysates relative to those in mock-treated cells. Error bars represent standard errors of the means (n = 3 replicates of 3).

FIG 9

Inhibition of infection by ouabain is rescued by inhibition of Src. HeLa cells were inoculated with luciferase-expressing MHV, FIPV, or VSV at an MOI of 0.1 for 2 h. Cells were treated with 50 nM ouabain (Ou), wortmannin (Wort), PP2, pNaKtide or a combination thereof as indicated from 30 min prior to (pre) or 2 h after (post) inoculation. The drugs were present until cell lysis at 7 h postinoculation. Infection levels were determined by measuring the luciferase activity in lysates of drug-treated cells relative to that in mock-treated cells. Error bars represent standard errors of the means (n = 3 replicates of 3).

FIG 10

Model of the effect of ATP1A1 knockdown and CTSs treatment on entry of CoVs and VSV. siRNA-mediated gene silencing of ATP1A1 encoding the α1 subunit of the Na⁺,K⁺-ATPase or treatment of cells with CTSs inhibits infection with CoVs and VSV at an early entry stage, resulting in reduced virus-cell fusion. In the presence of CTSs or after siRNA-mediated gene silencing of ATP1A1, virus particles accumulate in preendosomal invaginations that are not accessible to the membrane-impermeable HR2 peptide or trypsin. For VSV, this block in entry can be bypassed by low-pH shock. Knockdown of ATP1A1 leads to release of an Na⁺,K⁺-ATPase-bound subset of Src, Src activation, and increased Src signaling (20, 39, 63). Ouabain binding to the α1 subunit of Na⁺,K⁺-ATPase triggers a conformational change in this subunit, which also results in release of Src from Na⁺,K⁺-ATPase and its concomitant activation (20, 86). Activated Src induces yet unknown downstream signaling, which inhibits virus entry at an early stage upstream of the inhibitory effects of inhibitors of clathrin-mediated endocytosis.