Burkard Christine, Verheije Monique H, Haagmans Bart L, van Kuppeveld Frank J, Rottier Peter J M, Bosch Berend-Jan, de Haan Cornelis A M
Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands.
J Virol. 2015 Apr;89(8):4434-48. doi: 10.1128/JVI.03274-14. Epub 2015 Feb 4.
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.
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.
多亚基钠钾ATP酶除了转运离子外，还通过将强心甾（CTS）结合诱导的信号传递到细胞中发挥作用。在本研究中，我们分析了钠钾ATP酶，特别是其ATP1A1α亚基在冠状病毒（CoV）感染过程中的作用。作为对照，还包括水泡性口炎病毒（VSV）和甲型流感病毒（IAV）。使用基因沉默技术发现，ATP1A1蛋白对于小鼠肝炎病毒（MHV）、猫传染性腹膜炎病毒（FIPV）和VSV感染细胞至关重要，但对IAV感染细胞并非如此。缺乏ATP1A1并不影响病毒与宿主细胞的结合，但会导致MHV和VSV的进入受到抑制。同样，已知不影响钠钾ATP酶转运功能的纳摩尔浓度的强心甾哇巴因和蟾毒灵，在病毒接种期间存在时，可抑制细胞被MHV、FIPV、中东呼吸综合征（MERS）冠状病毒和VSV感染，但不影响IAV感染。强心甾被证明在早期阶段抑制MHV的进入，导致病毒粒子在细胞表面附近积累，从而减少融合。与感染早期受阻一致，低pH冲击可绕过CTS对VSV的抑制作用。在病毒进入后添加这些化合物时，病毒RNA复制不受影响。添加不同的Src激酶抑制剂可解除哇巴因的抗病毒作用，表明通过ATP1A1介导的Src信号在抑制CoV和VSV感染中起关键作用。
冠状病毒（CoV）是动物和人类的重要病原体，最近出现的人畜共患起源的新型人类CoV就证明了这一点。目前缺乏针对CoV感染的抗病毒药物。在本研究中，我们表明，作为离子转运体和信号转导器的钠钾ATP酶的ATP1A1亚基支持CoV感染。通过基因沉默或低浓度的与ATP1A1结合的强心甾哇巴因和蟾毒灵靶向ATP1A1，会在早期进入阶段抑制小鼠、猫和MERS冠状病毒的感染。对照病毒VSV的感染也受到抑制。由ATP1A1介导的Src信号在哇巴因和蟾毒灵抑制病毒进入中起关键作用。这些结果表明，使用强心甾靶向钠钾ATP酶，其中几种是FDA批准的化合物，可能是一种针对CoV和VSV感染的有吸引力的治疗方法。
