The envelope protein of SARS-CoV-2 increases intra-Golgi pH and forms a cation channel that is regulated by pH
The envelope protein of SARS-CoV-2 increases intra-Golgi pH and forms a cation channel that is regulated by pH
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DOI：
10.1113/jp281037
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发表时间：
2021-03-24
影响因子：
5.5
通讯作者：
Harrison, Neil L.
中科院分区：
文献类型：
--
作者：
Cabrera-Garcia, David;Bekdash, Ramsey;Harrison, Neil L.
Key pointsWe report a novel method for the transient expression of SARS-CoV-2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes.Intracellular expression of SARS-CoV-2 E protein increases intra-Golgi pH.By targeting the SARS-CoV-2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH.This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID-19.The envelope (E) protein of coronaviruses such as SARS-CoV-1 is proposed to form an ion channel or viroporin that participates in viral propagation and pathogenesis. Here we developed a technique to study the E protein of SARS-CoV-2 in mammalian cells by directed targeting using a carboxyl-terminal fluorescent protein tag, mKate2. The wild-type SARS-CoV-2 E protein can be trafficked to intracellular organelles, notably the endoplasmic reticulum-Golgi intermediate complex, where its expression increases pH inside the organelle. We also succeeded in targeting SARS-CoV-2 E to the plasma membrane, which enabled biophysical analysis using whole-cell patch clamp recording in a mammalian cell line, HEK 293 cells, and two-electrode voltage clamp electrophysiology in Xenopus oocytes. The results suggest that the E protein forms an ion channel that is permeable to monovalent cations such as Na+, Cs+ and K+. The E current is nearly time- and voltage-independent when E protein is expressed in mammalian cells, and is modulated by changes of pH. At pH 6.0 and 7.4, the E protein current is activated, whereas at pH 8.0 and 9.0, the amplitude of E protein current is reduced, and in oocytes the inward E current fades at pH 9 in a time- and voltage-dependent manner. Using this directed targeting method and electrophysiological recordings, potential inhibitors of the E protein can be screened and subsequently investigated for antiviral activity against SARS-CoV-2 in vitro and possible efficacy in treating COVID-19.
