key: cord-0696134-ceoltj1a
authors: Xia, Bingqing; wang, Yi; pan, Xiaoyan; Cheng, Xi; Ji, Hongying; Zuo, Xiaoli; Li, Jia; Gao, Zhaobing
title: Why SARS-CoV-2 Omicron variant is milder? A single high-frequency mutation of structural envelope protein matters
date: 2022-02-07
journal: bioRxiv
DOI: 10.1101/2022.02.01.478647
sha: e79b74af9c2ad003dd620dab37619c90f54e9356
doc_id: 696134
cord_uid: ceoltj1a

SARS-CoV-2 Omicron variant is highly transmissible and extensive morbidity, which has raised concerns for antiviral therapy. In addition, the molecular basis for the attenuated pathogenicity and replication capacity of Omicron remains elusive. Here, we report for the first time that a high-frequency mutation T9I on 2-E of SARS-CoV-2 variant Omicron forms a non-selective ion channel with abolished calcium permeability and reduced acid sensitivity compared to the WT channel. In addition, T9I caused less cell death and a weaker cytokine production. The channel property changes might be responsible for the Omicron variant releases less efficiently and induces a comparatively lower level of cell damage in the infected cells. Our study gives valuable insights into key features of the Omicron variant, further supporting 2-E is a promising drug target against SARS-CoV-2 and providing critical information for the COVID-19 treatment.

cell death, provoke cytokine storm and even cause acute respiratory distress syndrome 48 (ARDS)-like damages in vivo. Its inhibitor exhibits excellent antiviral activity in vivo 9 . 49 Therefore, we focus on the 2-E mutations of Omicron variant. 50 Three high quality E protein sequences of Omicron variants (B.1.1.529, BA1and BA2) 51 in the CNCB database as of January 18th, 2022 (https://ngdc.cncb.ac.cn/ncov/) were 52 comprehensively analyzed and a mutation 2-E T9I (T9I) caught our attention. Compared 53 with the original Wuhan reference strain, among more than one hundred identified 2-E 54 mutations in Omicron variant, T9I shows a much higher frequency than the rest 55 mutations. In addition, the mutation probability of T9I in all statistical samples is higher 56 than 99.5%. For the most common Omicron strain B.1.1.529, the mutation probability 57 is 100% (Fig.1a, b) . According to the solved NMR structure, T9 locates at the top of 58 the transmembrane domain (TMD) of 2-E proteins 10 . The influence of T9I has not been 59 studied yet completely. 60 First, we asked whether T9I still retains channel activity. We purified T9I protein and 61 reconstituted the protein on Planar Lipid Bilayer (BLM) as previously described 62 9 ( Supplementary Fig.S1 ). The observed T9I-induced typical single-channel currents 63 supported that T9I was able to form ion channels also as wild-type (WT) 2-E proteins 64 9 . However, the reversal potential of T9I channels was shifted to left, from 57 mV to 3 65 mV, under asymmetric KCl solutions. Similar reversal potential shifting was also 66 detected in asymmetric NaCl solutions ( Fig.1c.d, Supplementary Fig.S2) . A reversal 67 potential close to 0 mV suggests that T9I may have lost its selectivity to cations. To 68 determine whether T9I can permeate chloride，the channel activity of T9I was further 69 examined in asymmetric 50:500 mM choline chloride solutions. The quaternary 70 ammonium choline is not permeable to most cation channels and thus is always used to 71 detect anion permeability. As expected, outward currents were indeed observed when 72 the holding voltage potentials were higher than the theoretical reversal potential for 73 chloride (Erev> 70 mV). Besides, WT channels are permeable to Ca 2+ . As shown in 74 Figure S2c , for WT channels, the frequent and continuous inward potassium currents 75 in the asymmetric 50:500 mM (trans: cis) K + solutions indicated that the 2-E channels 76 were incorporated into the membranes. The membrane potential was then changed to 77 +65 mV (K + reverse potential) to eliminate the K + currents. Intriguingly, outward step-78 like signals appeared when 20 mM (final concentration) Ca 2+ was added to the trans 79 side. When the Ca 2+ concentration increased to 20 mM, the outward currents increased.

In contrast, under the identical conditions, T9I channels did not induce detectable 81 calcium currents ( Supplementary Fig.S2c ). These results revealed that T9I owns a 82 different ion selectivity and permeability from those of WT.

WT channels are pH sensitive 9 . The pH influences on T9I channels were evaluated on 84 a same channel using titration strategy. After the channels incorporated into the 85 membranes, HCl was titrated into either cis or trans side to alter the pH to 4 from 6.

Consistent to our previous results, reduction of pH, either in cis or trans side, induced 87 significant increase of amplitude and open probability of WT channels. In contrast, pH 88 reduction in cis side failed to increase the channel activity of T9I channels. Although 89 we didn't have direct evidences to explain how the threonine (T9) site is sensitive to 90 pH changes, the structure of 2-E channels supported the residues (Glu7 and Glu8) 91 around T9 were sensitive to pH change and their side chain carboxyl could be 92 deprotonated at neutral pH and protonated at acidic pH 10 . It is reported that SARS-

CoV-2 traffics to the lysosomes for egress by lysosome deacidification, instead of using 94 the conventional biosynthetic secretory pathway 11 . The E channels of coronavirus are 95 localized to intracellular organelles, including lysosome and Golgi apparatus 11, 12 . The 96 cellular localization and expression of WT and T9I were assessed using 97 immunocytochemistry and confocal microscopy. We found that both WT and T9I are 98 co-localized with the lysosomal marker LAMP (Fig.1g, supplementary Fig.S3 ). Given the localization on lysosome, the influences of WT and T9I on the luminal pH of 100 lysosome were compared. Flow cytometric was used to analyze pH probe (pHluorin) 101 ratio. This pH probe consists of green fluorescent protein (GFP) pHluorin molecule 102 fused with lysosome-resident protein CD63. To generate a pH calibration curve, un-103 transfected cells were subjected to treatment with buffers ranging from pH 5.5 to 7.5 in 104 the presence of the ionophores monensin and nigericin prior to flow analysis (Fig.1h,   105 i). Expression of WT channels robustly neutralized the lysosome pH, whereas T9I 106 channels exhibited less influences in luminal pH, which was consistent with our 107 electrophysiological results (Fig.1j) . Therefore, the capability of deacidification of WT 108 channels in lysosomes was significantly impeded by the mutation T9I. Based on above, Finally, the channel activity of the 2-E protein is a determinant of virulence. We used 118 Annexin V/PI staining and CCK8 assays to evaluate the cell lethality of T9I in Vero E6 119 cells. We found that although the expression level of T9I was far exceeding the 120 expression level of WT ( Fig. 1 l) , it caused similar cell death as WT did ( incubate the cells at a 1:1,000 dilution. Images were captured using the Leica TCS-SP8 

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β-Coronaviruses Use Lysosomes 187 for Egress Instead of the Biosynthetic Secretory Pathway

The Infectious Bronchitis Coronavirus Envelope 190

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Structure of M(pro) from SARS-CoV-2 and discovery of 196 its inhibitors

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