SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation.
SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation.
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DOI：
10.1128/jvi.00002-21
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发表时间：
2021-04-12
影响因子：
5.4
通讯作者：
Hahn AS
中科院分区：
文献类型：
--
作者：
Hörnich BF;Großkopf AK;Schlagowski S;Tenbusch M;Kleine-Weber H;Neipel F;Stahl-Hennig C;Hahn AS
细胞-细胞融合允许病毒感染邻近细胞而不需要产生游离病毒，并且通过产生病毒感染的合胞体而有助于组织损伤。我们的研究结果表明，S2′切割位点是TMPRSS 2激活所必需的，并揭示了SARS-CoV和SARS-CoV-2之间的重要差异，其中，SARS-CoV-2更依赖于ACE 2表达和金属蛋白酶激活细胞-细胞融合。严重急性呼吸综合征相关冠状病毒2型（SARS-CoV-2）通过其刺突蛋白（SARS 2-S）与血管紧张素转换酶2（ACE 2）的相互作用以及蛋白酶（特别是跨膜蛋白酶丝氨酸2（TMPRSS 2））的激活来感染细胞。病毒也可以通过感染细胞与未感染细胞的融合传播。我们比较了SARS 2-S介导的和SARS-CoV-S（SARS 1-S）介导的细胞-细胞融合对ACE 2表达、蛋白水解激活和对抑制剂的敏感性的要求。TMPRSS 2适度增加SARS 2-S驱动的融合，ACE 2强烈增加，而TMPRSS 2强烈增加SARS 1-S驱动的融合，ACE 2表达增加较少。与SARS 1-S相反，SARS 2-S介导的细胞-细胞融合被巴马司他敏感的金属蛋白酶有效激活。当ACE 2或TMPRSS 2受限时，S1/S2蛋白水解切割位点的突变减少了效应细胞-靶细胞融合，并使SARS 2-S驱动的细胞-细胞融合更加依赖于TMPRSS 2。当ACE 2和TMPRSS 2都丰富时，与野生型（wt）SARS 2-S相比，初始靶细胞-效应细胞融合没有改变，但合胞体仍然较小。S2切割（S2′）位点的突变特异性地消除了TMPRSS 2对细胞-细胞融合和SARS 2-S驱动的假颗粒进入的激活，但仍然允许金属蛋白酶激活细胞-细胞融合和组织蛋白酶激活颗粒进入。最后，我们发现TMPRSS 2抑制剂溴己新与抑制剂卡莫司他不同，不能减少SARS 1-S和SARS 2-S引起的TMPRSS 2激活的细胞-细胞融合。有趣的是，溴己新在TMPRSS 2存在下增强细胞-细胞融合，而其代谢物氨溴索在某些条件下表现出抑制活性。在Calu-3肺细胞上，氨溴索对SARS 2-S驱动的慢病毒假颗粒进入有微弱的抑制作用，两种物质对真正的SARS-CoV-2都表现出剂量依赖性的微弱抑制趋势。重要性细胞-细胞融合允许病毒感染邻近细胞而不需要产生游离病毒，并通过产生病毒感染的合胞体而导致组织损伤。我们的研究结果表明，S2′切割位点是TMPRSS 2激活所必需的，并揭示了SARS-CoV和SARS-CoV-2之间的重要差异，其中，SARS-CoV-2更依赖于ACE 2表达和金属蛋白酶激活细胞-细胞融合。据报道，溴己新是TMPRSS 2的抑制剂，目前正在2019年针对冠状病毒疾病的临床试验中进行测试。我们的研究结果表明，溴己新在某些条件下增强融合。因此，在更好地了解溴己新对SARS-CoV-2峰激活的影响之前，我们警告不要使用高剂量的溴己新。与溴己新相似，临床上用作祛痰剂的相关化合物氨溴索对细胞-细胞融合没有激活作用。这两种化合物在高浓度下对SARS-CoV-2感染均表现出较弱的抑制活性，这可能是氨溴索在临床上可达到的。
Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2′ cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2′) site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2. IMPORTANCE Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2′ cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol.