Stoneham Charlotte A, Singh Rajendra, De Leon Amalia, Tafelmeyer Petra, Acosta Francisco, Fuori Angus, Anderson Michael, Ramirez Peter W, Schwartzer-Sperber Hannah S, Pillai Satish, Lew-Inski Mary K, Guatelli John
VA San Diego Healthcare System, San Diego CA USA.
University of California San Diego, La Jolla CA USA.
bioRxiv. 2025 May 6:2025.05.04.652125. doi: 10.1101/2025.05.04.652125.
Like all coronaviruses, the infectivity of SARS-CoV-2 virus particles (virions) requires incorporation of the Spike glycoprotein. Yet, the mechanisms that support the virion-incorporation of Spike are incompletely defined. We noted an unusual feature of human sarbecovirus Spike proteins: their cytoplasmic domains (CDs) contain a stretch of acidic amino acids (DEDDSE). This sequence resembles a cluster of acidic residues, or acidic cluster (AC) motif, found in the cytoplasmic domain of the cellular endoprotease Furin. In Furin, the acidic cluster acts as a protein sorting signal, supporting its intracellular localization at the -Golgi network (TGN). We tested the contribution of the acidic cluster motif in the Spike CD to protein interactions and to the infectivity of SARS-CoV-2. We used virus-like particles (VLPs) as a model for viral "infection" (transduction). The SARS-CoV2 VLPs were produced by co-expressing Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins and deliver an RNA encoding luciferase to target cells expressing the ACE2 receptor. Remarkably, when all five acidic residues of the DEDDSE sequence were replaced with alanines, the VLPs were rendered non-infectious. The N-terminal DE residues provided most of the activity of the acidic cluster. These virologically-impaired Spike mutants were able to reach the cell surface and induce the formation of syncytia, indicating that they are fusogenic and capable of anterograde traffic through the biosynthetic pathway to the plasma membrane. Despite this, they failed to efficiently incorporate into virions. We observed acidic cluster motif-dependent interactions of the Spike CD with several cellular proteins that could potentially support its role in virion-incorporation, including the ERM proteins Ezrin, Radixin, and Moesin; the retromer subunit Vps35, and the medium subunits of the clathrin adaptor complexes AP1 and AP2. While the key cofactor and mechanism of action remains to be defined, this region of acidic residues in the Spike CD appears to be a novel determinant of SARS-CoV-2 infectivity.
与所有冠状病毒一样，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒颗粒（病毒体）的感染性需要刺突糖蛋白的掺入。然而，支持刺突蛋白掺入病毒体的机制尚未完全明确。我们注意到人类sarbecovirus刺突蛋白的一个不寻常特征：它们的细胞质结构域（CD）包含一段酸性氨基酸（DEDDSE）。该序列类似于在细胞内蛋白酶弗林蛋白酶（Furin）的细胞质结构域中发现的一簇酸性残基，即酸性簇（AC）基序。在弗林蛋白酶中，酸性簇作为一种蛋白质分选信号，支持其在反式高尔基体网络（TGN）中的细胞内定位。我们测试了刺突蛋白CD中酸性簇基序对蛋白质相互作用以及对SARS-CoV-2感染性的作用。我们使用病毒样颗粒（VLP）作为病毒“感染”（转导）的模型。通过共表达刺突蛋白（S）、膜蛋白（M）、包膜蛋白（E）和核衣壳蛋白（N）来产生SARS-CoV-2病毒样颗粒，并将编码荧光素酶的RNA递送至表达血管紧张素转换酶2（ACE2）受体的靶细胞。值得注意的是，当DEDDSE序列的所有五个酸性残基都被丙氨酸取代时，病毒样颗粒变得无感染性。N端的DE残基提供了酸性簇的大部分活性。这些病毒学功能受损的刺突蛋白突变体能够到达细胞表面并诱导多核巨细胞的形成，这表明它们具有融合能力，并且能够通过生物合成途径向顺式运输至质膜。尽管如此，它们未能有效地掺入病毒体。我们观察到刺突蛋白CD与几种细胞蛋白之间存在依赖于酸性簇基序的相互作用，这些细胞蛋白可能支持其在病毒体掺入中的作用，包括埃兹蛋白（Ezrin）、根蛋白（Radixin）和膜突蛋白（Moesin）等ERM蛋白；逆转录复合物亚基Vps35，以及网格蛋白衔接复合物AP1和AP2的中型亚基。虽然关键辅助因子和作用机制仍有待确定，但刺突蛋白CD中的这一酸性残基区域似乎是SARS-CoV-2感染性的一个新决定因素。
