Identification and validation of key host cellular factors directing SARS-CoV-2 assembly and consequences for neuronal proteostasis

我们之前已经证明，嗜神经病毒的复制会导致细胞宿主蛋白的募集，从而扰乱蛋白质稳态，从而释放神经退行性疾病的细胞病理学中已知的自我维持的病理信号级联反应。解剖SARS-CoV-2进入细胞后的确切分子途径是了解其发病机制的关键。在本研究中，我们通过纯化一种通过结合宿主细胞辅助因子有效抑制SARS-CoV-2组装的药物的靶蛋白，重点鉴定协助SARS-CoV-2组装的细胞宿主蛋白。通过药物亲和层析结合质谱鉴定的候选宿主蛋白将通过多种实验进行验证，包括CRISPR/Cas9敲除相应基因和测量病毒在基因缺陷细胞中的复制。此外，基于我们在神经退行性变中观察到的病毒可导致蛋白质聚集的观察结果，我们将研究病毒对神经元样细胞中蛋白质停滞的特定影响。我们从这些拟议的细胞研究中获得的见解将得到covid - 19患者人脑切片（共）染色的支持，以确认神经嗜性和长期影响的可能性。我们的研究将对有关SARS-CoV-2的公共卫生政策具有重要意义。

We have previously demonstrated that replication of neurotropic viruses leads to cellular host protein recruitment, thereby to disturbed proteostasis that can unleash self-sustaining pathological signaling cascades known from the cellular pathology of neurodegenerative diseases. Dissecting the exact molecular pathway that SARS-CoV-2 runs through after entering the cells is key for understanding its pathogenesis. In this proposal, we focus on identifying cellular host proteins aiding in SARS-CoV-2 assembly through purifying the target proteins of a drug that potently inhibits SARS-CoV-2 assembly by binding to host cell cofactors. Candidate host proteins identified by drug affinity chromatography combined with a mass spectrometric will be validated by a variety of experiemtns including CRISPR/Cas9 knockout of corresponding genes and measuring virus replication in gene-deficient cells. Furthermore, specific effects on proteostasis in neuron-like cells will be investigated based on our observations that viruses can lead to protein aggregation observed also in neurodegeneration. Our insights from these proposed cellular studies will be backed by (co-) staining of human brain sections of COVID19 patients to confirm neurotropism and the possibility of long term effects. Our studies will be important for public health polocies regarding SARS-CoV-2.