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

我们以前已经证明，嗜神经病毒的复制导致细胞宿主蛋白质的募集，从而干扰蛋白质稳态，可以释放自我维持的病理信号级联已知的神经退行性疾病的细胞病理。剖析SARS-CoV-2进入细胞后的确切分子途径是理解其发病机制的关键。在这个提议中，我们专注于通过纯化药物的靶蛋白来鉴定有助于SARS-CoV-2组装的细胞宿主蛋白，该药物通过与宿主细胞辅因子结合来有效抑制SARS-CoV-2组装。通过药物亲和色谱结合质谱鉴定的候选宿主蛋白将通过多种实验进行验证，包括相应基因的CRISPR/Cas9敲除和测量基因缺陷细胞中的病毒复制。此外，基于我们的观察结果，即病毒可导致在神经变性中也观察到的蛋白质聚集，将研究对神经元样细胞中蛋白质稳态的特定影响。我们从这些拟议的细胞研究中获得的见解将得到COVID 19患者的人脑切片（共）染色的支持，以确认神经向性和长期影响的可能性。我们的研究将对SARS-CoV-2的公共卫生政策非常重要。

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.