SARS-CoV-2 Nsp2 recruits GIGYF2 near viral replication sites and supports viral protein production [nsp2_del_rna_seq]

The SARS-CoV-2 genome encodes 16 non-structural proteins (Nsps), with Nsp2 being the least conserved and least understood. In this study, we found a crucial role of Nsp2 in the SARS-CoV-2 life cycle and demonstrated its interaction with GIGYF2, which causes the relocation of GIGYF2 to near double membrane vesicles (DMVs) and the enhancement of viral protein production. Deletion of the Nsp2-coding region from the viral genome led to a drastic reduction in viral RNA synthesis early in infection (3–4 hours post infection). Through interactome analysis of Nsp2 in virus-infected cells, we identified GIGYF2—a protein involved in translational regulation—as a key partner of Nsp2. We confirm the interaction between GIGYF2 and Nsp2 from both SARS-CoV-1 and SARS-CoV-2. Depleting GIGYF2 or its cofactor ZNF598 phenocopied the replication defects observed with Nsp2 deletion, suggesting their critical roles in viral reproduction. Moreover, GIGYF2 and ZNF598 relocate to areas near DMVs, viral replication sites, upon infection. This relocation does not happen with the Nsp2-deleted virus, indicating Nsp2's role in directing GIGYF2 to DMVs. Using formaldehyde crosslinking and immunoprecipitation-sequencing experiments (fCLIP-seq), we found that GIGYF2 interacts with viral RNAs, particularly in regions encoding Nsp3, M, and Orf6. Depletion of GIGYF2 resulted in decreased expression of these proteins. Our findings reveal the function of Nsp2 in supporting viral protein production by exploiting GIGYF2 as a host factor. Overall design: We employed formaldehyde immunoprecipitation and sequencing (fCLIP-seq) using anti-GIGYF2 antibody after SARS-CoV-2 infection. To investigate the function of GIGYF2 and NSP2 in SARS-CoV-2 replication, we performed RNA-sequencing after infection with WT and nsp2-deleted virus. We also performed RNA-sequencing in parental and GIGYF2 shRNA cells after SARS-CoV-2 virus infection.

严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的基因组编码16种非结构蛋白（non-structural proteins，Nsps），其中Nsp2是保守性最差且研究最为匮乏的蛋白。本研究揭示了Nsp2在SARS-CoV-2生命周期中的关键作用，并证实其与GIGYF2存在相互作用：该相互作用可使GIGYF2迁移至双膜囊泡（double membrane vesicles，DMVs）附近，并促进病毒蛋白的合成。从病毒基因组中敲除Nsp2编码区，会在感染早期（感染后3~4小时）导致病毒RNA合成大幅下降。通过对病毒感染细胞中Nsp2进行相互作用组分析，我们鉴定出GIGYF2——一种参与翻译调控的蛋白——是Nsp2的关键互作伴侣。我们证实GIGYF2与Nsp2的相互作用存在于SARS-CoV-1和SARS-CoV-2两种病毒中。敲低GIGYF2或其辅因子ZNF598，可重现Nsp2缺失所导致的病毒复制缺陷，表明二者在病毒增殖过程中发挥关键作用。此外，感染病毒后，GIGYF2与ZNF598会迁移至病毒复制位点双膜囊泡附近区域；而在感染缺失Nsp2的病毒时，该迁移现象不会发生，这表明Nsp2负责引导GIGYF2靶向至双膜囊泡。通过甲醛交联免疫沉淀测序（formaldehyde crosslinking and immunoprecipitation-sequencing，fCLIP-seq）实验，我们发现GIGYF2可与病毒RNA结合，尤其富集于编码Nsp3、M以及Orf6的区域。敲低GIGYF2会导致上述蛋白的表达水平下降。本研究结果揭示了Nsp2通过劫持宿主因子GIGYF2来促进病毒蛋白合成的功能机制。

实验整体设计：我们在SARS-CoV-2感染后，利用抗GIGYF2抗体开展了甲醛交联免疫沉淀测序（fCLIP-seq）实验。为探究GIGYF2与Nsp2在SARS-CoV-2复制过程中的功能，我们分别用野生型（wild type，WT）和Nsp2缺失型病毒感染细胞，并对感染后的样本进行RNA测序。此外，我们还分别用SARS-CoV-2感染亲本细胞与GIGYF2短发夹RNA（short hairpin RNA，shRNA）干扰细胞，并对其进行RNA测序。