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

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. 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.

SARS-CoV-2基因组编码16种非结构蛋白（non-structural proteins, Nsps），其中Nsp2是保守性最低、研究最不透彻的亚型。本研究揭示了Nsp2在新冠病毒生命周期中的关键作用，并证实其与GIGYF2存在相互作用：该相互作用可促使GIGYF2向双膜囊泡（double membrane vesicles, DMVs）附近移位，并增强病毒蛋白的合成。将病毒基因组中编码Nsp2的区域敲除后，感染早期（感染后3~4小时）的病毒RNA合成就会出现显著下降。通过对病毒感染细胞中Nsp2开展互作组分析，我们鉴定出GIGYF2——一种参与翻译调控的宿主蛋白——是Nsp2的关键互作伴侣。我们进一步证实，GIGYF2与SARS-CoV-1、SARS-CoV-2的Nsp2均存在相互作用。敲低GIGYF2及其辅因子ZNF598的表达，可重现Nsp2缺失所导致的病毒复制缺陷，提示二者在病毒增殖过程中发挥关键作用。此外，病毒感染宿主细胞后，GIGYF2与ZNF598会向病毒复制位点——双膜囊泡附近区域——移位；而在感染缺失Nsp2的病毒时，该移位现象并不会发生，这表明Nsp2负责引导GIGYF2向双膜囊泡募集。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，尤其偏好结合编码Nsp3、M以及Orf6的区域。敲低GIGYF2的表达会导致上述蛋白的表达量显著下降。本研究结果阐明了Nsp2通过劫持宿主因子GIGYF2来促进病毒蛋白合成的功能机制。实验层面，我们在SARS-CoV-2感染后，使用抗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，我们分别对野生型（WT）及Nsp2缺失型病毒感染后的样本进行了RNA测序；同时也对SARS-CoV-2感染后的亲本细胞与GIGYF2短发夹RNA（shRNA）敲低细胞开展了RNA测序。