The current work investigated SARS‐CoV‐2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC‐based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with the SG proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following oxidative stress and heat shock. Treatment of recombinant NCAP with RNA isolated from A549 cells exposed to oxidative stress‐stimulated NCAP to undergo liquid–liquid phase separation (LLPS). RNA degradation using RNase A treatment completely blocked the LLPS property of NCAP as well as its SG association. The RNA intercalator mitoxantrone also disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS‐CoV‐2 NCAP. We have identified novel interactors of the SARS‐CoV‐2 nucleocapsid (NCAP) protein in human lung epithelial cancer cells. NCAP showed specific interaction with the stress granule (SG) proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following stress treatments. NCAP exhibited RNA‐dependent phase separation. The RNA intercalator mitoxantrone disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the NCAP.
【저자키워드】 viral infection, SARS‐CoV‐2, nucleocapsid, stress granules, G3BP1, mitoxantrone, viral factory, liquid–liquid phase separation, NCAP, SILAC, 【초록키워드】 Treatment, Stress, oxidative stress, RNA, SARS‐CoV‐2, Protein, cells, human lung, N protein, epithelial, association, A549, Interaction, RNA degradation, RNase, Cancer cells, Heat shock, PKR, approach, G3BP2, oxidative, blocked, investigated, exhibited, undergo, provide, cancer cell, biological processe, A549 cell, translocated, USP10, YTHDF3, 【제목키워드】 droplet, nucleocapsid protein, SARS‐CoV‐2, stress granule, interact,