Dataset Information

Targeted in situ cross-linking for determination of structure and interactions of SARS-CoV-2 proteins in the cellular context

ABSTRACT: Discovery of host-pathogen intra-cellular interactions is critical for identification of mechanisms of infection and discovery. Affinity purification based on Mass Spectrometry (AP-MS) approaches can identify the host-pathogen protein-protein interaction network that includes hundreds of cellular proteins. For example, over 330 interactions were identified for SARS-CoV-2 major proteins. However, identification of the critical anchoring interactions directly induced by the viral proteins is still challenging. Here we developed a complete workflow of in-situ XL-MS followed by AP-MS, and employ it to investigate SARS-CoV2 protein inside the host cell. We focused on three Sars-Cov-2 proteins for which the structural information is either missing or incomplete: NSP1, NSP2, and the Nucleocapsid protein (N protein). AP-MS revealed that several human proteins were co-eluting with NSP1 and NSP2 while protein N eluted alone.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Severe Acute Respiratory Syndrome

SUBMITTER: Moriya Slavin

LAB HEAD: Nir Kalisman

PROVIDER: PXD023542 | Pride | 2021-10-06

REPOSITORIES: Pride

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Source:

			Action	DRS
	2020_06_16_01_C-143_16h_Strep_IP_NO_VEC_p2.raw	Raw
	2020_06_16_03_C-143_16h_Strep_IP_NO_XL_p2.raw	Raw
	2020_06_16_08_C-143_16h_Strep_IP_DSS_p2.raw	Raw
	2020_06_29_01_C-143_Strep_IP_No_Vec_p2.raw	Raw
	2020_06_29_03_C-143_Strep_IP_No_XL_p2.raw	Raw

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Project description:Human coronaviruses (hCoV) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite high sequence similarity between SARS-CoV-1 and -2, each strain has distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV non-structural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43 – an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologs from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identify nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response (UPR) associated proteins, and anti-viral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondrial-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed anti-viral therapeutics.

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Targeted in situ cross-linking for determination of structure and interactions of SARS-CoV-2 proteins in the cellular context

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