Dataset Information

Identification of host dependency factors involved in SARS-CoV-2 replication organelle formation through proteomics and ultrastructural analysis

ABSTRACT: Positive-strand RNA viruses subvert the cellular endomembrane system for the generation of distinct compartments termed replication organelles (ROs) that harbor the site where viral RNAs are generated. In this study, we corroborate that the SARS-CoV-2 non-structural proteins 3 and 4 (nsp3 and nsp4) suffice to remodel the endoplasmic reticulum to form double membrane vesicles similar to ROs observed in viral infection. Cellular membrane alterations induced by nsp3/4 expression were evaluated through electron tomography and confocal microscopy and nsp3/4 associated host factors were identified using mass spectrometry. The role of these host factors in virus infection was determined using gene silencing, identifying several host proteins involved in the SARS-CoV-2 replication cycle. Combining the gene silencing approach with ultrastructural analysis of nsp3/4-expressing cells, we found that the host dependency factors FAM149B1, CCAR2 and ZC3HAV1 play a role in the formation of double membrane vesicles in a replication-independent manner.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Pietro Scaturro

LAB HEAD: Pietro Scaturro

PROVIDER: PXD036206 | Pride | 2023-10-23

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	210312_PS_CB_IP_HA-nsp3-nsp4-V5_1_Slot1-14_1_1726.d.zip	Other
	210312_PS_CB_IP_HA-nsp3-nsp4-V5_1_Slot1-14_1_1762.d.zip	Other
	210312_PS_CB_IP_HA-nsp3-nsp4-V5_2_Slot1-20_1_1727.d.zip	Other
	210312_PS_CB_IP_HA-nsp3-nsp4-V5_2_Slot1-20_1_1763.d.zip	Other
	210312_PS_CB_IP_HA-nsp3-nsp4-V5_3_Slot1-26_1_1728.d.zip	Other

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Project description:Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, non-structural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV, and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) analysis for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance nuclear protein important for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.1 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3.1 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

Project description:Preclinical studies of primary cancer cells are always done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3-10% range, with most tumors in an hypoxic or 1-2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anti-cancer agents is routinely examined at ambient O2. Here, using both mouse models and human cancers, we demonstrate that tumors collected, processed and propagated at physiologic (physioxia) O2 compared to ambient air display very distinct differences in key signaling networks including Lgr5/Wnt, Yap, and Nrf2/Keap1, nuclear reactive oxygen species levels, alternative splicing, and sensitivity to several targeted therapies including PIK3CAalpha-specific and EGFR inhibitors. Significance: Extra-physiologic oxygen shock/stress (EPHOSS), as noted in cells collected/processed under ambient air, has been demonstrated to have significant impact on numbers and engrafting ability of hematopoietic stem cells. We report deleterious effects of EPHOSS on cancer cell behavior and EPHOSS-mediated effects on cancer cells give misleading information on signaling pathway activation that could severely impact the relevance of these findings. Cancer cells under EPHOSS show higher proliferation rate compared to cells under physioxia and thus are sensitive to anti-proliferative agents. Thus, drugs that show effectiveness on cancer cells collected in ambient air and subjected to EPHOSS may not be effective or as relevant in vivo, results that could partially explain the limited clinical translation of laboratory findings. Evaluating cell signaling and effects of drugs on cancer cells under physiologic O2 prior to in vivo studies could substantially reduce cost and aid in drug discovery relevant to the actual physioxia/pathological status of the tumor cells in vivo.

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Identification of host dependency factors involved in SARS-CoV-2 replication organelle formation through proteomics and ultrastructural analysis

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