Pdr Repair In Tracy Ca?

. 2021 Oct 13;13(10):2056.

doi: 10.3390/v13102056.

SARS-CoV-2 Spike Impairs Deoxyribonucleic acid Damage Repair and Inhibits V(D)J Recombination In Vitro

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• PMID: 34696485
• PMCID: PMC8538446
• DOI: 10.3390/v13102056

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SARS-CoV-2 Fasten Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro

Hui Jiang  et al. Viruses. 2021 .

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-two) has led to the coronavirus disease 2022 (COVID-19) pandemic, severely affecting public health and the global economy. Adaptive amnesty plays a crucial office in fighting against SARS-CoV-2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID-19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS-CoV-ii impedes adaptive amnesty remains unclear. Hither, by using an in vitro cell line, we report that the SARS-CoV-ii spike protein significantly inhibits DNA impairment repair, which is required for effective V(D)J recombination in adaptive amnesty. Mechanistically, we found that the fasten poly peptide localizes in the nucleus and inhibits Deoxyribonucleic acid damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.

Keywords: Deoxyribonucleic acid damage repair; SARS–CoV–ii; 5(D)J recombination; fasten; vaccine.

Conflict of interest argument

The authors have declared that no competing interests exist. The funders had no role in written report blueprint, data collection and assay, determination to publish, or preparation of the manuscript.

Figures

Figure ane

Issue of severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) nuclear-localized proteins on Dna damage repair. (A) Subcellular distribution of the SARS–CoV–2 proteins. Immunofluorescence was performed at 24 h after transfection of the plasmid expressing the viral proteins into HEK293T cells. Scale bar: 10 µm. (B) Schematic of the EJ5-GFP reporter used to monitor not-homologous stop joining (NHEJ). (C) Event of empty vector (E.V) and SARS–CoV–2 proteins on NHEJ DNA repair. The values correspond the mean ± standard divergence (SD) from three contained experiments (see representative FACS plots in Figure S2A). (D) Schematic of the DR-GFP reporter used to monitor homologous recombination (HR). (Eastward) Effect of E.V and SARS–CoV–2 proteins on HR DNA repair. The values correspond the mean ± SD from three contained experiments (see representative FACS plots in Figure S2B). The values represent the mean ± SD, n = 3. Statistical significance was determined using one-way assay of variance (ANOVA) in (C,Eastward). ** p < 0.01, *** p < 0.001, **** p <  0.0001.

Effigy two

Severe astute respiratory syndrome coronavirus 2 (SARS–CoV–2) spike protein inhibits DNA damage repair. (A) Schematic of the primary construction of the SARS–CoV–2 spike protein. The S1 subunit includes an N–terminal domain (NTD, xiv–305 residues) and a receptor–binding domain (RBD, 319–541 residues). The S2 subunit consists of the fusion peptide (FP, 788–806 residues), heptapeptide repeat sequence one (HR1, 912–984 residues), HR2 (1163–1213 residues), TM domain (TM, 1213–1237 residues), and cytoplasm domain (CT,1237–1273 residues). (B,C) Event of titrated expression of the spike protein on Deoxyribonucleic acid repair in HEK–293T cells. (D,E) Only full-length spike protein inhibits not-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair. The values represent the hateful ± SD from 3 contained experiments (come across representative FACS plots in Figure S4A,B). (F) Full–length spike (Due south–FL) poly peptide–transfected HEK293T cells exhibited more DNA damage than empty vector-, S1–, and S2–transfected cells under different Deoxyribonucleic acid harm atmospheric condition. For doxorubicin: 4 µg/mL, 2 h. For γ–irradiation: 10 Gy, 30 min. For H₂O₂: 100 µM, 1 h. Calibration bar: 50 µm. (G) Corresponding quantification of the comet tail moments from 20 different fields with due north > 200 comets of three contained experiments. Statistical significance was assessed using a two-way analysis of variance (ANOVA). NS (Non Significant): * p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Severe astute respiratory syndrome coronavirus two (SARS–CoV–ii) spike protein impedes the recruitment of Dna harm repair checkpoint proteins. (A) Membrane fraction (MF), cytosolic fraction (CF), soluble nuclear fraction (SNF), and chromatin-spring fraction (CBF) from HEK293T cells transfected with SARS–CoV–2 spike protein were immunoblotted for His-tag spike and indicated proteins. (B) Left: Immunoblots of Deoxyribonucleic acid damage marking γH2AX in empty vector (E.Five)– and spike protein–expressing HEK293T cells after 10 Gy γ-irradiation. Right: corresponding quantification of immunoblots in left. The values represent the mean ± SD (n = 3). Statistical significance was determined using Student's t-test. **** p < 0.0001. (C) Immunoblots of Deoxyribonucleic acid damage repair related proteins in spike poly peptide–expressing HEK293T cells. (D) Representative images of 53BP1 foci formation in E.V– and spike poly peptide-expressing HEK293 cells exposed to 10 Gy γ–irradiation. Scale bar: 10 µm. (E) Quantitative analysis of 53BP1 foci per nucleus. The values stand for the mean ± SEM, northward = 50. (F) BRCA1 foci formation in empty vector- and fasten protein-expressing HEK293 cells exposed to ten Gy γ–irradiation. Calibration bar: x µm. (K). Quantitative analysis of BRCA1 foci per nucleus. The values represent the mean ± SEM, north = fifty. Statistical significance was determined using Student'southward t-test. **** p < 0.0001.

Effigy 4

Spike protein impairs Five(D)J recombination in vitro. (A) Schematic of the 5(D)J reporter system. (B) Representative plots of menses cytometry show that the SARS–CoV–two spike protein impedes Five(D)J recombination in vitro. (C) Quantitative analysis of relative V(D)J recombination. The values represent the mean ± SD, due north = 3. Statistical significance was determined using Educatee's t-test. **** p < 0.0001.

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