Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Aug;13(8):602-614.
doi: 10.1007/s13238-022-00905-7. Epub 2022 Apr 6.

SARS-CoV-2 impairs the disassembly of stress granules and promotes ALS-associated amyloid aggregation

Yichen Li #  1   2 , Shuaiyao Lu #  3   4 , Jinge Gu #  5   6 , Wencheng Xia  5   6 , Shengnan Zhang  5   6 , Shenqing Zhang  1   2 , Yan Wang  7 , Chong Zhang  7 , Yunpeng Sun  5   6 , Jian Lei  7 , Cong Liu  5   6 , Zhaoming Su  8 , Juntao Yang  9 , Xiaozhong Peng  10   11 , Dan Li  12   13   14
Affiliations

SARS-CoV-2 impairs the disassembly of stress granules and promotes ALS-associated amyloid aggregation

Yichen Li et al. Protein Cell. 2022 Aug.

Abstract

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration.

Keywords: SARS-CoV-2; nucleocapsid protein; stress granule.

PubMed Disclaimer

Figures

Figure 1
Figure 1
SARS-CoV-2 infection results in the incorporation of N protein with SGs in mammalian cells. (A) Schematic workflow of the virus infection and SG induction. (B) Confocal images of mammalian cells infected by SARS-CoV-2. Infected cells were stressed with 100 μmol/L sodium arsenite for 1 h, and stained with DAPI, antibodies for N protein and SG marker proteins G3BP1. Arrows indicate SGs. Scale bar, 5 µm. (C) Confocal images of HeLa cells transfected with Flag-tagged N protein (Flag-N). Cells were stressed with 100 μmol/L sodium arsenite for 1 h, and stained with DAPI, anti-flag, and anti-G3BP1. Arrows indicate SGs. Scale bar, 5 µm
Figure 2
Figure 2
SARS-CoV-2 infection impairs the disassembly but enhances the cellular clearance of host SGs. (A) Schematic workflow of the experiment of SG self-disassembly. (B) Confocal images of ACE2-HeLa cells with or without (control) infection of SARS-CoV-2. Cells were stressed with 100 μmol/L sodium arsenite for 1 h, followed by washing out sodium arsenite. Cells were stained with antibodies for viral N protein and SG marker protein G3BP1. Arrows indicate SGs. Scale bar, 5 µm. Quantitative analysis of the images is shown on the right as the area of SGs per cell. Values are means ± SD, n > 150 cells from 3 replicates. Student’s t-test, *P < 0.05, **P < 0.01; ns, not significant. (C) Schematic workflow of the experiment of cellular SG clearance. (D) Confocal images of ACE2-HeLa cells with or without (control) SARS-CoV-2 infection. Cells were stressed with 100 μmol/L sodium arsenite for 1 h or 5 h. Cells were stained with antibodies for N and G3BP1 proteins. Arrows indicate SGs. Scale bar, 5 µm. Quantitative analysis of the images is shown on the right as the area of SGs per infected cell. Values are means ± SD, n > 150 cells from 3 replicates. Student’s t-test, *P < 0.05, **P < 0.01; ns, not significant. (E) In situ FRAP for SGs in HeLa cells overexpressing RFP-tagged N protein. Cells overexpressing RFP-tag were performed as a control. mEGFP-tagged G3BP1 was co-overexpressed with RFP or RFP-N to fluorescently label SGs. Scale bar, 5 µm. FRAP montages of an SG for each sample are shown. The arrows indicate the action of bleaching. (F) Fluorescent recovery curves of the SGs. Data shown are means ± S.D., n = 3 individual SGs. Student’s t-test
Figure 3
Figure 3
SARS-CoV-2 N protein co-phase separates with SG proteins and solidifies their liquid-like droplets. (A) Fluorescence images of co-phase separation of N protein with SG proteins hnRNPA1, FUS, and TDP43, respectively. SG protein concentrations and the molar ratio of SG proteins to N protein are indicated. LLPS buffer: 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 10% PEG 3,350 (no PEG for FUS). EGFP is a fluorescence tag. Alexa-488 and QSY7 are fluorescence dyes. Scale bar, 5 µm. (B) FRAP montages of FUS-EGFP droplets (left). The arrows indicate the action of bleaching. Protein concentrations are indicated. Buffer: 50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl. The droplets are incubated for 0 h and 12 h, respectively. Scale bar, 2 µm. The graphs (right) show the recovery fraction as the function of time. Data shown are means ± SD, n = 3. (C) Representative images of the morphological changes of FUS-EGFP droplets in the absence or presence of N protein over time. The phase separation condition is the same as (B). A FUS droplet with fibrils growing out is enlarged in the inset. Scale bar, 10 µm
Figure 4
Figure 4
SARS-CoV-2 N protein non-specifically interacts with LC domains of FUS and TDP-43. (A) Residue-specific intensity changes of signals in the 2D 1H-15N HSQC spectra of 25 μmol/L 15N-labeled FUS-LC in the presence of N protein at molar ratios (FUS-LC : N) of 1:0.5 (green) and 1:2 (orange). The x axis for the spectra on the top is shown according to the residue numbers; that at the bottom is according to the amino acid composition. Residue signals that dropped over 70% are shown in (B). (C) Residue-specific intensity changes of signals in the 2D 1H-15N HSQC spectra of 20 μmol/L 15N-labeled TDP43-LC in the presence of N protein at molar ratios (TDP43-LC : N) of 1:2.5 (pink) and 1:5 (grey). Residue signals that dropped over 30% are shown in (B)
Figure 5
Figure 5
SARS-CoV-2 N protein promotes the amyloid aggregation of SG proteins. (A) ThT kinetic assays for amyloid fibril formation of the LC domains of FUS, TDP43, and hnRNPA1. Protein concentrations and molar ratios are indicated. Buffer for FUS-LC and hnRNPA1-LC amyloid formation: 50 mmol/L Tris-HCl, pH 7.5 and 100 mmol/L NaCl. Buffer for TDP43-LC amyloid formation: 50 mmol/L Bis-Tris, pH 6.5 and 100 mmol/L NaCl. Data correspond to mean ± SD, n = 3 independent replicates. (B) TEM images of samples in (A) at the end time point. Scale bar, 500 nm. (C) Confocal images of CFP-FUS P525L aggregation puncta in ACE2-HeLa cells infected by SARS-CoV-2. Cells without treatment of the virus are used as a control. FUS P525L is visualized by CFP fluorescence. N protein is immunostained with anti-N. The arrows indicate FUS P525L aggregates. Scale bar, 5 μm. Quantitative analysis of the aggregation area per cell in the imaging data is shown on the right. Values are means ± SD, n > 150 cells from 3 replicates. Student’s t-test
Figure 6
Figure 6
Schematic diagram for the interplay between SARS-CoV-2 and host SGs. During SARS-CoV-2 replication in host cells, the viral N protein enters the host SGs and directly interacts with SG-related amyloid-forming proteins (e.g., FUS, hnRNPA1 and TDP43), which stimulates the liquid to solid phase transition (amyloid formation) of these host proteins. Inefficient maintenance of proteostasis may result in accumulation of the pathological amyloid fibrils and development of neurodegeneration
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Amraei R, Yin W, Napoleon MA, Suder EL, Berrigan J, Zhao Q, Olejnik J, Chandler K, Xia C, Feldman J, et al (2021) CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2. bioRxiv - PMC - PubMed
    1. Bellmann J, Monette A, Tripathy V, Sojka A, Abo-Rady M, Janosh A, Bhatnagar R, Bickle M, Mouland AJ, Sterneckert J. Viral infections exacerbate FUS-ALS phenotypes in iPSC-derived spinal neurons in a virus species-specific manner. Front Cell Neurosci. 2019;13:480. doi: 10.3389/fncel.2019.00480. - DOI - PMC - PubMed
    1. Bostanciklioglu M (2020) Severe acute respiratory syndrome coronavirus 2 is penetrating to dementia research. Curr Neurovasc Res. - PubMed
    1. Buchan JR, Kolaitis RM, Taylor JP, Parker R. Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. Cell. 2013;153:1461–1474. doi: 10.1016/j.cell.2013.05.037. - DOI - PMC - PubMed
    1. Burke KA, Janke AM, Rhine CL, Fawzi NL. Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II. Mol Cell. 2015;60:231–241. doi: 10.1016/j.molcel.2015.09.006. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances