The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

Abstract

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

SciScore for 10.1101/2020.07.30.228023: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
Lentivirus is produced in HEK293t cells by transfection of lentiviral plasmid and packaging plasmids pMD2.G and psPAX2.	HEK293t suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
After two days of transfection, the culture medium containing the lentivirus was passed through a 0.45 μm filter and was used to infect U2OS cell line.	U2OS suggested: CLS Cat# 300364/p489_U-2_OS, RRID:CVCL_0042)
Software and Algorithms
Sentences	Resources
Cloning …

SciScore for 10.1101/2020.07.30.228023: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
Lentivirus is produced in HEK293t cells by transfection of lentiviral plasmid and packaging plasmids pMD2.G and psPAX2.	HEK293t suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
After two days of transfection, the culture medium containing the lentivirus was passed through a 0.45 μm filter and was used to infect U2OS cell line.	U2OS suggested: CLS Cat# 300364/p489_U-2_OS, RRID:CVCL_0042)
Software and Algorithms
Sentences	Resources
Cloning and Protein Purification: SARS-CoV-2 N protein constructs (NFL, N1-364, N49-419, N49-364, N1-264, N247-419, N49-246, N49-209, N1-174, N49-174) were amplified by PCR from the IDT 2019-nCoV N positive control plasmid (IDT cat. # 10006625; NCBI RefSeq YP_009724397) and inserted by ligation-independent cloning into UC Berkeley Macrolab vector 2B-T (AmpR, N-terminal His6-fusion; Addgene #29666) for expression in E. coli.	RefSeq suggested: (RefSeq, RRID:SCR_003496)
The FRAP data were quantified using ImageJ or Zeiss Zen built-in profile model.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
The images were recorded on a K2 Summit direct detector (Gatan) in counting mode using SerialEM (64).	SerialEM suggested: (SerialEM, RRID:SCR_017293)
Motion-correction and dose-weighting, using MotionCor2 (66), was applied to the individual frames of the tilt-series images.	MotionCor2 suggested: (MotionCor2, RRID:SCR_016499)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

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