SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

  1. Amanda Jack
  2. Luke S. Ferro
  3. Michael J. Trnka
  4. Eddie Wehri
  5. Amrut Nadgir
  6. Xammy Nguyenla
  7. Douglas Fox
  8. Katelyn Costa
  9. Sarah Stanley
  10. Julia Schaletzky
  11. Ahmet Yildiz

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Abstract

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

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  1. Version published to 10.1371/journal.pbio.3001425
  2. Version published to 10.1101/2020.09.14.295824v3 on bioRxiv
  3. eLife

    Reviewer #3:

    Jack and colleagues report that SARS-CoV-2 interacts with RNA to form phase-separated liquid compartments, similar to P bodies and nucleoli, shown here as blobs. The authors then perturbed the system in numerous ways, showing that: i) different nucleic acids give rise to different blobs; ii) that protein cross-linking and mass spec suggests that the phase-separated N is in a different tertiary or quaternary conformation than the soluble N; iii) that some N domains (e.g., PLD, R2) are important for blob formation, particularly when the protein is phosphorylated (by an unknown kinase); and iv) some small molecules can affect the number and size of the blobs. Overall, this story is at a very early stage phenomenology and lacks clear demonstration of physiological relevance. Certainly, the claim that "nilotinib disrupts the association of the N protein into higher order structures in vivo and could serve as a potential drug candidate against packaging of SARS-CoV-2 virus [sic] in host cells" ought to be tested - it would be easy enough to do, though I don't think this would complete the story.

    Major comments:

    1. Figure 1 is difficult to interpret with the information provided. In panel A, the colors seem to be important, but readers are not given a clue as to what. In panel B, how were the Y axes calculated? What are we really looking at in Figs. 1C and D? Were these on glass slides? Plastic? Was the surface coated, passivized, or otherwise derivatized in any way? What kind of microscope was used? What do the white signals (blobs) come from? Is there a fluorescent label involved? Is this phase contrast? In panel D, please include a buffer only control (no protein) to demonstrate blobs are not simply a buffer artefact. Finally, what N:RNA molar ratios were used in this Figure?

    2. For the polymeric RNAs, what were the average chain lengths?

    3. In describing Figure 1, the authors state "The shapes of these asymmetric structures were consistent with remodeling of vRNPs into 'beads on a string', as observed by cryoEM." This is wishful thinking. I see blobs of different shapes, but there is no way to know whether these represent N protein "beads" on RNA "strings." Reference 6 cited in the manuscript and showing "beads on a string" model has a scale bar of 50 nm = 0.05 µm, and even there, the N:RNA complex is very obscure.

    4. My greatest concern of this work is that no information was provided about the N protein that was used for the in vitro studies. How pure was it? What steps were taken to remove co-purifying nucleic acids? Was it monodisperse? Aggregated? Please include DLS data and show silver stained SDS-PAGE.

    5. Similarly, how did the mutant forms of N (Fig. 3A) behave? Were they properly folded? Did the authors check them by CD or SEC? And what concentrations of mutant proteins were used? Without these data, the rest of Fig. 3 is uninterpretable.

    1. B. Could the authors please explain what the numbers on the Y axis are and how they were calculated. Also, their disorder prediction predicts dimerization regions to be highly disordered, would they consider a problem with the prediction method?

    1. C, D, E what is the N: RNA molar ratio?

    2. Could the authors please explain the calculation method used to calculate the % surface area covered by droplets?

    3. Fig, 4A and B. Why is [N] so low? In other experiments the authors usually used 18.5 µM, whereas here the concentration was 7.8 µM, almost invisible blobs as observed in other figures provided by the authors (and below ksat, or very close to it).

    4. Fig, 4C. What is 1.5 M N RNA? [N] is set to 57.6 µM, much higher than in Fig. 4 A-B assays. Is there a reason?

    5. Fig. 4D is missing control cells transfected with GFP only (no N).

  4. eLife

    Reviewer #2:

    This paper contributes to the large number of papers currently posted on BioRxiv showing that the N protein of SARS CoV2 can undergo liquid-liquid phase separation on its own and in the presence of RNA, and that this behavior can be modulated by phosphorylation. The work here is somewhat different from much of the other work in that the authors have generated the N protein from mammalian cells. The authors have also examined the effects of known drugs on the phase separation process. Given the importance of coronavirus it is imperative to get out information on its biology. But it is also imperative that the information be correct, interpreted with appropriate caution, and of sufficient depth to be valuable to others in the field and not potentially misdirect future research and clinical efforts. In this respect, I think the authors need to clean up some of their experiments and pull back on some of their claims, as I detail below.

    Major comments:

    1. In general, the authors' use of size, number and morphology of droplets to assess the effects of small molecules in figure 4 is problematic. The authors should be measuring the effects of the compounds on the phase separation threshold concentration (of N+RNA or of salt) to see whether the compounds stabilize or destabilize the droplets. Changes in size, number and morphology can be due to many factors, many of which are unlikely to be relevant to viral assembly.

    For example, the authors report that nelfinavir mesylate and LDK378 produced fewer but larger droplets, and conclude that these compounds could disrupt virion assembly. This is problematic for two reasons. Most importantly, it is almost impossible to interpret what fewer larger droplets means. Are they nucleating more slowly and/or growing more rapidly? Are they more viscous and thus less disrupted by handling? Are they denser and thus settling more rapidly? Has the thermodynamic threshold to phase separation changed? Secondarily, because of these uncertainties, it is an overinterpretation to state based on the data that these compounds could act by disrupting virion assembly.

    The class II molecules, which increase both size and number of droplets, are probably more relevant, since concomitant increases in both probably mean that the threshold concentration for LLPS has decreased, and thus the compound has stabilized the droplets.

    The changes in morphology induced by the class III molecules are also hard to interpret. Does the change reflect greater adhesion to and spreading on the slide surface (probably irrelevant to drug action)? Or changes in droplet dynamics--slowed fusion or increased viscosity? What does it mean that nilotinib causes the morphology of N+RNA condensates to become filamentous, and could this same effect account for the (modest) decrease in N protein foci in cells upon drug treatment?
    I honestly am concerned that the authors conclude the paper urging use of nilotinib in clinical trials, and the effects of drugs on phase separation as a proxy for vRNP formation, based on these very thin data.

    1. In Figure 1 (and beyond), it is not good practice to use fractional areas of droplets that have settled to a slide surface to quantify droplet formation in LLPS experiments. Droplets fall to the slide surface at different rates depending on their sizes, which in turn depend on many factors, some biochemical (the relative rates of nucleation and growth; density; all of which can vary with buffer conditions) and some technical (exactly how the sample was handled). Turbidity, which also is imperfect, is nevertheless a more reliable measure; so is microscopic assessment of the presence or absence of droplets. The authors should provide at least some additional measure in these initial experiments to show the numbers obtained from the fractional area are qualitatively correct.

    2. In figure 1C, the dissolution with salt is not a measure of liquid-like properties, as claimed at the bottom of page 3. The authors should look for evidence of droplet fusion, spherical shape (for droplets larger than the diffraction limit) and rapid exchange with solvent.

    3. The claims on page 4 that the condensates formed with viral RNA fragments are gel-like should be supported with some measure of dynamics, and not simply the shape of the objects that settle to the slide surface.

    4. In the CLMS experiments, how do the authors know that the changes observed are due to LLPS per se and not to differences in structure induced by differences in salt? It seems like additional controls are warranted to make this claim. Relatedly, the authors should state/examine whether higher salt affects dimerization of the dimerization domain.

    5. The analogy made on page 4 between the asymmetric structures observed upon mixing N and viral RNA fragments to the strings of vRNPs observed by cryoEM is quite a stretch. The vRNPs are 15 nm in diameter. The structures observed here are vastly larger. Such associated but non-fused droplets are often observed for solidifying phase separating systems. The superficial similarity of connected particles between the cellular vRNPs and the structures here is, in my opinion, unlikely to be meaningful.

  5. eLife

    Reviewer #1:

    This article proposes that the assembly of the Sars-CoV-2 capsid is mediated by liquid-liquid phase separation of the N protein and RNA. The strength of the manuscript is a series of in vitro experiments showing that N protein can undergo liquid-liquid phase separation (LLPS) in a manner enhanced by RNA. The authors also identify nilotinib as a compound that alters the morphology of assemblies consisting of RNA and the N protein. The primary weakness of the manuscript is that there is little data connecting the in vitro observations to intracellular events, or viral assembly. Taken together, I find the experiments interesting but, as detailed below, premature.

    Major comments:

    1. A key issue with any in vitro assembly process such as LLPS is a demonstration that same process occurs in the cell. This is an issue since many molecules can undergo LLPS in vitro in a manner unrelated to their biological function. In this work, the authors show that the N protein can undergo LLPS in vitro in a manner a) stimulated by RNA, b) enhanced by the R2 domain, and c) changed in morphology by nilotinib.

    Their argument that this LLPS is relevant to the viral life cycle rests on: a) the observation that over-expressed N protein forms foci in the cytosol, and b) the number of these foci (but not necessarily their morphology as seen in vitro) is somewhat reduced by nilotinib. In my opinion, this is not a very convincing argument for two main reasons.

    First, it is unclear why the N protein is forming foci in cells. Specifically:

    a) Is it being recruited to P-bodies, or some other existing subcellular assembly? (Which could be examined by staining with other markers).

    b) Is it forming a new assembly with RNA as they have proposed? (Which could be addressed by staining for either specific or generic RNAs, or purifying these assemblies and determining if they contain RNA)

    Second, it is unclear that the foci seen in cells are related to the LLPS they observe in vitro or relevant to the viral life cycle. Specifically:

    c) Is the assembly related to the LLPS they have observed in vitro beyond a poorly understood alteration with nilotinib ? (Which could be addressed by examining if the deletions they observe affect LLPS in vitro also affect the formation of N protein foci in cells).

    d) Is the nature of this assembly relevant to the viral life cycle? (Given the difficulty of working with COVID, this is hard. My suggestion here is at a minimum to discuss the issue, and ideally do an experiment with a related coronavirus to test their hypothesis). Frankly, the idea that coronavirus would trigger a LLPS of multiple viral RNAs would seem to be inhibitory to efficient packaging of individual virions. A discussion of how the virus would benefit from such a mechanism, as opposed to a cooperative coating of a viral genome initiated at a high affinity N protein binding site would be important to put the work in context.

    1. The manuscript would be improved by examining the presence of RNA in each LLPS, and the ability of RNA to undergo self-assembly under the conditions examined in the absence of the N protein. As it stands, in some cases, the authors could be studying RNA based self-assembly, that then recruits the N protein to the RNA LLPS by RNA binding (see Van Treeck et al., 2018, PNAS for specific example of this phenomenon). This may be particularly likely for some of the longer viral RNAs that can form more stable base-pairs and thereby promote more "tangled" assemblies (e.g. Tauber et al., 2020, Cell).

    2. I found the CLMS to not fit well in this manuscript for two reasons:

    a) As I understood the methods, the CLMS experiment is looking at cross linking in high and low salt, with some LLPS occurring under low salt. However, since the cross linking was not limited to the dense phase of the low salt condition, a significant fraction (perhaps majority?) of the N proteins will not be in the dense phase. Because of this, the cross linking is essentially mapping interactions that change between high and low salt. If the authors really want to do this experiment, they should separate the phases and examine the crosslinks forming in the dense and dilute phases under the same salt conditions.

    b) A second issue with this cross-linking experiment is that the regions that dominate the changes in cross linking are not ones that appear to be important in driving LLPS in vitro based on their deletion analysis. If the authors want to include this data, it should be related to the deletion experiments and connected to the work in a manner to make it meaningful.

    1. The work would be improved by comparing how alterations that impact LLPS affect specific biochemical interactions of the relevant molecules. In these experiments, the authors are examining assemblies that form through N-N, N-RNA, RNA-RNA interactions. In each case, biochemical assays could be used to examine which of these interactions are altered by deletions or compounds. By understanding the underlying alterations in molecular interactions, a greater understanding of the mechanism of the observed LLPS, and its relevance to the viral life cycle could be revealed.
  6. eLife

    Summary: Although there is a clear interest in SARS-CoV-2 biology and characterization of the physical properties of its viral proteins, ultimately the reviewers felt that the data was too preliminary and did not link it to physiological relevance even if the experimental concerns could be addressed. We hope that the reviewer's comments will be useful.

  7. ScreenIT

    SciScore for 10.1101/2020.09.14.295824: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    For protein expression, HEK GNTI cells were grown in suspension in Freestyle media (Gibco) supplemented with 2% FBS (VWR) and 1% penicillin-streptomycin (Gemini Bio-products) to 2 million cells/mL.
    HEK GNTI
    suggested: ATCC Cat# CRL-3022, RRID:CVCL_A785)
    Weighted t-tests were performed in R. Drug Screening and Image Processing: For the FDA-approved drug screen, 75 mL of N protein at 16 μM was purified from HEK cells.
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Vero E6 Cell Culture and Drug Testing: Vero E6 cells were cultured in phenol-negative DMEM media supplemented with 10% FBS and 1% PS at 37° with 5% CO2.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Crosslinked spectra were identified with Protein Prospector 6.2.23 (66) using the combination of DSS/DSS:2H12 at uncleaved Lys residues and protein N-terminus as the crosslinking reagents.
    Protein Prospector
    suggested: (Protein Prospector, RRID:SCR_014558)
    For quantitating the isotopically labeled crosslinks, peak areas were measured from the extracted precursor ion chromatograms (XICs) using the small molecule interface of Skyline (v20.1.0.155).
    Skyline
    suggested: (Skyline, RRID:SCR_014080)
    A Skyline transition list was generated containing the elemental composition of each distinct peptide pair with both light and heavy BS3 modification and in each charge state detected in the Prospector search.
    Prospector
    suggested: (Protein Prospector, RRID:SCR_014558)
    Solidity (area/convex area) was measured by Fiji.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)

    Results from OddPub: Thank you for sharing your code and data.

    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:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • 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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  8. Version published to 10.1101/2020.09.14.295824v1 on bioRxiv
  9. Version published to 10.1101/2020.09.14.295824v2 on bioRxiv