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Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

Edoardo Salladini, Claire Debarnot, Vincent Delauzun, Maria-Grazia Murrali, Priscila Sutto-Ortiz, Silvia Spinelli, Roberta Pierattelli, Christophe Bignon, Sonia Longhi

Preprint posted on December 14, 2018 https://www.biorxiv.org/content/early/2018/12/14/497024

Phase transitions are everywhere – a viral protein is able to form amyloid-like hydrogels, contributing to cellular toxicity

Selected by Tessa Sinnige

Categories: biophysics

Background

Henipaviruses are a genus of RNA viruses that can infect animals and humans, with potentially deadly consequences. In the search for therapeutic targets to develop vaccines and treatments, a detailed characterisation of the components of the virus is crucial. The phosphoprotein P is an essential co-factor of the viral polymerase,  and the P gene furthermore gives rise to two alternative proteins V and W, as a result of mRNA editing. P, V and W have an N-terminal region (PNT) of ~400 residues in common that was shown to be intrinsically disordered (1), but the functional relevance of this region is currently unclear.

 

Findings of the preprint

The preprint starts off with the serendipitous discovery that the V protein of a henipavirus species forms a hydrogel after thawing the purified protein from -20 °C. Aiming to find out which region is responsible for this behaviour, the authors test different fragments of the V protein’s PNT domain, and find only one fragment (PNT3) that forms hydrogels (Figure 1). This fragment comprises a low-complexity region enriched in acidic residues, in addition to a short motif containing three consecutive tyrosine residues that is predicted highly prone to form amyloid fibrils (Figure 1).

 

Figure 1. A) Domain architecture of the V protein and sequence of the PNT3 fragment with the amyloid-prone motif in yellow and the low-complexity region underlined. B,C,E,F) Hydrogels formed by the full-length V protein (B), the PNT domain (C), PNT3 (E) and PNT3-GFP (F). Reproduced from the preprint with permission from the authors.

 

The formation of solid hydrogels is typically preceded by liquid-liquid phase separation, as was demonstrated previously for disease-associated proteins such as FUS (2), hnRNPA1 (3) and tau (4). To investigate whether PNT3 may go through a similar process, the authors subject it to a range of conditions and find that it forms droplets given a combination of the right protein concentration, molecular crowding agent, salt concentration and temperature. The droplets look rather static and are unable to fuse, suggesting that they rapidly solidify. To examine whether the solidified protein droplets are amyloid-like, the authors turn to the typical amyloid-binding dyes Congo Red and Thioflavin T, both of which interact with PNT3 under the conditions resulting in phase separation. Circular dichroism and X-ray fiber diffraction provide further evidence that the protein adopts the characteristic β-sheet structure of amyloid fibrils.

Aiming to describe the amyloid formation of PNT3 over time, the authors use SAXS, NMR and TEM, and demonstrate the loss of disordered monomers and the appearance of larger, ordered structures, with a fibrillar morphology in the electron micrographs. The fibrils are however not as stable as some known pathogenic amyloids, and dissolve into smaller species upon treatment with the detergent SDS. Given the role of tyrosine residues in mediating liquid-liquid phase separation (5), and the predicted amyloidogenicity of the YYY motif in PNT3, the authors furthermore mutate these residues to alanine and observe that the resulting variant does not form fibrils.

Finally, the authors find that PNT3 forms fibrillar structures not only in vitro but also in mammalian cells, which show Congo Red fluorescence upon PNT3 expression. When subjecting the cells to stress by hydrogen peroxide treatment, PNT3-expressing cells die in larger numbers than control cells. The decrease in resistance is directly correlated to the propensity of the protein to form fibrils, since expressing the AAA mutant leaves the survival rates unaffected compared to control cells.

 

Why I chose this preprint

It has recently become clear that a plethora of proteins – typically containing disordered regions – can undergo phase transitions, and this phenomenon has been linked to the formation of membrane-less organelles, as well as pathogenic protein aggregation. The preprint describes the first detailed characterisation of a viral protein displaying this behaviour, which it possibly uses to the advantage of the virus. It is very exciting that a simple biological system like a virus may make functional use of phase separation and amyloid fibril formation, extending the impact of the recent discoveries in this field.

 

Questions

It is still debated whether amyloid formation is required for liquid-liquid phase separation to occur, i.e. whether liquid droplets are held together by transient amyloid-like interactions. The amyloid-prone YYY motif of PNT3 lies outside of the predicted low complexity region. The authors show that mutating these residues to alanine prevents PNT3 from forming fibrils, but is the mutant still capable of undergoing liquid-liquid phase separation?

How conserved is the YYY motif in this family of viruses?

The authors raise the point in the discussion that henipavirus may use its ability to undergo phase separation to form the ‘viral factories’ in which transcription and replication take place inside infected cells. These factories have recently been shown to have the properties of liquid droplets, in the case of the related rabies virus (6). Would it be feasible to examine if the AAA mutant fails to form these factories? Would this be sufficient for the virus to lose its infectivity?

 

References

  1. Habchi J, Mamelli L, Darbon H and Longhi S (2010) Structural Disorder within Henipavirus Nucleoprotein and Phosphoprotein: From Predictions to Experimental Assessment. PLoS One 5: e11684
  2. Patel A, Lee HO, Jawerth L, Maharana S, Jahnel M, Hein MY, Stoynov S, Mahamid J, Saha S, Franzmann TM, Pozniakovski A, Poser I, Maghelli N, Royer LA, Weigert M, Myers EW, Grill S, Drechsel D, Hyman AA, et al. (2015) A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 162: 1066–1077
  3. Molliex A, Temirov J, Lee J, Coughlin M, Kanagaraj AP, Kim HJ, Mittag T and Taylor JP (2015) Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization. Cell 163: 123–133
  4. Wegmann S, Eftekharzadeh B, Tepper K, Zoltowska KM, Bennett RE, Dujardin S, Laskowski PR, MacKenzie D, Kamath T, Commins C, Vanderburg C, Roe AD, Fan Z, Molliex AM, Hernandez‐Vega A, Muller D, Hyman AA, Mandelkow E, Taylor JP, et al. (2018) Tau protein liquid–liquid phase separation can initiate tau aggregation. EMBO J. e98049: 1–21
  5. Brangwynne CP, Tompa P and Pappu R V. (2015) Polymer physics of intracellular phase transitions. Nat. Phys. 11: 899–904
  6. Nikolic J, Le Bars R, Lama Z, Scrima N, Lagaudrière-Gesbert C, Gaudin Y and Blondel D (2017) Negri bodies are viral factories with properties of liquid organelles. Nat. Commun. 8: 58

Tags: amyloid fibrils, henipavirus, intrinsically disordered proteins, phase separation

Posted on: 11th January 2019

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