Defining basic rules for hardening influenza A virus liquid condensates

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1. 

   Version published to 10.7554/elife.85182 on eLife

   Apr 4, 2023
2. 

   eLife

   Mar 12, 2023

   Author Response

   Reviewer #1 (Public Review):

   The authors have tried to correlate changes in the cellular environment by means of altering temperature, the expression of key cellular factors involved in the viral replication cycle, and small molecules known to affect key viral protein-protein interactions with some physical properties of the liquid condensates of viral origin. The ideas and experiments are extremely interesting as they provide a framework to study viral replication and assembly from a thermodynamic point of view in live cells.

   The major strengths of this article are the extremely thoughtful and detailed experimental approach; although this data collection and analysis are most likely extremely time-consuming, the techniques used here are so simple that the main goal and idea of the article become elegant. A second …

   Author Response

   Reviewer #1 (Public Review):

   The authors have tried to correlate changes in the cellular environment by means of altering temperature, the expression of key cellular factors involved in the viral replication cycle, and small molecules known to affect key viral protein-protein interactions with some physical properties of the liquid condensates of viral origin. The ideas and experiments are extremely interesting as they provide a framework to study viral replication and assembly from a thermodynamic point of view in live cells.

   The major strengths of this article are the extremely thoughtful and detailed experimental approach; although this data collection and analysis are most likely extremely time-consuming, the techniques used here are so simple that the main goal and idea of the article become elegant. A second major strength is that in other to understand some of the physicochemical properties of the viral liquid inclusion, they used stimuli that have been very well studied, and thus one can really focus on a relatively easy interpretation of most of the data presented here.

   There are three major weaknesses in this article. The way it is written, especially at the beginning, is extremely confusing. First, I would suggest authors should check and review extensively for improvements to the use of English. In particular, the abstract and introduction are extremely hard to understand. Second, in the abstract and introduction, the authors use terms such as "hardening", "perturbing the type/strength of interactions", "stabilization", and "material properties", for just citing some terms. It is clear that the authors do know exactly what they are referring to, but the definitions come so late in the text that it all becomes confusing. The second major weakness is that there is a lack of deep discussion of the physical meaning of some of the measured parameters like "C dense vs inclusion", and "nuclear density and supersaturation". There is a need to explain further the physical consequences of all the graphs. Most of them are discussed in a very superficial manner. The third major weakness is a lack of analysis of phase separations. Some of their data suggest phase transition and/or phase separation, thus, a more in-deep analysis is required. For example, could they calculate the change of entropy and enthalpy of some of these processes? Could they find some boundaries for these transitions between the "hard" (whatever that means) and the liquid?

   The authors have achieved almost all their goals, with the caveat of the third weakness I mentioned before. Their work presented in this article is of significant interest and can become extremely important if a more detailed analysis of the thermodynamics parameters is assessed and a better description of the physical phenomenon is provided.

   We thank you for the comments and, in particular, for being so positive regarding the strengths of our manuscript and for raising concerns that will surely improve it. We have taken the following actions to address your concerns:

   1. Extensive revisions have been made to the use of English, particularly in the abstract and introduction. Key terms are defined as they are introduced in the text to enhance the clarity of the argument. This is a significant revision that is highlighted within the text, but it is too extensive to detail here.

   2. In the results section, we improved and extended the discussion of our graphs to the extent possible. However, we found that attempting to explain the graphs' meanings more thoroughly would detract from our manuscript's main focus: identifying thermodynamic changes that could potentially lead to alterations in material properties, specifically aspect ratio, size, and Gibbs free energy. As a result, we introduced the type of information we could obtain from our analyses in the introduction (Lines 112-125) and briefly commented on it in the ‘results’ section (Lines 304-306, sentences below).

   From introduction – lines 112-125:

   “In addition, other parameters like nucleation density determine how many viral condensates are formed per area of cytosol. Overall, the data will inform us if changing one parameter, e.g. the concentration, drives the system towards larger condensates with the same or more stable properties, or more abundant condensates that are forced to maintain the initial or a different size on account of available nucleation centres (Riback et al., 2020:Snead, 2022 #1152). It will also inform us if liquid viral inclusions behave like a binary or a multi-component system. In a binary mixture, Cdilute is constant (Klosin et al., 2020). However, in multi-component systems, Cdilute increases with bulk concentration (Riback et al., 2020). This type of information could have direct implications about the condensates formed during influenza infection. As the 8 different genomic vRNPs have a similar overall structure, they could, in theory, behave as a binary system between units of vRNPs and Rab11a. However, a change in Cdilute with concentration would mean that the system behaves as a multi-component system. This could raise the hypothesis that the differences in length, RNA sequence and valency that each vRNP has may be relevant for the integrity and behaviour of condensates.”.

   From results lines 304-306:

   This indicates that the liquid inclusions behave as a multi-component system and allow us to speculate that the differences in length, RNA sequence and valency that each vRNP may be key for the integrity and behaviour of condensates.

   1. The reviewer has drawn our attention to the absence of phase separation analysis in our study. We believe that the formation of influenza A virus condensates is governed by phase separation (or percolation coupled to phase separation). However, we must exercise caution at this point because the condensates we are studying are highly complex, and the physics of our cellular system may not be adequate to claim phase separation without being validated by an in vitro reconstitution system. IAV inclusions contain a variety of cellular membranes, different vRNPs, and Rab11a. While we have robust data to propose a model in which the liquid-like properties of IAV inclusions arise from a network of interacting vRNPs that bridge multiple cognate vRNP-Rab11 units on flexible membranes, similar to what occurs in phase-separated vesicles in neurological synapses, our model for this system still lacks formal experimental validation. As a note, the data supporting our model includes: the demonstration of the liquid properties of our liquid inclusions (Alenquer et al. 2019, Nature Communications, 10, 1629); and impairment of recycling endocytic activity during IAV infection Bhagwat et al. 2020, Nat Commun, 11, 23; Kawaguchi et al. 2012, J Virol, 86, 11086-95; Vale-costa et al. 2016, J Cell Sci, 129, 1697-710. This leads to aggregated vesicles seen by correlative light and electron microscopy (Vale-Costa et al., 2016 JCS, 129, 1697-710) and by immunofluorescence and FISH (Amorim et al. 2011,. J Virol 85, 4143-4156; Avilov et al. 2012, Vaccine 30, 7411-7417; Chou et al. 2013, PLoS Pathog 9, e1003358; Eisfeld et al. 2011, J Virol 85, 6117-6126 and Lakdawala et al. 2014, PLoS Pathog 10, e1003971.

   To be able to explore the significance of the liquid material properties of IAV inclusions, we used the strategy described in this current work. By developing an effective method to manipulate the material properties of IAV inclusions, we provide evidence that controlled phase transitions can be induced, resulting in decreased vRNP dynamics in cells and a negative impact on progeny virion production. This suggests that the liquid character of liquid inclusions is important for their function in IAV infection. We have improved our explanation addressing this concern in the limitations of our study (as outlined below in the box and in manuscript in lines 857-872).

   We are currently establishing an in vitro reconstitution system to formally demonstrate, in an independent publication, that IAV inclusions are formed by phase separation (or percolation coupled to phase separation). For this future work, we teamed up with Pablo Sartori, a theorical physicist to derive in-depth analysis of the thermodynamics of the viral liquid condensates in the in vitro reconstituted system and compare it to results obtained in the cell. This will provide means to establish comparisons. We think that cells have too many variables to derive meaningful physics parameters (such as entropy and enthalpy) and models that need to be complemented by in vitro systems. For example, increasing the concentration inside a cell is not a simple endeavour as it relies on cellular pathways to deliver material to a specific place. At the same time, the 8 vRNPs, as mentioned above, have different size, valency and RNA sequence and can behave very differently in the formation of condensates and maintenance of their material properties. Ideally, they should be analysed individually or in selected combinations. For the future, we will combine data from in vitro reconstitution systems and cells to address this very important point raised by the reviewer.

   From the paper on the section ‘Limitations of the study’:

   “Understanding condensate biology in living cells is physiological relevant but complex because the systems are heterotypic and away from equilibria. This is especially challenging for influenza A liquid inclusions that are formed by 8 different vRNP complexes, which although sharing the same structure, vary in length, valency, and RNA sequence. In addition, liquid inclusions result from an incompletely understood interactome where vRNPs engage in multiple and distinct intersegment interactions bridging cognate vRNP-Rab11 units on flexible membranes (Chou et al., 2013, Gavazzi et al., 2013, Sugita et al., 2013, Shafiuddin and Boon, 2019, Haralampiev et al., 2020, Le Sage et al., 2020). At present, we lack an in vitro reconstitution system to understand the underlying mechanism governing demixing of vRNP-Rab11a-host membranes from the cytosol. This in vitro system would be useful to explore how the different segments independently modulate the material properties of inclusions, explore if condensates are sites of IAV genome assembly, determine thermodynamic values, thresholds accurately, perform rheological measurements for viscosity and elasticity and validate our findings. The results could be compared to those obtained in cell systems to derive thermodynamic principles happening in a complex system away from equilibrium. Using cells to map how liquid inclusions respond to different perturbations provide the answer of how the system adapts in vivo, but has limitations.

   Reviewer #2 (Public Review):

   During Influenza virus infection, newly synthesized viral ribonucleoproteins (vRNPs) form cytosolic condensates, postulated as viral genome assembly sites and having liquid properties. vRNP accumulation in liquid viral inclusions requires its association with the cellular protein Rab11a directly via the viral polymerase subunit PB2. Etibor et al. investigate and compare the contributions of entropy, concentration, and valency/strength/type of interactions, on the properties of the vRNP condensates. For this, they subjected infected cells to the following perturbations: temperature variation (4, 37, and 42{degree sign}C), the concentration of viral inclusion drivers (vRNPs and Rab11a), and the number or strength of interactions between vRNPs using nucleozin a well-characterized vRNP sticker. Lowering the temperature (i.e. decreasing the entropic contribution) leads to a mild growth of condensates that does not significantly impact their stability. Altering the concentration of drivers of IAV inclusions impact their size but not their material properties. The most spectacular effect on condensates was observed using nucleozin. The drug dramatically stabilizes vRNP inclusions acting as a condensate hardener. Using a mouse model of influenza infection, the authors provide evidence that the activity of nucleozin is retained in vivo. Finally, using a mass spectrometry approach, they show that the drug affects vRNP solubility in a Rab11a-dependent manner without altering the host proteome profile

   The data are compelling and support the idea that drugs that affect the material properties of viral condensates could constitute a new family of antiviral molecules as already described for the respiratory syncytial virus (Risso Ballester et al. Nature. 2021)

   Nevertheless, there are some limitations in the study. Several of them are mentioned in a dedicated paragraph at the end of a discussion. This includes the heterogeneity of the system (vRNP of different sizes, interactions between viral and cellular partners far from being understood), which is far from equilibrium, and the absence of minimal in vitro systems that would be useful to further characterize the thermodynamic and the material properties of the condensates.

   There are other ones.

   We thank reviewer 2 for highlighting specific details that need improving and raising such interesting questions to validate our findings. We have addressed the comments of Reviewer 2, we performed the experiments as described (in blue) below each point raised.

   1. The concentrations are mostly evaluated using antibodies. This may be correct for Cdilute. However, measurement of Cdense should be viewed with caution as the antibodies may have some difficulty accessing the inner of the condensates (as already shown in other systems), and this access may depend on some condensate properties (which may evolve along the infection). This might induce artifactual trends in some graphs (as seen in panel 2c), which could, in turn, affect the calculation of some thermodynamic parameters.

   The concern of using antibodies to calculate Cdense is valid, and we thought it was very important. We addressed this concern by performing the same analyses using a fluorescent tagged virus that has mNeon Green fused to the viral polymerase PA (PA-mNeonGreen PR8 virus). Like NP, PA is a component of vRNPs and labels viral inclusions, colocalising with Rab11 when vRNPs are in the cytosol. However, per vRNP there is only one molecule of PA, whilst of NP there are 37-96 depending on the size of vRNPs. As predicted, we did observe changes in the Cdilute, Cdense and nucleation density. However, the measurements and values obtained for Gibbs free energy, size, aspect ratio detecting viral inclusions with fluorescently tagged vRNPs or antibody staining followed the same trend and allow us to validate our conclusion that major changes in Gibbs free energy occur solely when there is a change in the valency/strength of interactions but not in temperature or concentration (Figure 1 below). Given the extent of these data, we show here the results but, in the manuscript, we will describe the limitations of using antibodies in our study within the section ‘Limitations of the study’ from lines 881-894. Given the importance of the question regarding the pros and cons of the different systems for analysing thermodynamic parameters, we have decided to systematically assess and explore these differences in detail in a future manuscript.

   For more information. This reviewer may be asking why we did not use the PA-fluorescent virus in the first place to evaluate inclusion thermodynamics and avoid problems in accessibility that antibodies may have to get deep into large inclusions. Our answer is that no system is perfect. In the case of the PA-fluorescent virus, the caveats revolve around the fact that the virus is attenuated (Figure 1a below), exhibiting a delayed infection as demonstrated by reduced levels of viral proteins (Figure 1b below). Consistently, it shows differences in the accumulation of vRNPs in the cytosol and viral inclusions form later in infection and the amount of vRNPs in the cytosol does not reach the levels observed in PR8-WT virus. After their emergence, inclusions behave as in the wild-type virus (PR8-WT), fusing and dividing (Figure 1c below) and displaying liquid properties.

   As the overarching goal of this manuscript is to evaluate the best strategies to harden liquid IAV inclusions and given that one of the parameters we were testing is concentration, we reasoned that using PR8-WT virus for our analyses would be reasonable.

   In conclusions, both systems have caveats that are important to systematically assess, and these differences may shift or alter thermodynamic parameters such as nucleation density, inclusion maturation rate, Cdense, Cdilute in particular by varying the total concentration. As a note, to validate all our results using the PA-mNeonGreen PR8 virus, we considered the delayed kinetics and applied our thermodynamic analyses up to 20 hpi rather than 16 hpi.

   However, because of the question raised by this reviewer, on which is the best solution for mitigating errors induced by using antibodies, we re-checked all our data. Not only have we compared the data originated from attenuated fluorescently tagged virus with our data, but also made comparisons with images acquired from Z stacks (as used for concentration and for type/strength of interactions) with those acquired from 2D images. Our analysis revealed that there is a very good match using images acquired with Z-stacks and analysed as Z projections with between antibody staining and vRNP fluorescent virus. Therefore, we re-analysed all our thermodynamic data done with temperature using images acquired from Z stacks and altered entirely Figure 2. We believe that all these comparisons and analyses have greatly improved the manuscript and hence we thank all reviewers for their input.

   

   Figure 1 – The PA-mNeonGreen virus is attenuated in comparison to the WT virus and data obtained is consistent for Gibbs free energy with analyses done with images processed with antibody fluorescent vRNPs. A. Representation of the PA-mNeonGreen virus (PA-mNG; Abbreviations: NCR: non coding region). B. Cells (A549) were transfected with a plasmid encoding mCherry-NP and co-infected with PA-mNeonGreen virus for 16h, at an MOI of 10. Cells were imaged under time-lapse conditions starting at 16 hpi. White boxes highlight vRNPs/viral inclusions in the cytoplasm in the individual frames. The dashed white and yellow lines mark the cell nucleus and the cell periphery, respectively. The yellow arrows indicate the fission/fusion events and movement of vRNPs/ viral inclusions. Bar = 10 µm. Bar in insets = 2 µm. C-D. Cells (A549) were infected or mock-infected with PR8 WT or PA-mNG viruses, at a multiplicity of infection (MOI) of 3, for the indicated times. C. Viral production was determined by plaque assay and plotted as plaque forming units (PFU) per milliliter (mL) ± standard error of the mean (SEM). Data are a pool from 2 independent experiments. D. The levels of viral PA, NP and M2 proteins and actin in cell lysates at the indicated time points were determined by western blotting. (E-G) Biophysical calculations in cells infected with the PA-mNeonGreen virus upon altering temperature (at 10 hpi, evaluating the concentration of vRNPs (over a time course) in conditions expressing native amounts of Rab11a or overexpressing low levels of Rab11a and upon altering the type/strength of vRNP interactions by adding nucleozin at 10 hpi during the indicated time periods. All data: Ccytoplasm/Cnucleus; Cdense, Cdilute, area aspect ratio and Gibbs free energy are represented as boxplots. Above each boxplot, same letters indicate no significant difference between them, while different letters indicate a statistical significance at α = 0.05 using one-way ANOVA, followed by Tukey multiple comparisons of means for parametric analysis, or Kruskal-Wallis Bonferroni treatment for non-parametric analysis.

   1. Although the authors have demonstrated that vRNP condensates exhibit several key characteristics of liquid condensates (they fuse and divide, they dissolve upon hypotonic shock or upon incubation with 1,6-hexanediol, FRAP experiments are consistent with a liquid nature), their aspect ratio (with a median above 1.4) is much higher than the aspect ratio observed for other cellular or viral liquid compartments. This is intriguing and might be discussed.

   IAV inclusions have been shown to interact with microtubules and the endoplasmic reticulum, that confers movement, and undergo fusion and fission events. We propose that these interactions and movement impose strength and deform inclusions making them less spherical. To validate this assumption, we compared the aspect ratio of viral inclusions in the absence and presence of nocodazole (that abrogates microtubule-based movement). The data in figure 2 shows that in the presence of nocodazole, the aspect ratio decreases from 1.42±0.36 to 1.26 ±0.17, supporting our assumption.

   

   Figure 2 – Treatment with nocodazole reduces the aspect ratio of influenza A virus inclusions. Cells (A549) were infected with PR8 WT for 8 h and treated with nocodazole (10 µg/mL) for 2h, after which the movement of influenza A virus inclusions was captured by live cell imaging. Viral inclusions were segmented, and the aspect ratio measured by imageJ, analysed and plotted in R.

   1. Similarly, the fusion event presented at the bottom of figure 3I is dubious. It might as well be an aggregation of condensates without fusion.

   We have changed this (check Fig 5A and B in the manuscript), thank you for the suggestion.

   1. The authors could have more systematically performed FRAP/FLAPh experiments on cells expressing fluorescent versions of both NP and Rab11a to investigate the influence of condensate size, time after infection, or global concentrations of Rab11a in the cell (using the total fluorescence of overexpressed GFP-Rab11a as a proxy) on condensate properties.

   We have included a new figure, figure 5 with the suggested data.

   Read the original source
3. 

   eLife

   Jan 10, 2023

   Author Response

   We thank the reviewers for their positive feedback and thoughtful suggestions that will improve our manuscript. Here we summarise our plan for immediate action. We will resubmit our manuscript once additional experiments have been performed to clarify all the major and minor concerns of the reviewers and the manuscript has been revised. At that point, we will respond to all reviewer’s points and highlight the changes made in the text.

   Reviewer #1 (Public Review):

   The authors have tried to correlate changes in the cellular environment by means of altering temperature, the expression of key cellular factors involved in the viral replication cycle, and small molecules known to affect key viral protein-protein interactions with some physical properties of the liquid condensates of viral origin. The ideas and experiments …

   Author Response

   We thank the reviewers for their positive feedback and thoughtful suggestions that will improve our manuscript. Here we summarise our plan for immediate action. We will resubmit our manuscript once additional experiments have been performed to clarify all the major and minor concerns of the reviewers and the manuscript has been revised. At that point, we will respond to all reviewer’s points and highlight the changes made in the text.

   Reviewer #1 (Public Review):

   The authors have tried to correlate changes in the cellular environment by means of altering temperature, the expression of key cellular factors involved in the viral replication cycle, and small molecules known to affect key viral protein-protein interactions with some physical properties of the liquid condensates of viral origin. The ideas and experiments are extremely interesting as they provide a framework to study viral replication and assembly from a thermodynamic point of view in live cells.

   The major strengths of this article are the extremely thoughtful and detailed experimental approach; although this data collection and analysis are most likely extremely time-consuming, the techniques used here are so simple that the main goal and idea of the article become elegant. A second major strength is that in other to understand some of the physicochemical properties of the viral liquid inclusion, they used stimuli that have been very well studied, and thus one can really focus on a relatively easy interpretation of most of the data presented here.

   There are three major weaknesses in this article. The way it is written, especially at the beginning, is extremely confusing. First, I would suggest authors should check and review extensively for improvements to the use of English. In particular, the abstract and introduction are extremely hard to understand. Second, in the abstract and introduction, the authors use terms such as "hardening", "perturbing the type/strength of interactions", "stabilization", and "material properties", for just citing some terms. It is clear that the authors do know exactly what they are referring to, but the definitions come so late in the text that it all becomes confusing. The second major weakness is that there is a lack of deep discussion of the physical meaning of some of the measured parameters like "C dense vs inclusion", and "nuclear density and supersaturation". There is a need to explain further the physical consequences of all the graphs. Most of them are discussed in a very superficial manner. The third major weakness is a lack of analysis of phase separations. Some of their data suggest phase transition and/or phase separation, thus, a more in-deep analysis is required. For example, could they calculate the change of entropy and enthalpy of some of these processes? Could they find some boundaries for these transitions between the "hard" (whatever that means) and the liquid?

   The authors have achieved almost all their goals, with the caveat of the third weakness I mentioned before. Their work presented in this article is of significant interest and can become extremely important if a more detailed analysis of the thermodynamics parameters is assessed and a better description of the physical phenomenon is provided.

   We thank reviewer 1 for the comments and, in particular, for being so positive regarding the strengths of our manuscript and for raising concerns that will surely improve the manuscript. At this point, we propose the following actions to address the concerns of Reviewer 1:

   1. We will extensively revise the use of English, particularly, in the abstract and introduction, defining key terms as they come along in the text to make the argument clearer.

   2. We acknowledge the importance of discussing our data in more detail and we propose the following. We will discuss the graphs and what they mean as exemplified in the paragraph below.

   Regarding Figure 3 - As the concentration of vRNPs increases, we observe an increase in supersaturation until 12hpi. This means that contrary to what is observed in a binary mixture, in which the Cdilute is constant (Klosin et al., 2020), the Cdilute in our system increases with concentration. It has been reported that Cdilute increases in a multi-component system with bulk concentration (Riback et al., 2020). Our findings have important implications for how we think about the condensates formed during influenza infection. As the 8 different genomic vRNPs have a similar overall structure, they could, in theory, behave as a binary system between units of vRNPs and Rab11a. However, a change in Cdilute with concentration shows that our system behaves as a multi-component system. This means that the differences in length, RNA sequence and valency that each vRNP have are key for the integrity of condensates.

   1. The reviewer calls our attention to the lack of analysis of phase separations. We think that phase separation (or percolation coupled to phase separation) governs the formation of influenza A virus condensates. However, we think we ought to exert caution at this point as the condensates we are working with are very complex and that the physics of our system in cells may not be sufficient to claim phase separation without an in vitro reconstitution system. In fact, IAV inclusions contain cellular membranes, different vRNPs and Rab11a. So far, we can only speculate that the liquid character of IAV inclusions may arise from a network of interacting vRNPs that bridge several cognate vRNP-Rab11 units on flexible membranes, similarly to what happens in phase separated vesicles in neurological synapses. However, the speculative model for our system, although being supported by correlative light and electron microscopy, currently lacks formal experimental validation.

   For this reason, we thought of developing the current work as an alternative to explore the importance of the liquid material properties of IAV inclusions. By finding an efficient method to alter the material properties of IAV inclusions, we provide proof of principle that it is possible to impose controlled phase transitions that reduce the dynamics of vRNPs in cells and negatively impact progeny virion production. Despite having discussed these issues in the limitations of the study, we will make our point clearer.

   We are currently establishing an in vitro reconstitution system to formally demonstrate, in an independent publication, that IAV inclusions are formed by phase separation. For this future work, we teamed up with Pablo Sartori, a theorical physicist to derive in- depth analysis of the thermodynamics of the viral liquid condensates. Collectively, we think that cells have too many variables to derive meaningful physics parameters (such as entropy and enthalpy) as well as models and need to be complemented by in vitro systems. For example, increasing the concentration inside a cell is not a simple endeavour as it relies on cellular pathways to deliver material to a specific place. At the same time, the 8 vRNPs, as mentioned above, have different size, valency and RNA sequence and can behave very differently in the formation of condensates and maintenance of their material properties. Ideally, they should be analysed individually or in selected combinations. For the future, we will combine data from in vitro reconstitution systems and cells to address this very important point raised by the reviewer.

   From the paper on the section Limitations of the study: “Understanding condensate biology in living cells is physiologically relevant but complex because the systems are heterotypic and away from equilibria. This is especially challenging for influenza A liquid inclusions that are formed by 8 different vRNP complexes, which although sharing the same structure, vary in length, valency, and RNA sequence. In addition, liquid inclusions result from an incompletely understood interactome where vRNPs engage in multiple and distinct intersegment interactions bridging cognate vRNP-Rab11 units on flexible membranes (Chou et al., 2013; Gavazzi et al., 2013; Haralampiev et al., 2020; Le Sage et al., 2020; Shafiuddin & Boon, 2019; Sugita, Sagara, Noda, & Kawaoka, 2013). At present, we lack an in vitro reconstitution system to understand the underlying mechanism governing demixing of vRNP-Rab11a-host membranes from the cytosol. This in vitro system would be useful to explore how the different segments independently modulate the material properties of inclusions, explore if condensates are sites of IAV genome assembly, determine thermodynamic values, thresholds accurately, perform rheological measurements for viscosity and elasticity and validate our findings”.

   Reviewer #2 (Public Review):

   During Influenza virus infection, newly synthesized viral ribonucleoproteins (vRNPs) form cytosolic condensates, postulated as viral genome assembly sites and having liquid properties. vRNP accumulation in liquid viral inclusions requires its association with the cellular protein Rab11a directly via the viral polymerase subunit PB2. Etibor et al. investigate and compare the contributions of entropy, concentration, and valency/strength/type of interactions, on the properties of the vRNP condensates. For this, they subjected infected cells to the following perturbations: temperature variation (4, 37, and 42{degree sign}C), the concentration of viral inclusion drivers (vRNPs and Rab11a), and the number or strength of interactions between vRNPs using nucleozin a well-characterized vRNP sticker. Lowering the temperature (i.e. decreasing the entropic contribution) leads to a mild growth of condensates that does not significantly impact their stability. Altering the concentration of drivers of IAV inclusions impact their size but not their material properties. The most spectacular effect on condensates was observed using nucleozin. The drug dramatically stabilizes vRNP inclusions acting as a condensate hardener. Using a mouse model of influenza infection, the authors provide evidence that the activity of nucleozin is retained in vivo. Finally, using a mass spectrometry approach, they show that the drug affects vRNP solubility in a Rab11a-dependent manner without altering the host proteome profile.

   The data are compelling and support the idea that drugs that affect the material properties of viral condensates could constitute a new family of antiviral molecules as already described for the respiratory syncytial virus (Risso Ballester et al. Nature. 2021).

   Nevertheless, there are some limitations in the study. Several of them are mentioned in a dedicated paragraph at the end of a discussion. This includes the heterogeneity of the system (vRNP of different sizes, interactions between viral and cellular partners far from being understood), which is far from equilibrium, and the absence of minimal in vitro systems that would be useful to further characterize the thermodynamic and the material properties of the condensates.

   We thank reviewer 2 for highlighting specific details that need improving and raising such interesting questions to validate our findings. We will address all the minor comments of Reviewer 2. To address the comments of Reviewer 2, we propose the actions described in blue below each point raised that is written in italics.

   1. The concentrations are mostly evaluated using antibodies. This may be correct for Cdilute. However, measurement of Cdense should be viewed with caution as the antibodies may have some difficulty accessing the inner of the condensates (as already shown in other systems), and this access may depend on some condensate properties (which may evolve along the infection). This might induce artifactual trends in some graphs (as seen in panel 2c), which could, in turn, affect the calculation of some thermodynamic parameters.

   The concern of using antibodies to calculate Cdense is valid. We will address this concern by validating our results using a fluorescent tagged virus that has mNeon Green fused to the viral polymerase PA (PA-mNeonGreen PR8 virus). Like NP, PA is a component of vRNPs and labels viral inclusions, colocalising with Rab11 when vRNPs are in the cytosol without the need of using antibodies.

   This virus would be the best to evaluate inclusion thermodynamics, where it not an attenuated virus (Figure 1A below) with a delayed infection as demonstrated by the reduced levels of viral proteins (Figure 1B below). Consistently, it shows differences in the accumulation of vRNPs in the cytosol and viral inclusions form later in infection. After their emergence, inclusions behave as in the wild-type virus (PR8-WT), fusing and dividing (Figure 1C below) and displaying liquid properties. The differences in concentration may shift or alter thermodynamic parameters such as time of nucleation, nucleation density, inclusion maturation rate, Cdense, Cdilute. This is the reason why we performed the thermodynamics profiling using antibodies upon PR8-WT infection. For validating our results, and taking into account a possible delayed kinetics, and differenced that may occur because of reduced vRNP accumulation in the cytosol, this virus will be useful and therefore we will repeat the thermodynamics using it.

   As a side note, vRNPs are composed of viral RNA coated with several molecules of NP and each vRNP also contains 1 copy of the trimeric RNA dependent RNA polymerase formed by PA, PB1 and PB2. It is well documented that in the cytosol the vast majority of PA (and other components of the polymerase) is in the form of vRNPs (Avilov, Moisy, Munier, et al., 2012; Avilov, Moisy, Naffakh, & Cusack, 2012; Bhagwat et al., 2020; Lakdawala et al., 2014), and thus we can use this virus to label vRNPs on condensates to corroborate our studies using antibodies.

   

   Figure 1 – The PA- mNeonGreen virus is attenuated in comparison to the WT virus. A. Cells (A549) were infected or mock-infected with PR8 WT or PA- mNeonGreen (PA-mNG) viruses, at a multiplicity of infection (MOI) of 3, for the indicated times. Viral production was determined by plaque assay and plotted as plaque forming units (PFU) per milliliter (mL) ± standard error of the mean (SEM). Data are a pool from 2 independent experiments. B. The levels of viral PA, NP and M2 proteins and actin in cell lysates at the indicated time points were determined by western blotting. C. Cells (A549) were transfected with a plasmid encoding mCherry-NP and co-infected with PA-mNeonGreen virus for 16h, at an MOI of 10. Cells were imaged under time-lapse conditions starting at 16 hpi. White boxes highlight vRNPs/viral inclusions in the cytoplasm in the individual frames. The dashed white and yellow lines mark the cell nucleus and the cell periphery, respectively. The yellow arrows indicate the fission/fusion events and movement of vRNPs/ viral inclusions. Bar = 10 µm. Bar in insets = 2 µm.

   1. Although the authors have demonstrated that vRNP condensates exhibit several key characteristics of liquid condensates (they fuse and divide, they dissolve upon hypotonic shock or upon incubation with 1,6-hexanediol, FRAP experiments are consistent with a liquid nature), their aspect ratio (with a median above 1.4) is much higher than the aspect ratio observed for other cellular or viral liquid compartments. This is intriguing and might be discussed.

   IAV inclusions have been shown to interact with microtubules and the endoplasmic reticulum, that confers movement, and also undergo fusion and fission events. We propose that these interactions and movement impose strength and deform inclusions making them less spherical. To validate this assumption, we compared the aspect ratio of viral inclusions in the absence and presence of nocodazole (that abrogates microtubule-based movement). The data in figure 2 shows that in the presence of nocodazole, the aspect ratio decreases from 1.42±0.36 to 1.26 ±0.17, supporting our assumption.

   

   Figure 2 – Treatment with nocodazole reduces the aspect ratio of influenza A virus inclusions. Cells (A549) were infected PR8 WT and treated with nocodazole (10 µg/mL) for 2h time after which the movement of influenza A virus inclusions was captured by live cell imaging. Viral inclusions were segmented, and the aspect ratio measured by imageJ, analysed and plotted in R.

   1. Similarly, the fusion event presented at the bottom of figure 3I is dubious. It might as well be an aggregation of condensates without fusion.

   We will change this, thank you for the suggestion.

   1. The authors could have more systematically performed FRAP/FLAPh experiments on cells expressing fluorescent versions of both NP and Rab11a to investigate the influence of condensate size, time after infection, or global concentrations of Rab11a in the cell (using the total fluorescence of overexpressed GFP-Rab11a as a proxy) on condensate properties.

   We will try our best to be able to comply with this suggestion as we think it is important.

   Reviewer #3 (Public Review):

   This study aims to define the factors that regulate the material properties of the viral inclusion bodies of influenza A virus (IAV). In a cellular model, it shows that the material properties were not affected by lowering the temperature nor by altering the concentration of the factors that drive their formation. Impressively, the study shows that IAV inclusions may be hardened by targeting vRNP interactions via the known pharmacological modulator (also an IAV antiviral), nucleozin, both in vitro and in vivo. The study employs current state-of-the-art methodology in both influenza virology and condensate biology, and the conclusions are well-supported by data and proper data analysis. This study is an important starting point for understanding how to pharmacologically modulate the material properties of IAV viral inclusion bodies.

   We thank this reviewer for all the positive comments. We will address the minor issues brought to our attention entirely, including changing the tittle of the manuscript and we will investigate the formation and material properties of IAV inclusions in the presence and absence of nucleozin for the nucleozin escape mutant NP-Y289H.

   References

   Avilov, S. V., Moisy, D., Munier, S., Schraidt, O., Naffakh, N., & Cusack, S. (2012). Replication- competent influenza A virus that encodes a split-green fluorescent protein-tagged PB2 polymerase subunit allows live-cell imaging of the virus life cycle. J Virol, 86(3), 1433- 1448. doi:10.1128/JVI.05820-11

   Avilov, S. V., Moisy, D., Naffakh, N., & Cusack, S. (2012). Influenza A virus progeny vRNP trafficking in live infected cells studied with the virus-encoded fluorescently tagged PB2 protein. Vaccine, 30(51), 7411-7417. doi:10.1016/j.vaccine.2012.09.077

   Bhagwat, A. R., Le Sage, V., Nturibi, E., Kulej, K., Jones, J., Guo, M., . . . Lakdawala, S. S. (2020). Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association. Nat Commun, 11(1), 23. doi:10.1038/s41467-019-13838-3

   Chou, Y. Y., Heaton, N. S., Gao, Q., Palese, P., Singer, R. H., & Lionnet, T. (2013). Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis. PLoS Pathog, 9(5), e1003358. doi:10.1371/journal.ppat.1003358

   Gavazzi, C., Yver, M., Isel, C., Smyth, R. P., Rosa-Calatrava, M., Lina, B., . . . Marquet, R. (2013). A functional sequence-specific interaction between influenza A virus genomic RNA segments. Proc Natl Acad Sci U S A, 110(41), 16604-16609. doi:10.1073/pnas.1314419110

   Haralampiev, I., Prisner, S., Nitzan, M., Schade, M., Jolmes, F., Schreiber, M., . . . Herrmann, A. (2020). Selective flexible packaging pathways of the segmented genome of influenza A virus. Nat Commun, 11(1), 4355. doi:10.1038/s41467-020-18108-1

   Klosin, A., Oltsch, F., Harmon, T., Honigmann, A., Julicher, F., Hyman, A. A., & Zechner, C. (2020). Phase separation provides a mechanism to reduce noise in cells. Science, 367(6476), 464-468. doi:10.1126/science.aav6691

   Lakdawala, S. S., Wu, Y., Wawrzusin, P., Kabat, J., Broadbent, A. J., Lamirande, E. W., . . . Subbarao, K. (2014). Influenza a virus assembly intermediates fuse in the cytoplasm. PLoS Pathog, 10(3), e1003971. doi:10.1371/journal.ppat.1003971

   Le Sage, V., Kanarek, J. P., Snyder, D. J., Cooper, V. S., Lakdawala, S. S., & Lee, N. (2020). Mapping of Influenza Virus RNA-RNA Interactions Reveals a Flexible Network. Cell Rep, 31(13), 107823. doi:10.1016/j.celrep.2020.107823

   Riback, J. A., Zhu, L., Ferrolino, M. C., Tolbert, M., Mitrea, D. M., Sanders, D. W., . . . Brangwynne, C. P. (2020). Composition-dependent thermodynamics of intracellular phase separation. Nature, 581(7807), 209-214. doi:10.1038/s41586-020-2256-2

   Shafiuddin, M., & Boon, A. C. M. (2019). RNA Sequence Features Are at the Core of Influenza a Virus Genome Packaging. J Mol Biol. doi:10.1016/j.jmb.2019.03.018

   Sugita, Y., Sagara, H., Noda, T., & Kawaoka, Y. (2013). Configuration of viral ribonucleoprotein complexes within the influenza A virion. J Virol, 87(23), 12879- 12884. doi:10.1128/JVI.02096-13

   Read the original source
4. 

   eLife

   Jan 10, 2023

   eLife assessment

   The work presented is fundamental to the field of negative-sense RNA viruses. It showcases a compelling set of ideas and approaches to study some physicochemical properties of organelles in live cells during a viral infection. However, the conclusions could benefit by adding a deeper theoretical approach and additional experimental support.

   Read the original source
5. 

   eLife

   Jan 10, 2023

   Reviewer #1 (Public Review):

   The authors have tried to correlate changes in the cellular environment by means of altering temperature, the expression of key cellular factors involved in the viral replication cycle, and small molecules known to affect key viral protein-protein interactions with some physical properties of the liquid condensates of viral origin. The ideas and experiments are extremely interesting as they provide a framework to study viral replication and assembly from a thermodynamic point of view in live cells.

   The major strengths of this article are the extremely thoughtful and detailed experimental approach; although this data collection and analysis are most likely extremely time-consuming, the techniques used here are so simple that the main goal and idea of the article become elegant. A second major strength is that …

   Reviewer #1 (Public Review):

   The authors have tried to correlate changes in the cellular environment by means of altering temperature, the expression of key cellular factors involved in the viral replication cycle, and small molecules known to affect key viral protein-protein interactions with some physical properties of the liquid condensates of viral origin. The ideas and experiments are extremely interesting as they provide a framework to study viral replication and assembly from a thermodynamic point of view in live cells.

   The major strengths of this article are the extremely thoughtful and detailed experimental approach; although this data collection and analysis are most likely extremely time-consuming, the techniques used here are so simple that the main goal and idea of the article become elegant. A second major strength is that in other to understand some of the physicochemical properties of the viral liquid inclusion, they used stimuli that have been very well studied, and thus one can really focus on a relatively easy interpretation of most of the data presented here.

   There are three major weaknesses in this article. The way it is written, especially at the beginning, is extremely confusing. First, I would suggest authors should check and review extensively for improvements to the use of English. In particular, the abstract and introduction are extremely hard to understand. Second, in the abstract and introduction, the authors use terms such as "hardening", "perturbing the type/strength of interactions", "stabilization", and "material properties", for just citing some terms. It is clear that the authors do know exactly what they are referring to, but the definitions come so late in the text that it all becomes confusing. The second major weakness is that there is a lack of deep discussion of the physical meaning of some of the measured parameters like "C dense vs inclusion", and "nuclear density and supersaturation". There is a need to explain further the physical consequences of all the graphs. Most of them are discussed in a very superficial manner. The third major weakness is a lack of analysis of phase separations. Some of their data suggest phase transition and/or phase separation, thus, a more in-deep analysis is required. For example, could they calculate the change of entropy and enthalpy of some of these processes? Could they find some boundaries for these transitions between the "hard" (whatever that means) and the liquid?

   The authors have achieved almost all their goals, with the caveat of the third weakness I mentioned before. Their work presented in this article is of significant interest and can become extremely important if a more detailed analysis of the thermodynamics parameters is assessed and a better description of the physical phenomenon is provided.

   Read the original source
6. 

   eLife

   Jan 10, 2023

   Reviewer #2 (Public Review):

   During Influenza virus infection, newly synthesized viral ribonucleoproteins (vRNPs) form cytosolic condensates, postulated as viral genome assembly sites and having liquid properties. vRNP accumulation in liquid viral inclusions requires its association with the cellular protein Rab11a directly via the viral polymerase subunit PB2. Etibor et al. investigate and compare the contributions of entropy, concentration, and valency/strength/type of interactions, on the properties of the vRNP condensates. For this, they subjected infected cells to the following perturbations: temperature variation (4, 37, and 42{degree sign}C), the concentration of viral inclusion drivers (vRNPs and Rab11a), and the number or strength of interactions between vRNPs using nucleozin a well-characterized vRNP sticker. Lowering the …

   Reviewer #2 (Public Review):

   During Influenza virus infection, newly synthesized viral ribonucleoproteins (vRNPs) form cytosolic condensates, postulated as viral genome assembly sites and having liquid properties. vRNP accumulation in liquid viral inclusions requires its association with the cellular protein Rab11a directly via the viral polymerase subunit PB2. Etibor et al. investigate and compare the contributions of entropy, concentration, and valency/strength/type of interactions, on the properties of the vRNP condensates. For this, they subjected infected cells to the following perturbations: temperature variation (4, 37, and 42{degree sign}C), the concentration of viral inclusion drivers (vRNPs and Rab11a), and the number or strength of interactions between vRNPs using nucleozin a well-characterized vRNP sticker. Lowering the temperature (i.e. decreasing the entropic contribution) leads to a mild growth of condensates that does not significantly impact their stability. Altering the concentration of drivers of IAV inclusions impact their size but not their material properties. The most spectacular effect on condensates was observed using nucleozin. The drug dramatically stabilizes vRNP inclusions acting as a condensate hardener. Using a mouse model of influenza infection, the authors provide evidence that the activity of nucleozin is retained in vivo. Finally, using a mass spectrometry approach, they show that the drug affects vRNP solubility in a Rab11a-dependent manner without altering the host proteome profile.

   The data are compelling and support the idea that drugs that affect the material properties of viral condensates could constitute a new family of antiviral molecules as already described for the respiratory syncytial virus (Risso Ballester et al. Nature. 2021).

   Nevertheless, there are some limitations in the study. Several of them are mentioned in a dedicated paragraph at the end of a discussion. This includes the heterogeneity of the system (vRNP of different sizes, interactions between viral and cellular partners far from being understood), which is far from equilibrium, and the absence of minimal in vitro systems that would be useful to further characterize the thermodynamic and the material properties of the condensates.

   There are other ones.

   1. The concentrations are mostly evaluated using antibodies. This may be correct for Cdilute. However, measurement of Cdense should be viewed with caution as the antibodies may have some difficulty accessing the inner of the condensates (as already shown in other systems), and this access may depend on some condensate properties (which may evolve along the infection). This might induce artifactual trends in some graphs (as seen in panel 2c), which could, in turn, affect the calculation of some thermodynamic parameters.
   2. Although the authors have demonstrated that vRNP condensates exhibit several key characteristics of liquid condensates (they fuse and divide, they dissolve upon hypotonic shock or upon incubation with 1,6-hexanediol, FRAP experiments are consistent with a liquid nature), their aspect ratio (with a median above 1.4) is much higher than the aspect ratio observed for other cellular or viral liquid compartments. This is intriguing and might be discussed.
   3. Similarly, the fusion event presented at the bottom of figure 3I is dubious. It might as well be an aggregation of condensates without fusion.
   4. The authors could have more systematically performed FRAP/FLAPh experiments on cells expressing fluorescent versions of both NP and Rab11a to investigate the influence of condensate size, time after infection, or global concentrations of Rab11a in the cell (using the total fluorescence of overexpressed GFP-Rab11a as a proxy) on condensate properties.

   Read the original source
7. 

   eLife

   Jan 10, 2023

   Reviewer #3 (Public Review):

   This study aims to define the factors that regulate the material properties of the viral inclusion bodies of influenza A virus (IAV). In a cellular model, it shows that the material properties were not affected by lowering the temperature nor by altering the concentration of the factors that drive their formation. Impressively, the study shows that IAV inclusions may be hardened by targeting vRNP interactions via the known pharmacological modulator (also an IAV antiviral), nucleozin, both in vitro and in vivo. The study employs current state-of-the-art methodology in both influenza virology and condensate biology, and the conclusions are well-supported by data and proper data analysis. This study is an important starting point for understanding how to pharmacologically modulate the material properties of IAV …

   Reviewer #3 (Public Review):

   This study aims to define the factors that regulate the material properties of the viral inclusion bodies of influenza A virus (IAV). In a cellular model, it shows that the material properties were not affected by lowering the temperature nor by altering the concentration of the factors that drive their formation. Impressively, the study shows that IAV inclusions may be hardened by targeting vRNP interactions via the known pharmacological modulator (also an IAV antiviral), nucleozin, both in vitro and in vivo. The study employs current state-of-the-art methodology in both influenza virology and condensate biology, and the conclusions are well-supported by data and proper data analysis. This study is an important starting point for understanding how to pharmacologically modulate the material properties of IAV viral inclusion bodies.

   Read the original source
8. 

   Version published to 10.1101/2022.08.03.502602v2 on bioRxiv

   Nov 7, 2022
9. 

   Version published to 10.1101/2022.08.03.502602v1 on bioRxiv

   Aug 5, 2022