Myristoylation licenses disordered viral VP4 protein to anchor to and perforate the membrane through phase separation
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eLife Assessment
This valuable study combines multiscale molecular simulations with supporting biophysical experiments to investigate how the myristoylated VP4 peptide of non-enveloped viruses interacts with host membranes during viral entry. The authors show that myristoylation facilitates VP4 membrane anchoring, condensate formation, and membrane remodeling events linked to early stages of membrane breaching. The work provides a convincing biophysical framework for understanding myristoylation-dependence in membrane-penetrating proteins.
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Abstract
The VP4 protein of enteroviruses, such as Coxsackievirus B3, is a small, intrinsically disordered protein essential for perforating the host cell membrane during viral entry. A key feature of VP4 is its N-terminal myristoylation, which is required for infectivity in some enteroviruses but dispensable in others, suggesting a complex and context-dependent role that is not fully understood. The precise biophysical mechanisms by which this lipid anchor enables a disordered protein to breach a membrane remain unresolved. Here, using Coxsackievirus B3 VP4 as a model system and integrating multi-scale molecular dynamics simulations with confocal microscopy, we demonstrate that myristoylation is not a simple membrane tether but a multi-functional regulator that orchestrates VP4 activity through distinct, hierarchical roles. First, it provides the necessary hydrophobic anchor to recruit the disordered VP4 to the membrane interface. Second, the myristoyl group acts as a key molecular driver that promotes the liquid-liquid phase separation of VP4, leading to the formation of dynamic condensates on the membrane surface. These condensates actively remodel the membrane, generating substantial curvature that, in turn, lowers the free energy barrier for VP4 penetration. Furthermore, we find evidence that the myristoyl group plays a third role in stabilizing the final transmembrane pore. Our findings establish a novel paradigm where a single lipid modification empowers a disordered viral protein to form a functionally potent condensate that mechanically primes and physically breaches the target membrane, a mechanism that may explain the conditional myristoylation requirement across enteroviruses.
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eLife Assessment
This valuable study combines multiscale molecular simulations with supporting biophysical experiments to investigate how the myristoylated VP4 peptide of non-enveloped viruses interacts with host membranes during viral entry. The authors show that myristoylation facilitates VP4 membrane anchoring, condensate formation, and membrane remodeling events linked to early stages of membrane breaching. The work provides a convincing biophysical framework for understanding myristoylation-dependence in membrane-penetrating proteins.
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Reviewer #1 (Public review):
This manuscript investigates the conformational flexibility and membrane-interaction behavior of the N-terminal segment of the VP4 protein from non-enveloped viruses, such as Coxsackievirus B3, with particular emphasis on the role of myristoylation, an essential process implicated in viral entry and transmission. The authors employ a multiscale simulation framework, combining all-atom (AA) and coarse-grained (CG) molecular dynamics simulations, to characterize the behavior of VP4 peptides in both bulk aqueous and membrane environments.
AA simulations suggest that the VP4 N-terminus remains predominantly disordered in bulk water, whereas CG simulations highlight the importance of conformational flexibility during interactions with a POPC membrane. The CG approach is further used to demonstrate an enhanced …
Reviewer #1 (Public review):
This manuscript investigates the conformational flexibility and membrane-interaction behavior of the N-terminal segment of the VP4 protein from non-enveloped viruses, such as Coxsackievirus B3, with particular emphasis on the role of myristoylation, an essential process implicated in viral entry and transmission. The authors employ a multiscale simulation framework, combining all-atom (AA) and coarse-grained (CG) molecular dynamics simulations, to characterize the behavior of VP4 peptides in both bulk aqueous and membrane environments.
AA simulations suggest that the VP4 N-terminus remains predominantly disordered in bulk water, whereas CG simulations highlight the importance of conformational flexibility during interactions with a POPC membrane. The CG approach is further used to demonstrate an enhanced aggregation tendency of myristoylated VP4 monomers compared to non-myristoylated forms and to estimate the free-energy barriers associated with VP4 translocation across the membrane in monomeric and aggregated states. The study proposes a connection between VP4 aggregation, membrane remodeling, and peptide insertion into the membrane. Finally, well-tempered metadynamics simulations are used to explore changes in VP4 helicity during pore formation.
Overall, the study addresses an important problem and applies appropriate computational approaches. However, several aspects of the methodology, interpretation of results, and consistency with existing literature require clarification before the conclusions can be fully supported. The authors should revise the manuscript with due attention to the comments below.
(1) Disordered State of VP4 in Bulk Water
Figures 1(f-g, i-j) indicate that both myristoylated and non-myristoylated VP4 peptides adopt largely disordered conformations in bulk water. This finding appears to contradict prior experimental and computational reports discussed in the Introduction, which suggest partial or transient helicity in this region. A more detailed explanation is required to reconcile these differences with the existing literature. Additionally, since α-RMSD (aRMSD) is a direct and quantitative measure of helicity, the authors may consider reporting helical content explicitly using this metric to strengthen the analysis.
(2) Lack of Backmapped Atomistic Data for Membrane-Bound States
Figure 2 presents membrane-bound conformations of VP4 obtained from CG simulations. While this provides useful qualitative insight, the absence of backmapped all-atom representations limits the ability to extract detailed information regarding residue-level interactions, peptide conformations, and specific binding modes at the membrane interface. Inclusion and analysis of backmapped atomistic data would significantly strengthen the mechanistic interpretation of VP4-membrane interactions.
(3) VP4 Binding to Membrane
Figure 2(H): The key takeaway from the exercise using multiple different rigidity for the peptide was that the different sections of the peptide have reduced membrane contacts, particularly the N-terminus. However, the contribution from each membrane component is not very apparent due to stacked transparent plots. Re-plotting using bars placed side to side or using a line representation will help to make this clearer.
(4) Aggregation Stability in Bulk Versus Membrane Environments
The manuscript states that the aggregation rate and stability of VP4 20-mers in bulk water are weaker than in the presence of a membrane, as shown in Figure S5. However, no clear or significant reduction in aggregation stability is apparent from the figure as currently presented. The authors should clarify which quantitative metrics support this claim and, if necessary, provide additional analysis to substantiate the reported difference.
(5) Decoding the Role of MYR on the VP4 n-mer Aggregation
The authors have suggested that the MYR tail plays a key role in the recruitment of VP4 peptides into the aggregate. This is based solely on visual evidence from the simulation. This can be tested directly by using a combination of MYR and non-MYR VP4 molecules, with MYR VP4 acting as membrane anchors. The change in aggregation rate or the number of clusters will give a more complete picture of this phenomenon. In the case of 20 non-MYR VP4 peptides, the aggregate forms within 2 µs, which is comparable to the complete aggregation in the case of MYR-VP4 6-mer. This further brings into question whether the faster aggregation for MYR cases is due to the proximity to the membrane or due to the lipid recruitment aspect of the MYR group.
(6) Interpretation of Umbrella Sampling Results and Membrane Remodeling
Figure 4 reports CG umbrella sampling results indicating a reduced translocation free-energy barrier for VP4 in aggregated (condensate) form, which is linked to membrane curvature and remodeling. Additional methodological details are required to support this interpretation:
(a) What is the nature of the membrane used in the umbrella sampling simulations? Specifically, was the membrane initially flat or curved, and was the same membrane (with identical curvature and properties) used for the single, 6-mer, and 20-mer cases? Differences in membrane geometry would directly influence the translocation free-energy profiles.
(b) Additional details regarding the peptide models used in umbrella sampling simulations should be provided, including peptide length, aggregation state definition, restraints applied (if any), and reference configurations, to improve clarity.(7) VP4 n-mer Condensate Dynamics
The authors have performed an autocorrelation analysis of Rg of VP4 in the 6 and 20-mer condensates and found that the decay is slower in the 6-mer. This suggests a higher degree of rearrangement within the VP4 20-mer. This could be due to a faster relaxation time upon formation for the 6-mer compared to the 20-mer owing to its smaller size. It would be informative to look at whether these differences still hold when the 20-mer simulations are extended beyond 10 µs.
(8) Comparison Between Metadynamics and Backmapped Membrane-Bound Structures
Figure 5 presents Well-Tempered Metadynamics results for VP4 in a membrane environment. To strengthen the conclusions regarding peptide binding and conformational behavior, it would be valuable to directly compare the peptide conformations and interaction characteristics observed in the Metadynamics simulations with those obtained from the backmapped structures corresponding to Figure 2.
(9) Interpretation of the Z-Coordinate in Free-Energy Profiles
Figure 5(a) shows the free-energy landscape of the VP4 peptide as a function of reaction coordinates. However, the corresponding Z-position of the peptide relative to the membrane is not clearly defined. The authors should clarify whether the reported Z-values correspond to peptide conformations at the membrane surface, within the hydrophobic core, or fully translocated across the membrane, as this is essential for proper interpretation of the free-energy minima.
(10) Helicity in Bulk Water from Metadynamics Simulations
Figure 5(b) shows a free-energy minimum at relatively high helicity (~0.6) even at a peptide-membrane distance of approximately 3.6 nm, which appears to correspond to a bulk-water-like environment. This observation contradicts the predominantly disordered peptide behavior reported in bulk water simulations (Figure 1). The authors should provide a mechanistic explanation for this inconsistency between the bulk AA simulations and the Metadynamics results.
(11) Folding and Insertion Free Energy of VP4
The free energy calculation for folding of VP4 using metadynamics in the POPC membrane and the 2D free energy calculated using umbrella sampling do not show the same picture. As in the first case, the deeper insertion into the membrane promotes a higher helicity, which is not present in the 2D free energy landscape. Assuming the same scale bar for the free energy between the two plots, as that is not mentioned for the free energy obtained from the metadynamics simulations, we see a massive preference towards a helicity fraction of >0.6. This is absent, both in the aqueous and the membrane-embedded environment of the 2D free energy simulations. It will also be useful to mention the plane of the phosphate groups to demarcate the hydrophilic and hydrophobic sections of the membrane
Final Recommendation
The manuscript presents interesting and potentially impactful findings on the conformational dynamics and membrane interactions of VP4. However, substantial clarification and additional analysis addressing the points above are required to ensure consistency, rigor, and alignment with existing literature. I recommend major revisions.b
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Reviewer #2 (Public review):
Summary:
The authors Huang et al. studied how a small disordered VP4 protein present in the viral capsid of naked viruses, such as Coxsackievirus B3, enables the transfer of the viral genome into the host cell by breaching the host cell membrane. The authors show that post-translational myristoylation of VP4 plays a critical role in this process. Using computer simulations of VP4 and its interactions with the membrane, the authors show that myristoylated VP4 anchors to the membrane faster, aggregates faster to form dense phases via LLPS, and remodels the membrane, thereby lowering the energy barrier for the protein to insert into the membrane. The authors further showed, through simulations, that the myristoylated VP4 forms helices within the membrane with higher stability, which then form structured pores, …
Reviewer #2 (Public review):
Summary:
The authors Huang et al. studied how a small disordered VP4 protein present in the viral capsid of naked viruses, such as Coxsackievirus B3, enables the transfer of the viral genome into the host cell by breaching the host cell membrane. The authors show that post-translational myristoylation of VP4 plays a critical role in this process. Using computer simulations of VP4 and its interactions with the membrane, the authors show that myristoylated VP4 anchors to the membrane faster, aggregates faster to form dense phases via LLPS, and remodels the membrane, thereby lowering the energy barrier for the protein to insert into the membrane. The authors further showed, through simulations, that the myristoylated VP4 forms helices within the membrane with higher stability, which then form structured pores, disrupting the membrane and enabling the transfer of the viral genome into the host cell.
Strengths:
The strength of the manuscript is that different sets of unbiased and enhanced-sampling simulations using all-atom and coarse-grained models of the protein and membrane are performed to bridge multiple time and length scales involved in the transfer of the viral genome into the host cell. There is experimental support for most of the conclusions arrived at from the simulations.
Weaknesses:
The drawback is that experimental evidence was lacking to support the pore-formation proposal from the simulations.
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