Interactions between the Molecular Components of the Cowpea

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Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

Interactions between the Molecular Components of the Cowpea Chlorotic Mottle Virus Investigated by Molecular Dynamics Simulations Jingzhi Chen,† Yves Lansac,‡ and Guillaume Tresset*,† †

Laboratoire de Physique des Solides, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France GREMAN, UMR 7347, CNRS, Université de Tours, 37200 Tours, France

‡

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S Supporting Information *

ABSTRACT: The formation of a viral particle generally involves hundreds of proteins, making the assembly process intricate. Despite its intrinsic complexity, the production of a viral particle begins through the interaction between the basic assembly components. For the cowpea chlorotic mottle virus (CCMV), the first steps of the assembly process involve dimers of the capsid protein. Here, we carried out atomistic molecular dynamics simulations to investigate the initial assembly process of CCMV to get insight into the interactions at the molecular level. We found that salinity not only affects the electrostatic interactions between dimers but also changes the conformation of the positively charged N-terminal tails and can cause a serious steric hindrance for other dimers binding to the hydrophobic domains. An RNA rod was used to mimic a long segment of a viral genome and to study its interaction with dimers. We observed that the dimer with tails prefers to bind on the RNA rod with its positively charged inner side. The dimer−RNA interaction was found to be as strong as the dimer−dimer interaction, whereas the association energies between a dimer and a pentamer or a hexamer of dimers were high but strongly depended on the presence of the tails. Upon heating, the capsid experienced a shrinkage accompanied by a loss of order in the icosahedral crystal structure.

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INTRODUCTION Viruses are known for infecting all classes of living organisms on Earth, whether vegetal or animal. Virions consist of a nucleic acid genome protected by a single or multilayered protein shell called capsid and in some cases by an envelope of lipids. The viral capsid is generally made of hundreds or thousands of proteins forming ordered structures. Half of all known viruses exhibit an icosahedral symmetry, the rest are helical, prolate, or have a complex irregular structure.1 Recently, viral particles have attracted increasing attention due to their extremely regular structure and their potential use for fabricating nanostructures with various functions.2−8 Therefore, understanding the assembly mechanisms underlying the production of viral particles is not only helpful to the development of inhibitors for therapeutic purpose but it should also open new routes for the self-assembly of complex supramolecular materials. Cowpea chlorotic mottle virus (CCMV) is an icosahedral plant virus, with a single-stranded RNA genome packaged inside a single-layered capsid consisting of 180 chemically identical proteins. The assembly process of CCMV has been intensively investigated these last years, yet a number of issues remain unsolved, especially at the molecular level. The assembly of empty CCMV capsid is usually described by a nucleation-and-growth mechanism,9 where the assembly begins with the formation of a nucleus, followed by the sequential addition of free dimers in the solution until the © XXXX American Chemical Society

completion of a full capsid. The growth stage can be readily probed by experimental techniques, such as time-course static light scattering,10,11 small-angle scattering techniques,12−15 size exclusion chromatography,10,16−18 and mass spectrometry,19−21 whereas less attention has been devoted to the nucleation stage because of its very transient nature. Analyses performed on assembly kinetics10,11 speculated that the pentamer of dimers is the nucleus.22 Yet there has been little further investigation on the role of the pentamer of dimers in the nucleation process. The viral genome not only carries the genetic information but also directs the assembly of viral particles. For CCMV in particular, the genome acts as a heterogeneous nucleus and effectively lowers the energy barrier to the capsid formation.23−25 There is no doubt that the presence of the genome alters the assembly pathway26 and makes it more complex. There are already several works on the influence of the genome on the formation of viral particles, notably through the charge ratio between the genome and the assembly subunits,27−29 such as the dimer for CCMV, or via the RNA secondary structure.30 The interaction energy between the RNA genome and the subunits as well as the molecular processes taking place during assembly has been rarely investigated. The lack of these Received: August 17, 2018 Revised: September 21, 2018

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DOI: 10.1021/acs.jpcb.8b08026 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

The modified structure of a single dimer was used as the initial conformation to probe the behavior of a dimer in water. The effect of ionic strength on the conformation of dimer is analyzed by carrying out three independent simulations at different NaCl concentrations (0.1, 0.2, and 0.5 M). An energy optimization was performed and followed by a 100 ps NVT and a 100 ps NPT equilibration. Each production run lasted 500 ns. Root mean square deviation (RMSD) was calculated for both the N-terminal tail and the body of the protein (the rest of the residues excluding the N-terminal tail) independently by aligning the simulated structure with its initial conformation. The radius of gyration of the N-terminal tails was determined using the GROMACS analysis tools as well over the last 300 ns trajectory. In addition, the contact map between the whole series of residues of a dimer was constructed by the GROMACS mdmat tools, which established distance matrices consisting of the smallest distance between residue pairs within 1.5 nm. Dimer Pair. The equilibrated conformation of a single dimer was employed as the initial conformation of dimers in this section. Two dimers were aligned in parallel with a separation of 7 nm. The systems were energy optimized before a 100 ps NVT and a 100 ps NPT equilibration. The first simulation was performed at salinity of 0.2 M, and the production run lasted 2.125 μs at 300 K. The association energy between two dimers was determined by calculation of the potential of mean field (PMF). The PMF was calculated by umbrella sampling method and analyzed by the weighted histogram analysis method.56 The details of the methodology can be found in the Supporting Information, where you can find the initial conformation applying to product conformations for sampling windows (Figure S2) and umbrella histograms (Figure S3). To examine the stability of the structure formed at 300 K, the temperature of the system was increased to 400 K in one step and kept for 1 μs. RNA−Dimer Complex. The viral genome is obviously too large to be studied by atomistic simulations, even considering the shortest segment, i.e., RNA4, which consists of 824 nucleotides.57 Thus, a much shorter RNA segment (50 nucleotides) taken from the RNA4 in the form of either a single strand or a double strand was used to study the interactions with a single dimer. To further simplify the problem, these RNA strands were fixed as helical rods during simulations. The dimer was located at a position 9 nm away from the center of RNA rod to allow the dimer to adjust its orientation and conformation before being captured by the RNA. The added tails of the dimer were deliberately set in a stretched conformation. The initial conformation is shown in Figure S4. Three systems consisting of different components were studied at 0.1 M. The first system consists of a normal dimer and a single-stranded RNA rod and acts as a reference system. A mutated dimer without the N-terminal tails was implemented in the second system to investigate the role played by these tails in the interaction with RNA. At last, the influence of the RNA secondary structure form was evaluated by changing the RNA rod into a double-stranded form in the third system. The simulations of the first two systems lasted for 1 μs, whereas the third one lasted for 800 ns. The final conformations were employed in SMD simulations to measure the rupture force required to unbind the dimer from the RNA rod. Five pulling simulations were conducted for each system.

information causes difficulty in assigning proper values to the subunit−RNA and subunit−subunit interaction energies in many analytical models.24,31,32 Molecular dynamics (MD) simulation is a helpful and complementary method and has been widely used in the study of various viruses.33−36 It can partly supply the missing information mentioned previously. To date, MD simulations have mainly focused on physical processes in subassemblies and capsids, including the interaction between capsid proteins and small molecules, which might act as antiviral drugs,37−41 the properties of a whole capsid,33−36,41−44 and the thermodynamic properties of the subunits.45 However, investigations on the assembly of subunits are rather limited, even with coarse-grained (CG) methods. In this article, we carry out all-atom MD simulations to shed light on the initial stage of the self-assembly of empty CCMV capsid and on the interaction strength between the relevant components. First, a single dimer is investigated with different salinities to obtain its equilibrium conformation, which is subsequently employed for the simulations on the interaction between a pair of dimers. We try next to elucidate the way dimers interact with RNA. Several simulations are conducted involving an infinitely long RNA rod and a short RNA strand. Meanwhile, the interaction strengths between different components is estimated via their rupture force determined by steered molecular dynamics (SMD) simulations. In the last section, the thermal shrinkage of viral capsid is investigated by CG simulations.

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METHODS Simulation Protocol. All simulations were performed with the GROMACS 5.1.4 simulation package,46 the atomistic Amber99sb-ILDN force field,47 and the TIP3 water model at pH 7. Simulations were carried out in a rectangular box with a size large enough so that the biomacromolecules in neighboring boxes could not interact with each other. The system was neutralized and adjusted to a specific ionic strength by adding NaCl salt. The simulations were conducted in NPT ensemble and the temperature was maintained at 300 K by the Berendsen weak-coupling method48 with coupling constants τT = 0.1 ps while the pressure was kept at 1 bar by the Parrinello− Rahman barostat49 with a time constant τP = 2.0 ps. For the nonbonded interactions, the short-range van der Waals and Coulomb interactions were cut off and shifted at 1.0 nm whereas the long-range Coulomb interactions were calculated by the particle mesh Ewald method50 with a grid spacing of 0.16 nm. A time step of 2 fs was applied throughout the simulations, and all bonds were constrained by the LINCS algorithm.51 Single Dimer. The initial structures used in this article were extracted from the CCMV capsid crystal structure resolved by Speir and co-workers52 (PDB: 1CWP). There are three kinds of protein in this structure noted A, B, and C. However, several residues, making up the N-terminal tail of the proteins, could not be solved by X-ray crystallography (25 residues for proteins A and C and 39 residues for protein B) due to their flexibility. Considering that the charged Nterminal tails have an indispensable role in the interaction between dimers, as reported by experiments,53 the structure of the missing residues were generated54 and manually added to the PDB structure by a package in VMD.55 The modified structures for a single dimer, pentamer, and hexamer of dimers can be found in Figure S1. B



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RESULTS Conformations of a Single Dimer. The conformations of a single dimer at different ionic strengths are depicted in Figure 1. Both the positively charged N-terminal tails and the body of a dimer presented a structural change at different levels.

In addition, another simulation was carried out to examine the interactions between the dimer and a flexible RNA segment (46 nucleotides) that possesses both double-stranded and single-stranded domains. The sequence of this RNA segment and its conformation after 200 ns can be found in Figure S5. The interactions between the RNA segment and a dimer were probed for 500 ns. Steered Molecular Dynamics (SMD) Simulations. The initial conformations of subassemblies for SMD simulations were produced by various ways. The conformation of the dimer−RNA complex was obtained from a simulation of the interaction of a dimer with an RNA segment, as described in the previous section. The conformation of the other subassemblies was obtained after 500, 100, and 100 ns simulations for a pair, a pentamer, and a hexamer of dimers, respectively. The interaction strength of a pentamer and a hexamer was investigated both for intact proteins as well as for proteins missing N-terminal tails. In the latter case, all monomers constituting the pentamer had their residues 2−39 cleaved, whereas for the hexamer, the residues 2−25 of the “inner” monomers forming the barrel structure and the residues 2−39 of the outer monomers were cleaved (see Figure S1). The pulling force constant was 1000 kJ mol−1 nm−1, and the pulling velocity was 10 Å ps−1. In addition, heavy atoms on the RNA rod have been restrained during the pulling. The maximum force of pulling curves was used to estimate the rupture force to unbind the specific components from the subassemblies. Five pulling simulations with different initial velocities were performed for each subassembly to obtain an average rupture force. Coarse-Grained MD Simulations. If a complete capsid is still too large to get statistically meaningful information with an all-atom model, CG models can help improve our understanding of the capsid stability under various conditions and allow comparisons with experiments.44,54 The MARTINI CG force field58,59 is one of the optimal options, demonstrating a good compromise between molecular details and simulation speed, and it has been applied to investigate viral systems, including CCMV.60 A 17.5 μs MD simulation at 300 K was first carried out to test the stability of a CCMV capsid modeled with the MARTINI CG force field. To mimic the thermal dissociation process of viral capsids, the temperature was increased up to 400 K for an additional 17 μs. The stability of the capsid was evaluated by analyzing the average radius of capsid and the distance distribution between the monomers on the capsid. CG simulations with MARTINI force field were performed with the GROMACS simulation package. The missing residues of the tails were not added, and the CG beads were restrained by the ELNEDYN elastic network61 with a spring constant of 200 kJ mol−1 nm−2 to prevent the unfolding of the polypeptide chains. Temperature and pressure were maintained at 300 or 400 K and 1 bar using the same thermostat and barostat as the ones used in atomistic simulations with τT = 1 ps and τp = 12 ps. The time step was 20 fs. The short-range van der Waals interactions were cut off and shifted at 1.1 nm, whereas Coulomb interactions were treated by the reaction field method62 with a short-range truncation at 1.1 nm and the introduction of a relative permittivitty constant εr = 15. All bonds were constrained by the LINCS algorithm. All CG simulations went through a 10 000-step energy optimization and 25 000-step equilibration.

Figure 1. RMSD of the tails and the body of a dimer with respect to their initial conformation at different ionic strengths.

According to the calculations of the root mean square deviation (RMSD) in Figure 1, the conformation of the body of the dimer remained stable during the simulation at all ionic strengths, which was in sharp contrast with a large conformational adjustment of the N-terminal tails. Nevertheless, as indicated on Figure S6A,B, the relative position and orientation between the bodies in the same dimer changed significantly. The orientation angle increased from 37 to 81°, and the separation increased from 2.8 to 3.4 nm. This phenomenon is observed at all studied ionic strengths, suggesting that the structure of the free dimer is looser than within a capsid. The conformations of the dimer at different salinities are displayed in Figure 2A where the N-terminal tails are highlighted in red. To quantify the sensitivity of the Nterminal tails to salt concentration, the radius of gyration of the N-terminal tails and their contact map with the bodies were calculated and are plotted in Figure 2B,C. Salinity has a strong impact on the conformation of the tails. At low salinities, the N-terminal tails presented a stretched conformation and easily bound on the mainly negatively charged body, as shown in Figure 2A. There was no specific part of the tails bound on the body; instead, the residues in contact were distributed evenly along the tails, as indicated from Figure 2C. In addition, it is surprising to observe that part of the N-terminal tails even covered the β-sheets where hydrophobic residues concentrate, leading to a decrease of the available hydrophobic area (the snapshot of 0.1 M in Figure 2A). Interestingly, this phenomenon disappeared by increasing the salinity to 0.5 M where the N-terminal tails became more compact (Figure 2B) and less likely to bind on the body (Figure 2C). The response of the N-terminal tails to the salinity suggests that the change in the ionic strength might not only affect the electrostatic interactions between dimers but also lead to the adjustment of their conformation, especially that of the N-terminal segments, and subsequently alter the assembly pathway. Self-Assembly of a Dimer Pair. The full understanding at an atomistic level of the interactions between two dimers is imperative to unveiling the self-assembly mechanism behind the formation of viral capsid. The low spatial resolution inherent to most experimental techniques is far from being enough to gain insight into the interaction details during the assembly process. To date, the conformation of the N-terminal tails in the CCMV dimer is still unclear, even more so, when a C


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Figure 2. Conformation of the tails of a dimer at different salinities. (A) Snapshots of the conformation of a dimer for salinities of 0.1, 0.2, and 0.5 M. The tails of a dimer (residues 2−39) are in red. (B) Radius of gyration of the tails versus salinity. (C) Contact map between the residue pairs in a dimer at three salinity values. The yellow areas are the contacts between a residue in the tails (residues 2−39) and a residue in the body (residues 40−190), whereas the blue and white areas are the contacts between residues belonging to the tails only and to the body only, respectively.

Figure 3. Simulations of a pair of dimers at an ionic strength of 0.2 M. (A) Evolution of the orientation angle α and the separation between the two dimers (Dcontact) at 300 K (left of the red dot-dashed line) and at 400 K (right of the red dot-dashed line). (B) Snapshot of a dimer pair at the end of the simulation at 300 K (left) and the native conformation of two dimers in the capsid (right). (C) Potential of mean force calculated by starting with the conformation obtained at the end of the simulation at 300 K. The inserted snapshot points out the pulling direction. (D) Superimposition of the simulated conformation (in cyan for the body, in red for the tails, and in gray for the aligned dimer) and the native conformation in the capsid (in green, tails are not shown) at 300 K (left) and 400 K (right), from two different viewing angles, front view (top) and top view (bottom).

angle between the dimers fluctuating around an angle of 120° and a separation between the centers of mass (COM) of the contacted proteins around 4 nm (see Figure 3B). The simulated conformation demonstrates that the N-terminal tails play a bridging role by connecting the two dimers in this case. However, this conformation is very different from the probable equilibrium conformation, the native conformation, whose orientation angle and COM separation are 60° and 2.9

dimer interacts with another one or with the genome. In this section, we focused on the interactions between a CCMV dimer pair, which is a very first step but also an indispensable one to comprehend the whole assembly process. The evolution of the separation and orientation between the dimers at an ionic strength of 0.2 M is depicted in Figure 3A. The dimer pair experienced a severe reorientation before finding a “stable” binding conformation with an orientation D


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Figure 4. Simulations of the interactions between a dimer and a piece of RNA. (A) Snapshot of a dimer (in green for the body and in red for the tails) bound on a free RNA segment (46 nucleotides) with two quasi-double-stranded domains at the end of the chain (in yellow) and a singlestranded domain in the middle (in cyan). The initial conformation of the RNA segment is obtained by a 200 ns simulation. (B) Rupture force to unbind a dimer from an RNA rod at 0.1 M. The numbers in x-axis represent the interacting systems: a dimer with a single-stranded RNA rod (1), a tail-cleaved dimer with a single-stranded RNA rod (2), and a dimer with a double-stranded RNA rod (3). The snapshots of the conformations of these systems are depicted in the right side with their front view and left side view.

nm, respectively (Figure 3B), indicating that the pair of dimers failed to reach their equilibrium state even after 2 μs. The difference can be clearly indicated by the conformation superimposition of the simulated conformation and the native conformation in Figure 3D. The association energy between two dimers is reported to range from −4 to −10kBT0,63−65 where kB is the Boltzmann constant and T0 is the room temperature. The association free energy was determined by calculating the PMF between the COM of the two dimers while their relative orientation is free. The calculation is carried out from the final conformation obtained at 300 K. As shown in Figure 3C, the association free energy of a pair of dimers is −8kBT0, i.e., within the range reported experimentally. This finding makes plausible our hypothesis that a strong association energy between the dimers leads to kinetically trapped conformations. With the expectation of overcoming the energy barrier, the conformation simulated at 300 K was brought to 400 K for 1 μs to allow for larger energy fluctuations. The dimers were stable at 400 K, as revealed by the RMSD of the body of dimers in Figure S7. As shown in Figure 3A, an immediate conformational adjustment occurred with the orientation angle decreasing from 120 to 90° and a closer contact formed (the separation goes from 4 to 3.5 nm). Figure 3D depicts a superimposition of the simulated conformations with the native conformation showing that the conformation obtained at 400 K is closer from the native one even though some discrepancies persist. The obstruction for narrowing this difference may come from the tails occupying the hydrophobic domains and inhibiting the access of other dimers, which will increase the difficulty to reproduce the native conformation. Interaction between RNA and Dimers. The genome of viruses is a reservoir of negative charges, which can promote the assembly of viral particles in many cases. In our previous work on the self-assembly of CCMV particles in the presence of RNA genome, we found that the RNA genome captured free dimers in the solution and formed disordered nucleoprotein complexes, which subsequently relaxed into viral particles through a structural self-organization.66 Despite the identification of the process, the way the dimers interact with the viral genome at the molecular level is still unclear. In this section, several simulations were carried out to attempt to clarify the mechanisms.

The viral genome has in fact a complex secondary structure and includes locally double-stranded segments and singlestranded loops. To identify the domains of the genome that the dimer prefers to bind on, a specific RNA chain possessing both double-stranded and single-stranded domains in its equilibrium conformation was extracted from the viral RNA4 segment and employed to interact with a dimer. In this simulation, the RNA was permitted to move freely to sample its translational, orientational, and conformational degrees of freedom without any position constraints. The RNA chain was first equilibrated for 200 ns, leading to a conformation with two quasi-double-stranded segments formed at the two ends of the chain connected by a single-stranded segment in the middle, as shown in Figure S5. The equilibrated RNA chain was then allowed to interact with a dimer, and the resulting complex conformation is displayed in Figure 4A. We found out that the dimer preferred to bind on the double-stranded domains via the N-terminal tails, which can be explained by the fact that double-stranded domains have a higher charge density. A simplified genome model was used to obtain the first insight into the conformation and the interaction strength of a dimer bound to different regions of RNA. More specifically, an RNA rod was employed to mimic a random segment of the whole RNA4 chain. The heavy atoms of the RNA rod were restrained, whereas the dimer was allowed to move freely. The RNA−dimer complex formed after 1 μs was utilized in SMD simulations to measure the rupture force to unbind the dimer from the RNA rod. These simulations were carried out in different conditions, and the results are shown in Figure 4B. As indicated by the evolution of the orientation angle and distance in Figure S8, the binding between the RNA rod and the dimer when occurring remained stable over the simulations. It is not surprising to find that a dimer has a stronger interaction (∼1068 kJ mol−1 nm−1) with a doublestranded RNA than with a single-stranded one (∼750 kJ mol−1 nm−1) owing to the stronger electrostatic attraction caused by a higher negative charge density. This finding agrees well with the simulation performed with a flexible RNA chain in Figure 4A. However, the dimer−RNA interaction will be greatly weakened to ∼506 kJ mol−1 nm−1 by removing the N-terminal tails that are highly positively charged and strongly involved in the interaction mechanism. E


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RNA was moderate. This fact can be rationalized by noticing that it is important for a virus to release easily its genetic material in the host cell, and therefore a relatively weak binding energy between the dimers and the RNA genome may be necessary for its own survival. Thermal Shrinkage of CCMV Capsid. The major driving force for the capsid self-assembly is the hydrophobic interaction between subunits. Yet due to its nature, the temperature will affect the strength of the hydrophobic interaction. In our previous thermal dissociation experiment of CCMV capsid by small-angle neutron scattering, we observed that the size of the capsid decreased from 14.5 nm at 20 °C to 11.5 nm at 65 °C at pD 7.5 due to the strengthened hydrophobic interaction at high temperature.64 To gain a molecular insight into this phenomenon, a CG simulation using the MARTINI force field was performed at a higher temperature, 400 K, to accelerate the shrinkage process. Meanwhile, a reference simulation to probe the stability of the native capsid was performed at room temperature (300 K). The capsid was stable at 300 K with a radius around 11.4 nm (Figure 6A). The distribution of COM distances between monomers within the capsid shows a regular pattern with distinctive peaks with only a slight loss of the long-range order (around a separation of 25 nm between monomers) due to thermal fluctuations (Figure 6B). In addition, the symmetry of the capsid, such as the 5-fold and 3-fold symmetry centers, were still conserved (see the snapshots in Figure 6A). In contrast, at 400 K, an apparent radius shrinkage of the capsid from 11.8 to 10.2 nm was observed (Figure 6C). Moreover, the capsid quickly lost its symmetry with a distribution of distances between monomers fading away, leaving only a shortrange order (Figure 6D). The strengthened hydrophobic interactions induce strong fluctuations of the monomers on the surface of capsids, leading subsequently to a disordered structure that finally will break apart.

It is unexpected that the stretched N-terminal tails in the initial conformations preferred to bind to the body of dimers first, instead of the negatively charged RNA rods, which are supposed to have a strong interaction with the positively charged tails. Another interesting thing is that the dimer with tails prefers to contact the RNA rod with its inner interface that is more positively charged (see Figure S9), a phenomenon that is not observed when interacting with a flexible RNA chain or removing its tails. The orientation of the dimer in this way will increase the side-by-side contact probability between the dimers, and given the fact that the side face of CCMV dimers is majorly dominated by hydrophobic domains, this suggests that this orientation will facilitate the assembly of dimer. Interaction Strengths of Subassemblies. Quantifying the strength of interactions between a dimer and other viral subassemblies or components should be helpful to understand better the assembly process of viral particles. Due to the large size of the relevant subassemblies and the limitation in computational resources, it is difficult to determine the interaction energies directly by calculating their PMFs. Nevertheless, we can obtain an estimate of the interactions by determining their rupture forces through SMD simulations. In this section, five typical subassemblies were analyzed: a pair of dimers, a dimer−RNA complex, a pentamer of dimers, and a hexamer of dimers. Their rupture forces are given in Figure 5. The weakest rupture force (∼750 kJ mol−1 nm−1)

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CONCLUSIONS By using atomistic MD simulations, we have investigated the interactions between a dimer and various subassemblies. The structure of a dimer is looser in solution than within a capsid, and the conformation of its N-terminal tails is sensitive to ionic strength. At low salinity, the N-terminal tails present a stretched conformation and slightly bind on the body of dimer covering a part of the hydrophobic domains. Subsequently, the binding of another dimer will require a conformational change of the tails so as to expose the hydrophobic domains. This change may be triggered by the presence of RNA on which the tails will preferentially bind, allowing thus the dimers association. N-terminal tails are involved in the self-assembly of dimers at low ionic strength: on one hand, they accelerate the association by bridging the dimers, but on the other hand, they can lead to kinetic traps and misassembly caused by a strong interaction with the body of the other dimer. We observed that pentamer and hexamer of dimers are stable subassemblies with a dimer requiring a strong rupture force to be extracted from them. This result is in agreement with the fact that an icosahedral capsid is made up of pentamers and hexamers of dimers and the integrity of the capsid is preserved through the solidity of these subassemblies. Quite interestingly, the interaction between RNA and a dimer is moderate, which is consistent with our experimental measurements.66 This may be necessary for the survival of

Figure 5. Rupture force in different subassemblies. The number on the x-axis corresponds to the subassemblies: a pair of dimer in a native conformation within a capsid (1), a complex of a dimer and a flexible RNA piece (2), a pentamer of dimers (3) and a hexamer of dimers (4). The pulled component is colored in red.

was measured for separating two dimers, whereas the force to drag a dimer away from the flexible RNA segment comprising both double-stranded and single-stranded sections (see above) was a little higher and amounted to ∼986 kJ mol−1 nm−1. The dimer exhibited strong interactions within the pentamer (∼1752 kJ mol−1 nm−1) and the hexamer (∼2262 kJ mol−1 nm−1).The interaction strengths decrease sharply to a mediate level significantly (∼1170 kJ mol−1 nm−1 for the pentamer and ∼1354 kJ mol−1 nm−1 for the hexamer) when the N-terminal tails were removed. It is because the presence of the Nterminal tails linking the monomers in a pentamer or hexamer greatly increases the stability of these structures, emphasizing again the critical role played by the tails in stabilizing these structures. The high rupture forces show that the pentamer and hexamer of dimers are stable subassemblies, which are consistent with their pivotal role in the icosahedral structure of a capsid.53 Interestingly, the rupture force between dimer and F


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Figure 6. Shrinkage of CCMV capsids. (A, C) are the variations of the radius of the capsid at 300 and 400 K, respectively, whereas (B, D) show the distributions of COM distances between monomers within a capsid at different time steps. The inserted snapshots in (A, C) are conformation at different times.

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the virus since it needs to readily release its genome in the host cell. This may be also useful for the selection of the genome: with a weak binding energy, dimers are enabled to quickly unbind RNA if it does not belong to the viral genome and have thereby more chances to package the right segments to ensure the virus proliferation. A dimer binds also preferentially to the double-stranded domains of RNA because the charge density is higher. CG simulations have allowed us to probe the behavior of the whole capsid upon increase of temperature. In qualitative agreement with experiments,64 we observed a shrinkage of the capsid due to the enhancement of hydrophobic interactions between proteins. Furthermore, the icosahedral symmetry was lost and the protein array became disordered. Despite the size and complexity of the CCMV virus, we have been able to shed some light on the interactions between dimers and subassemblies at the atomistic level. Structures, forces, energies, and time scales become increasingly important to understand the dynamical phenomena underlying the virus self-assembly. Only a few of these quantities are accessible through experiments, and atomistic computer simulations can bring realistic, complementary information. We believe that experiments, theory, and atomistic simulations will nurture one another and illuminate the multiscale processes occurring in biological systems.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Guillaume Tresset: 0000-0002-3755-4109 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.C. acknowledges a scholarship from the China Scholarship Council (CSC). Most of molecular dynamics simulations were performed at the “Centre de Calcul Scientifique en Région Centre” facility (CCRS-Orléans, France) under the CASCIMODOT program and using HPC resources from GENCIIDRIS. G.T. acknowledges financial support from the Agence Nationale de la Recherche (contract ANR-16-CE30-0017-01).

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REFERENCES

(1) Roos, W. H.; Bruinsma, R.; Wuite, G. J. L. Physical Virology. Nat. Phys. 2010, 6, 733−743. (2) Brillault, L.; Jutras, P. V.; Dashti, N.; Thuenemann, E. C.; Morgan, G.; Lomonossoff, G. P.; Landsberg, M. J.; Sainsbury, F. Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery. ACS Nano 2017, 11, 3476−3484. (3) Comellas-Aragonès, M.; de la Escosura, A.; Dirks, A. J.; van der Ham, A.; Fuste-Cune, A.; Cornelissen, J. J. L. M.; Nolte, R. J. M. Controlled Integration of Polymers into Viral Capsids. Biomacromolecules 2009, 10, 3141−3147. (4) Kwak, M.; Minten, I. J.; Anaya, D. M.; Musser, A. J.; Brasch, M.; Nolte, R. J. M.; Mullen, K.; Cornelissen, J. J. L. M.; Herrmann, A. Virus-like Particles Templated by DNA Micelles: A General Method for Loading Virus Nanocarriers. J. Am. Chem. Soc. 2010, 132, 7834− 7835.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b08026. Initial conformations of subassemblies; details of PMF calculation by umbrella sampling; additional MD simulations (PDF) G


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Interactions between the Molecular Components of the Cowpea

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