Virus assembly crete

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts. The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric. Find more information on the Altmetric Attention Score and how the score is calculated. Simple RNA viruses self-assemble spontaneously and encapsulate their genome into a shell called the capsid.

This process is mainly driven by the attractive electrostatics interaction between the positive charges on capsid proteins and the negative charges on the genome. Despite its importance and many decades of intense research, how the virus selects and packages its native RNA inside the crowded environment of a host cell cytoplasm in the presence of an abundance of nonviral RNA and other anionic polymers has remained a mystery.

In this paper, we perform a series of simulations to monitor the growth of viral shells and find the mechanism by which cargo—coat protein interactions can impact the structure and stability of the viral shells.

We show that coat protein subunits can assemble around a globular nucleic acid core by forming nonicosahedral cages, which have been recently observed in assembly experiments involving small pieces of RNA. Sign up! Your notification has been saved.

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Two more coronavirus deaths reported in Hall County. Lincoln not included in state's loosening of coronavirus restrictions. Smithfield in Crete may close because of rising number of cases. Smithfield reverses course, 50 workers walk off the job; 'They're scared,' says union rep. The SEC experiments show that under optimal assembly conditions the only species present in detectable concentrations are either complete capsids or small protein oligomers which we refer to as the basic assembly unit.

The size of the basic assembly unit is virus dependent and ranges from 2—6 proteins. Under certain conditions, the intensity of light scattering signal is proportional to the mass-averaged molecular weight of species in solution, which closely tracks the fraction of subunits in capsids provided that intermediate concentrations remain small.

The image is reprinted with permission from Ref. B Assembly products at long times for a subunit icosahedral shell as a function of temperature i. Representative structures for several regions are shown on the right.

Figure adapted with permission from Ref. It is difficult to characterize assembly pathways with bulk techniques because most intermediates are transient. Techniques that monitor individual capsids have begun to address this limitation. Mass spectrometry has been used to characterize key intermediates and assembly pathways for several viruses 19 — Borodavka et al. Zlotnick and coworkers 11 , 27 , 28 developed an approach to describe capsid assembly kinetics with a system of rate equations for the time evolution of concentrations of intermediates.

The equations are made tractable by assuming one or a few structures for each intermediate size. Continuum-level descriptions of assembly dynamics with further simplifications have also been developed 30 , The assumption of one structure per intermediate size can be relaxed by enumerating pathways 32 , or in an alternative approach 33 — 37 , pathways consistent with a Master equation are stochastically sampled using the BKL or Gillespie algorithm 38 , The approaches described in the previous paragraph must pre-assume the state space i.

This limitation can be relaxed by performing simulations which explicitly track the dynamics of subunit positions and orientations using molecular dynamics, Brownian dynamics, or other equations of motion. Several groups have developed coarse-grained models for subunits, which have excluded-volume geometry and orientation-dependent attractions designed such that the lowest energy structure is a shell with icosahedral symmetry e.

A recent approach uses particle-based simulations to systematically derive Markov state models, which can then be simulated using methods from the previous paragraph We begin by analyzing the formation process of an empty capsid.

While this process is most relevant to viruses that first form empty procapsids during assembly, it also provides a useful starting point to understand co-assembly with nucleic acids, lipid membranes, or scaffolding proteins. Although we focus on icosahedral capsids, we note that many viruses have non-icosahedral capsids, and that the capsid proteins of some icosahedral viruses can form other structures including sheets, tubes, and multi-layered shells depending on solution conditions To simplify the presentation, we assume that there is one dominant intermediate species for each number of subunits n.

Here G n cap is the free energy due to subunit-subunit interactions for intermediate n. Under most conditions at equilibrium, almost all of the subunits are found in complete capsids or as free subunits 5 , This prediction arises from virtually any model for assembly of finite-size structures e. Under these conditions Eq. Zlotnick and coworkers have shown that the assembly of HBV 56 can be captured by Eq.

The requirement for weak interactions appears to be quite general, for reasons discussed in section 2. These interactions and changes in subunit rotational entropy are described by the factor G cap.

In many cases assembly is primarily driven by hydrophobic interactions, attenuated by electrostatics 58 , 59 with directional specificity imposed by electrostatic, van de Waals, and hydrogen bonding interactions. These interactions are short-ranged under assembly conditions, with scales ranging from a few angstroms Van der Waals interactions and hydrogen bonds to 0. In contrast, intermediates in the growth phase are relatively stable; thus, successive additions of subunits or small oligomers are independent and the timescale for a capsid to complete the growth phase has a low-order dependence on the free subunit concentration 5 , Schematic of the assembly mechanism for cowpea chlorotic mottle virus CCMV In the nucleation phase, addition of capsid protein dimers is unfavorable until reaching the critical nucleus.

Subsequent additions the growth phase are relatively favorable, though still reversible, until the capsid is completed. The diameter of the complete CCMV capsid is 28 nm. Due to the geometry of an icosahedral shell, the first few intermediates have relatively few subunit-subunit contacts and are thus relatively unstable.

The critical nucleus often corresponds to a small polygon Fig. As the subunit-subunit binding free energy or the free subunit concentration decreases, small intermediates become less stable and the critical nucleus size increases Therefore, as subunit supersaturation decreases over the course of an assembly reaction, the critical nucleus size increases, asymptotically approaching a half capsid 5.

Similar considerations apply to capsid disassembly. The first few subunits to disassemble must break many contacts, leading to a large activation barrier. There is therefore a pronounced hysteresis between assembly and disassembly at a given set of conditions 41 , This condition allows capsids to be highly metastable even at infinite dilution 64 , which is an important feature given that they must eventually leave their host cell to infect another.

Some capsids undergo post-assembly maturation processes which further increase their stability. Capsid assembly kinetics, whether measured by experiments 10 — 17 or calculated from theoretical or computational models 11 , 14 , 16 , 27 , 28 , 37 , 61 , are sigmoidal Fig. There is an initial lag phase during which capsid intermediates form, followed by rapid capsid production, and then an asymptotic approach to equilibrium during which assembly slows as nucleation barriers rise due to depletion of free subunits.

Increasing the subunit concentration c T or the strength of inter-subunit interactions g b typically by decreasing pH or increasing salt concentration initially leads to more rapid assembly. However, while thermodynamics Eq. These modeling and experimental studies show that there is a trade-off between interaction specificity and kinetic accessibility—more specific interactions increase selectivity of assembly for the target structure but decrease assembly rates due to decreased kinetic cross-sections 57 , For a given interaction specificity, the kinetic phase diagram can be classified into five regimes.

Finally, stronger-than-optimal interactions lead to suppressed yields due to two forms of kinetic traps. This condition occurs when the timescale required for capsids to complete the growth phase exceeds the typical nucleation timescale 5 , The presence of these two forms of kinetic traps iv and v explain the experimental 56 , 69 and computational 41 , 48 observation that weak interactions are required for productive capsid assembly.

These kinetic traps lead to similar constraints on interactions in other forms of assembly such as crystallization This section focuses on viruses for which the capsid assembles spontaneously around the viral genome during infection.

This category includes most ssRNA viruses and the Hepadnaviridae e. Electrostatic interactions between positive charges on capsid proteins and negative charges on RNA provide an important thermodynamic driving force for this process. Specific RNA sequences or chemistry are not essential for assembly, as demonstrated by early in vitro experiments in which ssRNA capsid proteins assembled around heterologous nucleic acids and even polyvinylsulfate 72 , 73 , and more recent experiments in which capsid proteins assembled around various negatively charged substrates e.

We begin this section by describing what these experiments and theoretical models have revealed about how assembly depends on the physical characteristics of RNA or other polyelectrolytes, such as charge, size, and structure.

Due to space limitations, we do not discuss studies on assembly around non-polymeric cores see Refs. We then discuss mechanisms by which virus-specific interactions can enhance co-assembly and enable selective packaging of the viral genome.

We consider a solution of capsid protein subunits and cores e. RNA molecules with respective total concentrations of c T and x T. Extending Eq. The core interaction energy G core includes, for example in the case of an RNA core, attractive protein-RNA interactions, intramolecular electrostatic repulsions, and base-pairing interactions. This capability is exploited by many ssRNA viruses, whose capsids assemble only in the presence of RNA or other polyanions at physiological conditions, thus ensuring that the genome is packaged during assembly.

On the other hand, unfavorable core contributions, such as would arise from the stiffness and electrostatic repulsions of dsDNA molecules, can direct assembly away from the capsid structure to other morphologies It has been proposed that viral genomes face a selective pressure to maintain a length which maximizes the stability of the nucleocapsid complex i.

In support of the importance of nonspecific electrostatics to driving RNA encapsidation, the total positive charge on the capsid inner surface correlates to the length of the genomic RNA for a diverse group of ssRNA viruses 90 , 91 Fig.

Furthermore, changing the capsid charge alters the amount of cargo encapsidated in cells and in vitro 92 — 96 , although the effect of mutations on the amounts and sequences of packaged RNA can depend on factors other than charge 95 , In vitro competition assays in which different species of RNAs competed for packaging demonstrated that longer RNAs up to the viral genome length are preferentially packaged over shorter RNAs 98 , indicating that overcharged genomes are optimal for packaging even in the absence of cell-specific factors.

Relationship between genome length and capsid charge. A Survey of the charge ratio, or number of nucleotides in the genome divided by total positive charge on the inner capsid surface, for ssRNA viruses. B The thermodynamic optimum charge ratio predicted from simulations 99 symbols is compared to actual charge ratios symbols for several viruses.

Predicted optimal charge ratios in the absence of base-pairing are also shown symbols. The thermodynamic optimum charge ratio is defined as the NA length which minimizes the free energy for encapsidating the genome divided by the positive capsid charge. Motivated by these observations, researchers theoretically and computationally calculated how the free energy F encap L to encapsulate a linear polyelectrolyte varies with its length L reviewed in 5 , Several works performed self-consistent field theory calculations in which F L is calculated from a continuum description of polymer conformational statistics coupled to the Poisson-Boltzmann equation.

While Ref. Ting et al. However, the subsequent in vitro competition assays 98 demonstrated that overcharging is optimal for assembly in the absence of a Donnan potential. Perlmutter et al. Optimal lengths predicted by the model for several specific viruses closely matched genome lengths for those viruses Fig.

Overcharging was found to arise because only a fraction of encapsulated polymer segments can closely interact with positive capsid charges i. Consequently, packaging of multiple short polyelectrolytes will lead to reduced or no overcharging.

The thermodynamic optimal lengths closely matched the lengths which optimized the yield of long but finite-time dynamical simulations, indicating a connection between the thermostability and optimal assembly of a viral particle.

Although a self-consistent field theory predicted no difference between the optimal lengths for linear polyelectrolytes and compact star architectures , subsequent theory and simulations 99 found that branching consistent with the structures of base-paired RNA increases the optimal genome length as compared to a linear polyelectrolyte, by compensating for intramolecular charge repulsions and by favoring compact conformations Fig.

Based on secondary structure predictions, Yoffe et al. The previous section showed that, for a given capsid protein and solution conditions, there is a length of RNA for which assembly is optimal. To understand the effect of perturbing parameters from these optimal values, in vitro assembly products were characterized by electron microscopy e. In addition, several groups have performed Brownian dynamics simulations in which coarse-grained triangular or pentameric subunits assemble around flexible polyelectrolytes 49 , 50 , 99 , — , semi-flexible polyelectrolytes , or model NAs In both experiments and simulations, parameters must be carefully tuned to achieve high yields of well-formed capsids.

An example simulation phase diagram illustrating some of the alternative products that form at non-optimal parameters is shown in Fig.

A Crystal structure of the SV40 basic assembly unit , which is a homopentamer of the capsid protein capsid subunit, and a coarse-grained model pentameric subunit. The locations of the positively charged ARMs are shown in yellow most of the ARM residues are not resolved in the crystal structure.

B The dominant products of assembly around a linear polyelectrolyte as a function of ionic strength and subunit-subunit interaction strength at thermodynamically optimal polyelectrolyte lengths, which vary from — depending on the ionic strength.

C Simulation snapshots which exemplify the dominant assembly outcomes. D A doublet formed in simulations around a polyelectrolyte with segments twice the optimal length.

Image provided by R. Garmann, C. Knobler and W. Assembly around polymers with non-optimal lengths leads to several outcomes.

The first is polymorphism, or formation of capsids with different T -numbers Fig. The favored polymorph depends on RNA length, the preferred curvature of capsid protein-protein interactions i. For RNA significantly below optimal length, multiple RNAs were packaged in each capsid , , as seen in coarse-grained dynamics simulations with short linear polyelectrolytes In the experiments, capsid formation required equilibrium between multiple disordered protein-RNA complexes, leading to highly cooperative assembly RNAs which were 2, 3, or 4 times the genome length lead respectively to predominantly doublets Fig.

An early study likely also observed doublets, although the structures could not be confirmed 72 and recently doublet dodedecadron capsids were observed for assembly of SV40 capsid proteins around certain lengths of RNA Simulations independently predicted the formation of doublets around polyelectrolytes with about twice the optimal length 49 , 99 , Fig.

At lengths only slightly greater than optimal, simulations predict malformed but single capsids 49 , 50 , However, these malformations may be difficult to resolve experimentally. Simulations 49 , show that two classes of assembly mechanisms occur around RNA or a linear polymer Fig. One closely resembles the nucleation-and-growth mechanism found for empty capsid assembly, except that the polymer stabilizes protein-protein interactions and can enhance the flux of proteins to the assembling capsid A small partial capsid first nucleates on the polymer, followed by a growth phase in which one or a few subunits sequentially and reversibly add to the partial capsid.

In the alternative mechanism, first proposed by McPherson and then Refs 49 , , , subunits adsorb onto the polymer en masse in a disordered fashion and then cooperatively rearrange to form an ordered capsid.

Simulations predict that the assembly mechanism can be tuned by solution conditions and capsid protein-protein interactions The nucleation-and-growth mechanism is favored by weak protein-polymer association high salt concentration and strong protein-protein interactions typically low pH 89 , while the en masse mechanism arises for lower salt and weaker protein-protein interactions. Two mechanisms for assembly around a polyelectrolyte A Low ionic strength strong subunit-polyelectrolyte interactions and weak subunit-subunit interactions lead to the en masse mechanism typified by disordered intermediates.

B High ionic strength weak subunit-polymer interactions and strong subunit-subunit interactions lead to the nucleation-and-growth mechanism in which an ordered nucleus forms on the polymer followed by sequential addition of subunits. Observations in vitro suggest that both mechanisms are viable. The absence of detectable intermediates suggested that assembly follows the nucleation-and-growth mechanism. In support of this conclusion, simulations showed that the disordered intermediates arising in en masse pathways lead to measurably different SAXS profiles, whereas profiles from nucleation-and-growth trajectories are consistent with the experimental observations.

Other observations suggest that virus-like particles can assemble through the en masse mechanism. First, at low salt strong protein-RNA interactions and neutral pH weak protein-protein interactions the proteins undergo extensive but disordered adsorption onto RNA. Subsequently, pH is reduced to enhance protein-protein binding, leading to the formation of ordered capsids Similarly, a recent observation of capsid protein assembly around charge-functionalized nanoparticles found that assembly initially proceeded through nonspecific aggregation of proteins and nanoparticles, followed by the gradual extrusion of complete capsids formed around nanoparticles The CCMV experiments , , found that complete encapsidation of all RNA present in solution requires a significant excess of capsid protein, such that the positive charges in protein ARMs balance the negative RNA charge recall that in the complete capsid the negative RNA charge significantly exceeds the positive ARM charge, section 3.

This criteria occurs because the disordered protein-RNA complexes occurring during the first step of assembly are charge-balanced. During capsid formation the second step , excess proteins are displaced to the exterior, where their positive ARM charges interact with negative residues on the outer surface of the capsid To be infectious, a virion must assemble specifically around the viral genome amidst a panoply of cellular RNA molecules.

Structure- and sequence-specific RNA-protein interactions may be a widespread mechanism of achieving specificity by promoting assembly around the viral genome although not all viruses are selective for their genomic RNA in vitro 96 , 98 , suggesting the importance of cell-specific factors. Several studies suggest that the specific folded structure of the genomic RNA may enhance assembly.

Based on the crystal structure of STMV, which shows 30 ds helical segments interacting with the capsid inner surface , , McPherson and coworkers proposed that during assembly the STMV genome forms a conformation comprising linearly connected stem loops which sequentially bind capsid proteins.

Using the crystal structure as constraints, Schroeder et al. Simulation of the complete STMV capsid with atomic resolution demonstrated that this secondary structure is consistent with the crystal structure These analyses were not restricted to stem-loops, and found secondary structures that differed significantly from the encapsidated, stem-loop structure. Interestingly though, the primary probing data is similar for the encapsidated and unencapsidated RNAs, suggesting that the same nucleic acids are base-paired in both cases.

Extensive evidence shows that packaging signals, or short RNA sequences that are specifically bound by capsid proteins, play significant roles in controlling assembly pathways for some viruses reviewed in , Combining identified packaging signals with geometric constraints derived from electron density maps of MS2 capsids led to a structure of the encapsidated genome Earlier work using mass spectrometry 19 , coarse-grained simulations , and kinetic models suggested that RNA binding drives a conformational switch in the MS2 capsid protein and identified two dominant pathways for MS2 assembly.

Using Gillespie algorithm simulations section 1. A striking observation supporting an active, sequence-specific role of the genome was made by Borodavka et al. Assembly around genomic RNAs was characterized by either constant R H or, in some trajectories, a collapsed complex followed by gradual increase to the size of an assembled capsid.

In contrast, assembly around heterologous RNA led to an increase in R H before eventually decreasing to the size of the capsid. The different assembly pathways were attributed to the presence of packaging signals in the genomic RNAs. The collapsed structures are reminiscent of a previous observation , in which incubation of CCMV RNA with sub-stoichiometric concentrations of capsid proteins led to a compact nucleocapsid complex that triggered rapid assembly upon introduction of additional capsid proteins.

Finally, we emphasize that sequence-specific protein-RNA interactions are not the only mechanism that drives selective genome packaging in vivo; other factors include, e. In this section we consider mechanisms by which the proteins of enveloped viruses assemble on lipid bilayers to drive budding. The passage of nanoscale particles through membranes is an extremely broad topic; we focus on viral budding driven by protein assembly. Enveloped viruses can be divided into two groups based on how they acquire their lipid membrane envelope.

For the first group, which includes influenza and type C retroviruses e.