Virus Capsid Dissolution Studied by Microsecond Molecular Dynamics Simulations

Citation: Larsson DSD, Liljas L, van der Spoel D ( Virus Capsid Dissolution Studied by Microsecond Molecular Dynamics Simulations Daniel S. D. Larsson 0 1 Lars Liljas 0 1 David van der Spoel 0 1 Michael Nilges, Institut Pasteur, France 0 Funding: This work was funded by the Swedish Research Council (http://vr.se) through the eSSENCE project, the Swedish National Infrastructure for Computing (SNIC 014/10-11) at the PDC Center for High Performance Computing in Stockholm, Sweden and initial simulations were performed in the context of a DEISA (Distributed European Infrastructure for Supercomputing Applications) project, XXLBIOMD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 1 Department of Cell and Molecular Biology, Uppsala University , Uppsala , Sweden Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis. - Non-enveloped icosahedral viruses often contain binding sites for divalent cations, usually Ca2z. The ions are typically bound between coat proteins or on the icosahedral symmetry axes. This is broadly observed in three plant virus taxa: the family Tombusviridae (and an associate satellite virus), the genus Sobemoviruses and the family Bromoviridae [14]. Binding sites for calcium ions have also been found in bacteriophages of the Leviviridae family [5], fish and insect viruses of the Nodaviridae family [6] and in the Picornaviridae family, e.g. several human rhinoviruses [7]. In many of the plant viruses it is possible to induce a conformational change in vitro by removing the ions, either by a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or by exhaustive dialysis against deionized water. Ion-deprived virions reversibly expand on the order of 510% at neutral or slightly alkaline pH. In the swollen state internal parts of the virion as well as the RNA molecule may become susceptible to degrading enzymes [8,9]. Chelation of the metal ions is also required for synthesis of virus proteins in cell-free translation systems [9]. Only two low-resolution crystal structures of expanded virons are available: tobacco bushy stunt virus (TBSV) at 8 A [10] and satellite tobacco necrosis virus (STNV) at 7.5 A [11]. The radial increases are about 11% and 4%, respectively. In addition, an expanded cowpea chlorotic mottle virus (CCMV) virion was imaged with cryo-electron microscopy at 29 A and interpreted using rigid body fitting of the high-resolution structures of the native proteins [4]. The dynamic nature of the swelling process as well as the limited resolution of swollen virus particles structures prompted us to perform a simulation study of the capsid of STNV, with and without bound Ca2z, over one microsecond. The simulations allowed us to reproduce the swelling behavior upon removal of the calcium in silico and develop an atomistic description of the process. The T = 1 capsid of STNV consists of 60 identical coat proteins with one protein per icosahedral asymmetric unit. The coat protein is 195 amino acid residues long where residues 25195 make up the main domain that constitutes the capsid shell. The virions readily crystallize and the major part of the coat protein has been resolved by X-ray crystallography [2,1214]. The shell domain at the C-terminus folds as a b-jelly roll similar to many other single-stranded RNA plant viruses. Residues 1224 form a helical structure that together with the helices of two neighboring subunits form a short stalk that projects inwards into the central cavity around the icosahedral 3-fold axis. The first 11 residues at the N-terminus are disordered and cannot be detected in the electron density maps in the simulations these residues were modeled as a helix as well. This N-terminal arm and the interior surface of the capsid are lined with positively charged residues that presumably interact with the single-stranded positive-sense RNA molecule [14]. The 1239 nucleotide long genome encompasses only one open reading frame that encodes the coat protein and hence STNV is dependent on the co-infection of a helper virus (tobacco necrosis virus) for copying its RNA genome. The capsid has three different types of Ca2z binding sites (Figure 1). Type I is between two subunits close to the 3-fold symmetry axis. The protein ligands are the carboxyl groups of Asp194 and Glu25 as well as the main chain carbonyl oxygens of Ser61 and Gln64. Type II is on the 3-fold symmetry axis 8.05 nm from the center of the virion. It is coordinated by the carboxyl We have studied the capsid of satellite tobacco necrosis virus using large scale molecular dynamics simulations, where the atomic motions of 1,2 million particles were tracked over one microsecond. We find that the capsid swells in the simulations, and that the permeability for water increases 10-fold upon removal of the structural calcium ions. The water leaks in predominantly near the three-fold symmetry axis, suggesting that this is the spot where capsid dissociation is initiated following infection. groups of three Asp55 residues. Type III is on the 5-fold symmetry axis 9.04 nm from the center. This Ca2z is coordinated by the main chain carbonyl oxygen of five Thr138 residues. In total the capsid can accommodate 92 Ca2z ions (60 at type I sites, 20 at type II sites and 12 at type III sites). Simulations were performed of the capsid with and without Ca2z at two different salt concentrations for one microsecond each (Table 1). The carboxyl groups of one of the three Asp55 residues at each of the type II calcium binding sites were protonated in the two simulations without Ca2z, effectively simulating the capsid at a slightly acidic pH to mimic the conditions of the expanded capsid in the 7.5 A crystal structure [11]. The RNA molecule was not included in our simulations since it cannot be modeled completely in the electron density maps [14,15]. The aim of this work was to probe the dynamic behavior of a virus capsid over timescales that are more than an order of magnitude longer than what (...truncated)