RESUMO
Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes.
Assuntos
Proteínas do Capsídeo/química , Engenharia Genética , Genoma Viral , RNA Viral/química , Vírus Satélite da Necrose do Tabaco/genética , Montagem de Vírus , Motivos de Aminoácidos , Sítios de Ligação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Expressão Gênica , Tamanho do Genoma , Sequências Repetidas Invertidas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , RNA Viral/genética , RNA Viral/metabolismo , Vírus Satélite da Necrose do Tabaco/metabolismo , Vírus Satélite da Necrose do Tabaco/ultraestrutura , Replicação ViralRESUMO
The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ~13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.
Assuntos
Substituição de Aminoácidos , Proteínas do Capsídeo/química , Corticoviridae/química , Modelos Moleculares , Espalhamento a Baixo Ângulo , Bromus/química , Bromus/ultraestrutura , Bromus/virologia , Proteínas do Capsídeo/ultraestrutura , Corticoviridae/ultraestrutura , Microscopia Crioeletrônica/métodos , Microscopia Crioeletrônica/tendências , Cristalização , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Vírus do Mosaico do Tabaco/química , Vírus do Mosaico do Tabaco/ultraestrutura , Vírus Satélite da Necrose do Tabaco/química , Vírus Satélite da Necrose do Tabaco/ultraestrutura , Tombusvirus/química , Tombusvirus/ultraestruturaRESUMO
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 10-fold 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.
Assuntos
Cálcio/química , Capsídeo/química , Capsídeo/ultraestrutura , Modelos Biológicos , Modelos Químicos , Vírus Satélite da Necrose do Tabaco/química , Vírus Satélite da Necrose do Tabaco/ultraestrutura , Sítios de Ligação , Simulação por Computador , Modelos Moleculares , Conformação MolecularRESUMO
The first experimental data from single-particle scattering experiments from free electron lasers (FELs) are now becoming available. The first such experiments are being performed on relatively large objects such as viruses, which produce relatively low-resolution, low-noise diffraction patterns in so-called "diffract-and-destroy" experiments. We describe a very simple test on the angular correlations of measured diffraction data to determine if the scattering is from an icosahedral particle. If this is confirmed, the efficient algorithm proposed can then combine diffraction data from multiple shots of particles in random unknown orientations to generate a full 3D image of the icosahedral particle. We demonstrate this with a simulation for the satellite tobacco necrosis virus (STNV), the atomic coordinates of whose asymmetric unit is given in Protein Data Bank entry 2BUK.
Assuntos
Imageamento Tridimensional/métodos , Vírus Satélite da Necrose do Tabaco/ultraestrutura , Algoritmos , Imageamento Tridimensional/estatística & dados numéricos , Lasers , Fenômenos Ópticos , Espalhamento de Radiação , Difração de Raios XRESUMO
This paper examines the interaction between a block copolymer and a virus. A poly(styrene-b-4-vinylpyridine) block copolymer was loaded with nickel, and cast from a selective solvent mixture to form a cylindrical microstructure (PS/P4VP-Ni). The nickel ions were confined within the P4VP block of the copolymer. The binding of tobacco mosaic virus (TMV) and tobacco necrosis virus on microphase-separated PS/P4VP-Ni was examined. A staining technique was developed to simultaneously visualize virus and block copolymer structure by transmission electron microscopy. Electron microscopy revealed virus particles associated with block copolymer microphase-separated domains, even after extensive washes with Tween. In contrast, virus associated with PS/P4VP block copolymers lacking Ni were readily removed by Tween. The cylinder long axis of the microstructure was oriented using a hot press and a cooled channel die for quenching, resulting in PS/P4VP cylinders that had a strong anisotropic directional preference. When exposed to flowing solutions of TMV, the PS/P4VP-Ni surface exhibited an ability to retain TMV in a partially aligned state, when the direction of flow coincided with the long axis of the PS/P4VP-Ni cylinders. These results suggest that Coulombic interactions provide a robust means for the binding of virus particles to block copolymer surfaces.