Complementarity in the Supramolecular Design of Arenaviruses and Retroviruses Revealed by Electron Cryomicroscopy and Image Analysis (original) (raw)
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Assembly of Arena virus an Ultra structural Perspective
The Arenaviredae are a family of viruses whose members are generally associated with rodent transmitted disease in humans which currently comprises 24 viral species. Arenavirus infections are relatively common in humans in some areas of the world and can cause severe illnesses including several haemorrhagic fevers. The virus particles vary in diameter from 60 to more than 300 nm. They are spherical and have a reported average diameter of 92 nanometres. All are enveloped in a lipid bilayer and have a bisegmented ambisense RNA genome, but relatively little is known about how virions are assembled and how virion structure relates to transmissibility. To investigate the role of each viral structural protein in forming and maintaining the structure of the virion, we have imaged particles of arenaviruses LCMV, PICV and TCRV, and compared their shape and structural characteristics to similar sized phospholipid vesicles. A very strong association between particle size and shape was found for all arenavirus particles: small virions were significantly rounder than vesicles of similar size, while large particles tended to be more elliptical in appearance. The natural variation in surface glycoprotein decoration and ribonucleoprotein incorporation was then measured. From this data it was concluded that there is no strong evidence relating particles size to decoration for arenaviruses as a group, but we did detect significant correlations between internal density and virion shape. Overall, we are able to conclude that small virions are round and relatively rigid compared to vesicles of the same size, while large virions are not. By comparing relative density of the membrane-proximal region it was discovered that arenavirus shape is controlled by complexes containing GPC, Z and NP at the surface of the virion, and that an unbroken inner shell of NP is essential for maintaining a rigid spherical shape. Furthermore, it was revealed that the inner leaflet of intact arenaviruses has a lower density than the inner leaflet of vesicles consistent with the interpretation that viral proteins are displaying lipid molecules from the inner leaflet of the viral membrane.These data provide a new way of assessing the function of viral protein interactions on virion structure and may be of use in designing antiviral drugs that act at the level of virion structure.
Journal of Virology, 2009
It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins G N and G C , organized on a T21؍ icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either G N and G C homodimers or G N -G C heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five-and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a G N -G C heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens.
Electron Cryotomography of Tula Hantavirus Suggests a Unique Assembly Paradigm for Enveloped Viruses
Journal of Virology, 2010
Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses that cause a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, G N and G C , are embedded in the viral membrane and form protrusions, or "spikes." The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein, and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryomicroscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane, and ribonucleoprotein are organized. The structure of the G N -G C spike complex was solved to 3.6-nm resolution by averaging tomographic subvolumes. Each spike complex is a square-shaped assembly with 4-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses.
A personal account of virus structure determination at the Indian Institute of Science, Bangalore
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Virus particles are excellent models for understanding the specificity of protein-protein and protein-nucleic acid interactions and mechanisms of biological assembly. At the Indian Institute of Science, we have carried out detailed investigations on the structure, stability and assembly of two isometric plant viruses, Sesbania mosaic virus (SeMV) and Physalis mottle virus (PhMV). The protein coat of SeMV consists of 180 protein subunits of molecular mass 29 kDa. It encapsidates a ss-RNA genome of 4149 bases. The genome of PhMV is a ss-RNA of size 6.67 kb encapsidated in an icosahedral shell of 180 identical coat protein (CP) subunits of molecular mass 20 kDa. The spatial arrangements of protein subunits in both the virus particles confirms to a T = 3 icosahedral lattice. The three dimensional X-ray structures of these viruses and a large number of their recombinant capsids have been determined. It was necessary to develop new algorithms and write a large number of programs for the determination of these structures. The stability of SeMV and PhMV particles are based on very different interactions. The capsid stability of SeMV depends on protein-protein, protein-nucleic acid and calcium mediated protein-protein interactions. In contrast, PhMV particles are stabilized predominantly by hydrophobic interactions between coat protein subunits. An analysis of the structural, biochemical and biophysical properties of the native SeMV, its recombinant capsid and several of its mutants has led to the understanding of the detailed pathway for the assembly of SeMV. Comparative structural analysis of the native and recombinant capsids of PhMV and studies on the assembly and stability properties of a large number of site specific and deletion mutants have suggested that the subunit folding and particle assembly are concerted events in this virus.
1989
We have solved the structure of the Mahoney strain of type 1 and the Sabin (attenuated vaccine) strain of type 3 poliovirus. These structures have provided considerable insight into the architecture, assembly and immune recognition of these viruses. Recent studies of a conformationally altered form of the virus which is produced upon association with susceptible cells have begun to provide insight into the mechanism of cell entry. Using synthetic peptide antibodies and specific proteolytic probes we have shown that this conformational change results in the extrusion of the aminoterminus of the capsid protein VP1 (which is normally inside the viral capsid), and that the exposed aminoterminus confers the ability to attach to lipid vesicles. We have also begun to develop several crystallographic models which we hope will clarify structural factors which contribute to the pathogenesis of poliovirus. These included studies of the structural factors effecting temperature sensitivity in the Sabin strain of type 3 poliovirus (in collaboration with Philip Minor, NIBSC), crystallographic studies of the mouse adapted Lansing strain of type 2 poliovirus (in collaboration with Vincent Racaniello, Columbia University), crystallographic studies of a chimera between the Lansing strain of type 2 and the Mahoney strain of type 1 poliovirus in which the substitution of the Lansing sequence for residues 94-105 into the Mahoney strain confers mouse adaptation on the chimer (in collaboration with Marc Girard, Pasteur Institute), and crystallographic studies of the DA strain of Theiler's virus (in collaboration with Robert Fujinami, UCSD).
Structure, 2003
The capsid shells of these viruses, however, exhibit striking architectural differences. Except for the single-Baylor College of Medicine Houston, Texas 77030 shelled cypoviruses such as the cytoplasmic polyhedrosis virus (CPV), all other viruses in the Reoviridae have 3 State Key Lab for Biocontrol Institute of Entomology additional protein shells, such as the double-shelled rice dwarf virus (RDV) (Lu et al., 1998), and triple-shelled Zhongshan University Guangzhou 510275 rotavirus (Shaw et al., 1993) and bluetongue virus (BTV) (Grimes et al., 1998). In addition to conferring host speci-China ficity and mediating cell entry, these additional layers are believed to play important structural roles in maintaining the stability of the thin inner shell and sequestering the Summary dsRNA genome (Lawton et al., 2000). The inner shells of the Reoviridae are more homogenous and can be The single-shelled cytoplasmic polyhedrosis virus divided into two major groups. Those in the first group (CPV) is a unique member of the Reoviridae. Despite have a smooth inner shell made up of 120 CSP molecules lacking protective outer shells, it exhibits striking capenclosed by one or two outer T ϭ 13 layers, as exemplisid stability and is capable of endogenous RNA tranfied by BTV, RDV, and rotavirus. Those in the second scription and processing. The 8 Å three-dimensional group also have an inner shell consisting of 120 CSP structure of CPV by electron cryomicroscopy reveals molecules, but this shell is decorated by turrets (the secondary structure elements present in the capsid mRNA capping complexes) on the icosahedral vertices proteins CSP, LPP, and TP, which have ␣ϩ folds. The and by molecular clamps (large protrusions) joining extensive nonequivalent interactions between CSP neighboring CSP molecules. In addition, these viruses and LPP, the unique CSP protrusion domain, and the either have incomplete outer T ϭ 13 layers (e.g., orthoperfect inter-CSP surface complementarities may acreovirus [Dryden et al., 1993; Reinisch et al., 2000] and count for the enhanced capsid stability. The slanted aquareovirus [Shaw et al., 1996]) or completely lack any disposition of TP functional domains and the stacking outer protein layer (e.g., CPV [Hill et al., 1999; Xia et of channel constrictions suggest an iris diaphragmal., 2003; Zhang et al., 1999]). In these viruses, mRNA like mechanism for opening/closing capsid pores and transcription and posttranscriptional processing take turret channels in regulating the highly coordinated place in a series of well-coordinated steps, beginning steps of mRNA transcription, processing, and release. with mRNA transcription at the transcriptional enzyme complexes underneath the vertices of the inner shell, Introduction followed by 5Ј end mRNA capping and subsequent release through the multifunctional turret (Bartlett et al., RNA transcription is a fundamental process involving a 1974; Bellamy and Harvey, 1976; Furuichi, 1974; Furuichi series of well-coordinated processes catalyzed by multiet al., 1976; Reinisch et al., 2000; White and Zweerink, functional enzymes, often embedded in multicompo-1976; Xia et al., 2003; Yazaki and Miura, 1980; Zhang et nent macromolecular complexes. Double-stranded (ds) al., 1999). RNA viruses in the family Reoviridae are extreme exam-Having only a single shell, CPV is structurally the simples of such multifunctional RNA transcriptional maplest member of the Reoviridae. Despite lacking the chines. Their hosts include plants, insects, mammals, outer protective layers existing in other dsRNA viruses, and humans, and their structural proteins have little to CPV virions are resistant to chemical treatments, includno recognizable sequence homologies (reviewed by ing cations, high pH, trypsin, chymotrypsin, ribo-Mertens et al., 2000). Still, viruses in the nine genera of nuclease A, deoxyribonuclease, phospholipase, and this family all contain a characteristic segmented dsRNA SDS, and retain infectivity for weeks at Ϫ15ЊC to 25ЊC genome and a highly conserved dsRNA-dependent sin-(Mertens et al., 2000; Zhang et al., 2002). The relative gle-stranded RNA polymerase enclosed in a capsid shell simplicity and unusual stability of CPV make it an attracmade up of 120 molecules of the inner capsid shell tive system for studying the structural basis of RNA protein (CSP) (reviewed by Lawton et al., 2000; Nibert transcription and posttranscriptional processing. While and Schiff, 2001; Patton and Spencer, 2000). The Reovirits infection of silkworms can have a negative economic idae are all capable of endogenous mRNA transcription impact in Asia, CPV is also recognized as an emerging within an intact virus particle, using viral-encoded enbiocontrol agent, serving as an environmentally friendly zymes for transcription initiation, elongation, 5Ј capping, pesticide for fruit and vegetable farming (Mertens et al., 2000). Previous low resolution electron cryomicroscopy (cryoEM) structures showed that CPV shares similar *Correspondence: z.h.zhou@uth.tmc.edu
J Mol Biol, 2000
Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 Å resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca2+ from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.
Structures of enveloped virions determined by cryogenic electron microscopy and tomography
Complementary Strategies to Understand Virus Structure and Function, 2019
Bundibugyo virus (BDBV) Charge coupled device (CCD) Chikungunya virus (CHIKV) Contrast transfer function (CTF) Cryogenic electron microscopy (cryo-EM) Dengue virus (DENV) Deoxyribonucleic acid (DNA) Direct electron detector (DED) Ebola virus (EBOV) Eastern equine encephalitis virus (EEEV) Endosomal sorting complex required for transport (ESCRT) Enterovirus 71 (EV71) Focused ion beam (FIB) Glycoprotein (GP) Hazara virus (HAZV) Hemagglutinin (HA) Hepatitis B virus (HBV) Human immunodeficiency virus (HIV) Human parainfluenza virus 3 (HPIV3) Japanese encephalitis virus (JEV) Lassa virus (LASV) Major capsid protein (MCP) Marburg virus (MARV) Measles virus (MeV) Mouse hepatitis virus (MHV) Mucin-like domain (MLD) Vesicular stomatitis virus (VSV) Virus-like particle (VLP) Volta phase plate (VPP) West Nile virus (WNV) Zika virus (ZIKV)
IUCrJ, 2014
For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.