Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.     

Atomic force microscopy investigation of a chlorella virus, PBCV-1

A virus PBCV-1, which infects certain fresh water algae and has been shown by transmission and cryo-electron microscopy to exist as a triskaidecahedron, was imaged using atomic force microscopy (AFM). From AFM the particles have diameters of about 190nm and the overall structure is in all important respects consistent with existing models. The surface lattice of the virion is composed of trimeric capsid proteins distributed according to p3 symmetry to create a honeycomb arrangement of raised edges forming quasi-hexagonal cells. At the pentagonal vertices are five copies of a different protein forming an exact pentagon, and this has yet another unique protein in its center. The apical protein exhibits some unusual mechanical properties in that it can be made to retract into the virion interior when subjected to AFM tip pressure. When PBCV-1 virions degrade, they give rise to small, uniform, spherical, and virus like particles (VLP) consistent with T=1 or 3 icosahedral products. Also observed upon disintegration are strands of linear dsDNA. Fibers of unknown function are also occasionally seen associated with some virions.     

鸭肠炎病毒CHv强毒株超微结构研究

将鸭成纤维细胞培养的鸭肠炎病毒(DEV),经超声处理、高速冷冻差速离心后,采用酒石酸钾—甘油非线性密度梯度超速离心,收集病毒蛋白带,3%磷钨酸负染后观察病毒粒子形态。结果表明:病毒粒子主要集中在40%~50%酒石酸钾—甘油缓冲液交界层。电镜下,病毒粒子纯净,具有疱疹病毒典型形态结构,剖面六角,外观轮廓清楚。成熟病毒粒子直径约150~266nm,病毒囊膜、核衣壳和核心清晰可见;囊膜外层较内层着色略深,且可见尚未形成完整囊膜的柄状拖尾结构。多数病毒粒子以单核衣壳为主,一定数量的病毒具有双核衣壳,偶见三核衣壳,核衣壳直径为100~150nm,呈现致密圆形、半圆形或马蹄形等类型。在核衣壳外和囊膜之间可见明显的亮晕。核心DNA电子染色较深,集中分布,直径40~65nm。本文获得的清晰DEV负染超微结构照片,为该病毒结构生物学的研究提供了重要依据。     

Specific Nucleoprotein Residues Affect Influenza Virus Morphology

Influenza virus strains are often pleiomorphic, a characteristic that is largely attributed to specific residues in matrix protein 1 (M1). Although the mechanism by which M1 controls virion morphology has not yet been defined, it is suggested that the M1 interaction with other viral proteins plays an important role. In this study, we rescued recombinant virus WSN-AichiM1 containing the spherical A/WSN/33 (WSN) backbone and the M1 protein from A/Aichi/2/68 (Aichi). Aichi M1 differs from WSN M1 by 7 amino acids but includes those identified to be responsible for filamentous virion formation. Interestingly, Aichi virus produced spherical virions, while WSN-AichiM1 exhibited a long filamentous morphology, as detected by immunofluorescence and electron microscopy. Additional incorporation of Aichi nucleoprotein (NP) but not the hemagglutinin (HA), neuraminidase (NA), or M2 gene to WSN-AichiM1 abrogated filamentous virion formation, suggesting that specific M1-NP interactions affect virion morphology. Further characterization of viruses containing WSN/Aichi chimeric NPs identified residues 214, 217, and 253 of Aichi NP as necessary and sufficient for the formation of spherical virions. NP residues 214 and 217 localize at the minor groove between the two opposite-polarity NP helical strands of viral ribonucleocapsids, and residue 253 also localizes near the surface of the groove. These findings indicate that NP plays a critical role in influenza virus morphology, possibly through its interaction with the M1 layer during virus budding.     

Morphology of the Nucleoprotein Component of Rabies Virus

The intracytoplasmic ground substance, or matrix, associated with the development of rabies virus and the nucleocapsid of the virus were investigated. The filaments of the matrix were identified as virus-specific by means of ferritin-labeled antibodies. In thin sections, the diameter was 15 nm and the strands seemed to be incorporated into virions during morphogenesis of the virus. The nucleocapsid was isolated from purified virus preparations and was studied in negative contrast. The rabies nucleocapsid appeared as a single-stranded helix with a diameter of 16 nm and a periodicity of 7.5 nm; its length was in excess of 1 mum.     

Some properties of a new virus of Pieris rapae

A new insect virus of Pieris rapae was purified using a chloroform-butanol treatment followed by two differential and sucrose gradient centrifugations. The sedimentation coefficient of the purified virion was approximately 132 S, and it banded at a density of 1.39 g/cm³ in cesium chloride. The virion has a nonenveloped capsid with icosahedral symmetry. Several virions were shown to have a regular hexagonal contour about 25 nm in diameter and to be composed of many capsomeres. Full and empty viral particles, with 12 capsomeres around the periphery of the capsid, were noted. In some particles a small core has been observed which is spherical, about 15 nm in diameter. Both purified virus and partially purified virus preparations from dead, infected larvae gave only one precipitin band with a reaction of identity when tested against the antiserum to partially purified virus. When crude extracts of uninfected larvae and purified virus were tested against antiserum to partially purified virus, the pure virus produced a precipitin band. The band was formed independently and did not join to the band of the uninfected insect producing a typical reaction of nonidentity.     

Physical and Biological Properties of Dengue-2 Virus and Associated Antigens

Dengue virus suspensions from mouse brain and cell culture were fractionated into three components by rate zonal centrifugation in sucrose gradients. Infectious virus sedimented in a single zone and possessed hemagglutinating (HA) and complement fixing (CF) activity. Electron micrographs showed the virion to be a spherical particle 48 to 50 nm in diameter with 7-nm spherical structures on its surface. Buoyant density in CsCl of virions from mouse brain was estimated at 1.22 g/cm(3) and from cell culture at 1.24 g/cm(3). During centrifugation of virions in CsCl, an additional HA component appeared with a buoyant density of 1.18 g/cm(3). It was shown in electron micrographs to consist of virion fragments. A noninfectious component with HA and CF activity sedimented in sucrose more slowly than intact virus, had a buoyant density of 1.23 g/cm(3) in CsCl, and appeared as "doughnut" forms measuring 13.8 to 14 nm in diameter. A third component, with CF activity and no HA activity, sedimented very little in sucrose gradients. Particles of the same size and shape as the spherical subunits on the surface of the virion were observed in electron micrographs.     

Electron tomography of nascent herpes simplex virus virions

Cells infected with herpes simplex virus type 1 (HSV-1) were conventionally embedded or freeze substituted after high-pressure freezing and stained with uranyl acetate. Electron tomograms of capsids attached to or undergoing envelopment at the inner nuclear membrane (INM), capsids within cytoplasmic vesicles near the nuclear membrane, and extracellular virions revealed the following phenomena. (i) Nucleocapsids undergoing envelopment at the INM, or B capsids abutting the INM, were connected to thickened patches of the INM by fibers 8 to 19 nm in length and < or =5 nm in width. The fibers contacted both fivefold symmetrical vertices (pentons) and sixfold symmetrical faces (hexons) of the nucleocapsid, although relative to the respective frequencies of these subunits in the capsid, fibers engaged pentons more frequently than hexons. (ii) Fibers of similar dimensions bridged the virion envelope and surface of the nucleocapsid in perinuclear virions. (iii) The tegument of perinuclear virions was considerably less dense than that of extracellular virions; connecting fibers were observed in the former case but not in the latter. (iv) The prominent external spikes emanating from the envelope of extracellular virions were absent from perinuclear virions. (v) The virion envelope of perinuclear virions appeared denser and thicker than that of extracellular virions. (vi) Vesicles near, but apparently distinct from, the nuclear membrane in single sections were derived from extensions of the perinuclear space as seen in the electron tomograms. These observations suggest very different mechanisms of tegumentation and envelopment in extracellular compared with perinuclear virions and are consistent with application of the final tegument to unenveloped nucleocapsids in a compartment(s) distinct from the perinuclear space.     

Studies on the Nucleocapsid Structure of a Group A Arbovirus

When Sindbis virus (273S) was treated with sodium desoxycholate, a nonhemagglutinating 136S particle was liberated from the virion, representing the viral nucleocapsid (core). Electron microscopically it appeared as a spherical particle 35 nm in diameter, showing ringlike morphological units 12 to 14 nm in diameter on its surface. When the one- and two-sided images of core particles were correlated, their structure could be demonstrated to have the T = 3 arrangement of 32 hexamer-pentamer morphological units within a symmetrical surface lattice. The core contained a further spherical structure (12 to 16 nm in diameter) which was designated as the central core component. Two proteins were found associated with the core, a third viral protein belonged to the hemagglutinating surface structures. The significance of these findings for virus classification is discussed.     

A second form of infectious bursal disease virus-associated tubule contains VP4.

Preparations of density gradient-purified infectious bursal disease virus (IBDV) were found to contain full and empty icosahedral virions, type I tubules with a diameter of about 60 nm, and type II tubules 24 to 26 nm in diameter. By immunoelectron microscopy we demonstrate that virions and both types of tubular structures specifically react with anti-IBDV serum. In infected cells intracytoplasmic and intranuclear type II tubules reacted exclusively with an anti-VP4 monoclonal antibody, as did type II tubules in virion preparations. The immunofluorescence pattern with the anti-VP4 antibody correlated with electron microscopical findings. Neither purified extracellular nor intracellular virions were labeled with the anti-VP4 MAb. Our data show that the type II tubules contain VP4 and suggest that VP4 is not part of the virus particle.     

Acute peristome edema disease in juvenile and adult sea cucumbers Apostichopus japonicus (Selenka) reared in North China

Acute peristome edema disease (APED) is a new disease that broke out in cultured sea cucumber along the Shangdong and Liaoning province coasts in China, PR, and has caused a great deal of death in Apostichopus japonicus (Selenka) since 2004. Here we report virus-like particles found in intestine epithelium of sea cucumbers reared in North China. It is the first time that sea cucumbers are reported to be infected by virus. Histological examinations showed that the viral inclusion bodies existed in intestine epithelium cells. Electron microscopic examinations show that the virions were spherical, 80-100nm in diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Detailed studies on the morphogenesis of these viruses found many characteristics previously described for coronaviruses. Virus particles always congregated, and formed a virus vesicle with an encircling membrane. The most obvious cellular pathologic feature is large granular areas of cytoplasm, relatively devoid of organelles. Tubular structures within virus-containing vesicles, nucleocapsid inclusions, and double-membrane vesicles are also found in the cytopathic cells. No rickettsia, chlamydia, bacteria, or other parasitic organisms were found.     

Compartmentalization of Pseudorabies Virus and Subvirion Components in BHK-21 Cells and in the Extracellular Fluid

The intracellular and extracellular localization of pseudorabies virions and subvirion components was determined at various stages in the replicative cycle. It was discovered that infectious pseudorabies virus appears first in the nucleus of the infected cell early in the infectious cycle but later accumulates in the cytoplasm. Subvirion components (nucleoids and nucleocapsids) are restricted to the nucleus, and only the complete virion is released from the infected cell.     

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.     

Structural responsiveness of filamentous bacteriophage Pf1: comparison of virion structure in fibers and solution. The effect of temperature and ionic strength.

X-ray diffraction from fibers and magnetically oriented solutions has been used to study the effect of changes in environment on the helical symmetry and radial structure of the Pf1 virus particle. Detailed analysis of equatorial scattering to a spacing of 8-10 A was used to identify small radial motions of structural elements in the virus particle. R-factor ratios were used to determine the statistical significance of observed changes. Comparison of the structure of virus particles in fibers with those in solution indicated that the helical symmetry of the virions remains unchanged during fiber formation. In most fibers the virions appear to be slightly distorted by the tight packing of virus particles. This distortion results in an apparent increase in the radius of the virus particle of approximately 0.6 A. A change in the radius of the DNA is also observed. Increase in the concentration of solvent molecules during fiber formation results in penetration of the virus interior by some solvent components. NaCl is also able to enter the virus interior. The change in the helical symmetry of the virions at approximately 8 degrees C appears to be the same whether observed by diffraction from fibers or from solutions. Only subtle changes in radial structure are associated with the temperature transition.