Cytoplasmic polyhedrosis virus structure at 8 A by electron cryomicroscopy: structural basis of capsid stability and mRNA processing regulation.

The single-shelled cytoplasmic polyhedrosis virus (CPV) is a unique member of the Reoviridae. Despite lacking protective outer shells, it exhibits striking capsid stability and is capable of endogenous RNA transcription and processing. The 8 A three-dimensional structure of CPV by electron cryomicroscopy reveals secondary structure elements present in the capsid proteins CSP, LPP, and TP, which have alpha+beta folds. The extensive nonequivalent interactions between CSP and LPP, the unique CSP protrusion domain, and the perfect inter-CSP surface complementarities may account for the enhanced capsid stability. The slanted disposition of TP functional domains and the stacking of channel constrictions suggest an iris diaphragm-like mechanism for opening/closing capsid pores and turret channels in regulating the highly coordinated steps of mRNA transcription, processing, and release.

A novel technique of three-dimensional reconstruction segmentation and analysis for sliced images of biological tissues.

A novel technique of three-dimensional (3D) reconstruction, segmentation, display and analysis of series slices of images including microscopic wide field optical sectioning by deconvolution method, cryo-electron microscope slices by Fourier-Bessel synthesis and electron tomography (ET), and a series of computed tomography (CT) was developed to perform simultaneous measurement on the structure and function of biomedical samples. The paper presents the 3D reconstruction segmentation display and analysis results of pollen spore, chaperonin, virus, head, cervical bone, tibia and carpus. At the same time, it also puts forward some potential applications of the new technique in the biomedical realm.

Structure of Cytoplasmic Polyhedrosis Virus from Bombyx mori.

The structures of full and empty capsids of CPV were studied by negative staining and electron cryomicroscopy and computer reconstruction techniques. By comparing the structures and biochemical compositions, the CPV was identified as a single-layered capsid with its five structural proteins located on it. This single capsid is arranged according to T=1 icosahedral symmetry with 12 turret-like spikes at its icosahedral vertices. The empty and full CPV show identical capsid but differ inside. The dense and ordered genomic dsRNA is located inside the full CPV. The internal space of the empty CPV has almost no electron density except for 12 electron densities attributed the transcriptional enzyme complexes extending inward from the base part of CPV spikes.

Morphology and morphogenesis of severe acute respiratory syndrome (SARS)-associated virus.

After infecting the Vero E6 cells by nasal/throat swabs collected from SARS patients, we studied the SARS-associated virus by electron microscopy and molecular biological technique. The results show that the diameter of newly isolated virus is about 50 nm without envelope or 100 nm with envelope. The virus was proved to be a new coronavirus by RT-PCR and it responded positively to convalescent-phase serum specimen from SARS patients, which is the evidence that this new virus is etiologically linked to the outbreak of SARS. The morphogenesis and distribution of the virus are also discussed in this article.

[Adsorption of cyromazine on five typical soils in China].

Batch equilibrium experiments were used to reveal cyromazine adsorption on five kinds of soils, namely Ali-Perudic Ferrosols collected from Yingtan of Jiangxi, Udic Argosols collected from Nanjing and Gleyic-Stagnic Anthrosols collected from Changshu of Jiangsu, Ustic Cambosols collected from Fengqiu of Henan, and Udic Isohumosols collected from Hailun of Heilongjiang. Results show that the experimental data are best described by the Freundlich and Langmuir model, while fitted successfully by the linear model. Different adsorption behaviors of Cyromazine are observed in the five tested soils, with the lgK(f) values varying from 1.6505 (cambosols), 1.6715 (argosols) and 1.7153 (ferrosols) to 2.4579 (anthrosols) and 2.6557 (isohumosols). Moreover, the Kf values are in a positive correlation to the OM of the soil (r = 0.989) but significantly negative correlated to soil pH (r = -0.938). The free energy of sorption ranged from -20.8 to -23.0 kJ/mol, indicated that the adsorption could be largely attributed to the physical adsorption.

Three-dimensional display and arbitrary region interactive segmentation of high-resolution virus capsids from cryo-electron microscopy single particle reconstruction.

Recent advances in cryo-electron microscopy (cryo-EM) instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3-D) structure determination of virus. In order to visualize and effectively understand the 3-D structure, we present a display method based on surface rendering, which has the function of 3-D arbitrary region interactive segmentation and quantitative analysis, and integrate them into a software package called CEM-3DVDSS (cryo-EM 3-D virus display and arbitrary region segmentation system). CEM-3DVDSS consists of a complete set of modular programs for 3-D display and segmentation of icosahedral virus, which is organized under a graphical user interface and provides user-friendly options. First, we convert volume data in the MRC format obtained by cryo-EM single particle reconstruction to the format of our own software; in the preprocessing step, the original volume data are compressed and a better vector dimension is found for controlling the speed and detail of display. Then, the new volume data can be displayed and segmented using CEM-3DVDSS. We demonstrate the applicability of CEM-3DVDSS by displaying the 3-D structures of 2.5 nm (resolution) BmCPV (Bombyx mori cytoplasmic polyhedrosis virus), 2.5 nm CSBV (Chinese Sacbrood bee virus) and 1.4 nm C6/36DNV (Densonucleosis virus). As a result, both the 3-D display speed and signal-to-noise ratio of CEM-3DVDSS are improved compared with the original method, and the segmentation results become precise and more intact with additional function of quantitative analysis of 3-D structure.

Structural comparisons of empty and full cytoplasmic polyhedrosis virus. Protein-RNA interactions and implications for endogenous RNA transcription mechanism.

Viruses in the family Reoviridae are capable of transcription within the intact capsids. As the only single-shelled and thus the simplest member of the Reoviridae, cytoplasmic polyhedrosis virus (CPV) provides an attractive system for studying endogenous transcription. We report the structures of the full and empty CPV determined at 13-A resolution by electron cryomicroscopy. The structure of the empty CPV reveals a density attributed to the transcription enzyme complex, which is attached to the internal surface of the capsid shell below each of the 12 turrets. The full capsid has an identical capsid shell but contains additional internal densities contributed by the genomic double-stranded (ds) RNA. The RNA densities proximal to the capsid shell are organized into layers with a dodecahedral appearance, suggesting a genome organization of dsRNA segments each having a cone shape spooling around a transcription enzyme complex. Our structures also suggest that the capsid shell serves as a scaffold for appropriate positioning of the RNA genome, whereas nascent mRNA release takes place through the constricted central channel of the turret. Based on these observations, a detailed moving template transcription mechanism is proposed that may provide insight into the well coordinated and highly efficient endogenous RNA transcription of dsRNA viruses.

Molecular interactions and viral stability revealed by structural analyses of chemically treated cypovirus capsids.

Cytoplasmic polyhedrosis virus (CPV, genus Cypovirus) is a unique member of the family Reoviridae which lacks the outer protective shells that exist in all other members, yet exhibits unusual stability. We have analyzed the effects of different acidic, basic, detergent, and urea treatments on CPV capsids. The integrity of the CPV capsids was unaffected under high-pH conditions that disrupted the orthoreovirus inner core, consistent with its ability to maintain structural integrity in extremely alkaline environments during infection. However, it was sensitive to low pH, detergents, and urea, similarly to other viruses in this family. The three-dimensional structure comparisons by electron cryomicroscopy of the intact empty CPV capsid with the "spikeless" capsid whose turrets were removed by chemical treatments revealed the interaction footprint of the turret on the capsid shell. The observed structural changes associated with the removal of the turret suggest critical structural roles of the turret in maintaining capsid integrity in addition to its enzymatic activities.

Entry of Bombyx mori cypovirus 1 into midgut cells in vivo.

In vivo entry of Bombyx mori cypovirus 1 into the silkworm midgut was studied by electron microscopy of ultrathin sections of midguts from silkworm larvae that had been administered virus-contaminated leaves. In 3 h, virions were observed outside and inside midgut cells, including columnar cells, goblet cells and muscle cells. Virions were seen adhering to the plasma membrane of microvilli, embedding in the membrane and settling themselves intact inside the microvilli of the columnar cells. These results suggested that intact virions entered columnar cells by means of direct penetration through the cell membrane. In 12, 24 and 48 h, virogenic stromata and progeny virions were observed in columnar cells, but not in other midgut cells.