Biomedical subjects

Giovanni Cardone

Publications and source records attributed to Giovanni Cardone.

3 recordsLinked to original sources

PubMed2006

Visualization of the herpes simplex virus portal in situ by cryo-electron tomography.

Herpes simplex virus type 1 (HSV-1), the prototypical herpesvirus, has an icosahedral nucleocapsid surrounded by a proteinaceous tegument and a lipoprotein envelope. As in tailed bacteriophages, the icosahedral symmetry of the capsid is broken at one of the 12 vertices, which is occupied by a dodecameric ring of portal protein, UL6, instead of a pentamer of the capsid protein, UL19. The portal ring serves as a conduit for DNA entering and exiting the capsid. From a cryo-EM reconstruction of capsids immuno-gold-labeled with anti-UL6 antibodies, we confirmed that UL6 resides at a vertex. To visualize the portal in the context of the assembled capsid, we used cryo-electron tomography to determine the three-dimensional structures of individual A-capsids (empty, mature capsids). The similarity in size and overall shape of the portal and a UL19 pentamer--both are cylinders of approximately 800 kDa--combined with residual noise in the tomograms, prevented us from identifying the portal vertices directly; however, this was accomplished by a computational classification procedure. Averaging the portal-containing subtomograms produced a structure that tallies with the isolated portal, as previously reconstructed by cryo-EM. The portal is mounted on the outer surface of the capsid floor layer, with its narrow end pointing outwards. This disposition differs from that of known phage portals in that the bulk of its mass lies outside, not inside, the floor. This distinction may be indicative of divergence at the level of portal-related functions other than its role as a DNA channel.

Animals↗

PubMed2006

Influenza virus pleiomorphy characterized by cryoelectron tomography.

Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging many identical particles. In this study, we used cryoelectron tomography to visualize the 3D structures of 110 individual virions of the X-31 (H3N2) strain of influenza A. The tomograms distinguish two kinds of glycoprotein spikes [hemagglutinin (HA) and neuraminidase (NA)] in the viral envelope, resolve the matrix protein layer lining the envelope, and depict internal configurations of ribonucleoprotein (RNP) complexes. They also reveal the stems that link the glycoprotein ectodomains to the membrane and interactions among the glycoproteins, the matrix, and the RNPs that presumably control the budding of nascent virions from host cells. Five classes of virions, four spherical and one elongated, are distinguished by features of their matrix layer and RNP organization. Some virions have substantial gaps in their matrix layer ("molecular fontanels"), and others appear to lack a matrix layer entirely, suggesting the existence of an alternative budding pathway in which matrix protein is minimally involved.

Animals↗

PubMed2005

A resolution criterion for electron tomography based on cross-validation.

Despite much progress in electron tomography, quantitative assessment of resolution has remained a problematic issue. The criteria that are used in single particle analysis, based on gauging the consistency between density maps calculated from half data sets, are not directly applicable because of the uniqueness of a tomographic volume. Here, we propose two criteria based on a cross-validation approach. One, called FSC(e/o), is based on a Fourier shell correlation comparison between tomograms calculated from the even and odd members of a tilt series. The other, called noise-compensated leave-one-out (NLOO), is based on Fourier ring correlation comparisons between an original projection and the corresponding reprojection of the tomogram calculated from all the other projections, taking into account the differing noise statistics. Plotted as a function of tilt angle, they allow assessment of the angular dependence of resolution and quality control over the series of projections. Integrated over all projections, the results give a global figure for resolution. Tests on simulated tomograms established consistency between these criteria and the FSC(ref), a correlation coefficient calculated between a known reference structure and the corresponding portion of a tomogram containing that structure. The two criteria-FSC(e/o) and NLOO-are mutually consistent when residual noise is the major resolution-limiting factor. When the size of the tilt increment becomes a significant factor, NLOO provides a more reliable criterion, as expected, although it is computationally intensive. Applicable to entire tomograms or selected structures, NLOO has also been tested on experimental tomographic data.

Capsid↗