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Cryotomography of budding influenza A virus reveals filaments with diverse morphologies that mostly do not bear a genome at their distal end.

Vijayakrishnan S, Loney C, Jackson D, Suphamungmee W, Rixon FJ, Bhella D - PLoS Pathog. (2013)

Bottom Line: Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends.Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length.The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Virus Research, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

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Related in: MedlinePlus

Segmentation of an Archetti body.(A) Stereo images of a segmented and isosurface rendered terminal varicosity, viewed perpendicular to the vitreous ice layer and (B) at 55° to the viewing direction in (A). Density within the bulb showed single or paired sheets (pink, green, orange, light blue, yellow) in close proximity to the membrane (grey). These features were attributed to M1. The gold fiducial markers (mustard) trace the outside edge and extent of the particle. (C) A slice through the same tomogram illustrating the presence of M1 density closely associated with the particle envelope. (D and E) Transverse sections showing that these features appear bracket shaped (black arrow) and are most likely tubes, supporting the view that these are composed of M1. See also Movie S3.
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ppat-1003413-g005: Segmentation of an Archetti body.(A) Stereo images of a segmented and isosurface rendered terminal varicosity, viewed perpendicular to the vitreous ice layer and (B) at 55° to the viewing direction in (A). Density within the bulb showed single or paired sheets (pink, green, orange, light blue, yellow) in close proximity to the membrane (grey). These features were attributed to M1. The gold fiducial markers (mustard) trace the outside edge and extent of the particle. (C) A slice through the same tomogram illustrating the presence of M1 density closely associated with the particle envelope. (D and E) Transverse sections showing that these features appear bracket shaped (black arrow) and are most likely tubes, supporting the view that these are composed of M1. See also Movie S3.

Mentions: To examine Archetti bodies and other cell associated filamentous structures in more detail and in three-dimensions, we performed CET of infected frozen hydrated cells (Fig. 4, Movie S2). Filamentous structures and Archetti bodies were densely covered in surface spikes confirming their viral origin and had a contiguous matrix layer. The filamentous regions of these particles had a diameter of 74.7±0.78 nm (mean +/− SEM, measured to the tips of the glycoproteins) and extended beyond 10 µm in length (Fig. 4C). Diameters of the bulbous heads ranged from approximately 200 nm to over 550 nm. Of 41 Archetti bodies imaged 25 (61%) were found to be empty while the remaining 16 (39%) had contents within the termini. Segmentation of such particles (Fig. 5A–B, Movie S3) and close inspection of the reconstructed density (Fig. 5C–E) strongly suggested that the contents were tubules formed from M1. These features were seen to be single or paired curved sheets of density (resembling a bracket when viewed in cross-section, Figs. 5D and E). Single sheets lay parallel and closely apposed to the particle envelope (spaced between 25 and 35 nm from the envelope) while paired sheets were 30–35 nm apart. The most likely interpretation of these structures is that they are tubes in which the top and bottom are not well resolved owing to the missing-wedge artefact (features in the z-axis are poorly resolved in electron tomographic reconstructions owing to incomplete sampling caused by the geometry of the transmission electron microscope that prevents tilting the specimen to +/−90°). The missing-wedge also complicated efforts to segment the ‘top’ and ‘bottom’ portions of the viral envelope, however the extent of the bulb was rendered visible by the presence of the gold fiducial markers, revealing that the particle is substantially flattened in the vitreous ice-layer. Furthermore the position of the fiducial markers (which will not have entered the particle) indicates that those putative M1 tubes that are seen as pairs of sheets are also closely apposed to the viral envelope. In all cases the measured internal diameter of the tubes is very similar to the interior diameter of the filamentous particles, supporting our view that these structures are most likely composed of the matrix protein M1.


Cryotomography of budding influenza A virus reveals filaments with diverse morphologies that mostly do not bear a genome at their distal end.

Vijayakrishnan S, Loney C, Jackson D, Suphamungmee W, Rixon FJ, Bhella D - PLoS Pathog. (2013)

Segmentation of an Archetti body.(A) Stereo images of a segmented and isosurface rendered terminal varicosity, viewed perpendicular to the vitreous ice layer and (B) at 55° to the viewing direction in (A). Density within the bulb showed single or paired sheets (pink, green, orange, light blue, yellow) in close proximity to the membrane (grey). These features were attributed to M1. The gold fiducial markers (mustard) trace the outside edge and extent of the particle. (C) A slice through the same tomogram illustrating the presence of M1 density closely associated with the particle envelope. (D and E) Transverse sections showing that these features appear bracket shaped (black arrow) and are most likely tubes, supporting the view that these are composed of M1. See also Movie S3.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675018&req=5

ppat-1003413-g005: Segmentation of an Archetti body.(A) Stereo images of a segmented and isosurface rendered terminal varicosity, viewed perpendicular to the vitreous ice layer and (B) at 55° to the viewing direction in (A). Density within the bulb showed single or paired sheets (pink, green, orange, light blue, yellow) in close proximity to the membrane (grey). These features were attributed to M1. The gold fiducial markers (mustard) trace the outside edge and extent of the particle. (C) A slice through the same tomogram illustrating the presence of M1 density closely associated with the particle envelope. (D and E) Transverse sections showing that these features appear bracket shaped (black arrow) and are most likely tubes, supporting the view that these are composed of M1. See also Movie S3.
Mentions: To examine Archetti bodies and other cell associated filamentous structures in more detail and in three-dimensions, we performed CET of infected frozen hydrated cells (Fig. 4, Movie S2). Filamentous structures and Archetti bodies were densely covered in surface spikes confirming their viral origin and had a contiguous matrix layer. The filamentous regions of these particles had a diameter of 74.7±0.78 nm (mean +/− SEM, measured to the tips of the glycoproteins) and extended beyond 10 µm in length (Fig. 4C). Diameters of the bulbous heads ranged from approximately 200 nm to over 550 nm. Of 41 Archetti bodies imaged 25 (61%) were found to be empty while the remaining 16 (39%) had contents within the termini. Segmentation of such particles (Fig. 5A–B, Movie S3) and close inspection of the reconstructed density (Fig. 5C–E) strongly suggested that the contents were tubules formed from M1. These features were seen to be single or paired curved sheets of density (resembling a bracket when viewed in cross-section, Figs. 5D and E). Single sheets lay parallel and closely apposed to the particle envelope (spaced between 25 and 35 nm from the envelope) while paired sheets were 30–35 nm apart. The most likely interpretation of these structures is that they are tubes in which the top and bottom are not well resolved owing to the missing-wedge artefact (features in the z-axis are poorly resolved in electron tomographic reconstructions owing to incomplete sampling caused by the geometry of the transmission electron microscope that prevents tilting the specimen to +/−90°). The missing-wedge also complicated efforts to segment the ‘top’ and ‘bottom’ portions of the viral envelope, however the extent of the bulb was rendered visible by the presence of the gold fiducial markers, revealing that the particle is substantially flattened in the vitreous ice-layer. Furthermore the position of the fiducial markers (which will not have entered the particle) indicates that those putative M1 tubes that are seen as pairs of sheets are also closely apposed to the viral envelope. In all cases the measured internal diameter of the tubes is very similar to the interior diameter of the filamentous particles, supporting our view that these structures are most likely composed of the matrix protein M1.

Bottom Line: Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends.Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length.The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Virus Research, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

Show MeSH
Related in: MedlinePlus