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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

Time course immunofluorescence imaging of filament formation in MDCK cells infected with Influenza A/Udorn/72.DAPI was used to stain cell nuclei (blue) while phalloidin was used to detect actin (red). Monoclonal antibodies were used to detect viral proteins; NP is shown in green and HA is shown in white. Budding virus is seen from as early as 6 hours post infection. From 8 hours we can see long filaments and Archetti bodies at the cell surface.
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ppat-1003413-g002: Time course immunofluorescence imaging of filament formation in MDCK cells infected with Influenza A/Udorn/72.DAPI was used to stain cell nuclei (blue) while phalloidin was used to detect actin (red). Monoclonal antibodies were used to detect viral proteins; NP is shown in green and HA is shown in white. Budding virus is seen from as early as 6 hours post infection. From 8 hours we can see long filaments and Archetti bodies at the cell surface.

Mentions: To achieve an overview of the formation of viral filaments we performed confocal immunofluorescence imaging of MDCK cells infected with influenza virus (A/Udorn/72) at low multiplicity of infection (MOI). This revealed an abundance of viral filaments, many of which had bulbous heads (Archetti bodies). Our images demonstrated the position of these heads to be at the leading (distal) end of the filaments (Fig. 1). Some filaments appeared to have varicosities along their lengths, however such features were not seen in subsequent CET analysis, strongly suggesting that these features were a consequence of several filaments of different lengths lying in close proximity. Viral filaments were extremely long, some measuring greater than 10 µm. A time course was performed revealing the presence of virions as early as 6 hours post-infection (p.i.) (Fig. 2) while filaments and Archetti bodies were seen from 8 hours p.i. (Fig. 2–3, movie S1). Fluorescence imaging of unpermeabilised infected-cells confirmed that the filamentous and Archetti structures seen were not artefacts of preparation (Fig S1). Further control experiments were performed to compare these data with patterns of fluorescence seen in A549 cells infected with Udorn (Fig S2). This revealed that fewer and shorter filaments were produced although the filamentous trait was still in evidence. MDCK cells infected with the spherical Influenza (A/WSN/33) strain on the other hand did not show any filamentous forms (Fig S3).


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)

Time course immunofluorescence imaging of filament formation in MDCK cells infected with Influenza A/Udorn/72.DAPI was used to stain cell nuclei (blue) while phalloidin was used to detect actin (red). Monoclonal antibodies were used to detect viral proteins; NP is shown in green and HA is shown in white. Budding virus is seen from as early as 6 hours post infection. From 8 hours we can see long filaments and Archetti bodies at the cell surface.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675018&req=5

ppat-1003413-g002: Time course immunofluorescence imaging of filament formation in MDCK cells infected with Influenza A/Udorn/72.DAPI was used to stain cell nuclei (blue) while phalloidin was used to detect actin (red). Monoclonal antibodies were used to detect viral proteins; NP is shown in green and HA is shown in white. Budding virus is seen from as early as 6 hours post infection. From 8 hours we can see long filaments and Archetti bodies at the cell surface.
Mentions: To achieve an overview of the formation of viral filaments we performed confocal immunofluorescence imaging of MDCK cells infected with influenza virus (A/Udorn/72) at low multiplicity of infection (MOI). This revealed an abundance of viral filaments, many of which had bulbous heads (Archetti bodies). Our images demonstrated the position of these heads to be at the leading (distal) end of the filaments (Fig. 1). Some filaments appeared to have varicosities along their lengths, however such features were not seen in subsequent CET analysis, strongly suggesting that these features were a consequence of several filaments of different lengths lying in close proximity. Viral filaments were extremely long, some measuring greater than 10 µm. A time course was performed revealing the presence of virions as early as 6 hours post-infection (p.i.) (Fig. 2) while filaments and Archetti bodies were seen from 8 hours p.i. (Fig. 2–3, movie S1). Fluorescence imaging of unpermeabilised infected-cells confirmed that the filamentous and Archetti structures seen were not artefacts of preparation (Fig S1). Further control experiments were performed to compare these data with patterns of fluorescence seen in A549 cells infected with Udorn (Fig S2). This revealed that fewer and shorter filaments were produced although the filamentous trait was still in evidence. MDCK cells infected with the spherical Influenza (A/WSN/33) strain on the other hand did not show any filamentous forms (Fig S3).

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