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Purification of infectious human herpesvirus 6A virions and association of host cell proteins.

Hammarstedt M, Ahlqvist J, Jacobson S, Garoff H, Fogdell-Hahn A - Virol. J. (2007)

Bottom Line: Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions.Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions.Cellular proteins are associated with HHV-6A virions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition at Novum, Karolinska Institutet, Huddinge, Sweden. maria.hammarstedt@med.lu.se

ABSTRACT

Background: Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A) in multiple sclerosis (MS).

Results: We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions.

Conclusion: Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated.

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Related in: MedlinePlus

EM thin section analyses of infected cells and of particles in iodixanol gradient fractions. A. HHV-6A infected cells at 3 dpi. Indicated are viral nucleocapsids (thin arrows) in cell nuclei and virions released into the extracellular milieu (thick arrow and insert). The bar is 1000 nm. B and C. Material in gradient fractions 11–15. Note the intact and morphologically preserved HHV-6A particles in material from HHV-6A-infected cells (B) and their absence in samples from mock-infected cells (C). The bars are 500 nm.
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Figure 4: EM thin section analyses of infected cells and of particles in iodixanol gradient fractions. A. HHV-6A infected cells at 3 dpi. Indicated are viral nucleocapsids (thin arrows) in cell nuclei and virions released into the extracellular milieu (thick arrow and insert). The bar is 1000 nm. B and C. Material in gradient fractions 11–15. Note the intact and morphologically preserved HHV-6A particles in material from HHV-6A-infected cells (B) and their absence in samples from mock-infected cells (C). The bars are 500 nm.

Mentions: To further analyze the production and purity of the HHV-6A particles, EM-analyses were performed. Shown in Fig. 4A is an HHV-6A-infected cell with a number of nucleocapsids dispersed in the nucleus (thin arrows) and a complete viral particle located extracellularly (thick arrow). About 70 of 100 counted cells contained viral particles at 3 dpi. To analyse the cellular material in iodixanol gradient fractions we pooled fractions 11–15, concentrated the samples by centrifugation, embedded the pellets in gelatine and performed EM-analyses. The analyses of the gradient peak fractions 11–15 showed apparently intact and spherical virus particles in the HHV-6A sample. Importantly, no obvious cellular material was visible in the peak fractions of HHV-6A or in the corresponding mock sample (Fig. 4B and 4C). The integrity of the purified virions was further verified by negative staining (data not shown). We conclude that purification in iodixanol gradients efficiently removes cellular contamination in form of vesicles and preserves the morphology of the virions.


Purification of infectious human herpesvirus 6A virions and association of host cell proteins.

Hammarstedt M, Ahlqvist J, Jacobson S, Garoff H, Fogdell-Hahn A - Virol. J. (2007)

EM thin section analyses of infected cells and of particles in iodixanol gradient fractions. A. HHV-6A infected cells at 3 dpi. Indicated are viral nucleocapsids (thin arrows) in cell nuclei and virions released into the extracellular milieu (thick arrow and insert). The bar is 1000 nm. B and C. Material in gradient fractions 11–15. Note the intact and morphologically preserved HHV-6A particles in material from HHV-6A-infected cells (B) and their absence in samples from mock-infected cells (C). The bars are 500 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2164960&req=5

Figure 4: EM thin section analyses of infected cells and of particles in iodixanol gradient fractions. A. HHV-6A infected cells at 3 dpi. Indicated are viral nucleocapsids (thin arrows) in cell nuclei and virions released into the extracellular milieu (thick arrow and insert). The bar is 1000 nm. B and C. Material in gradient fractions 11–15. Note the intact and morphologically preserved HHV-6A particles in material from HHV-6A-infected cells (B) and their absence in samples from mock-infected cells (C). The bars are 500 nm.
Mentions: To further analyze the production and purity of the HHV-6A particles, EM-analyses were performed. Shown in Fig. 4A is an HHV-6A-infected cell with a number of nucleocapsids dispersed in the nucleus (thin arrows) and a complete viral particle located extracellularly (thick arrow). About 70 of 100 counted cells contained viral particles at 3 dpi. To analyse the cellular material in iodixanol gradient fractions we pooled fractions 11–15, concentrated the samples by centrifugation, embedded the pellets in gelatine and performed EM-analyses. The analyses of the gradient peak fractions 11–15 showed apparently intact and spherical virus particles in the HHV-6A sample. Importantly, no obvious cellular material was visible in the peak fractions of HHV-6A or in the corresponding mock sample (Fig. 4B and 4C). The integrity of the purified virions was further verified by negative staining (data not shown). We conclude that purification in iodixanol gradients efficiently removes cellular contamination in form of vesicles and preserves the morphology of the virions.

Bottom Line: Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions.Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions.Cellular proteins are associated with HHV-6A virions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition at Novum, Karolinska Institutet, Huddinge, Sweden. maria.hammarstedt@med.lu.se

ABSTRACT

Background: Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A) in multiple sclerosis (MS).

Results: We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions.

Conclusion: Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated.

Show MeSH
Related in: MedlinePlus