Limits...
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.

Show MeSH

Related in: MedlinePlus

Metabolic labelling of proteins in HHV-6A. HHV-6A- or mock-infected cells were metabolically labelled with [35S]methionine between 24.5 and 28.5 hpi or 72.5 and 76.5 hpi and virions were collected without further labelling from 28.5 to 32.5 hpi or 76.5 to 80.5 hpi. HHV-6A virions and corresponding mock sample were purified, equalized based on the number of living cells in the two cultures at the end of collection period and analyzed by 6–15% SDS-PAGE. The 1 and 3 dpi media represented 2% of the total sample volume and fractions 11–15 corresponded to 98%. Estimated molecular weights in kD of the detected proteins are indicated. H, M and Mw abbreviations as in Fig. 3. Asterisks indicate cellular proteins 88 kD and 44 kD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2164960&req=5

Figure 3: Metabolic labelling of proteins in HHV-6A. HHV-6A- or mock-infected cells were metabolically labelled with [35S]methionine between 24.5 and 28.5 hpi or 72.5 and 76.5 hpi and virions were collected without further labelling from 28.5 to 32.5 hpi or 76.5 to 80.5 hpi. HHV-6A virions and corresponding mock sample were purified, equalized based on the number of living cells in the two cultures at the end of collection period and analyzed by 6–15% SDS-PAGE. The 1 and 3 dpi media represented 2% of the total sample volume and fractions 11–15 corresponded to 98%. Estimated molecular weights in kD of the detected proteins are indicated. H, M and Mw abbreviations as in Fig. 3. Asterisks indicate cellular proteins 88 kD and 44 kD.

Mentions: In Fig. 3, the protein patterns of purified material from infected and mock cells, isolated from the iodixanol gradient peak fractions 11–15, were compared to each other and to 2% aliquots of the non-purified collection media. At 1 dpi there was no significant difference in the protein pattern between the material in non-purified collection media from HHV-6A- and mock-infected cells (Fig. 3, lane 1 and 2). All detected proteins, e.g. 44 and 88 kD, were regarded to be cellular proteins.


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)

Metabolic labelling of proteins in HHV-6A. HHV-6A- or mock-infected cells were metabolically labelled with [35S]methionine between 24.5 and 28.5 hpi or 72.5 and 76.5 hpi and virions were collected without further labelling from 28.5 to 32.5 hpi or 76.5 to 80.5 hpi. HHV-6A virions and corresponding mock sample were purified, equalized based on the number of living cells in the two cultures at the end of collection period and analyzed by 6–15% SDS-PAGE. The 1 and 3 dpi media represented 2% of the total sample volume and fractions 11–15 corresponded to 98%. Estimated molecular weights in kD of the detected proteins are indicated. H, M and Mw abbreviations as in Fig. 3. Asterisks indicate cellular proteins 88 kD and 44 kD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Metabolic labelling of proteins in HHV-6A. HHV-6A- or mock-infected cells were metabolically labelled with [35S]methionine between 24.5 and 28.5 hpi or 72.5 and 76.5 hpi and virions were collected without further labelling from 28.5 to 32.5 hpi or 76.5 to 80.5 hpi. HHV-6A virions and corresponding mock sample were purified, equalized based on the number of living cells in the two cultures at the end of collection period and analyzed by 6–15% SDS-PAGE. The 1 and 3 dpi media represented 2% of the total sample volume and fractions 11–15 corresponded to 98%. Estimated molecular weights in kD of the detected proteins are indicated. H, M and Mw abbreviations as in Fig. 3. Asterisks indicate cellular proteins 88 kD and 44 kD.
Mentions: In Fig. 3, the protein patterns of purified material from infected and mock cells, isolated from the iodixanol gradient peak fractions 11–15, were compared to each other and to 2% aliquots of the non-purified collection media. At 1 dpi there was no significant difference in the protein pattern between the material in non-purified collection media from HHV-6A- and mock-infected cells (Fig. 3, lane 1 and 2). All detected proteins, e.g. 44 and 88 kD, were regarded to be cellular proteins.

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