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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

Identification of cellular proteins associated with HHV-6A particles. Media from HHV-6A- and mock-infected JJHAN cells were collected at 3 dpi and 2% aliquots were used directly for SDS-PAGE and western blot analyses and compared to the further purified material in iodixanol gradient fractions 11–15. The amounts of the mock (M) and HHV-6A (H) samples were equalized based on the number of living cells in the two cultures at the end of the collection. Cell lysates were analyzed in two amounts, 1× = 0.35 × 104 cells and 10× = 3.5 × 104 cells.
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Figure 6: Identification of cellular proteins associated with HHV-6A particles. Media from HHV-6A- and mock-infected JJHAN cells were collected at 3 dpi and 2% aliquots were used directly for SDS-PAGE and western blot analyses and compared to the further purified material in iodixanol gradient fractions 11–15. The amounts of the mock (M) and HHV-6A (H) samples were equalized based on the number of living cells in the two cultures at the end of the collection. Cell lysates were analyzed in two amounts, 1× = 0.35 × 104 cells and 10× = 3.5 × 104 cells.

Mentions: In an attempt to analyze the protein content of purified HHV-6A virions, with focus on cellular proteins, we performed western blot analysis on samples balanced to each other on basis of the number of living cells at the end of collection. We used a set of antibodies directed towards cellular proteins, including cytoplasmic, cytoskeletal and surface proteins. We decided to use only those antibodies that tested clearly positive in cell lysates, which excluded the antibodies directed towards for example CD4 and CD55. However anti-clathrin, -ezrin (also to some extent cross reactive to radixin and moesin), -Tsg101, -actin and -CD46 antibodies representing cellular vesicle, cytoskeletal, cytoplasmic and surface proteins gave a positive signal in cell lysates of infected and mock cells (Fig. 6, lanes 1 to 4). Next, we analyzed whether the cellular proteins were detected in aliquots of the non-purified collection media at 3 dpi. It was found that all selected proteins were to various degree detected in media collected from HHV-6A cells, but only Tsg101 and actin gave significant signals in comparable mock sample (Fig. 6, lanes 5 and 6). The latter finding indicates that at least Tsg101 and actin are present in background material released from cells, regardless if the cells were infected or not. Finally, we analyzed whether the proteins were present in gradient purified material from HHV-6A- and mock-infected cells, respectively (Fig. 6, lane 7 and 8). The results show that CD46 is clearly found with purified HHV-6A virions, but is virtually undetectable in the corresponding mock material. This suggests an association of CD46 with HHV-6A. Clathrin, ezrin and Tsg101 also appear to be concentrated to higher extent in HHV-6A particles compared to the equally purified mock sample and thus suggesting association of these cellular proteins with purified HHV-6A virions. This was confirmed by quantifications of at least two independent experiments yielding about 30 times more of CD46 in purified HHV-6A sample than in the corresponding mock sample. The numbers for clathrin was 4 times, for ezrin 13 times and for Tsg101 4 times. Actin was also detected in the purified HHV-6A virions but only 2 times more when compared to the corresponding mock sample. However, due to low level of signal to noise ratios, the quantifications were only approximate.


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)

Identification of cellular proteins associated with HHV-6A particles. Media from HHV-6A- and mock-infected JJHAN cells were collected at 3 dpi and 2% aliquots were used directly for SDS-PAGE and western blot analyses and compared to the further purified material in iodixanol gradient fractions 11–15. The amounts of the mock (M) and HHV-6A (H) samples were equalized based on the number of living cells in the two cultures at the end of the collection. Cell lysates were analyzed in two amounts, 1× = 0.35 × 104 cells and 10× = 3.5 × 104 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Identification of cellular proteins associated with HHV-6A particles. Media from HHV-6A- and mock-infected JJHAN cells were collected at 3 dpi and 2% aliquots were used directly for SDS-PAGE and western blot analyses and compared to the further purified material in iodixanol gradient fractions 11–15. The amounts of the mock (M) and HHV-6A (H) samples were equalized based on the number of living cells in the two cultures at the end of the collection. Cell lysates were analyzed in two amounts, 1× = 0.35 × 104 cells and 10× = 3.5 × 104 cells.
Mentions: In an attempt to analyze the protein content of purified HHV-6A virions, with focus on cellular proteins, we performed western blot analysis on samples balanced to each other on basis of the number of living cells at the end of collection. We used a set of antibodies directed towards cellular proteins, including cytoplasmic, cytoskeletal and surface proteins. We decided to use only those antibodies that tested clearly positive in cell lysates, which excluded the antibodies directed towards for example CD4 and CD55. However anti-clathrin, -ezrin (also to some extent cross reactive to radixin and moesin), -Tsg101, -actin and -CD46 antibodies representing cellular vesicle, cytoskeletal, cytoplasmic and surface proteins gave a positive signal in cell lysates of infected and mock cells (Fig. 6, lanes 1 to 4). Next, we analyzed whether the cellular proteins were detected in aliquots of the non-purified collection media at 3 dpi. It was found that all selected proteins were to various degree detected in media collected from HHV-6A cells, but only Tsg101 and actin gave significant signals in comparable mock sample (Fig. 6, lanes 5 and 6). The latter finding indicates that at least Tsg101 and actin are present in background material released from cells, regardless if the cells were infected or not. Finally, we analyzed whether the proteins were present in gradient purified material from HHV-6A- and mock-infected cells, respectively (Fig. 6, lane 7 and 8). The results show that CD46 is clearly found with purified HHV-6A virions, but is virtually undetectable in the corresponding mock material. This suggests an association of CD46 with HHV-6A. Clathrin, ezrin and Tsg101 also appear to be concentrated to higher extent in HHV-6A particles compared to the equally purified mock sample and thus suggesting association of these cellular proteins with purified HHV-6A virions. This was confirmed by quantifications of at least two independent experiments yielding about 30 times more of CD46 in purified HHV-6A sample than in the corresponding mock sample. The numbers for clathrin was 4 times, for ezrin 13 times and for Tsg101 4 times. Actin was also detected in the purified HHV-6A virions but only 2 times more when compared to the corresponding mock sample. However, due to low level of signal to noise ratios, the quantifications were only approximate.

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