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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

Purification of HHV-6A. A and B. Material in pooled iodixanol gradient fractions were concentrated by centrifugation, separated by 6–15% SDS-PAGE and visualized by silver nitrate staining or western blot using the viral specific (gp60/110) antibody. C. DNA analyses of iodixanol gradient fractions. The number of viral DNA copies in iodixanol gradient fractions of two independent experiments was measured by TaqMan based real-time PCR and the densities of the fractions by refractometer. D. The proteins in gradient purified material of HHV-6A- and mock-infected cultures were compared by SDS-PAGE stained with silver nitrate. Fresh culture medium was analyzed as a control. Estimated molecular weights in kD of the detected proteins are indicated. All samples in each separate analysis were equalized to each other based on sample volume. H, M and Mw indicate HHV-6A, mock and molecular weight marker.
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Figure 2: Purification of HHV-6A. A and B. Material in pooled iodixanol gradient fractions were concentrated by centrifugation, separated by 6–15% SDS-PAGE and visualized by silver nitrate staining or western blot using the viral specific (gp60/110) antibody. C. DNA analyses of iodixanol gradient fractions. The number of viral DNA copies in iodixanol gradient fractions of two independent experiments was measured by TaqMan based real-time PCR and the densities of the fractions by refractometer. D. The proteins in gradient purified material of HHV-6A- and mock-infected cultures were compared by SDS-PAGE stained with silver nitrate. Fresh culture medium was analyzed as a control. Estimated molecular weights in kD of the detected proteins are indicated. All samples in each separate analysis were equalized to each other based on sample volume. H, M and Mw indicate HHV-6A, mock and molecular weight marker.

Mentions: Media was collected from HHV-6A-infected cells and mock-infected control cells from 1 to 3 dpi. Importantly, the mock control was included in order to estimate the background level of contaminations during purification and analyses of HHV-6A particles. The virus and mock sample were purified in several steps as detailed in material and methods. Briefly, the collection media were clarified by short centrifugations, concentrated by ultra filtration and filtered through a 0.45 μm filter. The viral particles, and mock material, were finally purified by sedimentation on a 5–25% w/v iodixanol gradient. The iodixanol gradient fractions were concentrated into pellets by centrifugation and analyzed by SDS-PAGE followed by silver staining. As seen in lanes 1 and 2 in Fig. 2A and 2B, a considerable amount of protein containing material from both HHV-6A and mock preparations was detected in the top fractions of the iodixanol gradient while the bottom fractions contained much less proteins. A large difference in protein pattern between HHV-6A and mock samples is seen in fractions 11–15 (Fig 2A and 2B, lane 4). A number of clearly concentrated probable viral proteins are displayed in the HHV-6A sample while the mock shows a diffuse background. The presence of HHV-6A in fractions 11–15 was confirmed by parallel western blot analyses using the monoclonal HHV-6 antibody gp60/110 (Fig. 2A, lower). A strong signal for viral protein gp60 and a weak signal for gp110 were detected in these fractions. As expected, gp60/110 was not detected in the parallel mock analyses (Fig. 2B, lower). The faint bands detected in the top fractions of the mock sample gradient were most likely the result of unspecific reactions between the antibodies and serum proteins or cellular proteins (Fig 2B, lower, lanes 1 and 2). Corresponding bands were also seen in the HHV-6A blot (Fig. 2A, lower, lanes 1 and 2). Furthermore, real-time PCR analyses of viral DNA in the gradient fractions revealed a peak of HHV-6A DNA in fractions 11–15 as shown in Fig. 2C. The density of these fractions was determined to be between 1.09–1.12 g/ml. Although the majority of the initial mock material was removed during the purification procedure, a background was still present in the gradient fractions 11–15 (Fig. 2B, lane 4). We hypothesized that the background represented mostly proteins from the serum in the culture medium rather than contaminating host proteins. As a control for solely medium proteins an equal volume of fresh culture media was also concentrated, filtered and sedimented in iodixanol gradient and fractions 11–15 were analyzed in parallel with HHV-6A and mock preparations (Fig. 2D). The medium control clearly shows that the background in purified mock corresponded to culture media proteins. This background was increased if the virions were harvested in culture media containing 10% serum (data not shown). We concluded that the purification of HHV-6A virions removed a substantial amount of cellular contaminating material, but that the virions were still to some extent contaminated with soluble serum 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)

Purification of HHV-6A. A and B. Material in pooled iodixanol gradient fractions were concentrated by centrifugation, separated by 6–15% SDS-PAGE and visualized by silver nitrate staining or western blot using the viral specific (gp60/110) antibody. C. DNA analyses of iodixanol gradient fractions. The number of viral DNA copies in iodixanol gradient fractions of two independent experiments was measured by TaqMan based real-time PCR and the densities of the fractions by refractometer. D. The proteins in gradient purified material of HHV-6A- and mock-infected cultures were compared by SDS-PAGE stained with silver nitrate. Fresh culture medium was analyzed as a control. Estimated molecular weights in kD of the detected proteins are indicated. All samples in each separate analysis were equalized to each other based on sample volume. H, M and Mw indicate HHV-6A, mock and molecular weight marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Purification of HHV-6A. A and B. Material in pooled iodixanol gradient fractions were concentrated by centrifugation, separated by 6–15% SDS-PAGE and visualized by silver nitrate staining or western blot using the viral specific (gp60/110) antibody. C. DNA analyses of iodixanol gradient fractions. The number of viral DNA copies in iodixanol gradient fractions of two independent experiments was measured by TaqMan based real-time PCR and the densities of the fractions by refractometer. D. The proteins in gradient purified material of HHV-6A- and mock-infected cultures were compared by SDS-PAGE stained with silver nitrate. Fresh culture medium was analyzed as a control. Estimated molecular weights in kD of the detected proteins are indicated. All samples in each separate analysis were equalized to each other based on sample volume. H, M and Mw indicate HHV-6A, mock and molecular weight marker.
Mentions: Media was collected from HHV-6A-infected cells and mock-infected control cells from 1 to 3 dpi. Importantly, the mock control was included in order to estimate the background level of contaminations during purification and analyses of HHV-6A particles. The virus and mock sample were purified in several steps as detailed in material and methods. Briefly, the collection media were clarified by short centrifugations, concentrated by ultra filtration and filtered through a 0.45 μm filter. The viral particles, and mock material, were finally purified by sedimentation on a 5–25% w/v iodixanol gradient. The iodixanol gradient fractions were concentrated into pellets by centrifugation and analyzed by SDS-PAGE followed by silver staining. As seen in lanes 1 and 2 in Fig. 2A and 2B, a considerable amount of protein containing material from both HHV-6A and mock preparations was detected in the top fractions of the iodixanol gradient while the bottom fractions contained much less proteins. A large difference in protein pattern between HHV-6A and mock samples is seen in fractions 11–15 (Fig 2A and 2B, lane 4). A number of clearly concentrated probable viral proteins are displayed in the HHV-6A sample while the mock shows a diffuse background. The presence of HHV-6A in fractions 11–15 was confirmed by parallel western blot analyses using the monoclonal HHV-6 antibody gp60/110 (Fig. 2A, lower). A strong signal for viral protein gp60 and a weak signal for gp110 were detected in these fractions. As expected, gp60/110 was not detected in the parallel mock analyses (Fig. 2B, lower). The faint bands detected in the top fractions of the mock sample gradient were most likely the result of unspecific reactions between the antibodies and serum proteins or cellular proteins (Fig 2B, lower, lanes 1 and 2). Corresponding bands were also seen in the HHV-6A blot (Fig. 2A, lower, lanes 1 and 2). Furthermore, real-time PCR analyses of viral DNA in the gradient fractions revealed a peak of HHV-6A DNA in fractions 11–15 as shown in Fig. 2C. The density of these fractions was determined to be between 1.09–1.12 g/ml. Although the majority of the initial mock material was removed during the purification procedure, a background was still present in the gradient fractions 11–15 (Fig. 2B, lane 4). We hypothesized that the background represented mostly proteins from the serum in the culture medium rather than contaminating host proteins. As a control for solely medium proteins an equal volume of fresh culture media was also concentrated, filtered and sedimented in iodixanol gradient and fractions 11–15 were analyzed in parallel with HHV-6A and mock preparations (Fig. 2D). The medium control clearly shows that the background in purified mock corresponded to culture media proteins. This background was increased if the virions were harvested in culture media containing 10% serum (data not shown). We concluded that the purification of HHV-6A virions removed a substantial amount of cellular contaminating material, but that the virions were still to some extent contaminated with soluble serum 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