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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

Infections of JJHAN cells with HHV-6A (U1102). A. Quantifications of cellular (diamonds) and extracellular (squares) HHV-6A DNA. The log10 of the viral DNA copy number per ml medium or per 106cells as normalized to actin, in inoculum, after 3 hpi and 1, 3, 5 and 7 dpi are shown. Results are based on three experiments and presented as mean ± standard deviation (error bars). B and C. Light microscopic analyses of HHV-6A- and mock-infected JJHAN cells at 3 dpi. Note the CPE, i.e. enlargement of cells, in HHV-6A infected cells. D and E. Detection of HHV-6 antigen gp60/110 in HHV-6A infected cells. HHV-6A-and mock-infected cells were fixed and stained for gp60/110 (red) and counterstained with DAPI (blue) to reveal cell nuclei. Light microscopy pictures are magnified 20× and fluorescent pictures 40×.
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Figure 1: Infections of JJHAN cells with HHV-6A (U1102). A. Quantifications of cellular (diamonds) and extracellular (squares) HHV-6A DNA. The log10 of the viral DNA copy number per ml medium or per 106cells as normalized to actin, in inoculum, after 3 hpi and 1, 3, 5 and 7 dpi are shown. Results are based on three experiments and presented as mean ± standard deviation (error bars). B and C. Light microscopic analyses of HHV-6A- and mock-infected JJHAN cells at 3 dpi. Note the CPE, i.e. enlargement of cells, in HHV-6A infected cells. D and E. Detection of HHV-6 antigen gp60/110 in HHV-6A infected cells. HHV-6A-and mock-infected cells were fixed and stained for gp60/110 (red) and counterstained with DAPI (blue) to reveal cell nuclei. Light microscopy pictures are magnified 20× and fluorescent pictures 40×.

Mentions: During purification and characterisation of virions, a major obstacle is contaminations of the viral preparations. The contaminations can stem from soluble proteins derived from the producer cells or from serum in the culture medium. An additional source is cellular proteins in released and co-purified cellular vesicles. To decrease the contaminations we optimised the production and collection of HHV-6A. Our first concern was the presence of high concentration of serum in the culture medium. Routinely, 10% serum was used for propagation of HHV-6A [19]. We tested whether infections could be performed with only 2% serum present. However, this led to 2 log reduction of viral DNA copies in medium as determined by real-time PCR (data not shown). We then changed from 10% serum at 24 h post infection to 2% serum and found that the viral DNA copy number in medium remained equivalent to infections grown in 10% serum. Our second concern was that release of contaminating material from the producer cells would increase with time. Therefore, the growth characteristics of HHV-6A were investigated and ideal time point for collection of viral particles as early as possible after infection was determined. Infections of JJHAN caused an increase of viral DNA copies in cells and medium over time (Fig. 1A). After 3 days, the viral DNA copy number in the medium was about 1.4 × 107 per ml (= 7.1 log10) and further production gave only an insignificant increase as measured by real-time PCR analyses (Fig. 1A). This meant that at 3 days post infection (dpi), the infected cells had released 279 ± 103 viral DNA copies/infected cell (n = 3). The release of virion DNA corresponded well with the accumulation of intracellular viral DNA, which also increased rapidly to 3 dpi (Fig. 1A). Cells infected with HHV-6A displayed cytopathic effects (CPE) as enlargement of cells at day 3 (Fig. 1B), in comparison to mock-infected cells (Fig. 1C) as shown by light microscopic analyses. Altogether, we decided to perform infections in 10% serum and collect HHV-6A in 2% serum media from 1 dpi to 3 dpi. A third concern was whether the majority of cells in culture were infected with HHV-6A and thereby contributing to efficient release of virions. Immunofluorescence analysis showed that approximately 80% of the infected cells were stained by the HHV-6 specific monoclonal antibody gp60/110 at day 3 (Fig. 1D). A low number of mock-infected cells showed a diffuse red staining, but were negative for nuclear staining by DAPI and therefore most likely had non-specifically taken up rhodamine (Fig. 1E). We concluded that most cells were infected and contributed to HHV-6A production.


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)

Infections of JJHAN cells with HHV-6A (U1102). A. Quantifications of cellular (diamonds) and extracellular (squares) HHV-6A DNA. The log10 of the viral DNA copy number per ml medium or per 106cells as normalized to actin, in inoculum, after 3 hpi and 1, 3, 5 and 7 dpi are shown. Results are based on three experiments and presented as mean ± standard deviation (error bars). B and C. Light microscopic analyses of HHV-6A- and mock-infected JJHAN cells at 3 dpi. Note the CPE, i.e. enlargement of cells, in HHV-6A infected cells. D and E. Detection of HHV-6 antigen gp60/110 in HHV-6A infected cells. HHV-6A-and mock-infected cells were fixed and stained for gp60/110 (red) and counterstained with DAPI (blue) to reveal cell nuclei. Light microscopy pictures are magnified 20× and fluorescent pictures 40×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Infections of JJHAN cells with HHV-6A (U1102). A. Quantifications of cellular (diamonds) and extracellular (squares) HHV-6A DNA. The log10 of the viral DNA copy number per ml medium or per 106cells as normalized to actin, in inoculum, after 3 hpi and 1, 3, 5 and 7 dpi are shown. Results are based on three experiments and presented as mean ± standard deviation (error bars). B and C. Light microscopic analyses of HHV-6A- and mock-infected JJHAN cells at 3 dpi. Note the CPE, i.e. enlargement of cells, in HHV-6A infected cells. D and E. Detection of HHV-6 antigen gp60/110 in HHV-6A infected cells. HHV-6A-and mock-infected cells were fixed and stained for gp60/110 (red) and counterstained with DAPI (blue) to reveal cell nuclei. Light microscopy pictures are magnified 20× and fluorescent pictures 40×.
Mentions: During purification and characterisation of virions, a major obstacle is contaminations of the viral preparations. The contaminations can stem from soluble proteins derived from the producer cells or from serum in the culture medium. An additional source is cellular proteins in released and co-purified cellular vesicles. To decrease the contaminations we optimised the production and collection of HHV-6A. Our first concern was the presence of high concentration of serum in the culture medium. Routinely, 10% serum was used for propagation of HHV-6A [19]. We tested whether infections could be performed with only 2% serum present. However, this led to 2 log reduction of viral DNA copies in medium as determined by real-time PCR (data not shown). We then changed from 10% serum at 24 h post infection to 2% serum and found that the viral DNA copy number in medium remained equivalent to infections grown in 10% serum. Our second concern was that release of contaminating material from the producer cells would increase with time. Therefore, the growth characteristics of HHV-6A were investigated and ideal time point for collection of viral particles as early as possible after infection was determined. Infections of JJHAN caused an increase of viral DNA copies in cells and medium over time (Fig. 1A). After 3 days, the viral DNA copy number in the medium was about 1.4 × 107 per ml (= 7.1 log10) and further production gave only an insignificant increase as measured by real-time PCR analyses (Fig. 1A). This meant that at 3 days post infection (dpi), the infected cells had released 279 ± 103 viral DNA copies/infected cell (n = 3). The release of virion DNA corresponded well with the accumulation of intracellular viral DNA, which also increased rapidly to 3 dpi (Fig. 1A). Cells infected with HHV-6A displayed cytopathic effects (CPE) as enlargement of cells at day 3 (Fig. 1B), in comparison to mock-infected cells (Fig. 1C) as shown by light microscopic analyses. Altogether, we decided to perform infections in 10% serum and collect HHV-6A in 2% serum media from 1 dpi to 3 dpi. A third concern was whether the majority of cells in culture were infected with HHV-6A and thereby contributing to efficient release of virions. Immunofluorescence analysis showed that approximately 80% of the infected cells were stained by the HHV-6 specific monoclonal antibody gp60/110 at day 3 (Fig. 1D). A low number of mock-infected cells showed a diffuse red staining, but were negative for nuclear staining by DAPI and therefore most likely had non-specifically taken up rhodamine (Fig. 1E). We concluded that most cells were infected and contributed to HHV-6A production.

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