Purification of infectious human herpesvirus 6A virions and association of host cell proteins
- Maria Hammarstedt†1,
- Jenny Ahlqvist†2,
- Steven Jacobson3,
- Henrik Garoff1 and
- Anna Fogdell-Hahn2Email author
© Hammarstedt et al; licensee BioMed Central Ltd. 2007
Received: 15 August 2007
Accepted: 19 October 2007
Published: 19 October 2007
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).
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.
Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated.
Human herpesvirus 6A and 6B (HHV-6A and 6B) are members of the betaherpesvirus subfamily. HHV-6B is a ubiquitous virus and causes the common childhood disease exanthem subitum , whereas the seroprevalence rate and pathological features for HHV-6A is unknown. Both variants are neurotropic and can cause neurological disorder [2–6] and might be potential pathologic agents in multiple sclerosis (MS), though the mechanism(s) is not understood . Putatively, incorporation of host proteins into herpes virions could have implications for autoimmunity as indicated by studies of human cytomegalovirus (HCMV) [8, 9]. Several reports demonstrate that host proteins are incorporated into enveloped viral particles [10–13]. However, the purity of the virus preparations and if host proteins are truly incorporated have been debated since cellular vesicles might contaminate the viral preparations during purification by sedimentation in sucrose gradients [14, 15]. The drawbacks with sucrose gradients have been overcome by a switch to iodixanol gradients [16–18].
We have set up a purification protocol, including iodixanol gradient, for HHV-6A that result in carefully purified, intact and infectious viral particles. To control for cellular contaminants, the background produced by mock-infected cells was determined for every step in the purification scheme and during the characterisation of the HHV-6A virions. The cellular proteins CD46, clathrin, ezrin and Tsg101 were found to be associated with the purified HHV-6A virions. Actin was also, to a lower extent, found in the purified HHV-6A virion samples.
Purification of HHV-6A particles
Purification of HHV-6A relative to cellular material
Infected cells and parallel mock cultures were metabolically labelled during a few hours with 35S-methionine to label synthesizing cellular and viral proteins. By this approach the purification can be followed relative to cellular material, disregarding the non-labelled serum contamination. However, the JJHAN cells were sensitive to the toxic effects of the isotope, which was manifested as an increase in background material over time. Hence, the labelling period was minimized to only four hours and thereafter virus particles were collected for four hours without additional labelling. Two time points for labelling and collection of particles after infection were chosen. The first time point was at 1 dpi when viral DNA is detected in the producer cells, but yet no viral particles are released into the media (Fig. 1A). Thus, labelled proteins present in media at 1 dpi should represent the background level of cellular proteins released from both infected and mock-infected cells. The second time point was at 3 dpi when the production level of virions was high (Fig. 1A). To enable quantitative comparisons of the amount of proteins in HHV-6A and mock preparations, the samples were equalized based on the number of living cells in the cultures at the end of collection. This is crucial as the mock-infected cells, but not HHV-6A-infected cells, divided during the collection period and hence would have given an overestimation of released cellular material.
In contrast to at 1 dpi, there is a significant difference in the overall protein pattern between the non-purified collection media samples from HHV-6A-infected cells and the corresponding mock samples at 3 dpi (Fig. 3, lanes 3 and 6). The HHV-6A samples contain a number of strongly labelled proteins, which are not present in the mock sample. This difference is even more pronounced in the purified material in gradient fractions 11–15 (Fig. 3, lanes 4 and 5). Since these proteins had no equivalents in the 1 dpi collection media or in the 3 dpi mock samples we assumed that they likely are proteins of the viral particles. Examples are proteins of 220 kD, 158 kD, 106 kD, 76/72 kD, 50 kD and 36 kD. Notable is that the protein pattern of the metabolically labelled and purified HHV-6A virions (Fig. 3, lane 4) was very similar to the pattern found in silver stain analyses of HHV-6A virions (Fig. 2D).
Recovery and purity of HHV-6A preparations
Viral protein (kD)
Virus to host ratio2
3 dpi Medium
Electron microscopy analyses
Purified HHV-6A particles are infectious
The results show that both non-purified and purified virions gave similar fold increase in DNA viral copies/cell at 3 dpi, which suggests that both samples contained infectious particles. The same result was obtained at 7 dpi when comparing non-diluted inocula (Fig. 5). However, a lower fold increase in DNA viral copies/cell was noticed in diluted purified inoculum, especially at 7 dpi. This probably reflects the 80 times lower initial viral copy number in purified inoculum and at the 1:4 dilution the number of virions per cell might have reached a critical low point for successful infections to occur. Another consequence is that a lower amount of cells were initially infected although with same efficiency. Thus, less viral particles were released at 3 dpi that could contribute to a second round infection. When we analyzed the viral DNA copy numbers at 1 dpi, 3 dpi, 5 dpi and 7 dpi, we found that the growth curves for the non-purified inoculum and its dilutions reached a plateau after 5 dpi as in Fig. 1, which suggests that almost all cells in the culture had become infected (data not shown). In contrary, the growth curves for the purified virions and its dilutions were still increasing even at 7 dpi and probably a third round of infection would have been necessary in order to infect all cells in the culture.
In conclusion, the purified and morphological intact HHV-6A virions retained full infectivity during the purification procedure.
Detection of cellular proteins in purified HHV-6A preparations
The goal of this study was to establish a purification method enabling protein analyses, with focus on associated cellular proteins, of highly purified, morphology preserved and still infectious HHV-6A virions. For these analyses it is important that the isolated particles are as free as possible from cellular contaminations. To this end, we modified a purification method previously shown to yield highly purified retroviral particles [17, 18]. First, to avoid extensive contaminations, we collected the HHV-6A particles during a short time interval soon after the infection. Second, the collected virus particles were sedimented in iso-osmotic iodixanol gradients which efficiently separate soluble proteins, cellular vesicles and viral particles from each other in contrast to sucrose gradients, which can lead to viral preparations contaminated by cellular vesicles [14–16]. Another disadvantage with sucrose gradients is demonstrated by extensive aggregation of HCMV particles during sedimentation, which may influence the infectivity of the purified virus [23, 24]. Third and most importantly, controls for release of cellular material and contamination of purified preparations were included. For this purpose we analyzed material released from comparable mock-infected cells and fresh culture media.
The efficiency of purification was followed by thin section EM analyses to investigate the overall content of the viral preparations . The result showed that the viral preparations were purified from contaminations in form of large aggregates or cellular vesicles. Besides, the viral particles had intact morphology since no stain penetrated the particles in negative stain analyses. Despite efficient purification, the virions were to some extent contaminated by serum proteins as seen in sensitive silver stain analyses . We made an effort to further reduce the level of serum contamination by slowly reducing the level of serum in cell culture to only 0.2%. However, the production of virus particles was decreased and the method was therefore abandoned. It should be noted, that HCMV , Epstein-Barr virus (EBV)  and Human herpesvirus 8 (HHV-8)  can be purified to levels where serum bands are not detected by the 100 times less sensitive Coomassie Brilliant blue staining . However, comparable control samples have seldom been shown, which makes the purity difficult to estimate. Purifications of HHV-6B have often included time consuming sedimentations in cesium chloride gradients [29, 30]. Notable is also that purification protocols for other viruses have given higher purification folds and recovery rates than those we obtained. We have, on the other hand, aimed to reduce the contamination already by short collection times at a suitable time point. Previous purification protocols have often not assessed the infectivity capacity of the final product. We demonstrate that HHV-6A particles purified in iodixanol gradients are infectious. Our assay might be an alternative method, if fast and mild one-day purification of viable viral particles is required.
The HHV-6A viral preparations were to low extent contaminated by cellular proteins as seen in metabolic labelling experiments. However, the cells were sensitive to the toxic effects of the isotope, which was manifested in increased background with prolonged labelling times. Hence, the protein background found in purified preparations during metabolic labelling might not be fully representative of the protein contamination level of non-labelled HHV-6A preparations. It should be noted that the background level of metabolically labelled material in these analyses could be influenced by three parameters. First, increase of cell number in the mock culture compared to the HHV-6A-infected culture may result in overestimating of released cellular material from mock culture. Therefore, the analyses were based on the number of living cells in the cultures at the end of collection of virus particles. Second, cells are dying during the experiment and material is released into the culture media. The cells were counted throughout the experiments and the number of dead cells in HHV-6A- and mock-infected cultures did not differ extensively (data not shown). Also, that the cells were washed at every step of the experiment including at the start of labelling and before collection of particles, which reduce the amount of released soluble material in the collection media. Third, HHV-6A-infected cells might react differently from mock-infected cells and due to the infection release a higher extent of cellular material or a different set of proteins into the collection media, which may result in an increase of cellular proteins in purified virions. However, two representative cellular proteins, 44 kD and 88 kD, are found at similar levels in the purified samples of both HHV-6A and mock at both 1 dpi (data not shown) and 3 dpi, indicating that our control consisting of material released from mock-infected cells is comparable to the proportion of material released from HHV-6A-infected cells, Therefore, we conclude that the cellular proteins CD46, clathrin heavy chain, ezrin, and Tsg101 are associated with the purified HHV-6A virions. Actin might also be associated with purified HHV-6A since it was found at a level of 2 times more than in the corresponding mock sample. However, it is doubtful if 2 times more is significant and therefore we just conclude that actin was present in the purified HHV-6A sample. CD46 is the receptor for HHV-6A  and as such it can be discussed whether soluble or vesicle bound CD46 released from the infected cells might bind to the produced HHV-6A virions and account for the high association of CD46 with purified HHV-6A virions. However, the issue of unspecific attachment of released proteins to produced virions has been examined before and found to not significantly contribute to the number of associated proteins . Association of CD46 with HHV-6A viral particles has previously been indirectly shown in MS patient samples. In that study, HHV-6A particles from 4 out of 42 MS patient sera were isolated using an immunoaffinity column comprised of immobilized monoclonal antibody towards CD46 . Our present results confirm a direct association of CD46 with HHV-6A virions.
Incorporation of host proteins, like complement proteins, into viral particles may exert beneficial effects for the virus as protection from complement mediated lysis [13, 33, 34]. However, incorporation of host material may also result in detrimental immune responses, such as autoreactive B- and T cells [9, 35]. For instance, addition of myelin basic protein to the enveloped virus VV was shown to be important for autoimmunity and induction of encephalomyelitis . Given that HHV-6A forms a latent infection in the brain  and that reactivation of the virus has been detected in oligodendrocytes in MS patients , it is of high relevance to investigate the overall protein content of any HHV-6A particles and especially in those released from human oligodendrocytes and to analyze the subsequent immunological events. However, such a study is impeded due to difficulties in obtaining and propagating sufficient amount of human oligodendrocytes. Our present study is a first attempt to investigate these issues and the results show that a number of diverse cellular proteins are associated with purified HHV-6A particles produced in JJHAN cells. This opens up for the possible incorporation of other cellular proteins, such as myelin in HHV-6A particles produced in oligodendrocytes, and further investigations of mechanisms for induction of autoimmune reactions.
HHV-6A virions were purified using iodixanol gradient, which efficiently separate cellular vesicles and virions. The purification yielded morphology intact and infectious particles. Purity was assessed in each step of the purification procedure by comparing with a control consisting of material released from mock-infected cells. CD46, clathrin, ezrin and Tsg101 were found to be several times more concentrated in the purified virus sample than in the similarly purified sample from mock infected cells. This suggests that these cellular proteins are specifically associated with the virions.
Viruses and cell lines
HHV-6A (U1102) was propagated in the Human T-cell lymphoblastoid cell line JJHAN as previously described .
JJHAN cells were washed with phosphate buffered saline (PBS) and infected with clarified inocula containing about 1.3 × 108 DNA viral copies of HHV-6A (U1102) per 106 cells. After 3 h incubation, cells were washed and maintained in RPMI containing 10% FCS for 24 h. The cells were washed and RPMI containing 2% FCS was added and incubation continued. At time points 3 h, 1, 3, 5 and 7 days post infection (dpi), cells and media were harvested for DNA extraction. Samples for immunofluorescence assay and electron microscopy were taken at 3 dpi. Mock infection was carried out using clarified culture media of uninfected JJHAN cells.
Production, isolation and purification of viral particles
HHV-6 particles and mock material were collected in RPMI containing 2% FCS media at chosen time intervals, mostly 1 dpi to 3 dpi. Media was clarified by centrifugations twice for 10 min at 2000 × g in a Heraeus Labofuge 400R centrifuge and once for 20 min at 39 813 × g and 10°C in a Beckman JA17 rotor and then concentrated by ultra filtration in Millipore Amicon Ultra-15 tubes (Millipore Corporation, Bedford MA, USA) at 3939 × g for repeated 10 min intervals at 20°C in a Heraeus Labofuge 400R centrifuge until about 1 ml remained. The concentrated media were filtered through a Millipore low protein binding Durapore 0.45 μm filter (Millipore Corporation, Bedford MA, USA), layered on top of a 5 to 25% (w/v) iodixanol gradient (Axis-Shield PoC AS, Oslo, Norway) and particles were purified by sedimentation for 1.5 h at 160 000 × g at 4°C in a Beckman SW41 rotor. The gradients were fractionated from the top (700 μl/fraction) and virus containing fractions were detected by real time PCR, pooled, diluted by TNE (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.5 mM EDTA) and concentrated by centrifugation in a Beckman SW41 rotor at 151 260 × g at 4°C for 1.5 h. Alternatively, individual gradient fractions were diluted in TNE and concentrated by centrifugation at 34 000 × at 10°C for 1.5 h in a Beckman JA18.1 rotor.
The refractive index (Ri) of the gradient fractions were measured and their densities (δ) calculated by the formula δ = 3.362 × Ri-3.483.
Cells were lysed in 1% Nonidet P-40, 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 1 μg/ml phenyl methyl sulfonyl fluoride on ice and the lysate was clarified by a 5 min 6000 rpm (3709 × g) centrifugation in a table top Eppendorf centrifuge.
DNA extraction and quantitative real-time TaqMan PCR
Extraction of DNA and determination of viral DNA copies for HHV-6A was performed as previously described . The viral DNA copy number (NHHV-6A) per one million cells was calculated by the following formula: (NHHV-6A) × (Nβ-actin-1) × 0.5 × 106. Final number of viral DNA copies in collected culture media was expressed as viral DNA copies/ml.
Analysis by SDS-PAGE and silver staining
Samples were separated on SDS 6–15% gradient polyacrylamide gel electrophoresis (PAGE) as described . The samples were normalized to each other by volumes of the samples or by the number of living cells from which the samples were produced. Silver stain was performed essentially as described . The gel was fixed in 40% (v/v) ethanol and 10% (v/v) acetic acid for 1 h, washed with water for 15 min, incubated twice for 30 min with 0.05% (w/v) 2-,7-napfthalenedisulfonic acid disodium salt, washed with water four times for 15 min and incubated for 30 min in a 0.8% (w/v) AgNO3, 0.34% (v/v) NH3 and 18 mM NaOH mixture. The gel was washed with water, developed with 0.01% (w/v) citric acid, 0.01% (v/v) formaldehyde mixture and the reaction was stopped with 5% (v/v) acetic acid. The gel was washed with water, dried and scanned by a CanoScan 8400F (Canon Svenska AB, Solna, Sweden).
Western blot analyses
The primary antibodies used were anti-gp60/110 (MAB8537) and anti-actin (MAB1501-R) (Chemicon International, Temecula, CA, USA), the anti-Tsg101 (sc-6037), anti-ezrin (sc-6407), anti-clathrin HC (sc-6579) and anti-CD46 (sc-9098) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The secondary antibodies used were horseradish peroxidase conjugated donkey anti-rabbit IgG (NA934), sheep anti-mouse IgG (NA931) (Amersham Pharmacia Biotech, Uppsala, Sweden) and donkey anti-goat IgG (sc-2020, Santa Cruz Biotechnology). Western blot were performed as described . Quantifications of detected proteins were performed by using a Versa Doc Imaging system, model 4000 and the QuantityOne program from Bio-Rad Laboratories (Hercules, CA, USA).
Indirect Immunofluorescent Assay
Fluorescence microscopy analysis for gp60/110 was conducted as previously described .
Transmission Electron Microscopy
Negative staining of virions in gradient fractions and preparation of JJHAN cells was done as described [38, 40]. Material in iodixanol gradient fractions 11–15 were concentrated by centrifugation and the pellets were embedded in a droplet of warm 10% gelatine in PBS (37°C) for 10 min. The samples were fixed, postfixed, sectioned and stained . Sections were examined in a Tecnai 10 (Fei Company, Eindhoven, The Netherlands) microscope operated at 80 kV equipped with a MegaView 3 digital camera. The images were acquired and analyzed with the image processing software Analysis (Soft Imaging system GmbH, Munster, Germany).
The cells were infected for 3 h and maintained in RPMI containing 5% FCS for 21 or 69 h. The cells were washed with PBS and incubated for 30 min in low-methionine DMEM medium (3.0 μg of methionine/ml) (medium no. 991303; National Veterinary Institute, Uppsala, Sweden) supplemented with phosphate up to the regular concentration of 125 μg/ml, 2 mM glutamine, 5% FCS, 20 mM HEPES, 100 U of penicillin/ml and 100 μg of streptomycin/ml. The cells were labelled for 4 h in fresh similar media supplemented with 100 μCi/ml of [35S]methionine. The cells were washed with PBS, RPMI containing 2% FCS supplemented with an excess of unlabeled methionine (300 μg/ml) was added and virus collected from 28.5 to 32.5 or 76.5 to 80.5 hpi. The particles were collected, purified and analysed by SDS-PAGE. The gel was fixed in 10% trichloroacetic acid-40% methanol for 30 min at RT before being dried and exposed to a BAS-MS2025 image plate from Fujifilm (Science Imaging Scandinavia, Nacka, Sweden). The amount of radioactivity in proteins was measured using a Molecular Imager FX, and the QuantityOne program from Bio-Rad Laboratories (Hercules, CA, USA).
We thank the Electron Microscopy facility at the Karolinska Institutet for technical assistance and Mathilda Sjöberg for helpful discussions. This work was supported by grants from the Sven Gard foundation and the Karolinska Institutet/NIH graduate program to J.A., Swedish Research Council grant 621-2003-2778 to H.G. and the Swedish Association of Persons with Neurological Disabilities and Karolinska Institutet's grant office to A.F-H.
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