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1 Department of Microbiology, Osaka University, Graduate School of Medicine, Suita, Osaka 565-0871, Japan
2 Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan
3 The Research Foundation for Microbial Diseases of Osaka University, 2-9-41 Yahata-Cho, Kanonji, Kagawa 768-0061, Japan
Correspondence
Yasuko Mori
ymori{at}nibio.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-cyclodextrin (MCD) depletion. When cholesterol was removed from HHV-6 virions with MCD, infectivity was abolished, but it could be rescued by the addition of exogenous cholesterol. HHV-6 binding was affected slightly by MCD treatment. In contrast, envelope cholesterol depletion markedly affected HHV-6 infectivity and HHV-6-induced cell fusion. These results suggest that the cholesterol present in the HHV-6 envelope plays a prominent role in the fusion process and is a key component in viral entry. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Waarts et al., 2002). Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus entry also requires cholesterol in both the target and the viral membranes (Bender et al., 2003; Campbell et al., 2001, 2002; Graham et al., 2003; Guyader et al., 2002; Viard et al., 2002). Envelope cholesterol is also a crucial factor in the fusion process of influenza virus (Sun & Whittaker, 2003).
Human herpesvirus 6 (HHV-6) is a betaherpesvirus and a human pathogen of emerging clinical significance. HHV-6 was first isolated from the peripheral blood lymphocytes of patients with lymphoproliferative disorders and AIDS (Salahuddin et al., 1986). HHV-6 isolates can be categorized into two variants, A (HHV-6A) and B (HHV-6B); HHV-6B is the causative agent of exanthem subitum (Yamanishi et al., 1988). Therefore, HHV-6 was called roseola virus. Roseola occurs in a minority of infected patients and febrile seizures are associated infrequently with primary HHV-6 infection (Zerr et al., 2005b). There is increasing evidence of HHV-6-associated disease in organ-transplant recipients. HHV-6 reactivation is common after allogeneic haematopoietic stem-cell transplantation (Zerr et al., 2005a). Human CD46 is reported to be a cellular receptor for HHV-6 (Santoro et al., 1999) and the cell–cell fusion induced by HHV-6A requires human CD46 in the target cells (Mori et al., 2002). Recently, we found that the HHV-6A glycoprotein H–glycoprotein L (gH–gL) complex interacts with the glycoprotein Q1–glycoprotein Q2 (gQ1–gQ2) complex and identified the gH–gL–gQ1–gQ2 complex as the viral ligand for human CD46 (Akkapaiboon et al., 2004; Mori et al., 2003a, b). Santoro et al. (2003) have also reported that HHV-6 gH associates with CD46 by co-immunoprecipitation.
Here, we examined the role of cholesterol in the HHV-6 envelope by using methyl--cyclodextrin (MCD) depletion. MCD efficiently depleted the envelope cholesterol and significantly reduced HHV-6 entry. Virus binding was affected only slightly, whereas depleting the envelope of cholesterol markedly affected virus fusion. Our findings suggest that cholesterol in the viral envelope plays an important role in the viral-entry process.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HHV-6A strain GS was propagated in CBMCs and the viral titres were estimated by the TCID50 method using HSB-2 cells. HHV-6 cell-free virus was prepared as described previously (Dhepakson et al., 2002). When HHV-6-infected CBMCs showed evidence of >80 % infection by immunofluorescence assay (IFA), the cells were spun at 2500 g for 15 min and the supernatant was used as cell-free virus. Partially purified virions were prepared as follows (Mori et al., 2004). HSB-2 cells were infected with HHV-6 and, at 72–96 h post-infection (p.i.), the cells were spun at 2500 g for 15 min at 4 °C. The supernatant was then subjected to centrifugation at 70 000 g for 2 h at 4 °C through a 20 % sucrose cushion. Virions were collected from the bottom. The collected virions were then passed through a 0·22 µm filter (Millipore). Sucrose gradient-purified virions were obtained as follows. Supernatant containing the virus from infected cells was collected and virus was precipitated with 10 % polyethylene glycol (molecular mass 20 kDa) in the presence of NaCl. Virus was resuspended, layered over a gradient of 15–60 % sucrose and spun for 1 h at 70 000 g. The virus was collected from the band in the gradient and concentrated by sedimentation at 70 000 g for 2 h. The pellet was suspended in RPMI 1640 medium containing 10 % FCS and passed through a 0·22 µm filter.
Antibodies.
HHV-6A monoclonal antibodies (mAbs) anti-gQ1 (AgQ1-119), anti-gL (AgL-3), anti-IE1 (AIE1) and anti-gB (OHV-1) (Mori et al., 2002) have been described previously (Akkapaiboon et al., 2004). The B subunit of cholera toxin conjugated to fluorescein isothiocyanate (FITC) was obtained from List Biological Laboratories.
Western blotting.
Cells were lysed with sample buffer containing 32 mM Tris/HCl (pH 6·8), 1·5 % SDS and 5 % glycerol. Lysed proteins were resolved by SDS-PAGE and electrotransferred onto a PVDF membrane for immunoblotting. After blocking with 10 mM Tris/HCl (pH 7·2), 0·15 M NaCl, 3 % skimmed milk and 0·75 % Tween 20 for 1 h, membranes were incubated for 1 h with blocking buffer containing the mAbs. Reactive bands were visualized by using a horseradish peroxidase-linked secondary conjugate and enhanced chemiluminescence detection reagents (Amersham Biosciences).
Immunohistochemistry.
An IFA was performed as described previously (Akkapaiboon et al., 2004).
Cholesterol depletion.
MCD and filipin III were obtained from Sigma. HHV-6 was mixed with PBS or with various concentrations of MCD or filipin III and incubated for 1 h at 37 °C. Virus treated with MCD or filipin III was subjected to ultracentrifugation through a 20 % sucrose cushion at 70 000 g for 2 h to remove the MCD or filipin III. Virus was resuspended in 500 µl RPMI 1640 medium containing 10 % FCS and passed through a 0·22 µm filter before being used to infect cells.
Cholesterol replenishment of MCD-treated HHV-6.
Dihydrocholesterol was used in this study and obtained from Sigma. The exchange of virion-associated cholesterol with exogenous cholesterol required the initial removal of cholesterol from HHV-6 particles by MCD and subsequent replenishment of the cholesterol-depleted virus with exogenous cholesterol. Virus was treated with 2·5 mM MCD for 1 h at 37 °C. This step was followed by the addition of 50, 100 or 200 µM exogenous cholesterol to the virus suspension containing MCD and the sample was incubated again for 1 h at 37 °C. The treated virus was subjected to ultracentrifugation through a 20 % sucrose cushion at 70 000 g for 2 h to remove MCD, resuspended in 500 µl RPMI 1640 medium containing 10 % FCS and passed through a 0·22 µm filter before being used to infect cells.
Binding assay.
To monitor binding, HSB-2 cells were washed twice with PBS and kept on ice for 30 min. Sucrose gradient-purified virions were treated with 10 mM MCD and the treated virus was subjected to ultracentrifugation through a 20 % sucrose cushion at 70 000 g for 2 h to remove the MCD. Repurified virions were suspended in RPMI 1640 medium and kept on ice until used. Repurified virions were incubated with cells at 4 °C for 60 min and cells with bound virus were either fixed immediately to observe binding or incubated at 37 °C for 40 min to allow infection. Cells were then washed twice, fixed and processed as described below for immunofluorescence microscopy or flow cytometry.
Flow cytometry.
For flow cytometry, cells were washed twice with PBS, fixed with 4 % paraformaldehyde and incubated with primary antibody for 30 min, followed by incubation with secondary antibody for 20 min. Cells were analysed on a FACSCalibur cytometer (Becton Dickinson Immunocytometry Systems). At least 10 000 cells were analysed for each sample.
Construction of the soluble form of CD46.
The soluble form of the CD46 or CD4 ectodomain was produced from baculovirus-infected cells by using recombinant baculoviruses as described previously (Mori et al., 2003b). Briefly, the CD46 or CD4 ectodomain with six histidine codons added was amplified by PCR. The PCR product was inserted into the plasmid pFastBac-Msp-Fc (Mori et al., 2003b). Recombinant baculovirus was prepared according to the manufacturer's protocol (Invitrogen). Hi5 cells were infected with the recombinant baculovirus (bac-CD46 or bac-CD4). At 72 h p.i., the supernatant was clarified by centrifugation. The supernatant was concentrated 10–30-fold by using a Centricon apparatus (Millipore). The concentrated supernatant was used for co-sedimentation of HHV-6 proteins.
Co-sedimentation of HHV-6 proteins with soluble CD46.
Soluble CD46 (sCD46) or soluble CD4 (sCD4) was incubated with immobilized cobalt chelate resin (ProFound Pull-Down PolyHis Protein : Protein Interaction kit; Pierce) (Mori et al., 2003b, 2004). After the resin had been washed, it was incubated with lysates of 10 mM MCD-treated or untreated HHV-6 virions. After extensive washing, proteins were eluted in elution buffer containing 290 mM imidazole and the eluted proteins were detected by Western blotting with anti-gL or anti-gQ1 mAbs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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CD to deplete cholesterol reduces virus entry significantlyCD and incubated. The treated virus was then subjected to ultracentrifugation through a 20 % sucrose cushion to remove MCD, as described in Methods. Jurkat cells were infected with the treated virus and expression of the HHV-6A IE1 protein (Mori et al., 2002) at 20 h p.i. and fusion from without (FFWO) at 6 h p.i. were examined by indirect IFA and Western blotting and by microscopy, respectively. As shown in Fig. 1(a), exposure to MCD resulted in decreased HHV-6 IE1 expression and fusion. The percentage of large cells formed by cell–cell fusion was lower when MCD-treated virus was added, compared with untreated virus (Fig. 1a). Treatment of virus with 2 mM MCD caused a significant decrease in IE1 expression (Fig. 1a, b). Treatment of HHV-6 with 3 mM MCD resulted in little detectable IE1 expression in the cells by IFA (Fig. 1a) or Western blotting (Fig. 1b). We used Jurkat cells in these experiments to observe cell–cell fusion. To confirm the block in infection caused by MCD treatment, we repeated the experiments using HSB-2 cells, which are sensitive to HHV-6A strain GS infection. When HSB-2 cells were infected with MCD-treated virus, IE1 expression in cells was lower than when untreated virus was used, similar to Jurkat cells (data not shown). Furthermore, similar inhibition of IE1 expression and FFWO was observed when the virus was treated with another cholesterol-depleting drug, filipin III (Fig. 1c–e). These results indicated that cholesterol depletion decreased the ability of HHV6 to infect cells, probably by inhibiting viral entry. The mAb for IE1 showed non-specific bands of around 120 kDa on Western blots if the film was exposed for a long time, as shown in Fig. 7(b). Therefore, in Fig. 1(e), although there appeared to be a faint band of around 120 kDa in the lane of 4 µg filipin III ml–1, this band did not indicate the presence of IE1.
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CD was subjected to ultracentrifugation through a 20 % sucrose cushion and suspended in RPMI 1640 medium. The purified virions were used to infect HSB-2 or Jurkat cells (data not shown) for 40 min and the cells were stained with FITC-conjugated cholera toxin. Cholera toxin bound similarly to cells infected with MCD-treated and untreated viruses (Fig. 2), suggesting that MCD was removed from the virions and that the envelope cholesterol is important for virus entry. We confirmed that these cells were infected by untreated but not by MCD-treated virus (data not shown).
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CD has a slight effect on HHV-6 bindingCD at 4 °C or was untreated. Cells were washed, fixed and processed for flow cytometry with the anti-gB and anti-gQ1 mAbs. Virus binding was observed by flow cytometry (Fig. 3b). The staining of gQ1 or gB on the MCD-treated virions was decreased slightly, indicating that there were slight differences between the ability of MCD-treated and untreated viruses to bind to cells by flow cytometry. By IFA (Fig. 3a), binding of the MCD-treated virions also appeared to be different from that of untreated virions, although both could bind to cells. At the same time, HSB-2 cells were incubated with the 10 mM MCD-treated virus for 40 min at 37 °C, as described in Methods. After incubation for 18 h at 37 °C, cells were harvested and expression of IE1 was observed by Western blotting (Fig. 3d) and IFA (Fig. 3c). As shown in Fig. 3(d), no IE1 was detected in cells infected with MCD-treated virus. These results indicated that MCD-treated virus could bind to the cell surface, even after treatment with 10 mM MCD, but could not enter cells.
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CD; Mori et al., 2003b, 2004). In this study, we examined the effect of MCD on the interaction of this complex and human CD46. To investigate whether the gH–gL–gQ1–gQ2 complex associated with human CD46, sCD46 and sCD4 were prepared and binding experiments were performed as described previously (Akkapaiboon et al., 2004; Mori et al., 2003b, 2004). As shown in Fig. 4, the gL and gQ1 proteins were detected in both untreated and MCD-treated virion lysates. However, of the proteins eluted from the CD46-bound resin, the amount of protein eluted from MCD-treated virion lysates (Fig. 4a and b, lane 4) was lower than that of proteins eluted from untreated virion lysates (Fig. 4a and b, lane 2), although the amount of gQ1 or gL detected in MCD-treated virion lysates was nearly equal to that detected in untreated virion lysates (Fig. 4a and b, lanes 6 and 5, respectively). These results indicated that, in spite of 10 mM MCD treatment of virions, gH–gL–gQ1–gQ2 complexes could bind to CD46, but the CD46-binding ability of the complex was decreased by depletion of cholesterol in the viral envelope.
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CDCD and untreated virus were lysed with sample buffer and immunoblotted with anti-gQ1 or anti-gL mAb under reducing and non-reducing conditions. The gQ1 and gL proteins were detected in both MCD-treated and untreated viral lysates at similar levels (Fig. 5). Furthermore, the gH–gL complexes formed by disulfide bonds were also detected in both viral lysates (Fig. 5b), indicating that, even with cholesterol depletion of the viral envelope, the glycoproteins were maintained on the envelope. We also confirmed that there was no detectable IE1 expression in cells infected with MCD-treated virus (data not shown), indicating that the drug-treated virus did not infect the cells, even though the virions contained envelope glycoproteins.
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CD-treated HHV-6CD treatment. Sucrose gradient-purified virus was treated with various concentrations of MCD for 30 min at 37 °C and the cholesterol content was determined by using an Amplex Red Cholesterol Assay kit (Molecular Probes), according to the manufacturer's protocol. The virus showed a dose-dependent drop in the level of cholesterol (Fig. 6), indicating that MCD treatment produced a specific and efficient depletion of envelope cholesterol.
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CD was permanent or reversible and to confirm that the effects of MCD were solely due to cholesterol depletion, exogenous dihydrocholesterol was used to replenish the envelopes of MCD-treated HHV-6. The exchange of virion-associated cholesterol with exogenous cholesterol required the initial removal of cholesterol from the HHV-6 particles by MCD and subsequent replenishment of the cholesterol-depleted viruses with exogenous cholesterol. This procedure restored the expression of HHV-6A IE1 and HHV-6A-induced FFWO in Jurkat cells (Fig. 7a, b
), indicating that it restored HHV-6A infectivity. The addition of 50 µM cholesterol to virus treated with 2·5 mM MCD restored infectivity. We performed the same experiment using HSB-2 cells and analysed the expression of IE1 in the cells by Western blotting. IE1 expression in HSB-2 cells was restored by adding 50 µM exogenous cholesterol, as in the Jurkat cells (Fig. 7c). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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CD, HHV-6 virions still bound to target cells, but could not enter them. In all of the experiments in this study, after the virus was treated with MCD, the drug was removed by ultracentrifugation through a 20 % sucrose cushion, as reported elsewhere (Sun & Whittaker, 2003); therefore, the inhibition of virus infection by the drug was unlikely to be due to the effects of MCD itself on the cellular membrane.
Both MCD-treated and untreated virus bound to cells, as assessed by IFA and flow cytometry, although on visual inspection the level of binding of MCD-treated virions appeared to be lower than that of untreated virions. However, the addition of 10 mM MCD to purified virions inhibited the expression of IE1 and cell–cell fusion completely, indicating that, after depletion of cholesterol from the viral envelope, at least some virus could still bind to cells, although no virus could enter the target cells, as the envelope–cell fusion process was inhibited specifically.
Furthermore, the replenishment of envelope cholesterol was partially able to restore HHV-6 infectivity and FFWO, indicating that HHV-6 envelope cholesterol may be important for the virus-induced fusion process, as reported for other enveloped viruses (Guyader et al., 2002; Sun & Whittaker, 2003; Viard et al., 2002). As shown in Fig. 7, the restoration of infectivity after replenishment of cholesterol to the viral envelope did not appear to be perfect. It may be difficult to restore the composition of the envelope glycoproteins completely simply by adding exogenous cholesterol, or it may be that other molecules affected by the cholesterol removal are also required to restore the viral-envelope composition.
Previously, we showed that the HHV-6A gH–gL–gQ1–gQ2 complex binds to human CD46 and that this binding may be important for virus–cell fusion, but not for virus–cell binding (Mori et al., 2002, 2003b). Here, we investigated the CD46 binding of the gH–gL–gQ1–gQ2 complex itself on virus envelope treated with 10 mM MCD. As shown in Fig. 4, CD46 binding of the complex was decreased by the addition of MCD, but the complex still bound to CD46, although the virus could not enter the cells under these conditions. CD46 binding of the complex may require the steric conformation of the complex itself, which may be destroyed by cholesterol depletion of the envelope. However, the result may not reflect virus–cell binding directly, as CD46 binding of the complex may be important for virus–cell fusion, but not for virus–cell attachment; thus, CD46 binding of the complex may occur after virus attachment to the cell surface. gQ1 and gB staining shown in Fig. 3(a, b) indicated binding of virus itself to the cell surface.
To investigate the role of cholesterol in virus entry, we next examined the expression of HHV-6 envelope glycoproteins gQ1 and gL in MCD-treated and untreated virions by Western blotting. The levels of gQ1and gL expression in MCD-treated and untreated virions appeared similar, indicating that the glycoproteins remained on the envelope even after the depletion of cholesterol. Under non-reducing conditions, the bands of the gL proteins, which form complexes by disulfide bonds with gH and gQ2 or gH and gO, but not with gQ1, shifted to a high molecular mass, indicating that the complexes of glycoproteins joined by disulfide bonds were not destroyed by the depletion of cholesterol in the envelope. In this experiment, we could not use anti-gB and anti-gH mAbs, because they cannot bind to the proteins in Western blots.
For the virus–cell binding experiments shown in Fig. 3, before treatment with MCD, the viruses were purified over a sucrose gradient; therefore, the finding that the drug-treated virions bound to the cell surface was not due to soluble glycoproteins binding to the cell surface.
Why was the MCD-treated virus unable to induce the viral envelope–cell fusion required for entry? One possible answer is that the depletion of cholesterol makes the envelope itself less rigid, loosening its support of the glycoproteins. Even though the glycoproteins may still be attached to the envelope by other membrane-organization factors, the conformational change in the glycoproteins required for fusion may not occur because of the looseness of the envelope base. Thus, the virus may not enter the target cells, even though it binds to them. This might also explain why there was a visual difference in the cell-surface binding between the MCD-treated and untreated virus.
Our results support the idea that cholesterol in the viral envelope plays a role in the conformational changes accompanying the glycoprotein complex-mediated fusion in a manner similar to that reported for HIV-1 (Guyader et al., 2002).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 23 September 2005;
accepted 24 October 2005.
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