Department of Medical Virology1, and Department of Molecular Pathology2, University of Tübingen, D-72076 Tübingen, Germany
Department of Pathology, University of Heidelberg, D-69120 Heidelberg, Germany3
Institute of Virology, University of Mainz, D-55101 Mainz, Germany4
Author for correspondence: Christian Sinzger. Fax +49 7071 295790. e-mail christian.sinzger{at}med.uni-tuebingen.de
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Abstract |
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Introduction |
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To date, little is known about the underlying mechanisms of HCMV interstrain cell tropism variations. It has been suggested that the infectivity of HCMV strains in macrophages might depend on the efficiency of virus entry (Minton et al., 1994 ). Unfortunately, difficulties in propagating cultured macrophages in vitro limit the availability of these cells for more detailed analyses. In contrast, EC can be subpassaged after primary isolation and grown to higher cell numbers, thus making them a more accessible model for investigating HCMV cell tropism. The phenotype of endotheliotropic versus nonendotheliotropic HCMV strains has been well defined (MacCormac & Grundy, 1999
; Sinzger et al., 1999b
; Waldman et al., 1991
). However, there are no reports of interstrain comparative analyses to address the issue of which step of the virus life-cycle is critical for expression of this phenotype. We thus chose to investigate the EC culture model in order to assess the step during virus replication that limits HCMV infection in these cells. Evidence is presented demonstrating that nuclear translocation of virus particles that have penetrated the cell is a critical event that determines EC tropism of HCMV.
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Methods |
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Virus strains.
Strains VHL/E and VHL/F, initially propagated from a bone marrow transplant recipient on EC and fibroblasts, respectively, were kindly provided by J. Waldman (Waldman et al., 1991 ). Strains TB40/E and TB40/F were derived in our laboratory from a bone marrow transplant recipient by 22 passages in EC and fibroblasts, respectively (Sinzger et al., 1999b
). AD169 is a highly passaged fibroblast-adapted laboratory strain of HCMV. Strain KSA16/3 was isolated following coinfection of EC by strain AD169 and a clinical isolate (Sinzger et al., 1999b
). For preparation of virus stocks, HFF were infected at an m.o.i. of 0·1. Supernatants of infected cultures were harvested 6 days post-infection (p.i.) and stored at -80 °C after removal of cell debris by centrifugation for 10 min at 2800 g. For preparation of purified virions, viral particles were pelleted for 70 min at 80000 g and purified on glyceroltartrate gradients (Irmiere & Gibson, 1983
). The infectious titre in HCMV preparations was determined by TCID50 assays (Mahy & Kangro, 1996
) in fibroblasts on 96-well-plates.
For cell-free infection of cell cultures, medium was removed and replaced by fresh MEM5 60 min prior to infection. Virus preparations were then added for 90 min at 37 °C. Subsequently, cells were washed with fresh medium and maintained at 37 °C. For virus adsorption, cells were incubated with virus preparations on ice to prevent virus entry. To allow for virus penetration and replication, cells were shifted to 37 °C after a 90 min adsorption period. After infection, cells were washed and maintained at 37 °C in the appropriate medium. For cell-associated propagation of virus, infected cultures were subpassaged when they had grown to confluence.
For single-step growth curves, HUVEC grown to subconfluence in 75 cm2 culture flasks were infected with HCMV preparations as described before at a virus concentration of 106 TCID50/ml (m.o.i.=1). After 90 min of incubation, cultures were washed six times with medium to remove residual virus and were then cultured for 10 days at 37 °C in RPMI 1640 without heparin or ECGS. Starting at day 1 p.i., 2 ml supernatant was removed daily from infected cell cultures and replaced by 2 ml of fresh medium; the supernatant samples were stored at -80 °C prior to determination of the infectious titre.
Transfection of UL122/123 plasmid pRR47.
Plasmid pRR47 is a pUC18-based plasmid containing the complete UL122/123 gene region of AD169 in a 6·7 kb EcoRISalI insert (Stamminger et al., 1991 ). HFF or HUVEC were seeded into six-well culture plates at a density of 200000 cells per well 24 h prior to transfection. For transfection 1 µg of plasmid DNA was introduced into the respective cell culture using Superfect reagent (Qiagen). At 48 h after transfection, transfected cells were trypsinized, cytocentrifuged onto glass slides, and fixed with acetone at room temperature for 5 min. Viral IE antigens were detected by indirect immunoperoxidase staining as described below.
Radiolabelling of HCMV.
For use in binding assays HCMV was radioactively labelled in culture by incorporation of [35S]methionine. Infected HFF grown in 175 cm2 plastic flasks at 30% CPE were incubated with 15 ml medium containing 6·25 MBq [35S]methionine (Amersham) for 2 days. Cell debris in the supernatant was removed by centrifugation at 2800 g for 10 min and [35S]methionine-labelled virus was collected from the medium by centrifugation at 80000 g for 70 min in a Beckman ultracentrifuge. The radioactively labelled virus preparations were washed three times by ultracentrifugation to minimize the amount of unincorporated [35S]methionine and resuspended in 1 ml MEM.
Monoclonal antibodies, immunoblotting and immunoperoxidase staining.
To analyse viral gene expression, monoclonal antibodies (MAbs) against viral proteins from different phases of the HCMV replicative cycle were used. In detail, MAbs were reactive against the immediate early (IE) proteins IE72 and IE86 (pUL122/123, MAb E13; Biosoft, Paris, France), the early protein p52 (pUL44, MAb BS510; Biotest, Dreieich, Germany), the early late protein pp65 (pUL83, MAb 28-77; kindly provided by W. Britt, Birmingham, AL, USA), the late major capsid protein (pUL86, MAb 28-4; kindly provided by W. Britt), and the late tegument protein pp150 (pUL32, MAb XP1; Behringwerke, Marburg, Germany) (Jahn et al., 1990 ). MAbs against vimentin (Dako) and lamin B (Calbiochem) were used to detect cytoskeleton components and nuclear components, respectively.
For immunoblotting, protein samples were prepared by lysis of cells in sample buffer containing 2% SDS and 1·5% dithiothreitol, separated by SDSPAGE, blotted on nitrocellulose, and detected with the ECL Western blotting detection system (Amersham).
For in situ-detection of viral antigens in infected cells, indirect immunoperoxidase staining was done as follows. At various time-points after infection, cells grown in 24-well dishes were fixed with 80% acetone for 5 min at room temperature. Fixed cells were reacted with antibodies against viral antigens for 60 min at 37 °C, followed by incubation with peroxidase-conjugated goat anti-mouse IgFab'2 polyclonal sera (De Beer Medicals, Hilvarenbeek, Netherlands). Finally, antigens were detected by staining with diaminobenzidine (DAB; Sigma) and observation with a light microscope.
Analysis of virus adsorption and penetration.
For adsorption assays, HFF and HUVEC grown in 24-well dishes were incubated with [35S]methionine labelled virus at 4 °C for various times in triplicate. Negative controls contained heparin (100 IU/ml) to prevent virus adsorption. After incubation with virus the cells were washed three times with medium and lysed in 200 µl 1 M NaOH prior to scintillation counting of radioactivity in a -counter (Wallac 1409) to determine the extent of virus binding. For penetration assays, HFF and HUVEC grown in 75 cm2 plastic flasks were incubated with 35S-labelled virus for various times at 37 °C. Subsequently, cells were washed twice with ice-cold trypsinEDTA and were incubated with trypsinEDTA for 1 h on ice to eliminate adsorbed virus. Cells were pelleted by centrifugation and washed with MEM. Cells were then lysed in 1 M NaOH and radioactivity determined by scintillation in a
-counter. For analyses of penetration at low levels of infection, cells were treated as described above at m.o.i.=0·1. Trypsin-treated cells were harvested by centrifugation and after lysis of cells with buffer containing 0·5% SDS and 0·1 mg/ml proteinase K, DNA was extracted with phenolchloroform (Ausubel et al., 1989
) and analysed by quantitative HCMV-DNA-PCR.
Electron microscopy.
Cells were rinsed in PBS and fixed in 1·5% glutaraldehyde in 0·2 mol/l PBS for 30 min at 4 °C. After three washes for 10 min each in PBS containing 7·5% sucrose at room temperature cells were post-fixed in 1% osmium tetroxide in PBS for 60 min, dehydrated in a graded series of ethanol and embedded in Araldite (Merck). Ultrathin sections (70 nm) were picked up on 300 mesh nickel grids and stained with 1% uranyl acetate and 1·5% lead citrate. Sections were examined with a Zeiss EM 902 transmission electron microscope.
Analysis of nuclear localization of viral DNA.
For quantification of nuclear localization of viral DNA, HFF and HUVEC were infected at low m.o.i. (0·010·1). After incubation with virus preparations for 90 min, cells were harvested by trypsinEDTA treatment and washed with MEM5 to inactivate trypsin. After additional washings with PBS and hypotonic buffer (10 mmol/l HEPES, 1·5 mmol/l MgCl2, 10 mmol/l KCl, pH 7·6), cells were incubated with hypotonic buffer on ice for up to 90 min until 100% of cells were swollen. Nuclei were then liberated by 1015 strokes with a Dounce homogenizer, pelleted by centrifugation at 2800 g, resuspended in nuclei buffer (0·25 mol/l sucrose, 5 mmol/l MgCl2, 25 mmol/l KCl, 20 mmol/l TricineNaOH pH 7·8), mixed with an equal volume of 50% iodixanol solution (Optiprep; Gibco), and purified by centrifugation for 20 min at 10000 g through a 30/35% iodixanol gradient. Purified nuclei at the interface were collected and lysed with buffer containing 0·5% SDS and 0·1 mg/ml proteinase K. DNA was extracted from these preparations with phenolchloroform (Ausubel et al., 1989 ). The viral DNA content in nuclear DNA preparations was determined by quantitative competitive HCMV-DNA-PCR.
Quantitative PCR assays.
Viral nucleic acids within cellular or nuclear DNA probes were quantified by competitive PCR using primers P1 (5' GGT CAC TAG CGC TTG TAT GAT GAC CA 3') and P2 (5' TTC TCA GCC ACA ATT ACT GAG GAC AGA GGG A 3') within exon 4 of the HCMV IE gene UL123. An aliquot of 1000 copies of plasmid pHM 471 (kindly provided by T. Stamminger) was added to each sample of a logarithmic dilution series of the DNA preparations. pHM 471 carries a 100 bp deletion within the amplified gene region. The reactions were done in a total volume of 50 µl consisting of 2·0 mM MgCl2, 0·25 mM each dNTP, 10 pmol each primer, 1x PCR buffer (Boehringer Mannheim) and 1 U Taq polymerase (Boehringer Mannheim). Thermal cycling was performed as follows: 35 cycles of 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 1 min. Amplification products were visualized by electrophoresis in agarose gels, ethidium bromide staining and UV light illumination. Equivalence of the natural HCMV IE amplification product and the pHM471 plasmid amplification product indicated the presence of 1000 HCMV-DNA copies in the analysed sample.
Immunostaining of viral particles after penetration.
Cells were grown to subconfluence in 25 cm2 culture flasks in appropriate culture medium. Prior to infection, cells were preincubated for 30 min with MEM5. Cells were then incubated with cell-free virus preparations for 0·54 h at 37 °C. To remove adsorbed virus particles that had not penetrated, cells were treated with trypsinEDTA (Gibco) for 20 min. Trypsin was inactivated by washing with MEM5 and, after resuspension in PBS, cells were cytocentrifuged onto glass slides. Cells were fixed with acetone for 5 min at room temperature and immunostained. For detection of viral particles, MAb XP1, directed against the viral tegument protein pp150, was used as a primary antibody. Subsequently, rabbit anti-mouse Ig polyclonal antiserum, peroxidaseantiperoxidase complexes (PAP; Dako) from mouse, biotinylated swine anti-mouse Ig antibodies (Dako) and peroxidase-conjugated streptavidinbiotin complex (Dako) were added. Signals were detected using DAB or metal-enhanced (Co-)DAB (Sigma) as chromogens. These staining procedures resulted in punctate brown (DAB) or bluish-black (Co-DAB) visualization of viral particles. If simultaneous detection of viral IE antigen in the same cell preparations was desired, an indirect immunoalkaline phosphatase staining of pUL122/123 was performed subsequent to Co-DAB staining of the pp150 antigen. Incubation with MAb E13 was followed by incubation with peroxidase-conjugated goat anti-mouse IgFab'2 polyclonal sera (De Beer Medicals). Signals were then detected using DAB as chromogen, resulting in gold-brown nuclear staining. Slides were mounted with glycerolgelatin and staining observed with a Polyvar microscope (Cambridge Instruments) using interference contrast (DIC).
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Results |
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However, for analysis of the mechanism underlying this phenotype, focal expansion of HCMV in cell monolayers was unsuitable, and synchronized supernatant-associated infections were desired for dissection of distinct steps of the virus replication cycle. Therefore, the endotheliotropic phenotype of the various HCMV strains was quantitatively determined after infection of cell monolayers with cell-free virus preparations (Fig. 1, Table 1
). Fibroblast cultures and EC cultures were infected in parallel with each virus strain at an m.o.i. of 0·5 and the fraction of infected cells was determined at 24 h p.i. by immunodetection of viral IE antigen. Again, HCMV strains KSA16/3, TB40/E and VHL/E were found to be endotheliotropic, whereas infectivity in EC was dramatically reduced with HCMV strains AD169, TB40/F and VHL/F (Fig. 1
). Taken together, the infectivity of nonendotheliotropic strains was about 1001000-fold reduced in HUVEC as compared to HFF (Table 1
). In contrast, endotheliotropic strains displayed only a slight reduction of infectivity in HUVEC as compared to HFF. This dramatic phenotypic difference of HCMV variants in easy-to-standardize supernatant-associated infectivity assays could now be subjected to detailed analysis of dissected events during the virus life-cycle.
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Efficiency of nuclear translocation of viral DNA is correlated with the endotheliotropic phenotype of HCMV variants
Following virus penetration, nuclear transport of viral particles and release of viral DNA are prerequisites for the initiation of virus replication. As the phenotype of nonendotheliotropic HCMV strains could neither be explained by inefficient penetration nor by inefficiency of the IE gene in EC, we hypothesized that inefficient nuclear translocation of viral DNA might determine this phenotype. A major prerequisite for the investigation of the nuclear translocation of viral DNA was the preparation of pure nuclear fractions of infected cells. In particular, the cytoskeleton had to be separated from nuclei, as herpesviral particles are known to bind to the cytoskeleton after penetration (Sodeik et al., 1997 ). To this end, nuclei were liberated from infected cells with a Dounce homogenizer after swelling in hypotonic buffer and subsequently separated from cytoskeleton contaminants by density gradients. Western blot assays of the resulting subcellular fractions were done to prove the purity of the preparations. Absence of vimentin immunoreactivity in the presence of nuclear marker lamin B indicated the purity of the nuclear fractions. While standard protocols like sucrose-gradient centrifugation of Dounce-homogenized cell preparations failed to fulfil this criterion, iodixanol gradients did (Fig. 5A
). For all subsequent experiments, iodixanol-gradient centrifugation of Dounce-homogenized cell preparations was used. The viral DNA content in nuclei of infected cells was determined by quantitative competitive DNA PCR (Sinzger et al., 1999a
). This sensitive approach was preferred because other methods like Southern blotting or in situ-hybridization would entail infections at abnormally high multiplicities, which in turn might favour abnormal routes of virus trafficking. Three nonendotheliotropic strains (AD169, TB40/F and VHL/F) and two endotheliotropic strains (TB40/E and VHL/E) were included in this analysis. The nuclear HCMV-DNA content of all endotheliotropic variants was about equal in fibroblasts and EC (Fig. 5B
). In contrast, all nonendotheliotropic strains displayed 1001000-fold decreased HCMV-DNA titres in the nuclei of infected EC as compared to fibroblasts (Fig. 5B
).
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In summary, the EC tropism of HCMV variants appeared to be determined by the efficiency of nuclear transport of virus particles after successful penetration.
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Discussion |
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Regarding the mechanism underlying the inefficiency of nuclear DNA import, our results differ from the observations reported by Slobbe van Drunen et al. (1998) . While these authors described accumulation of signals around the nucleus, we found that in contrast viral particles of nonendotheliotropic HCMV strains accumulate in the cytoplasm of EC and do not reach the nucleus (Fig. 6
). Thus, while the former report would imply that release of viral DNA at the nucleus is blocked our data strongly suggest that the transport of virus particles towards the nucleus is impaired. This discrepancy might be partially explained by the use of different assays for virus detection. Condensed genomes within penetrated but still intact capsids might be undetectable by in situ hybridization and the application of very high m.o.i.s might favour aberrant entry pathways.
In our approach, combining immunostaining of particles with the detection of viral IE antigen, we could directly correlate the nuclear transport of penetrated virus with the successful initiation of viral gene expression even at low to moderate m.o.i.s. Using this approach an unusual mechanism for the determination of virus cell tropism was found. Nonendotheliotropic HCMV variants are distinguished from endotheliotropic HCMV variants by their dramatic inefficiency in nuclear translocation of penetrated virus particles in EC, although all HCMV variants are efficient in fibroblasts. Thus there appears to be both an interstrain difference between HCMV variants regarding initial events in EC and a cell type-specific difference between EC and fibroblasts regarding transport processes.
It is tempting to speculate about the possible interactions between viral and cellular structures that mediate the efficiency of nuclear transport of virus particles. During the transport of capsids from the cellular membrane to the nucleus, interactions of the viral tegument or capsid with components of the microtubule system might be important, as has been shown previously for herpes simplex virus (Sodeik et al., 1997 ). The use of different motor proteins in HUVEC and HFF would provide an explanation for the cell type differences observed. At present, these considerations are still speculative. However, a viruscell system is now available for detailed analyses of the nuclear transport processes involved in initiation of HCMV infection. Our finding that the transport of penetrated HCMV particles is a critical event in the virus life-cycle might furthermore indicate that this step is a potential target for future antiviral strategies.
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Acknowledgments |
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References |
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Received 30 May 2000;
accepted 22 August 2000.