Download Tropism of human cytomegalovirus for endothelial cells is

Journal
of General Virology (2000), 81, 3021–3035. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Tropism of human cytomegalovirus for endothelial cells is
determined by a post-entry step dependent on efficient
translocation to the nucleus
C. Sinzger,1 M. Kahl,1 K. Laib,1 K. Klingel,2 P. Rieger,3 B. Plachter4 and G. Jahn1
Department of Medical Virology1, and Department of Molecular Pathology2, University of Tu$ bingen, D-72076 Tu$ bingen, Germany
Department of Pathology, University of Heidelberg, D-69120 Heidelberg, Germany
4
Institute of Virology, University of Mainz, D-55101 Mainz, Germany
1, 2
3
Marked interstrain differences in the endothelial cell (EC) tropism of human cytomegalovirus
(HCMV) isolates have been described. This study aimed to define the step during the replicative
cycle of HCMV that determines this phenotype. The infection efficiency of various HCMV strains in
EC versus fibroblasts was quantified by immunodetection of immediate early (IE), early and late
viral antigens. Adsorption and penetration were analysed by radiolabelled virus binding assays
and competitive HCMV-DNA-PCR. The translocation of penetrated viral DNA to the nucleus of
infected cells was quantified by competitive HCMV-DNA-PCR in pure nuclear fractions. The
intracytoplasmic translocation of capsids that had penetrated was followed by immunostaining of
virus particles on a single particle level ; this was correlated with the initiation of viral gene
expression by simultaneous immunostaining of viral IE antigens. The infectivity of nonendotheliotropic HCMV strains in EC was found to be 100–1000-fold lower when compared to
endotheliotropic strains. The manifestation of this phenotype at the level of IE gene expression
indicated the importance of initial replication events. Surprisingly, no interstrain differences were
detected during virus entry. However, dramatic interstrain differences were found regarding the
nuclear translocation of penetrated viral DNA. With nonendotheliotropic strains, the content of
viral DNA in the cell nucleus was 100–1000-fold lower in EC when compared to endotheliotropic
strains, thereby reflecting the strain differences in IE gene expression. Simultaneous staining of
viral particles and viral IE antigen revealed that interstrain differences in the transport of
penetrated capsids towards the nucleus of endothelial cells determine the EC tropism of HCMV.
Introduction
A hallmark of human cytomegalovirus (HCMV) is its broad
host-cell range during acute infection in vivo, including
epithelial cells, fibroblasts, smooth muscle cells, endothelial
cells (EC) and macrophages (Ng Bautista & Sedmak, 1995 ;
Roberts et al., 1988, 1989 ; Sinzger et al., 1993, 1995, 1996 ;
Wiley & Nelson, 1988). EC and macrophages were suggested
to play a major role in the haematogeneous dissemination of
the virus (Grefte et al., 1995 ; Ibanez et al., 1991 ; Lathey &
Spector, 1991 ; Sinzger & Jahn, 1996). In cell culture significant
Author for correspondence : Christian Sinzger.
Fax j49 7071 295790.
e-mail christian.sinzger!med.uni-tuebingen.de
0001-7175 # 2000 SGM
interstrain differences have been described regarding the
infectivity of HCMV variants in EC and macrophages (Minton
et al., 1994 ; Sinzger et al., 1999 b ; Waldman et al., 1989).
Interestingly, the non-pathogenic fibroblast-adapted strains
like AD169 or Towne were less efficient in these cell types as
compared to low passage isolates. Therefore it can be
hypothesized that interstrain differences in HCMV infection of
these cell types reflect mechanisms that determine virulence
in vivo.
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
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C. Sinzger and others
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., 1999 b ; 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.
Methods
Cells. Human foreskin fibroblasts (HFF) were cultured in MEM
containing 2n4 mmol\l glutamine, 100 µg\ml gentamicin and 5 % foetal
calf serum (MEM5). Fibroblasts were used for experiments between
passages 10 and 25. Human umbilical vein endothelial cells (HUVEC)
were cultured in RPMI 1640 containing gentamicin (100 µg\ml), heparin
(5 IU\ml), ECGS (Becton Dickinson ; 50 µg\ml) and human serum (10 % ;
seronegative for HCMV). HUVEC were used for experiments between
passages 4 and 7. Contamination of cells by mycoplasma strains was
tested by enzyme immunoassay (Boehringer Mannheim). Cells were
discarded if mycoplasma contamination was detected.
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., 1999 b). 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.,
1999 b). For preparation of virus stocks, HFF were infected at an m.o.i. of
0n1. Supernatants of infected cultures were harvested 6 days postinfection (p.i.) and stored at k80 mC 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 80 000 g and purified on
glycerol–tartrate gradients (Irmiere & Gibson, 1983). The infectious titre
in HCMV preparations was determined by TCID assays (Mahy &
&!
Kangro, 1996) in fibroblasts on 96-well-plates.
Forcell-freeinfectionofcellcultures,mediumwasremovedandreplaced
by fresh MEM5 60 min prior to infection. Virus preparations were then
added for 90 min at 37 mC. Subsequently, cells were washed with fresh
medium and maintained at 37 mC. 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 mC after a
90 min adsorption period. After infection, cells were washed and
maintained at 37 mC 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 cm# culture flasks were infected with HCMV preparations as described
before at a virus concentration of 10' TCID \ml (m.o.i. l 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 mC 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
k80 mC 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 6n7 kb EcoRI–SalI insert (Stamminger et al., 1991).
HFF or HUVEC were seeded into six-well culture plates at a density of
200 000 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 [$&S]methionine.
Infected HFF grown in 175 cm# plastic flasks at 30 % CPE were incubated
with 15 ml medium containing 6n25 MBq [$&S]methionine (Amersham)
for 2 days. Cell debris in the supernatant was removed by centrifugation
at 2800 g for 10 min and [$&S]methionine-labelled virus was collected
from the medium by centrifugation at 80 000 g for 70 min in a Beckman
ultracentrifuge. The radioactively labelled virus preparations were
washed three times by ultracentrifugation to minimize the amount of
unincorporated [$&S]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 1n5 % dithiothreitol, separated
by SDS–PAGE, 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 mC, followed by incubation with
peroxidase-conjugated goat anti-mouse Ig–Fabh polyclonal sera (De
#
Beer Medicals, Hilvarenbeek, Netherlands). Finally, antigens were
detected by staining with diaminobenzidine (DAB ; Sigma) and observation with a light microscope.
Fig. 1. Phenotypic differences between endotheliotropic and nonendotheliotropic HCMV strains. Expression of viral IE antigen
(UL122/123) was detected by immunoperoxidase staining 24 h after infection of fibroblasts (HFF) and EC (HUVEC) with cellfree virus preparations at an m.o.i. of 0n5. Efficiency of infection of HCMV strains VHL/E, TB40/E and KSA16/3 was only
slightly reduced in EC as compared to fibroblasts. In contrast, the efficiency of infection of strains AD169, VHL/F and TB40/F
was dramatically decreased in EC.
DACC
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Determinants of human cytomegalovirus cell tropism
Fig. 1. For legend see opposite.
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A
B
Fig. 2. For legend see facing page.
Analysis of virus adsorption and penetration. For adsorption
assays, HFF and HUVEC grown in 24-well dishes were incubated with
[$&S]methionine labelled virus at 4 mC 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 β-counter (Wallac 1409) to determine the
extent of virus binding. For penetration assays, HFF and HUVEC grown
DACE
in 75 cm# plastic flasks were incubated with $&S-labelled virus for various
times at 37 mC. Subsequently, cells were washed twice with ice-cold
trypsin–EDTA and were incubated with trypsin–EDTA 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 β-counter. For analyses of penetration at
low levels of infection, cells were treated as described above at m.o.i. l
0n1. Trypsin-treated cells were harvested by centrifugation and after lysis
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Determinants of human cytomegalovirus cell tropism
C
Fig. 2. Course of HCMV infection in EC. (A) Kinetics of viral antigen expression by HCMV strains AD169 and TB40/E in EC
after infection at an m.o.i. of 10. IE antigen (MAb E13, UL122/123, 4 h p.i.), early antigen p52 (MAb BS510, UL44, 24 h p.i.)
and late antigen MCP (MAb 28-4, UL86, 72 h p.i.) were detected in EC by indirect immunoperoxidase staining. The timecourse of antigen expression in infected EC is indicated by bars. (B) Single-step growth curve of HCMV strain AD169 after
infection of EC monolayers at an m.o.i. of 1. Infectivity in supernatants from infected EC culture was determined by TCID50
assays. (C) Electron micrograph of an EC 5 days p.i. with HCMV strain AD169. Numerous viral capsids are visible in the
nucleus of the infected cell.
of cells with buffer containing 0n5 % SDS and 0n1 mg\ml proteinase K,
DNA was extracted with phenol–chloroform (Ausubel et al., 1989) and
analysed by quantitative HCMV-DNA-PCR.
Electron microscopy. Cells were rinsed in PBS and fixed in 1n5 %
glutaraldehyde in 0n2 mol\l PBS for 30 min at 4 mC. After three washes
for 10 min each in PBS containing 7n5 % 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 1n5 % lead citrate. Sections were examined
with a Zeiss EM 902 transmission electron microscope.
Analysis of nuclear localization of viral DNA. For quantification
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of nuclear localization of viral DNA, HFF and HUVEC were infected at
low m.o.i. (0n01–0n1). After incubation with virus preparations for 90 min,
cells were harvested by trypsin–EDTA treatment and washed with
MEM5 to inactivate trypsin. After additional washings with PBS and
hypotonic buffer (10 mmol\l HEPES, 1n5 mmol\l MgCl , 10 mmol\l
#
KCl, pH 7n6), cells were incubated with hypotonic buffer on ice for up to
90 min until 100 % of cells were swollen. Nuclei were then liberated by
10–15 strokes with a Dounce homogenizer, pelleted by centrifugation at
2800 g, resuspended in nuclei buffer (0n25 mol\l sucrose, 5 mmol\l
MgCl , 25 mmol\l KCl, 20 mmol\l Tricine–NaOH pH 7n8), mixed with
#
an equal volume of 50 % iodixanol solution (Optiprep ; Gibco), and
purified by centrifugation for 20 min at 10 000 g through a 30\35 %
iodixanol gradient. Purified nuclei at the interface were collected and
lysed with buffer containing 0n5 % SDS and 0n1 mg\ml proteinase K.
DNA was extracted from these preparations with phenol–chloroform
(Ausubel et al., 1989). The viral DNA content in nuclear DNA
preparations was determined by quantitative competitive HCMV-DNAPCR.
Quantitative PCR assays. Viral nucleic acids within cellular or
nuclear DNA probes were quantified by competitive PCR using primers
P1 (5h GGT CAC TAG CGC TTG TAT GAT GAC CA 3h) and P2 (5h
TTC TCA GCC ACA ATT ACT GAG GAC AGA GGG A 3h) 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 2n0 mM
MgCl , 0n25 mM each dNTP, 10 pmol each primer, 1i PCR buffer
#
(Boehringer Mannheim) and 1 U Taq polymerase (Boehringer Mannheim). Thermal cycling was performed as follows : 35 cycles of 94 mC for
1 min, 57 mC for 1 min, and 72 mC 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 cm# 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 0n5–4 h at 37 mC. To remove adsorbed virus particles that had not
penetrated, cells were treated with trypsin–EDTA (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, peroxidase–antiperoxidase complexes (PAP ;
Dako) from mouse, biotinylated swine anti-mouse Ig antibodies (Dako)
and peroxidase-conjugated streptavidin–biotin 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 Ig–Fabh polyclonal sera
#
(De Beer Medicals). Signals were then detected using DAB as chromogen,
resulting in gold-brown nuclear staining. Slides were mounted with
DACG
glycerol–gelatin and staining observed with a Polyvar microscope
(Cambridge Instruments) using interference contrast (DIC).
Results
The endotheliotropic phenotype of HCMV variants is
determined by the initial events of virus replication
Cytomegalovirus strains included in our analysis displayed
marked interstrain differences in the ability to grow in EC. Both
with a cell-associated and with a cell-free mode of virus
propagation, the attempt to grow a particular strain continuously in EC sharply distinguished two groups of viruses.
Non-endotheliotropic viruses like AD169, TB40\F and VHL\F
failed to increase the fraction of initially infected cells in the EC
culture. Thus they were unable to disseminate, and continuous
passaging of inoculated cultures eventually resulted in complete loss of infectious virus. In contrast, endotheliotropic
strains like KSA16\3, TB40\E and VHL\E increased the
number of infected cells by focal expansion (Sinzger et al.,
1999 b). These viruses were able to disseminate in endothelial
monolayers, yielding progressively higher titres of infectious
virus and subsequently reaching 100 % CPE.
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 0n5 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 100–1000-fold reduced in HUVEC as
compared to HFF (Table 1). In contrast, endotheliotropic
Table 1. Comparison of supernatant-associated infection
efficiency of various HCMV strains in fibroblasts (HFF) and
umbilical vein EC (HUVEC) at an m.o.i. of 0n5
Percentage of infected cells
Virus preparation
TB40\E
TB40\F
VHL\E
VHL\F
KSA16\3
AD169
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HFF
HUVEC
Ratio HFF/HUVEC
60
50
60
70
60
60
40
0n5
40
0n5
30
0n1
1n5
100
1n5
140
2n0
600
Determinants of human cytomegalovirus cell tropism
Fig. 3. Analysis of IE antigen expression after transfection of a UL122/123 plasmid into fibroblasts (A) and EC (B). Plasmid
pRR47 containing the UL122/123 gene of AD169 was transfected into subconfluent monolayers of the respective cell type. At
48 h p.i., IE antigen expression was detected by an indirect immunoperoxidase technique (DAB ; brown nuclear staining).
Counterstaining was done with haematoxylin. With each cell type, about 10 % of cells expressed the antigen.
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.
The finding that this phenotype is manifest at the IE stage
of the replicative cycle (Fig. 1) suggested that initial events are
critical for infectivity in this cell type. However, this did not
exclude an additional block later in the virus replication cycle.
Therefore we analysed the kinetics of viral gene expression to
follow the course of virus replication in those cells that had
started viral gene expression (Fig. 2 A). EC were infected by
HCMV strains AD169 and TB40\E at an m.o.i. of 10.
Subsequently, IE, early and late viral proteins were detected at
various times p.i. After infection with nonendotheliotropic
strain AD169, IE antigen became detectable at 4 h p.i. in a few
cells and was detectable in 1 % of cells at 24 h p.i. IE antigen
remained detectable throughout the whole replicative cycle.
Early antigen became detectable at 24 h p.i. and late antigen
became detectable at 48 h p.i. (Fig. 2 A) in 1 % of cells. After
infection with endotheliotropic strain TB40\E, antigens were
expressed with the same kinetics but in up to 100 % of cells
(Fig. 2 A). This indicated that (i) those cells that started viral
gene expression also proceeded through the early and late
stages of virus replication and (ii) that the kinetics of viral gene
expression in EC resembled the kinetics as known from
fibroblast cultures. Finally, the permissive and productive
nature of HCMV infection in EC was demonstrated by singlestep growth curves together with electron microscopical
detection of capsids in late stage infected EC, even for nonendotheliotropic strains like AD169 (Fig. 2 B, C). Endotheliotropic and nonendotheliotropic HCMV variants were only
distinguished by the efficiency to initiate infection in EC. Once
infection was initiated it was permissive on a single cell level
irrespective of the virus strain. Together, these experiments
emphasized the critical role of initial replication events for
expression of the endotheliotropic phenotype of HCMV
variants.
IE promoter function is independent of cell type
The inefficiency of IE antigen expression by HCMV strain
AD169 in EC might be caused by inefficient functioning of the
respective promoter in this cell type. To test this possibility,
we transfected a plasmid containing the complete UL122\123
gene of AD169 into fibroblasts and EC (Fig. 3). The fraction of
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Fig. 4. (A) Quantitative analysis of HCMV adsorption to EC (HUVEC, #) versus fibroblasts (HFF, $). Subconfluent monolayers
of HUVEC or HFF were incubated with 35S-labelled virus at 4 mC for various intervals. Virus inocula were normalized to result
in an m.o.i. of 50, with the total radioactivity of the inocula varying with labelling efficiency. After several washings, cells were
lysed and adsorbed virus was determined by counting of bound radioactivity in a β-counter. (B) Quantitative analysis of HCMV
penetration into EC (HUVEC, #) versus fibroblasts (HFF, $). Subconfluent monolayers of HUVEC or HFF were incubated with
labelled virus at 37 mC for various intervals. Virus inocula were normalized to result in an m.o.i. of 50, with the total radioactivity
of the inocula varying with labelling efficiency. After extensive washings, unpenetrated virus was removed by trypsin treatment.
Cells were lysed, and penetrated virus was analysed by counting of bound radioactivity in a β-counter. (C) Quantitative
comparison of HCMV penetration into EC (HUVEC) vs. fibroblasts (HFF) at low m.o.i. Subconfluent monolayers of HUVEC or
HFF were infected for 90 min with various virus strains at an m.o.i. of 0n1. Unpenetrated virus was removed by trypsin
treatment. The intracellular content of viral DNA was quantified by competitive PCR using primers within exon 4 of the HCMV IE
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Determinants of human cytomegalovirus cell tropism
cells expressing IE antigens was detected by immunoperoxidase staining at 48 h p.i. Detection of IE antigen in nuclei
of transfected cells would indicate activity of the IE promoter
in the respective cells. Both EC and fibroblast cultures
expressed IE antigen at an efficiency of 10 % of cells. This
demonstrated that the IE promoter is functional in EC and
indicated that the block in AD169 replication in EC is located
prior to IE gene expression.
No interstrain differences occur during HCMV entry
into EC
The cell tropism of many viruses is regulated at the level of
adsorption to and penetration into target cells. Therefore, we
analysed entry of HCMV variants into EC and fibroblasts in
order to determine whether interstrain differences in EC
tropism are associated with interstrain differences in
adsorption\penetration into EC. For quantitative analysis of
entry events, we performed binding studies with $&S-labelled
virus preparations (Fig. 4 A, B). Analysis of virus adsorption
was done at 4 mC to prevent virus entry. For analysis of virus
penetration, cells were incubated at 37 mC for various times.
Unpenetrated virus was removed by trypsin treatment.
Adsorption of HCMV to fibroblasts was 2–6-fold more
efficient as compared with EC. Interstrain differences, if present
at all, were scant (Fig. 4 A). Penetration into fibroblasts was
6–10-fold more efficient as compared to EC. Again, no dramatic
interstrain difference was found between endotheliotropic and
nonendotheliotropic HCMV variants (Fig. 4 B). For analysis
of virus entry at low m.o.i., we repeated the penetration
experiments at infection multiplicities of 0n1, employing
quantitative DNA PCR assays as a read-out system. Under
these conditions penetration into fibroblasts was again slightly
more efficient as compared to EC (factor 2–10) but no
interstrain differences were found (Fig. 4 C).
These findings might explain that even with endotheliotropic HCMV strains infection of fibroblasts is slightly
more efficient as compared to EC (Table 1). However, the
dramatic inefficiency of strains AD169, TB40\F and VHL\F in
infecting EC could not be explained at the level of virus entry.
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. 5 A). 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., 1999 a). 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. 5 B). In contrast, all
nonendotheliotropic strains displayed 100–1000-fold decreased HCMV-DNA titres in the nuclei of infected EC as
compared to fibroblasts (Fig. 5 B).
Efficiency of nuclear transport of penetrated viral
particles determines the endotheliotropism of HCMV
variants
The inefficient nuclear translocation of nonendotheliotropic
HCMV genomes in EC could be explained by impairment of
transport of viral particles from the cell periphery towards the
nucleus. Alternatively, uncoating of viral DNA from properly
translocated particles could be disturbed leading to a stalling of
DNA-containing capsids at the nuclear membrane. To test the
‘ transport ’ hypothesis versus the ‘ uncoating ’ hypothesis, we
sought to visualize single penetrated virus particles by
immunostaining. When reagents against various structural
proteins were tested we found that antibodies against
tegument protein pp150 were most suitable for this purpose.
While antibodies against tegument protein pp65 resulted in
gene UL123. An aliquot of 1000 copies of plasmid pHM 471 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. Equivalence (white arrowheads) of the
natural HCMV IE amplification product and the plasmid amplification product indicated the presence of 1000 HCMV-DNA
copies in the analysed sample dilution. Lanes in each gel image represent (from left to right) marker DNA and 10−1–10−5-fold
fractions of total DNA samples from infected cells.
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C. Sinzger and others
M2
Fig. 5. (A) Flow chart of the nuclear localization assay. Nuclear fractions of infected cells were prepared as indicated in the flow
chart. In the left-hand part of the figure, phase contrast micrographs of swollen cells, fractionated cells and purified nuclei are
presented. In the right-hand part of the figure Western blot analyses of the nuclear fraction from sucrose gradients and
iodixanol gradients are compared. Reactivity of the vimentin antibody indicates remnants of the cytoskeleton in the sucrose
fraction, while the iodixanol fraction is free of cytoskeleton signals. Reactivity of the lamin B antibody indicates the presence of
the nuclear membrane. (B) Quantitative analysis of nuclear translocation of viral DNA after virus entry. Nuclear fractions of
DADA
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Determinants of human cytomegalovirus cell tropism
diffuse cytoplasmic staining at initial times after penetration
and no signals were obtained with antibodies against the major
capsid protein MCP, pp150 staining resulted in well-defined
punctate signals. Three lines of control experiment indicated
that pp150 immunostaining actually detected capsid particles.
(i) When cells were incubated with high titre virus preparations
for 90 min under adsorption conditions, signals were located at
the cell surface. These signals were removed when cells were
trypsinized subsequent to virus adsorption but were found
within the cytoplasm when the temperature was shifted to
37 mC for 20 min subsequent to virus adsorption, thus allowing
for virus penetration (Fig. 6 A). (ii) The number of these pp150
signals correlated well with the m.o.i., whereas the intensity of
single signals was independent of the m.o.i. (Fig. 6 C). This
pattern can be explained by particle-associated protein rather
than by solubilized protein. (iii) Moreover, numerous signals
were obtained with gradient-purified virions (Fig. 6 B, C)
whereas only very few signals were detected with dense body
fractions containing only remnants of infectious virus (data not
shown). Together, this indicated that pp150 staining signals
represented penetrated virus capsids. This is in accordance
with previous reports about a tight association of pp150 with
capsids and dissociation of pp65 from the capsid after detergent
treatment (Benko et al., 1988 ; Chen et al., 1999 ; Gibson, 1996).
Failure to immunostain MCP in penetrated virus might indicate
that the respective epitope is not accessible at the surface
of intact capsids. As a result of these pretestings pp150
immunostaining was employed for detection of penetrated
capsids. As we observed that infectivity\particle efficiency was
superior with unmanipulated infectious supernatants as compared with ultracentrifuged and gradient-purified preparations,
we preferred unmanipulated preparations for subsequent
assays.
Using this approach we analysed the localization of viral
particles from various HCMV strains in HFF versus HUVEC
after virus penetration. Cells were incubated with virus
preparations at 37 mC for 4 h to allow for penetration and for
IE gene expression as a marker for initiation of infection. HFF
were additionally incubated with 10-fold dilutions of the
respective virus to normalize the different degree of virus
penetration (see Fig. 4). After the incubation period, cells were
trypsinized to remove residual adsorbed but unpenetrated
virus and then cytocentrifuged onto glass slides. Double
immunodetection of structural protein pp150 (pUL32) and
nonstructural newly synthesized IE protein (pUL122\123) was
performed to follow the route of virus particles after penetration and to correlate virus particle staining patterns with
the initiation of viral gene expression. Confirming the
aforementioned penetration data (Fig. 4), the level of virus
penetration was normalized between HUVEC and HFF when
10-fold diluted virus preparations were used for HFF cultures
(Fig. 6 D). Under conditions of normalized penetration, endotheliotropic strains VHL\E and TB40\E displayed efficient
nuclear translocation of virus capsids and initiation of viral
gene expression as evidenced by IE antigen detection in both
cell types. In sharp contrast, nonendotheliotropic HCMV
variants AD169, VHL\F and TB40\F failed to translocate
virus particles towards the nucleus in HUVEC cells, and no
viral IE gene expression was detectable in these cells. Nuclear
localization of particles and viral gene expression occurred in
only very few cells with nonendotheliotropic strains. Despite
their inefficiency in HUVEC, these strains were efficient in
nuclear localization and gene expression in HFF. Data are
shown for TB40\E versus TB40\F (Fig. 6 D) but were identical
for VHL\E versus VHL\F and AD169.
In summary, the EC tropism of HCMV variants appeared to
be determined by the efficiency of nuclear transport of virus
particles after successful penetration.
Discussion
The cell tropism of viruses is commonly determined during
the initial stage of the replicative cycle (Tyler & Fields, 1996).
For example, the coreceptors CCR5 and CXCR4 determine the
tropism of HIV variants for lymphocytes and macrophages,
respectively (Dittmar et al., 1997 ; Grivel & Margolis, 1999 ;
Penn et al., 1999). Another critical step seems to be the
initiation of viral gene transcription, mediated by cell typespecific transcription factors (Cripe et al., 1987 ; Gloss et al.,
1987 ; Kyo et al., 1995 ; Ori & Shaul, 1995). In this study we
show that the EC tropism of HCMV variants is also determined prior to IE gene expression, but surprisingly neither
adsorption\penetration nor the IE promoter function contribute significantly to cell tropism differences. Transfection of
the isolated IE gene of nonendotheliotropic variant AD169
into fibroblasts and EC yielded efficient IE gene expression in
both cell types, despite 1000-fold reduced infectivity in EC
after infection with the complete virus particle. Regarding the
efficiency of adsorption\penetration, virus entry appeared to
be slightly reduced in EC as compared to fibroblasts but no
infected cells were prepared as indicated in (A). The nuclear content of viral DNA 90 min p.i. was quantified by competitive
PCR using primers within exon 4 of the HCMV IE gene UL123. An aliquot of 1000 copies of plasmid pHM 471 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. Equivalence (white arrowheads) of the natural HCMV IE amplification product and the plasmid amplification
product indicated the presence of 1000 HCMV-DNA copies in the analysed sample dilution. Lanes in each gel image represent
(from left to right) 123 bp marker and 100–10−4-fold fractions of total nuclear DNA samples from infected cells. With
nonendotheliotropic strains AD169 and TB40/F, nuclear transport of viral DNA appears to be about 100–1000-fold reduced in
HUVEC as compared to HFF. With endotheliotropic strains VHL/E and TB40/E, nuclear transport of viral DNA is about equal in
the two cell types.
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C. Sinzger and others
A
B
C
Fig. 6. For legend see facing page.
interstrain differences occurred at that level. The slightly
reduced virus entry into HUVEC might explain why even
endotheliotropic HCMV strains are slightly more efficient in
fibroblasts. However, the dramatic interstrain differences in EC
tropism could not be explained by different adsorption or
penetration efficiencies of these viruses. The question was,
why fibroblast-adapted HCMV strains like AD169 fail to start
gene expression in EC despite their ability to penetrate and to
DADC
activate their IE promoter upon transfection. This could only
be explained by the inefficient release of viral DNA into the
cell nucleus after penetration into the cells. Actually, we found
dramatic interstrain differences in the efficiency of nuclear
translocation of viral DNA in EC as compared to fibroblasts.
While all strains efficiently released viral DNA to the nuclei in
fibroblasts, only EC-propagated strains were effective in EC.
Fibroblast-adapted strains were about 100–1000-fold less
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Determinants of human cytomegalovirus cell tropism
D
Fig. 6. (A) Immunostaining of viral particles with antibody XP1 against the viral tegument protein pp150 after incubation of
HUVEC with HCMV strain TB40/E at an m.o.i. of 10 under conditions allowing for adsorption of virus (90 min, 4 mC),
adsorption and removal of adsorbed virus (90 min, 4 mC ; 20 min trypsin) or adsorption and penetration of virus (90 min, 4 mC ;
20 min, 37 mC). Cell spots were prepared by cytocentrifugation. Signals were amplified by a five-step indirect
immunoperoxidase technique and visualized with DAB as chromogen (brown punctate staining). Counterstaining of nuclei was
done with haematoxylin. (B) Immunostaining of viral particles with antibody XP1 against the viral tegument protein pp150 after
incubation of HFF with gradient-purified virions of HCMV strain TB40/F at an m.o.i. of 1 for various time intervals at 37 mC.
Remnants of adsorbed virus were removed by trypsin treatment for 20 min at 37 mC and cells were cytocentrifuged onto glass
slides. Signals were amplified by a five-step indirect immunoperoxidase technique and visualized with Co-DAB as chromogen
(black punctate staining). A slight counterstaining of nuclei was done with haematoxylin. Examples of stained virus particles are
indicated by the arrowheads. (C) Double immunodetection of viral particles and viral antigen expression using antibody XP1
against the viral tegument protein pp150 and antibody E13 against IE antigens of HCMV after incubation of HFF with
increasing concentrations of gradient-purified virions of HCMV strain TB40/F. Cells were incubated at the m.o.i.s indicated for
4 h at 37 mC. Remnants of adsorbed virus were removed by trypsin treatment for 20 min at 37 mC and cells were
cytocentrifuged onto glass slides. Signals were amplified by a five-step indirect immunoperoxidase technique and visualized
with Co-DAB as chromogen (black punctate staining). Viral IE antigens were visualized by a two-step immunoperoxidase
staining using antibody E13 and DAB as chromogen (brown nuclei). No counterstaining was done. Examples of stained virus
particles are indicated by the arrowheads. (D) Double immunodetection of viral particles and viral antigen expression using
antibody XP1 against the viral tegument protein pp150 and antibody E13 against IE antigens of HCMV. EC (HUVEC) and
fibroblasts (HFF) were incubated at infection multiplicities of 1 and 0n1, respectively, with HCMV strains TB40/E and TB40/F
for 4 h at 37 mC. Remnants of adsorbed virus were removed by trypsin treatment for 20 min at 37 mC and cells were
cytocentrifuged onto glass slides. Viral particles were visualized by a five-step indirect immunoperoxidase technique using
antibody XP1 and Co-DAB as chromogen (black punctate staining). Viral IE antigens were visualized by a two-step
immunoperoxidase staining using antibody E13 and DAB as chromogen (brown nuclei). Examples of stained virus particles are
indicated by the arrowheads.
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DADD
C. Sinzger and others
efficient in EC and this was accompanied by a 100–1000-fold
reduced infectivity. Inefficient nuclear import of AD169 DNA
in EC had been demonstrated by in situ-hybridization (Slobbe
van Drunen et al., 1998). However, as these data were not
quantified and no comparisons with other HCMV strains were
made, this observation could not be directly linked to
interstrain differences in the EC tropism of HCMV variants.
Our quantitative comparisons of initial replication events now
provide direct evidence that HCMV interstrain differences in
EC tropism are actually due to inefficient translocation of the
viral genome into the nucleus of the infected cells.
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 typespecific 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 virus–cell 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
DADE
furthermore indicate that this step is a potential target for
future antiviral strategies.
We thank Dr James Waldman for providing HCMV strains VHL\E
and VHL\F, Dr Nuri Gueven for helpful advice with preparation of
nuclear fractions, Dr Thomas Stamminger for the pHM471 plasmid, and
the members of the collaborative BMBF project ‘ Herpesviruscomplications of organ transplantation ’ for stimulating discussions. This
work was supported by the Bundesministerium fu$ r Bildung und
Forschung (Projektnummer 01 KI 9602).
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Received 30 May 2000 ; Accepted 22 August 2000
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