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Journal of Virology, March 2001, p. 2435-2443, Vol. 75, No. 5
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.5.2435-2443.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Kaposi's Sarcoma-Associated Herpesvirus Can
Productively Infect Primary Human Keratinocytes and Alter Their
Growth Properties
Francesca
Cerimele,1,2
Francesca
Curreli,1
Scott
Ely,3
Alvin E.
Friedman-Kien,1,4
Ethel
Cesarman,3 and
Ornella
Flore1,*
Departments of
Microbiology1 and
Dermatology,4 New York University School
of Medicine, New York, New York 10016; Department of Pathology,
Weill Medical College of Cornell University, New York, New York
100213; and Istituto di Microbiologia,
Università Cattolica del Sacro Cuore, Rome,
Italy2
Received 30 June 2000/Accepted 15 November 2000
 |
ABSTRACT |
Previous studies have shown the presence of Kaposi's
sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in
keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and
in the epithelial cells of eccrine glands within KS lesions. We
infected primary cell cultures of human keratinocytes with KSHV/HHV8.
At 6 days post infection, transcription of viral genes was detected by
reverse transcriptase PCR (RT-PCR), and protein expression was
documented by an immunofluorescence assay with an anti-LANA monoclonal
antibody. To determine whether the viral lytic cycle was inducible by
chemical treatment, KSHV/HHV8-infected keratinocytes were treated with
12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was
performed to confirm the transcription of lytic genes such as open
reading frame 26, (which encodes a capsid protein). Finally, to assess
infectious viral production, other primary human cells (human umbilical
vein endothelial cells), were infected with concentrated supernatant of
KSHV-infected, TPA-induced keratinocytes and the presence of viral
transcripts was confirmed by RT-PCR. The uninfected keratinocytes
senesced 3 to 5 weeks after mock infection, while the
KSHV/HHV8-infected keratinocytes continued to proliferate and to date
are still in culture. However, 8 weeks after infection, viral genomes
were no longer detectable by nested PCR. Although the previously
KSHV/HHV8-infected keratinocytes still expressed epithelial markers,
they acquired new characteristics such as contact inhibition loss,
telomerase activity, anchorage-independent growth, and changes in
cytokine production. These results show that KSHV/HHV8, like other
herpesviruses, can infect and replicate in epithelial cells in vitro
and suggest that in vivo these cells may play a significant role in the
establishment of KSHV/HHV8 infection and viral transmission.
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INTRODUCTION |
Kaposi's sarcoma (KS) is a
multicentric vascular neoplasm involving the skin and mucosal surfaces
and in aggressive cases may involve visceral organs and lymph nodes. KS
lesions contain distinctive proliferating spindle cells, activated
endothelial cells, fibroblasts, smooth muscle cells, and infiltrating
inflammatory cells (35, 39). Four different epidemiologic
forms of Kaposi's sarcoma have been described, i.e., the classic,
endemic, iatrogenic, and AIDS-related forms (22, 47).
The gamma-2 herpesvirus KSHV, also known as human herpesvirus 8 (HHV8),
has been detected in >95% of KS patients with all clinical forms of
KS (22, 34, 47). KSHV/HHV8 DNA sequences are also present
in primary effusion lymphomas (PEL) (8), a subset of
B-cell lymphomas occurring more frequently in AIDS patients and in a
significant percentage of patients with multicentric Castelman's
disease (51). By in situ hybridization (5)
and immunohistochemistry (7, 15), the results of several
studies have documented the presence of KSHV/HHV8 in spindle and
endothelial cells in KS lesions. Reed et al. (38) by
analysis of the expression of the latently expressed v-cyclin gene,
noted the presence of KSHV/HHV8 DNA in scattered keratinocytes of the
epidermis overlying cutaneous lesions. Positive signals were also
detected in eccrine ductular epithelial cells and in spindle and
endothelial cells lining the well-formed blood vessels within and
surrounding KS lesions (38). Similar results were
described by Foreman et al. (20), who detected
KSHV/HHV8-positive signals by in situ PCR in basal keratinocytes in the
area immediately above some of the later-stage (i.e., plaque or tumor)
KS lesions. Epstein-Barr virus (EBV), the human herpesvirus most
closely related to KSHV/HHV8, has been detected both serologically and
histologically in epithelial cells (42, 49, 56).
Epithelial cells of many tissues, including salivary glands and
kidneys, support the replication of cytomegalovirus, another
herpesvirus that can also infect human keratinocytes (58).
In this study we investigated whether KSHV/HHV8 was capable of
infecting primary cultures of human keratinocytes in vitro and analyzed
the biological consequences of this infection. We demonstrate that
KSHV/HHV8 can establish a productive infection in primary human
keratinocytes and that viral particles purified from the supernatant of
infected keratinocytes can infect other primary cells, such as human
endothelial cells. Infection by KSHV/HHV8 resulted in alterations of
the keratinocytes, including the development of a spindle-shaped cell
morphology similar to that observed in other immortalized cell lines of
epithelial origin (24), and the ability to form colonies
in soft agar. We also observed an induced telomerase activity, which is
one of the characteristic factors of transformed human cells
(27).
While rodent cells from several normal tissues can undergo spontaneous
neoplastic transformation after variable periods in culture
(46), human cells are rather resistant in this respect (14). To exclude the possibility of spontaneous
immortalization, the infection experiment was repeated eight times, and
four out of eight KSHV-infected cultures did not senesced and to date
are still proliferating. Moreover, if such cells are the outcome of spontaneous transformation, they might also be expected to arise in
uninfected cultures; however, these were never observed and the
uninfected cultures did not proliferate longer than 3 to 5 weeks.
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MATERIALS AND METHODS |
Cell cultures.
Primary normal human epidermal keratinocytes
(NHEK) derived from newborn foreskin were obtained from Clonetics Corp.
(San Diego, Calif.). Primary cultures of keratinocytes were established from histologically normal adult body skin. The homogeneity of the
cultures was examined by immunohistochemistry staining using cytokeratin-specific antibodies for epithelial cells (CK AE1/AE3 and
CAM 5.2). Four separate cultures of keratinocytes from Clonetics and
four separate cultures derived from skin biopsy samples were used for
the experiments.
Cells were grown in low-calcium, serum-free Keratinocyte-SFM medium
(GIBCO BRL, Gaithersburg, Md.) supplemented with 100 U of penicillin
per ml, 100 µg of streptomycin per ml, 2 mM glutamine, bovine
pituitary extract (30 µg/ml), and recombinant epidermal growth factor
(0.2 ng/ml) (GIBCO BRL). According to the manufacturer, this medium is
selective for propagation of human keratinocytes.
BC-3, a KSHV/HHV8-positive, EBV-negative primary effusion lymphoma
(PEL)-derived cell line (
2), was cultured in RPMI 1640
supplemented with 20% fetal bovine serum. Primary human umbilical
vein
endothelial cells (HUVECs) were isolated from umbilical cord
veins by
collagenase treatment as described previously (
6).
Cells
were grown in gelatin-coated flasks containing M199 medium
(Bio-Whittaker, Walkersville, Md.) supplemented with 10 ng of
vascular
endothelial growth factor (VEGF) (Perprotech Inc., Rockville,
Md.) per
ml, 10 ng of basic fibroblast growth factor (bFGF) (R&D
Systems Inc.,
Minneapolis, Minn.), per ml, and 10 U of heparin
(Sigma, St. Louis,
Mo.) per
ml.
Viral infection.
Concentrated supernatant and cell-free
lysates from 4 × 108
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced BC-3
cells were used as a source of KSHV/HHV8 for the infection of eight
NHEK cultures: four derived from skin biopsy samples and four from Clonetics. Concentrated supernatant and cell-free lysates from KSHV-infected, TPA-induced NHEK cultures were used as a source of
KSHV/HHV8 for the infection of two HUVEC cultures. Primary keratinocytes supplied by Clonetics are specified to be at passage 3. The cells were split once before the infection; therefore, all primary
cell cultures (Clonetics and skin biopsy sample) we used were infected
at passage 4. To release the virus in the medium, cells were treated
for 48 h with 20 ng of TPA (Sigma) per ml and centrifuged and
supernatant was collected. The cell pellet was lysed by three
freeze-thaw cycles, centrifuged to eliminate cell debris, pooled with
the supernatant, filtered through a 0.45-µm-pore-size filter, and
concentrated at 4°C overnight with 7% polyethylene glycol 6000. The
precipitate was collected by centrifugation at 15,000 × g for 2 h, resuspended in a small amount of
phosphate-buffered saline (PBS), and purified through a 25% sucrose
cushion (26,000 rpm for 3 h in a Beckman SW27 rotor). The pellet
was allowed to soak overnight at 4°C in 1 ml of PBS with 0.1% bovine
serum albumin, and the suspension was treated with DNase (1 U/ml;
Promega) and RNase (5 µg/ml; Quiagen), layered over a 5 to 45%
sucrose gradient in PBS, and centrifuged at 14,000 rpm for 1 h at
10°C in a Beckman SW41 rotor. The visible band at the center of the
gradient was collected, dialyzed against PBS, and then frozen at
70°C. Since no protocol for determining the PFU of KSHV currently
exists, viral DNA from purified particles was extracted with
phenol-chloroform and the optical density was measured by
spectrophotometry. From the optical density value, the DNA quantity was
assessed in micrograms per milliliter, it was converted to moles per
milliliter, and the number of molecules of viral genome was calculated
from the number of moles. We estimated a yield in the range of 1.5 × 1010 to 2 × 1010 KSHV/HHV8 genome
equivalents (DNA molecules) per ml of concentrated supernatant,
depending on the viral preparation. Human primary keratinocytes were
infected at passage 4 with 5 to 10 viral genome equivalents/cell.
Supernatant and cell pellet lysates from 10
6 KSHV-infected
and TPA-induced NHEK cells were concentrated and purified as described
above and used to infect two parallel primary HUVEC
cultures.
Cells were incubated for 1 h at 37°C with purified viral
particles, and then the specific medium was added: Keratinocyte-SFM
in
the keratinocyte cultures or M199 medium in the HUVEC cultures.
After
incubation for 48 h, the monolayers were washed twice to
eliminate
the inoculum, fresh medium was added, and the cells
were maintained at
37°C. Viral infection experiments were repeated
eight
times.
PCR amplification.
To calculate the number of viral DNA
molecules in infected keratinocytes, semiquantitative PCR was performed
using the purified viral DNA from BC-3 cells as a standard. A fragment
of 233 bp was amplified with KS330233 BamHI
primers (10) and quantitated in serial dilutions from 100 to 10
8 amol/µl by electrophoresis on an ethidium
bromide agarose gel followed by Southern blot hybridization with an
internal probe. The range of DNA concentration measurable by regular
PCR was from 100 to 10
5 amol/µl.
Genomic DNA was extracted from the 10
6 KSHV/HHV8-infected
and uninfected NHEK cells by the phenol-chloroform method
(
45).
The presence of KSHV/HHV8-specific DNA in infected
NHEK cells
was verified by amplifying the 233-bp fragment with
KS330
233 primers.
BC-3 cells and uninfected keratinocytes
were used as positive
and negative controls, respectively. PCR products
were loaded
onto a 1.5% agarose gel, transferred to a nylon membrane,
and
hybridized with the same internal probe used for the standard
DNA.
Radiolabeling of the probe was performed with the RediPrime
DNA-labeling system (Amersham International, Little Chalfont,
England)
and [
32P]dCTP as specified by the manufacturer. Because
the molar quantity
of the standard viral DNA was known, the actual
number of viral
DNA molecules present in KSHV-infected keratinocytes
was calculated
by comparing the intensity of the band with that of the
standard
viral DNA. The total quantity of cellular genomic DNA
extracted
from 10
6 cells was quantitated, 100 ng was used
for PCR, and this was
multiplied back to obtain the total number of
viral DNA
molecules.
RT-PCR.
Total RNA was extracted from 106
infected and uninfected keratinocytes with Tri-reagent (Molecular
Research Center, Inc., Cincinnati, Ohio) as specified by the
manufacturer, and the extracted RNA was treated with RNase-free DNase
(Boeringher Mannheim, Indianapolis, Ind.). RNA was reverse transcribed
with the reverse transcription system (Promega, Madison, Wis.), and PCR
was performed. The cellular
-actin gene was amplified from each
sample as a control. The KSHV/HHV8 genes open reading frame 72 (ORF 72)
(encoding v-cyclin) (18), ORF K12 (encoding kaposin), and
ORF 26 (encoding the minor capsid protein) (10) were used
as reverse transcriptase PCR (RT-PCR) targets. The primers used for K12
detection were 5'-GGATAGAGGCTTAACGGTGTTTGTG-3' and (reverse)
5'-TGCAACTCGTGTCCTGAATGC-3', with the following amplification protocol: 94°C for 3 min, then 35 cycles of 94°C for
1 min, 62°C for 1 min, and 72°C for 1 min. RT-PCR was also performed targeting the VEGF (12) and bFGF transcripts in
infected and uninfected keratinocytes. The primers used to amplify bFGF were 5'-GGCCACTTCAAGGACCCCAAG-3' and reverse
5'-TCAGCTCTTAGCAGACA-3' with the following protocol: 94°C
for 3 min, then 35 cycles of 94°C for 1 min, 58°C for 1 min, and
72°C for 1 min.
PCR products were loaded onto 1.5% agarose gel, transferred to a nylon
membrane, and hybridized with a
32P-labeled internal
probe.
Nested PCR.
The first PCR was performed with the following
set of outer primers to amplify a fragment of 327 bp in the KSHV/HHV8
major capsid protein (MCP) gene: 5'-AGGCAACGTCAGATGTGAC-3'
and 5'-GAAATTACCCACGAGATCGC-3'. The conditions used
for amplification were a 10-min denaturation at 94°C followed by 25 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, with a single 5-min extension at 72°C. The inner primers used
for nested PCR were 5'-CATGGGAGTACATTGTCAGGACCTC-3' and
5'-GGAATTATCTCGCAGGTTGCC-3', and the condition for
amplification were a 10-min denaturation at 94°C followed by 35 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for
45 s, with a single 5-min extension at 72°C to amplify a 212-bp
fragment. The enzyme used for the reaction was Taq Gold
polymerase from Perkin-Elmer. Standard viral DNA was diluted 1/10 from
2 to 2 × 10
8 amol./µl.
IFA.
An immunofluorescence assay (IFA) was performed using
serum from a KS patient as a source of primary antibody or a monoclonal antibody (MAb) against ORF 73 (Advance Biotechnology, Inc., Columbia, Md.) for latent antigens and a monoclonal antibody against ORF 59 (generous gift from Bala Chandran) for lytic antigens.
The cells were detached with trypsin, counted, spotted on slides
(10
5/slide), air dried, and fixed in acetone for 10 min.
The slides
were blocked with 20% normal goat serum in PBS for 30 min
and
then incubated overnight at 4°C with the primary antibody.
Fluorescein
isothiocyanate-conjugated goat anti-mouse and anti-human
sera
were used as secondary antibodies (ICN/Cappel, Aurora, Ohio).
Staining controls included omission of the primary antibody and
staining of noninfected keratinocytes. Cell nuclei were counterstained
with DAPI (4',6-diamino-2-phenylindole dihydrochloride; Boehringer
Mannheim).
Growth in soft agar.
Uninfected and KSHV/HHV8-infected
keratinocytes were trypsinized and resuspended in medium at
concentrations of 103, 104, and 105
cells/ml. A 5-ml volume of 0.6% melted agar, made up by mixing 9 parts
of Keratinocyte-SFM standard medium and 1 part of 6% agar, was poured
into each 60-mm plate to form a bottom layer. Then 2 ml of 0.6% melted
agar-medium was added to 1 ml of the cell suspension, and 1 ml of this
mixture was poured over the bottom layer to form a top layer. Each
serial dilution was plated in triplicate. Cells were fed every week
with 3 ml of 0.4% agar-medium and routinely observed by microscopy for
colony formation.
Telomerase activity.
The enzymatic activity was measured by
using a telomeric repeat amplification protocol (4) (TRAP
assay kit; BD PharMingen, San Diego, Calif.).
Cytokine assay.
Cytokines were measured by enzyme-linked
immunosorbent assays in a commercial laboratory (Cytokine Core
Laboratory, Baltimore, Md.).
Immunohistochemistry staining.
Cells were processed by a
standard method of immunohistochemistry. Briefly, the cells were fixed
in phosphate-buffered formalin and embedded in paraffin. Deparaffinized
sections were treated with 0.1% pepsin (Sigma) for 7 min and were
reacted overnight at 4°C with the MAbs (Boeringer-Mannheim and DAKO,
Carpinteria, Calif.) listed in Table 1. The sections were rinsed in
PBS, and the bound antibodies were localized by the
avidin-biotin-peroxidase method (Elite kit; Vector Laboratories,
Burlingame, Calif.) using diaminobenzidine as the chromogen. Sections
stained with normal mouse serum were used as negative controls.
Cell proliferation assay.
Cells were plated in a T-25 flask
(5 × 105/flask), allowed to grow for 3 days,
harvested with trypsin, diluted in trypan blue to assess viability, and
counted in a Burker hemocytometer chamber.
 |
RESULTS |
KSHV can infect primary human keratinocytes.
To determine
whether primary human keratinocytes were susceptible to KSHV/HHV8
infection, 106 cells were exposed to purified viral
particles. The presence of KSHV/HHV8-specific DNA was confirmed by
performing a PCR with the KS330233 primers 6 days
postinfection on 100 ng of total DNA purified from infected cells (Fig.
1A). The results showed that without TPA
treatment the number of viral DNA molecules/100 ng of template was 500 to 600, depending on the particular culture. The number of KSHV genomes
in the cultures was small, suggesting that only about 5 to 6% of the
cells in these culture were infected, assuming one copy per infected
cell. This estimate was calculated as described in Materials and
Methods.

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FIG. 1.
PCR and RT-PCR of 106 keratinocytes infected
with KSHV/HHV8. (A) PCR with KS330 primers to test for the presence of
viral DNA 6 days postinfection. Lanes: 1 and 2, two different cultures
infected in parallel; 3, uninfected keratinocytes; 4, positive control
(BC-3 cells). (B) PCR with KS330 primers to test the presence of
KSHV/HHV8 genomes in infected keratinocytes 25 days postinfection.
Lanes: 1 and 2, two different cultures infected in parallel; 3, uninfected keratinocytes; 4, BC-3 cells. (C) RT-PCR 6 days
postinfection with primers specific for v-cyclin. Lanes: 1 and 3, RNA
from two cell cultures infected in parallel; 2 and 4, RNA controls
without reverse transcription showing no viral DNA contamination; 5, positive control (BC-3 cells); 6, uninfected keratinocytes; 7, primers
with no template. (D) RT-PCR 6 days postinfection with primers specific
for ORF K12. Lanes: 1 and 2, RNA from two cell cultures infected in
parallel; 3, primers with no template; 4, positive control (BC-3
cells).
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To determine whether the viral DNA was also transcribed, RT-PCR assays
were performed. Reactions using primers specific for
v-cyclin and K12
showed positive results in all infected cultures,
documenting the
presence of viral gene transcripts (Fig.
1C and
D show two parallel
infected cultures). The infected cells were
maintained in culture and
passaged every 3 to 5 days depending
on the particular culture. After 4 weeks, the infected cell cultures
were tested again by PCR with several
primer sets scattered along
the viral genome. Figure
1B shows PCR using
KS330
233 primers for
amplification of viral DNA from two
cultures infected in parallel.
These analyses showed that the number of
viral DNA molecules/100
ng of template was about 250, suggesting that
at this time postinfection
only about 2 to 3% of the cells contained
detectable viral DNA,
assuming one copy per infected cell. Comparison
between the number
of viral DNA in the cultures 6 days and 4 weeks
postinfection
indicated that the rate of decline was about 50 to 60%
during
this period. These experiments were repeated six additional
times
with similar results. After 8 weeks in culture, we could no
longer
detect any KSHV/HHV8 DNA by regular PCR. To ascertain whether
the viral genome was not detectable because direct PCR was not
sensitive enough, we performed nested PCR with outer and inner
MCP
primer sets. These primer sets amplify a region in the major
capsid
protein gene. Figure
2A shows nested
PCR-Southern blot
analyses of serial dilutions from 2 × 10
3 to 2 × 10
8 amol of viral DNA per
µl extracted from purified KSHV/HHV8 particles.
This analysis was
used as a standard to quantitate the viral DNA
concentration and to
measure the sensitivity of the method. The
lower detection limit was
2 × 10
6 amol/µl or approximately 1.2 KSHV
equivalent molecules. Figure
2B shows the results of a nested
PCR-Southern blot analysis of
DNA extracted from three separate
cultures of keratinocytes, 8
weeks postinfection. All nested PCRs that
we performed with the
keratinocyte cultures 8 weeks postinfection were
negative. This
means that the copy number of viral DNA relative to
total cellular
genome was smaller then the detection limit. Nested PCR
is a very
sensitive assay, and by this method we have been able to
detect
1.2 KSHV DNA molecules in our standard. Therefore, it is
unlikely
that there are any viral genomes in the proliferating
keratinocytes
8 weeks postinfection.

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FIG. 2.
Nested PCRs with MCP outer and inner sets of primers.
(A) Quantification by nested PCR of viral DNA extracted from purified
particles. Lanes: 1 to 6, 10-fold serial dilutions from 2 × 10 3 to 2 × 10 8 amol/µl; 7, negative
control (primers with no template). (B) Nested PCR of 106
keratinocytes 8 weeks after infection with KSHV. Lanes: 1 to 3, three
keratinocytes cultures independently infected; 4 and 5, uninfected
keratinocytes; 6, negative control (primers with no template); 7, positive control (BC-3 cells).
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Expression of viral latent nuclear antigen.
To further
document the in vitro infection of primary keratinocytes with KSHV, the
expression of latent viral antigens was examined by immunofluorescence.
Serum from a KS patient (data not shown) or a MAb against ORF 73 was
used as the primary antibody, and in both cases a punctate nuclear
pattern was detected in 2 to 5% of cells in the infected culture (Fig.
3A and B). This result demonstrated the
expression of viral proteins in KSHV/HHV8-infected keratinocytes. The
IFA has been performed in all infected cultures 6 days and 4 weeks
postinfection, and similar results have been obtained.

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FIG. 3.
IFA of KSHV/HHV8-infected keratinocytes. (A and B)
Detection of latent viral antigen. (A) Cells stained with anti-LANA
MAb. Positive cells show speckled nuclear pattern. (B) Cells
counterstained with DAPI to localize the nuclei. (C and D) Detection of
lytic viral antigen after TPA induction. (C) Cells stained with MAb
11D1, raised against the ORF 59 product, DNA replication protein.
Positive cells show characteristic nuclear staining. (D) Cells
counterstained with DAPI.
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Induction of KSHV lytic replication and viral serial
transmission.
To determine whether KSHV/HHV8 could establish a
productive infection in keratinocytes, infected cells were treated with
TPA to induce lytic replication of KSHV/HHV8 and release viral
particles into the medium (41). After 48 h of TPA
treatment, induction of lytic gene transcription was detected by RT-PCR
analysis of ORF 26 (encoding capsid protein) (Fig.
4A). Lytic gene expression was also
confirmed by immunostaining of TPA-induced cells with a MAb against the
ORF 59 product, a DNA-replication protein. The percentage of induced
cells that expressed viral lytic antigens never exceeded 40% of the
positive infected cells (Fig. 3C and D).

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FIG. 4.
(A) RT-PCR analysis of 106
KSHV/HHV8-infected keratinocytes after treatment with TPA for 48 h. Primers are specific for ORF 26, a late lytic gene encoding a capsid
protein. Lanes: 1 and 3, two different cultures infected in parallel
and TPA induced; 2 and 4, the same cultures without TPA induction; 5, uninfected keratinocytes; 6 to 10, the same samples without reverse
transcription; 11, positive control (BC-3 cells). (B) PCR analysis to
confirm the release of viral progeny in the supernatants of TPA-induced
keratinocytes. Viral DNA was amplified with KS330 primers. Lanes: 1 and
2, supernatants from two parallel cultures infected and TPA induced; 3 and 4, supernatants from uninfected keratinocytes; 5, primers with no
template; 6, positive control (BC-3 cells). (C) Serial transmission.
RT-PCR analysis of HUVECs infected with KSHV/HHV8 particles obtained
from TPA-stimulated keratinocytes. DNA was amplified with primers
specific for v-cyclin. Lanes: 1, KSHV/HHV8-infected HUVECs; 2, control
with no RT; 3, primers with no template; 4, positive control (BC-3
cells).
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To determine if productive viral replication had occurred, the
supernatant from parallel cultures of KSHV/HHV8-infected, TPA-induced
keratinocytes was collected and concentrated as described in Materials
and Methods. Virus was purified on a sucrose gradient, and the
presence
of KSHV/HHV8 genome was confirmed by PCR analysis using
primers KS330
as shown in Fig.
4B. To demonstrate that the isolated
viral particles
were infectious, new primary cultures of HUVECs
were infected with the
virus purified from the infected keratinocytes.
At 6 days after
infection, we performed an RT-PCR amplification
using primers within
the v-cyclin gene of KSHV, and positive RT-PCR
demonstrated that viral
transmission in the HUVEC culture was
achieved (Fig.
4C). At 2 weeks
postinfection, the HUVECs acquired
the typical spindle shape observed
in KSHV/HHV8-infected endothelial
cells (reference
19 and
data not
shown).
Effect of KSHV infection on the survival and growth properties of
keratinocytes.
Infected and uninfected cells were passaged at the
same rate. After seven passages (3 to 5 weeks, depending on the
particular culture) the uninfected cell cultures (Fig.
5A) stopped proliferating and trypsinized
cells did not adhere to fibronectin-coated flasks as well as the
majority of the cells in the infected cultures. However, a new
population of cells in the same infected flask began to proliferate
after developing adherent foci (Fig. 5B). In these new cultures derived
from adherent foci, KSHV/HHV8 was still detectable by PCR 4 weeks
postinfection, and the cells showed a spindle-shaped morphology, loss
of contact inhibition (Fig. 5C), and independence of growth factors
specific for keratinocytes (epidermal growth factor and bovine
pituitary extract). These cells were also capable of forming colonies
in soft agar (Fig. 5D). Of 104 cells plated, 168 colonies
(range, 105 to 256) were obtained; thus, only~2% of the cells plated
resulted in colony formation (Fig. 5E).

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FIG. 5.
Spindle shape acquisition of KSHV/HHV8
infected-keratinocytes as shown by phase contrast microscopy. (A)
Primary human keratinocytes. (B) Infected keratinocytes after 4 weeks
in culture (focus formation). (C) Infected keratinocytes after 4 weeks
in culture (loss of contact inhibition). (D) Infected
keratinocyte-forming colonies in soft agar. (E) Number of colonies
grown in agar after plating 104 cells per plate
(experiments were done in triplicate).
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Several studies have demonstrated that telomerase is essential to
maintain telomere length and the ability of cells to proliferate
indefinitely (
27). Although the viral genome was no
longer detectable
by nested PCR after 8 weeks in culture, the
previously KSHV/HHV8-infected
keratinocytes continued to proliferate
and to date are still in
culture. Therefore, we measured the telomerase
activity of all
cultures previously infected with KSHV/HHV8 and
maintained in
culture for 8 weeks and one culture of mock-infected
keratinocytes,
using a TRAP. We could not test any mock-infected cells
after
8 weeks in culture because, as we stated above, uninfected cells
did not survive longer then 3 to 5 weeks, depending on the particular
culture. Only the cell cultures previously positive for KSHV/HHV8
exhibited telomerase activity, which was not detectable in
mock-infected
keratinocytes (Fig.
6).

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FIG. 6.
Telomerase activity in previously KSHV/HHV8-infected
keratinocytes after 8 weeks in culture. Lanes: 1 to 3, telomerase-positive cell lines (positive controls); 4, positive control
after heat treatment; 5, telomeric DNA; 6, normal keratinocytes
(negative control); 7 and 10, two parallel cultures of previously
infected keratinocytes showing the presence of telomerase activity; 8 and 9, same cultures after heat treatment.
|
|
Immunohistochemistry.
Since the long-term-proliferating
keratinocytes established from cultures previously infected by
KSHV/HHV8 displayed changes in their morphology, it was important to
exclude the possibility that these cells represented contamination with
other cell types in the original cultures. By immunohistochemical
staining we investigated whether the long-term-proliferating
keratinocytes established from cultures previously infected by
KSHV/HHV8 still retained the markers specific for keratinocytes or
expressed other cellular surface molecules. Panels of antibodies
specific for keratinocytes (AE1-AE3 mixture, CAM 5.2, MNF116
[37]), endothelial cells (CD31, CD34), dendritic cells
(CD1A), melanocytes (S100-protein, HMB 45), and fibroblasts (type 4 collagen) were tested. Immunohistochemical detection showed positive
staining for the AE1-AE3 mixture and for cytokeratin 5, 6, 8, 17, and
19 (MNF116 antibody), while molecular markers for other cell types were
negative, except for vimentin, which is expressed by most spindle cells
(23) (Table 1).
Human cytokine production.
Recently it has been demonstrated
that inflammatory cytokines can modulate KSHV/HHV8 replication
(9). Since there were differences in the proliferative
capacity of uninfected keratinocytes and previously KSHV/HHV8-infected
long-term-proliferating keratinocytes, possibly due to higher
production or responsiveness to endogenous cytokines, we analyzed the
cytokine expression pattern of both uninfected and previously
KSHV/HHV8-infected keratinocytes 3 months postinfection. The latter
cells expressed higher levels of IL-6 and lower levels of IL-8 than did
the uninfected controls (Table 2). The
levels of other cytokines (macrophage inflammatory protein 1
[MIP-1
], MIP-1
, gamma interferon (IFN-
), tumor necrosis factor alpha [TNF-
], soluble TNF-R1, soluble TNF-R2, and
RANTES) were not significantly different in uninfected and
previously KSHV/HHV8-infected cells.
VEGF and bFGF gene transcription.
VEGF and bFGF play a key
role in the growth of KS lesions (13, 32, 44), and normal
human keratinocytes are a prominent source of VEGF (55).
To evaluate the levels of VEGF and bFGF transcripts in the previously
KSHV/HHV8-infected long-term-proliferating keratinocytes, we performed
RT-PCR assays after 3 months of infection. Figures
7A (bFGF) and B (VEGF) show no
significant differences in the transcription levels of the previously
KSHV/HHV8-infected, long-term-proliferating keratinocytes and of normal
primary keratinocytes.

View larger version (46K):
[in this window]
[in a new window]
|
FIG. 7.
Transcription levels of bFGF and VEGF genes in
106 infected and uninfected keratinocytes. (A) bFGF. Lanes:
1 and 3, two parallel cultures of previously KSHV/HHV8-infected
keratinocytes, 3 months after the infection; 5, normal primary
keratinocytes; 2, 4, and 6, controls with no RT; 7, primers with no
template. (B) VEGF. Lanes: 1 and 2, previously KSHV/HHV8-infected
keratinocytes in culture for 3 months; 3, normal primary keratinocytes;
4, primers with no template; 5, positive control (BC-3 cells). No
differences in transcription were seen.
|
|
 |
DISCUSSION |
This report describes the in vitro infection of primary human
keratinocytes with KSHV/HHV8. Other authors have shown, by either in
situ hybridization or in situ PCR, the presence of KSHV/HHV8 DNA and
RNA in scattered keratinocytes in the epidermis overlying KS lesions
(20, 38). These observations suggest that different human
cells, other then endothelial cells and lymphocytes, are permissive for
KSHV/HHV8 infection and replication. Moreover, detection of infectious
viral particles in the saliva of KSHV/HHV8-infected patients suggests
that the epithelial cells in the oral mucosa or in salivary glands are
a source of the virus (36, 54). Other studies have
demonstrated that the human embryonal-kidney epithelial cell line 293 and the owl monkey kidney OMK 637 cell line (21, 40) can
support KSHV/HHV8 infection in vitro. Since these cell lines are of
epithelial origin but are immortalized, we decided to assay primary
human epithelial cells, in particular keratinocytes, for their
susceptibility to viral infection and to study the biological
consequence of the infection. In the work presented here we demonstrate
that KSHV/HHV8 can indeed infect and replicate in primary human
keratinocytes. On treatment with TPA, PEL-derived cell lines and in
vitro-infected endothelial cells undergo the complete program of
KSHV/HHV8 gene expression, resulting in viral replication and release
of mature virions (19, 29, 35, 50). In primary
keratinocytes, KSHV/HHV8 conforms to this pattern as well. We show that
in latently KSHV/HHV8-infected keratinocytes, TPA treatment can induce
the viral lytic cycle and lead to the production of mature virions that
can be serially transmitted to other primary human cells such as
HUVECs. As previously reported for endothelial cells (19),
the cells surviving the initial viral infection continued to
proliferate. We have continued to propagate these cells, and they are
still in culture, although the rate of proliferation 9 months
postinfection is lower then it was 3 months postinfection. On the other
hand, uninfected controls senesced after seven passages in culture (3 to 5 weeks, depending on the particular culture). The
long-term-proliferating keratinocytes displayed several changes:
modification of cell morphology with acquisition of a spindle shape,
described also in several cell lines of epithelial origin
(24), loss of contact inhibition, and acquisition of
anchorage-independent growth. The presence of telomerase activity in
keratinocytes 8 weeks after infection with KSHV/HHV8 provides
additional evidence of a role for this virus in the long-term survival
of keratinocytes in culture (27). These results are
consistent with those obtained with KSHV/HHV8-infected primary
endothelial cells (19). Similar observations were made for
KSHV-infected dermal microvascular endothelial cells (35), although these cells were already immortalized by infection with a
recombinant retrovirus and telomerase activity could not be tested.
Examination of the phenotype of KSHV/HHV8-infected cells by
immunohistochemical analyses excluded infection and expansion of other
contaminating cell types, since all long-term-proliferating keratinocytes were cytokeratin positive, especially with MAb MNF116, which stains mainly basal keratinocytes (37). All other
cellular markers were negative except vimentin. Phenotypic changes,
such as vimentin expression, resemble an epithelial-to-mesenchymal transition that can occur when epithelial cells undergo malignant transformation such as in human squamous carcinoma (1, 23, 31,
53).
Cytokines produced by epidermal keratinocytes may play a significant
role as regulators in inflammation and host immune response. Long-term-proliferating keratinocytes exhibited a different pattern of
human cytokine production from normal primary human keratinocytes. Interestingly, expression of human interleukin-6 IL-6, a
multifunctional cytokine that plays a role in neovascularization and
wound healing and has also been implicated in the pathogenesis of KS
(30, 33), was highly upregulated. By contrast, expression
of IL-8, a proinflammatory cytokine that plays a role in the host
defense mechanism through its effects on neutrophil activation
(3), was downregulated. These results suggest that viral
factors may play a primary role in modification of the biologic
behavior of different cell types. Since bFGF and VEGF are potent
angiogenic growth factors that are highly expressed in KS spindle cells
and in primary KS lesions (16, 17, 43, 57), we
investigated the transcript levels of bFGF and VEGF genes. No
significant differences were found between infected and uninfected
cells, suggesting that neither bFGF nor VEGF is particularly involved
at this stage in the proliferating mechanisms of the previously
KSHV/HHV8-infected keratinocytes. We hypothesize that the ability of
KSHV/HHV8 to replicate in primary keratinocytes, in particular in
mucosal sites, is probably one of the first steps in clinical
infection. From there, the virus may reach the endothelial cells
locally or via circulating hematopoietic cells and contribute to the
formation of the vascular lesions typical of KS. This hypothesis is in
line with the infection modalities of the closest related known human herpesvirus, EBV, which is associated with lymphoid and epithelial lesions (25, 26, 28). EBV infects epithelial cells in vivo (49, 52, 56), transforms epithelial cells, and inhibits cell differentiation (48). Several studies have also
demonstrated that nasopharyngeal carcinoma cell lines and keratinocyte
cell lines can be infected with EBV by cell-to-cell contact
(11). Recently it has been shown that mucosal shedding of
KSHV/HHV8 occurred in men who were infected with this virus but did not have KS. This suggests that oral-oral contact might play a significant role in transmission (36).
In conclusion, we have shown that KSHV/HHV8, like other herpesviruses,
can productively infect epithelial cells. These results indicate that
keratinocytes can support KSHV/HHV8 infection and replication and are
consistent with the notion that epithelial cells, particularly those in
mucosal sites, are likely to be a primary site of infection from which
the virus may reach the endothelial cells or can be amplified in the
locally infiltrating B lymphocytes and then spread to other tissues.
 |
ACKNOWLEDGMENTS |
We thank Claudio Basilico and Jan T. Vilcek for helpful
discussions, Michael Bouchard for critical reading of the manuscript, and Amy Chadburn and Elizabeth M. Hyjek for assistance with the immunohistochemistry. We also thank Iraida Sharra-Pagano and Ron Liebman for providing keratinocytes and endothelial cells,
respectively, and Lily Ying for technical help.
Francesca Curreli was supported by a fellowship from FIRC (Federazione
Italiana per la Ricerca sul Cancro). This work was supported by the
Howard Gilman Foundation and by the Center for AIDS Research (CFAR).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, NYU School of Medicine, 550 First Ave., New York, NY
10016. Phone: (212) 263-5313. Fax: (212) 263-7933. E-mail:
ornella.flore{at}med.nyu.edu.
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Journal of Virology, March 2001, p. 2435-2443, Vol. 75, No. 5
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.5.2435-2443.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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