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Journal of Virology, June 2000, p. 5300-5309, Vol. 74, No. 11
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Downregulation of Major Histocompatibility Complex
Class I Molecules by Kaposi's Sarcoma-Associated Herpesvirus K3
and K5 Proteins
Satoshi
Ishido,1
Chunyang
Wang,1
Bok-Soo
Lee,1
George B.
Cohen,2 and
J. U.
Jung1,*
Department of Microbiology and Molecular
Genetics, New England Regional Primate Research Center, Harvard Medical
School, Southborough, Massachusetts 01772,1 and
AIDS Research Center, Massachusetts General Hospital, Harvard
Medical School, Charleston, Massachusetts 021292
Received 10 January 2000/Accepted 29 February 2000
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ABSTRACT |
The T-cell-mediated immune response plays a central role in the
defense against intracellular pathogens. To avoid this immune response,
viruses have evolved elaborate mechanisms that target and modulate many
different aspects of the host's immune system. A target common to many
of these viruses is the major histocompatibility complex (MHC) class I
molecules. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes K3
and K5 zinc finger membrane proteins which remove MHC class I molecules
from the cell surface. K3 and K5 exhibit 40% amino acid identity to
each other and localize primarily near the plasma membrane. While K3
and K5 dramatically downregulated class I molecules, they displayed
different specificities in downregulation of HLA allotypes. K5
significantly downregulated HLA-A and -B and downregulated HLA-C only
weakly, but not HLA-E, whereas K3 downregulated all four HLA allotypes.
This selective downregulation of HLA allotypes by K5 was partly due to
differences in amino acid sequences in their transmembrane regions.
Biochemical analyses demonstrated that while K3 and K5 did not affect
expression and intracellular transport of class I molecules, their
expression induced rapid endocytosis of the molecules. These results
demonstrate that KSHV has evolved a novel immune evasion mechanism by
harboring similar but distinct genes, K3 and K5, which target MHC class I molecules in different ways.
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INTRODUCTION |
A major immune defense against viral
infection is mediated by cytotoxic T lymphocytes (CTLs), which
recognize and lyse infected cells upon engagement of the T-cell
receptor with major histocompatibility complex (MHC) class I molecules
presenting viral peptides (10, 26). Viral proteins are
degraded to peptides by the proteasomes in the cytosol. These peptides
are translocated by the transporter associated with antigen processing
(TAP) to the endoplasmic reticulum (ER), where they assemble with the
MHC class I heavy chain and 
2 microglobulin to form a trimeric
complex. This mature complex is then transported from the ER to the
plasma membrane, where the MHC class I complexes present the peptide to
CTLs (20, 35).
Herpesviruses establish lifelong infections despite the presence of an
active immune system. To achieve this, herpesviruses encode gene
products that affect MHC class I expression, either at the level of
transcription or of expression at the cell surface (26). For
example, the herpes simplex virus type 1 US12 gene product, called
ICP47, binds to TAP and prevents the delivery of cytosolic antigen
peptides to assembling class I molecules in the ER (7, 11,
37). Epstein-Barr virus-encoded EBNA-1 protein inhibits antigen
processing, thus affecting presentation of certain peptides by MHC
class I complexes (18). Human cytomegalovirus (HCMV) and
murine CMV contain numerous lytic glycoproteins, each of which is
sufficient to cause increased turnover of MHC class I molecules. Two
ER-resident glycoproteins, US2 and US11, of HCMV are sufficient to
induce the rapid dislocation of newly synthesized MHC class I proteins
from the ER to the cytosol, where the class I heavy chains are degraded
by the proteasome (26, 34). HCMV US6 prevents peptide
loading of MHC class I molecules by inhibiting TAP-mediated peptide
translocation into the ER (1). In addition, the HCMV US3 and
the murine CMV m152 proteins prevent the transport of MHC class I
molecules to the cell surface by retaining the molecules within the ER
(12, 38).
DNA sequences of a member of the herpesvirus group, Kaposi's
sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8, have been
consistently identified in Kaposi's sarcoma tumors from human
immunodeficiency virus (HIV)-positive and HIV-negative patients (2, 3, 19). KSHV has also been identified in body
cavity-based lymphoma and some forms of Castleman's disease (2,
3, 27). The genomic sequence indicates KSHV to be a gamma-2
herpesvirus that is closely related to herpesvirus saimiri (HVS)
(21a, 29) and the recently isolated rhesus monkey
rhadinovirus (5, 32) and retroperitoneal fibromatosis
herpesvirus (28). Despite the colinearity of genomic
structures and the similarity of genomic sequences among these gamma-2
herpesviruses, each virus contains nonconserved, unique regions
(29). DNA sequence analysis of a 13-kb nonconserved region
of the KSHV genome revealed a number of cellular homologs (21a,
24, 29). These include a virus-encoded interleukin 6 (21,
22, 25), MIP1-
and - chemokines (13, 21,
25), Bcl-2 (30), dihydrofolic reductase, and
thymidylate synthetase. In addition, this region contains several
unique open reading frames, called K3, K4.2, K5, and K7, which do not
have apparent homology with any known cellular genes (24,
29). Interestingly, K3 and K5 exhibit 40% amino acid identity
with each other (24, 29) and are expressed during the early
lytic cycle of viral replication (24, 33). Both the K3 and
K5 gene products are related to the immediate-early gene (IE1) product of bovine herpesvirus 4 and ORF12 of HVS (24). All of these open reading frames contain two C4HC3 zinc
finger motifs at the amino terminus and hydrophobic transmembrane
regions in the center but are of various sizes in the carboxyl-terminal
tail (24).
In this report, we demonstrate that K3 and K5 downregulate the surface
expression of MHC class I molecules with different activities and
specificities. Biochemical analyses demonstrate that downregulation of
class I molecules induced by K3 and K5 is likely due to their rapid
endocytosis. Thus, KSHV encodes similar but distinct genes, K3 and K5,
which may protect the infected cells from destruction by immune
effector cells.
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MATERIALS AND METHODS |
Cell culture and transfection.
BJAB and 221 cells were grown
in RPMI medium supplemented with 10% fetal calf serum. COS-1 cells
were grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum. 221 cells harboring defined HLA allotypes have
been described previously (4). A Fusin lipofection
(Boehringer Mannheim) transfection procedure was used for transient
expression in COS-1 cells. The pEF1-derived expression vector (20 µg)
was introduced into BJAB cells by electroporation at 250 V and 960 µF
in serum-free Dulbecco's modified Eagle's medium. After a 48-h
incubation, the cells were cultured with selection medium containing 2 mg of neomycin/ml for 5 weeks.
Plasmid construction.
Based on the KSHV genomic sequence
(29), DNA containing the KSHV K3, K4.2, or K5 open reading
frame was amplified from BCBL-1 genomic DNA by PCR using a 5' primer
which corresponds to the amino-terminal sequence of each gene and a 3'
primer which corresponds to the carboxyl-terminal sequence of each gene
(15). The primers used for PCR contain EcoRI and
XbaI recognition sequences for subsequent cloning. Amplified
DNA was ligated into the EcoRI and XbaI cloning
sites of the pEpiTag vector (Invitrogen, Carlsbad, Calif.) for six-His
tagging at the carboxyl terminus of each gene. To construct green
fluorescent protein (GFP) expression vectors, each gene was subcloned
into the pQBIGFP vector. PCR-amplified DNA fragments were completely
sequenced to verify the presence of expected sequences and the absence
of aberrant alterations.
Metabolic labeling, immunoprecipitation, and endo H
digestion.
For metabolic labeling, cells were rinsed three times
with phosphate-buffered saline (PBS), washed once with labeling medium (RPMI minus methionine and cysteine plus 10% dialyzed fetal calf serum), and then incubated with 5 ml of the same medium containing 200 µCi of [35S]methionine and [35S]cysteine
(New England Nuclear, Boston, Mass.) for 16 h. For pulse-chase
analysis, cells were labeled for 15 min and chased for 20 and 40 min.
For immunoprecipitation, cells were harvested and lysed with lysis
buffer (0.15 M NaCl, 1% Nonidet P-40, and 50 mM HEPES buffer [pH
8.0]) containing 1 mM Na2VO3, 1 mM NaF, and
protease inhibitors (leupeptin, aprotinin, phenylmethylsulfonyl fluoride, and bestatin). Immunoprecipitation was performed with a
1:500-diluted anti-six-His antibody (Santa Cruz Biotech, Santa Cruz,
Calif.) together with 30 µl of protein A- and protein G-agarose beads. Washed immunoprecipitates were resuspended in 20 µl of 50 mM
sodium citrate (pH 5.5)-0.2% sodium dodecyl sulfate, heated for 5 min
at 95°C, and incubated for 6 h at 37°C with
endo--N-acetylglucosaminidase H (endo H).
Immunofluorescence.
Cells were fixed with 4%
paraformaldehyde for 15 min, permeabilized with 70% ethanol for 15 min, blocked with 10% goat serum in PBS for 30 min, and reacted with
1:100-diluted primary anti-six-His antibody (Santa Cruz Biotech) for
K3, K4.2, and K5 staining in PBS for 30 min at room temperature. After
incubation, the cells were washed extensively with PBS, incubated with
1:100-diluted secondary antibody (Vector, Burlingame, Calif.) in PBS
for 30 min at room temperature, and washed three times with PBS. For Golgi staining, cells were metabolically labeled with 5 µg of BODIPY-FL C5-ceramide (Molecular Probes, Eugene, Oreg.)/ml for 30 min
as recommended by the manufacturer's protocol. Finally, the cells were
mounted in mounting medium (Vector). Confocal microscopy was performed
using a TCS SP laser scanning microscope (Leica Microsystems, Exton,
Pa.) fitted with a 100× Leica objective (PL APO; 1.4 NA) and using the
Leica image software. Images were collected at 512- by 512-pixel
resolution. The stained cells were optically sectioned in the
z axis, and the images in the different channels (photomultiplier tubes) were collected simultaneously. The step size in
the z axis varied from 0.2 to 0.5 µm to obtain 30 to 50 slides/imaged field. The images were transferred to a Macintosh G3
computer (Apple Computer, Cupertino, Calif.), and NIH Image version
1.61 software was used to render the images.
Immunofluorescence-based endocytosis.
Cells were stained
with a fluorescein isothiocyanate (FITC)-conjugated class I antibody
and a Texas red-conjugated transferrin receptor antibody at 4°C for
30 min, washed twice with cold RPMI culture medium, and incubated at
37°C to allow for the internalization of antibody-bound class I
molecules and transferrin receptor. At various time points, the
localization of class I molecules and transferrin receptor was
determined with a Leica confocal immunofluorescence microscope.
Flow cytometry analysis.
Cells (5 × 105)
were washed with RPMI medium containing 10% fetal calf serum and
incubated with FITC- or phycoerythrin (PE)-conjugated monoclonal
antibodies for 30 min at 4°C. After being washed, each sample was
fixed with 1% paraformaldehyde solution and flow cytometry analysis
was performed with a FACScan (Becton Dickinson Co., Mountainview, Calif.). W6/32 antibody for MHC class I, B43 antibody for CD19, RPA-T8
antibody for CD8, M-A712 antibody for transferrin receptor, and TU39
antibody for MHC class II used for fluorescence-activated cell sorter
and confocal immunofluorescence analyses were obtained from PharMingen
Becton Dickinson Company.
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RESULTS |
Downregulation of surface expression of MHC class I molecules by
KSHV K3 and K5.
HVS viral superantigen is expressed in the
immediate-early lytic phase and powerfully induces the proliferation of
CD4-positive cells (6, 14, 36). At a position equivalent to
that of the HVS viral superantigen, KSHV contains unique membrane
proteins called K3, K4.2, and K5 (25). While none of these
membrane proteins demonstrated a detectable level of superantigenic
activity (data not shown), we found that either K3 or K5 expression
significantly downregulated surface expression of MHC class I molecules
(Fig. 1). However, the level of class I
downregulation by K3 was much higher than that by K5 (Fig. 1). In
contrast, K4.2 did not affect class I surface expression under the same
conditions (Fig. 1). Furthermore, the downregulation of MHC class I
molecules by K3 and K5 was specific; their expression did not affect
the surface expression of other lymphocyte antigens, including MHC
class II and immunoglobulin M under the same conditions (Fig. 1 and
data not shown).
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FIG. 1.
Downregulation of surface expression of MHC class I
molecules by K3 and K5. BJAB cells were electroporated with GFP
reporter, GFP-K3, GFP-K4.2, or GFP-K5 vector. The cell surface levels
of MHC class I molecules were assessed 48 h posttransfection by
staining the cells with a W6/32 antibody for MHC class I or TU39
antibody for MHC class II (y axis) and gating the
GFP-positive cell population (x axis) by flow cytometry.
Three populations of cells were distinguished by the applied gates; in
the upper left quadrant are the untransfected cells, in the upper right
quadrant are the GFP-positive transfected cells which have not
downregulated class I or class II, and in the lower right quadrant are
the GFP-positive transfected cells that have downregulated class I or
class II. The data were reproduced in three independent experiments.
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To further demonstrate the specific downregulation of class I molecules
by K3 and K5, BJAB cells were used to establish cell
lines stably
expressing the K3, K4.2, or K5 gene. Full-length
K3, K4.2, and K5 genes
were modified to encode a six-histidine
epitope tag at their carboxyl
termini and cloned into the expression
vector pEpiTag (EF), which
contains the elongation factor 1 promoter
for gene expression. After
the electroporation of the K3, K4.2,
or K5 expression vector, cells
were selected by growth in medium
containing 2 mg of G418/ml and
examined for MHC class I surface
expression by flow cytometry. The
stable expression of K3 and
K5 in BJAB cells induced a dramatic
downregulation of MHC class
I molecules on their surfaces, whereas K4.2
expression did not
affect MHC class I surface expression (Fig.
2A). Consistent with
results from a
transient-expression assay (Fig.
1), K3 downregulated
class I molecules
to a greater extent than K5 (Fig.
2A). The downregulation
of MHC class
I by K3 and K5 was specific; their expression did
not affect the
surface levels of MHC class II, immunoglobulin
M, and CD19 under the
same conditions (Fig.
2A and data not shown).
When G418-resistant cells
were examined for K3 or K5 expression
by radioactive
immunoprecipitation with an anti-six-His antibody,
a 50-kDa protein for
K3 and a 38-kDa protein for K5 were detected
(Fig.
2B, lanes 2 and 3).
In contrast, these proteins were not
detected in control BJAB cells
transfected with an empty vector
(Fig.
2B, lane 1).
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FIG. 2.
Downregulation of MHC class I molecules on cells stably
expressing K3 or K5. (A) Downregulation of MHC class I molecules by K3
or K5. G418-resistant cells were stained with a PE-conjugated W6/32
antibody or a PE-conjugated pan-class I antibody and analyzed by flow
cytometry. Two hundred thousand events were collected on a FACScan flow
cytometer. As a control, a histogram of each cell line (shaded) is
overlaid with a dotted-line histogram of an isotype antibody control.
The mean value of the relative level of class I and II surface
expression is presented inside each graph. (B) Expression of K3 and K5.
BJAB cells were labeled with [35S]methionine and
[35S]cysteine overnight. Radioactive cell lysates were
used for immunoprecipitation with an anti-six-His antibody. Lane EF,
BJAB/EF; lane K3, BJAB/K3; lane K5, BJAB/K5. The arrows indicate the K3
and K5 proteins.
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Subcellular localization of K3 and K5.
To investigate the
roles of K3 and K5 in the downregulation of MHC class I surface
expression, we examined their subcellular localization by
indirect-immunofluorescence tests. COS-1 cells were transfected with an
expression vector containing a six-His epitope-tagged K3, K4.2, or K5
gene. Two days posttransfection, the cells were costained with a Texas
red-conjugated anti-six-His antibody and a FITC-conjugated
Golgi-specific dye and examined under a confocal immunofluorescence
microscope. The K3, K4.2, and K5 proteins were all primarily present in
the cytoplasm. While a high degree of overlapping staining between K4.2
and the Golgi complex was detected, no overlapping staining or a
minimal level of overlapping staining between K3 or K5 and the Golgi
complex was observed (Fig. 3A).
Furthermore, cross sections of transfected cells demonstrated that K3
and K5 were localized near the plasma membrane and that a large portion
of K4.2 was localized at the Golgi complex (Fig. 3A).
Immunofluorescence tests with BJAB cells stably expressing K3 and K5
also indicated their presence in the cytoplasm and near the plasma
membrane (Fig. 3B).
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FIG. 3.
Localization of K3 and K5 near the plasma membrane. (A)
Localization in COS-1 cells. COS-1 cells were transfected with
pEF-K3-6xHis (K3), pEF-K4.2-6xHis (K4.2), or pEF-K5-6xHis (K5) DNA. The
cells were permeabilized with ethanol and reacted with a Texas
red-conjugated anti-six-His antibody (red) and FITC-conjugated
Golgi-specific dye (green). Immunofluorescence was examined with a
Leica confocal immunofluorescence microscope. To determine their
specific locations, stained cells were cross-sectioned and viewed under
the Leica confocal immunofluorescence microscope and are presented in
the side boxes. The overlapping staining was visualized as yellow
color. (B) Localization in BJAB cells. BJAB/EF, BJAB/K3, and BJAB/K5
cells were permeabilized with ethanol and reacted with a Texas
red-conjugated anti-six-His antibody. Immunofluorescence was examined
with a Leica confocal immunofluorescence microscope.
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Endo H digestion.
Carbohydrate moieties of MHC class I
complexes contain high-mannose-type oligosaccharides in the ER which
are susceptible to endo H digestion. After transport to the medial
Golgi, however, their carbohydrate moieties acquire resistance to
cleavage by endo H (23). To investigate the effect of K3 and
K5 on the intracellular transport of MHC class I complexes, BJAB/EF,
BJAB/K3, and BJAB/K5 cells were metabolically labeled with
[35S]methionine and [35S]cysteine for 15 min and chased for 20 or 40 min. MHC class I complexes were then
precipitated with a monoclonal W6/32 antibody and subjected to endo H
digestion. The heavy chain of MHC class I in BJAB/K3 and BJAB/K5 cells
acquired resistance to endo H cleavage equivalent to that in control
BJAB/EF cells (Fig. 4). In addition, a
similar level of class I molecules was expressed in BJAB/EF, BJAB/K3,
and BJAB/K5 cells (Fig. 4 and data not shown). These results indicate
that K3 and K5 do not affect the expression and acquisition of endo H
resistance by MHC class I molecules at a detectable level.
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FIG. 4.
Endo H sensitivity of class I molecules. BJAB/EF,
BJAB/K3, and BJAB/K5 cells were metabolically labeled with
[35S]methionine and [35S]cysteine for 15 min and chased for 0, 20, or 40 min. Radioactively labeled lysates were
used for immunoprecipitation with an anti-class I antibody.
Immunoprecipitates were treated with endo H prior to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. The portion of the gel
displaying the heavy chain of class I molecules is shown. *, the endo
H-resistant heavy chain of class I molecules.
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Acceleration of endocytosis of class I molecules by K3 and K5.
HIV Nef has been shown to downregulate MHC class I molecules by
accelerating their endocytosis (9, 16). To examine whether K3 and K5 affected the internalization of class I molecules, we used an
immunofluorescence-based endocytosis assay. After being stained with
anti-class I and anti-transferrin receptor antibodies at 4°C for 30 min, BJAB/EF, BJAB/K3, and BJAB K5 cells were washed twice with cold
culture medium and incubated at 37°C to allow for the internalization
of antibody-bound class I molecules and transferrin receptor. At
appropriate time points, the localization of class I and transferrin
receptor was determined by confocal immunofluorescence microscope
analysis. Unlike control cells, in which an apparent endocytosis of
class I molecules was detected after approximately 120 min of
incubation, K3-expressing cells exhibited a rapid endocytosis of class
I molecules; the internalization of class I molecules was evident
within 30 min and was almost completed after 60 min of incubation (Fig.
5A). K5-expressing cells also showed an
accelerated endocytosis of class I molecules compared to control cells:
however, the rate of endocytosis in K3-expressing cells was much higher
than that in K5-expressing cells (Fig. 5A). This is consistent with our
observation that the effect of K3 on downregulation of class I
molecules is more pronounced than that of K5, as shown in Fig. 1 and 2.
In contrast, similar rates of endocytosis of the transferrin receptor
were detected in all three cell lines under the same conditions (Fig. 5B). Furthermore, class I molecules were predominantly organized into
punctate patterns on the surfaces of K3- and K5-expressing cells,
whereas they were evenly distributed on the surfaces of control BJAB/EF
cells (Fig. 5A). These results suggest that the downregulation of class
I molecules induced by K3 and K5 is likely due to their rapid
endocytosis.
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FIG. 5.
Rapid endocytosis of class I molecules by K3 and K5.
BJAB/EF, BJAB/K3, and BJAB/K5 cells were stained with a FITC-conjugated
class I antibody (A) and a Texas red-conjugated transferrin receptor
antibody (B) at 4°C for 30 min, washed twice with cold culture
medium, and incubated at 37°C to allow for the internalization of
antibody-bound class I molecules and transferrin receptor. At the
indicated time points (in minutes), the localization of class I and
transferrin receptor was determined with a Leica confocal
immunofluorescence microscope.
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Selective downregulation of HLA allotypes by K5.
HLA-A and
HLA-B proteins are primarily responsible for presenting processed
antigen peptides to CTLs, whereas HLA-C and HLA-E are the predominant
class I alleles responsible for preventing natural killer (NK) cells
from killing lymphoid cells (17). To investigate potential
selectivity of downregulation of HLA allotypes by K3 and K5, 221 cells
stably expressing defined HLA allotypes (4) were
electroporated with GFP, GFP-K3, or GFP-K5 expression vector. The
effect of K3 and K5 expression on the cell surface levels of HLA
allotypes was assessed 48 h posttransfection by staining for the
MHC class I protein and gating of the GFP-positive cell population by
flow cytometry. A dramatic downregulation of HLA-A2, HLA-B2705,
HLA-Cw4, and HLA-E was observed in the K3-expressing GFP-positive cell
population (Fig. 6). The K5-expressing
GFP-positive cell population exhibited approximately four- to fivefold
downregulation of HLA-A2 and HLA-B2705 (Fig. 6). However, K5 induced
only twofold downregulation of HLA-Cw4 and it caused almost no
downregulation of HLA-E (Fig. 6). Transfection with the GFP reporter
vector resulted in no effect on surface expression of HLA allotypes
under the same conditions (Fig. 6).
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FIG. 6.
Selective downregulation of HLA allotypes by K3 and K5.
221 cells stably expressing defined HLA allotypes were electroporated
with GFP, GFP-K3, or GFP-K5 vector. Cell surface levels of MHC class I
molecules were assessed 48 h posttransfection by staining the
cells with PE-conjugated pan-class I W6/32 antibody (y axis)
and gating the GFP-positive cell population (x axis) by flow
cytometry. The numbers inside the boxes indicate the average mean
values of class I surface expression of the GFP-positive cell
population. The data were reproduced in three independent
experiments.
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Specific regions of HLA required for K3- and K5-mediated
downregulation.
To identify the region of HLA-A2 that accounted
for K3- and K5-dependent downregulation, 221 cells containing a
chimeric protein between human CD8 and HLA-A2 were
electroporated with GFP, GFP-K3, and GFP-K5 expression vectors.
This chimera contained the extracellular region of CD8 joined to the
transmembrane (Tm) and cytoplasmic tail region of HLA-A2
(CD8/A2-Tm+tail) (4). Upon transfection, wild-type CD8
did not undergo appreciable downregulation by K3 and K5, whereas
CD8/A2-Tm+tail was drastically downregulated in a K3- and
K5-dependent manner (Fig. 7). This
indicates that the transmembrane and cytoplasmic regions of HLA-A2 are
responsible for the downregulation by K3 and K5.
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FIG. 7.
Downregulation of CD8 chimera containing transmembrane
and cytoplasmic regions of HLA-A2 by K3 and K5. 221 cells stably
expressing CD8 or the CD8/A2-Tm+tail chimera were electroporated
with GFP reporter, GFP-K3, or GFP-K5 vector. The cell surface level of
CD8 molecules was assessed 48 h posttransfection by staining the
cells with an anti-CD8 antibody (y axis) and gating the
GFP-positive cell population (x axis) by flow cytometry. The
numbers inside the boxes indicate the average mean values of CD8
surface expression of the GFP-positive cell population. The data were
reproduced in three independent experiments.
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Since K5 induced a minimal level of downregulation of HLA-Cw4, we
further investigated whether a transfer of the transmembrane
and
cytoplasmic tail (A2-Tm+tail) or the cytoplasmic tail (A2-tail)
of
HLA-A2 to HLA-Cw4 conferred K5-dependent downregulation. 221
cells
expressing the HLA-Cw4/A2-Tm+tail or HLA-Cw4/A2-tail chimera
were
electroporated with GFP, GFP-K3, or GFP-K5 expression vector,
and the
levels of surface expression of these chimeras were examined
by flow
cytometry. The replacement of the cytoplasmic region of
HLA-Cw4 with
that of HLA-A2 did not affect the level of K5-mediated
downregulation;
K5 induced approximately twofold downregulation
of HLA-Cw4 and
HLA-Cw4/A2-tail (Fig.
8). In addition,
the replacement
of the cytoplasmic region of HLA-Cw4 with that of
HLA-B27 did
not affect the level of K5-mediated downregulation (data
not shown).
However, a transfer of both transmembrane and cytoplasmic
regions
of HLA-A2 moderately augmented the K5-mediated downregulation
of HLA-Cw4/A2 chimeras (Fig.
8). The level of K5-mediated
downregulation
of Cw4/A2-Tm+tail was comparable to that of HLA-A2 (Fig.
6 and
8). In contrast, K3 was able to drastically downregulate all
forms
of HLA proteins under the same conditions (Fig.
8). These studies
indicate that K5 downregulates class I molecules in a way that
is
similar to but distinct from that of K3. In addition, the transmembrane
regions of HLA-A and HLA-B potentially contribute to their
susceptibilities
to K5-mediated downregulation.
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FIG. 8.
Regions of class I molecules required for downregulation
by K3 and K5. 221 cells stably expressing class I chimeric molecules
were electroporated with GFP reporter, GFP-K3, or GFP-K5 vector. The
level of MHC class I surface expression was assessed 48 h
posttransfection by staining the cells with a PE-conjugated W6/32
antibody for class I (y axis) and gating the GFP-positive
cell population (x axis) by flow cytometry. The numbers
inside the boxes indicate the average mean values of MHC class I
surface expression of the GFP-positive cell population. The data were
reproduced in three independent experiments.
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DISCUSSION |
To establish lifelong infection in the host, herpesviruses have
evolved mechanisms for avoiding the host immune response. Among these
mechanisms is the downregulation of the surface expression of MHC class
I molecules (26). In this report, we add two KSHV proteins
to an increasing list of viral proteins which apparently interfere with
cellular immunity. KSHV is closely related to other gamma-2
herpesviruses by virtue of the colinearity of their genomic structures
and their common possession of cellular homologs. However, the K3 and
K5 genes are unique to KSHV. These genes are adjacent in the viral
genome and are expressed during the early lytic cycle of viral
replication, and their gene products exhibit 40% amino acid identity
(24). These properties suggest that one of the genes has
arisen via a gene duplication event. The advantage of a virus
simultaneously expressing two proteins with similar functions is not
immediately clear. However, HCMV uses an even more complex strategy to
prevent class I presentation. HCMV has four glycoproteins, US2, US3,
US6, and US11, all of which downregulate class I-mediated antigen
presentation (20, 26). One hypothesis for the overlapping kinetics and functions of these proteins is that they may assure complete prevention of class I-mediated antigen presentation and thus
T-cell recognition of virus-infected cells.
Despite their sequence identity and functional similarity, K3 and K5
exhibit differences in the activity and specificity of class I
downregulation. For example, K3 drastically downregulates HLA-A, -B,
-C, and -E, whereas K5 exclusively downregulates HLA-A and -B. This
suggests that K3 and K5 downregulate class I molecules in similar but
distinct ways. In addition, studies of HLA-Cw4/A2 and HLA-Cw4/B27
chimeras indicate that the transmembrane regions of HLA-A2 and HLA-B27
potentially contribute to their susceptibilities to K5-mediated downregulation.
While the dramatic downregulation of HLA allotypes by K3 likely confers
a protection from killing by CTLs, its indiscriminate downregulation of
HLA-C and -E may invite NK cells to attack infected cells. To protect
against this, a virus may employ additional tactics: a dominant
selective activity of K5 over K3 in HLA downregulation, differential
expression between K3 and K5 in the viral lytic cycle, or a
yet-to-be-discovered, novel mechanism. Further study will be directed
to providing evidence for or against this hypothesis. Nevertheless,
this study suggests that KSHV has evolved a novel immune evasion
mechanism by harboring similar but distinct genes, K3 and K5, which
target MHC class I molecules with different activities and specificities.
A diversity of mechanisms of viral interference with cellular immunity
is becoming increasingly apparent. HIV Nef has been shown to
downregulate MHC class I molecules by accelerating their endocytosis
(9, 16). Immunofluorescence tests indicated a redistribution
of MHC class I molecules on the surfaces of K3- and K5-expressing
cells: the molecules were predominantly organized into a punctate
pattern. This staining pattern is similar to that obtained when the
clathrin-coated regions of the plasma membrane are visualized. This
suggests that, similar to HIV Nef, K3 and K5 may redistribute class I
molecules to clathrin-coated regions of the plasma membrane, resulting
in an accelerated endocytosis. Furthermore, similar to K5, HIV Nef has
been shown to downregulate HLA-A and -B but not HLA-C and -E
(4). Thus, despite the absence of discernible homology
between KSHV K5 and HIV Nef, they share the ability to induce the
selective downregulation of HLA allotypes. This also suggests that KSHV
K5 and HIV Nef may target similar cellular proteins but with different
mechanism. Kaposi's sarcoma is a complex tumor, and its progression is
consistently associated with the suppression of various host immune
controls (8, 31). Thus, a detailed study of molecular
mechanisms of immune evasion by KSHV K3 and K5 will not only lead to a
better understanding of viral persistence and disease progression but
also provide a novel means for investigating cellular immune regulatory systems.
| |
ACKNOWLEDGMENTS |
We especially thank B. Damania, L. Alexander, and R. Means for
critical reading of the manuscript, K. Toohey for photography support,
X. Alvarez for confocal microscope analysis, M. DeMaria for flow
cytometry analysis, and B. Roy for manuscript preparation.
This work was supported by U.S. Public Health Service grants CA31363,
CA82057, CA86841, AI38131, ACS99295, and RR00168.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Tumor Virology
Division, New England Regional Primate Research Center, Harvard Medical School, 1 Pine Hill Dr., Southborough, MA 01772. Phone: (508) 624-8083. Fax: (508) 786-1416. E-mail:
jae_jung{at}hms.harvard.edu.
| |
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Abstract
Introduction
