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J. Exp. Med. Vol. 207 No. 9 2033-2041
Cytotoxic T-lymphocytes (CTL) are essential for
limiting and clearing viral infections (Doherty
et al., 1992). CTLs are restricted by class I MHC
molecules, which are a frequent target of viral
strategies for their down-regulation or even elim-
ination. The unique short region of human cyto-
megalovirus (HCMV) genome contains the
US2-US11 genes, a region predicted to encode at
least eight small glycoproteins of only limited ho-
mology (Weston and Barrell, 1986; Kouzarides
et al., 1988). Several of them interfere with class I
MHC–restricted antigen presentation, through
inhibition of the MHC-encoded TAP peptide
transporter (HCMV US6; Ahn et al., 1997; Jun
et al., 2000), retention of newly synthesized class I
MHC products at their site of synthesis (HCMV
US3; Jones et al., 1996; Jun et al., 2000), or dislo-
cation of class I MHC products from the endo-
plasmic reticulum (HCMV US2 and HCMV
US11; Jones et al., 1996; Wiertz et al., 1996a,b;
Machold et al., 1997; Schust et al., 1998). The co-
ordinate regulation of the genes contained in the
unique short region protein (US) region and the
common theme of interference with class I
MHC–restricted antigen presentation suggest the
possibility that other members of the family, with
as yet poorly defined functions, may affect class I
MHC antigen presentation as well.
For US8 and US10, a physical interaction
with classical class I MHC products occurs
(Furman et al., 2002; Tirabassi and Ploegh, 2002),
but neither show significant ER retention or
down-regulation of class I MHC products to the
extent seen for US3, US2, and US11. Although
both US8 and US10 bind to classical class I
MHC products, only the expression of US10
imposes a delay on their egress from the ER,
without affecting overall turnover of assembled
class I MHC complexes or free class I MHC
heavy chains. Based on our experience with the
US2 and US11 products, the observation win-
dow of these experiments was limited to short
periods only, and was thus biased against the
possibility of documenting changes that occur
Hidde L. Ploegh:
Abbreviations used: 2m,
cytotoxic T-lymphocyte; endo H,
endoglycosidase H; HCMV,
human cytomegalovirus; shRNA,
short hairpin RNA; US, unique
short region protein.
B. Park’s present address is Dept. of Biology, College of
Life Science and Biotechnology, Yonsei University, Seoul
120-749, South Korea.
The HCMV membrane glycoprotein US10
selectively targets HLA-G for degradation
Boyoun Park,1 Eric Spooner,1 Brandy L. Houser,2 Jack L. Strominger,2
and Hidde L. Ploegh1
1Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02115
2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident trans-
membrane glycoprotein, US10, expressed early in the replicative cycle of HCMV as part of
the same cluster that encodes the known immunoevasins US2, US3, US6, and US11.
We show that US10 down-regulates cell surface expression of HLA-G, but not that of
classical class I MHC molecules. The unique and short cytoplasmic tail of HLA-G (RKKSSD)
is essential in its role as a US10 substrate, and a tri-leucine motif in the cytoplasmic tail
of US10 is responsible for down-regulation of HLA-G. Both the kinetics of HLA-G degrada-
tion and the mechanisms responsible appear to be distinct from those used by the US2 and
US11 pathways, suggesting the existence of a third route of protein dislocation from the
ER. We show that US10-mediated degradation of HLA-G interferes with HLA-G–mediated
NK cell inhibition. Given the role of HLA-G in protecting the fetus from attack by the
maternal immune system and in directing the differentiation of human dendritic cells to
promote the evolution of regulatory T cells, HCMV likely targets the HLA-G–dependent
axis of immune recognition no less efficiently than it interferes with classical class I
MHC–restricted antigen presentation.
© 2010 Park et al. This article is distributed under the terms of an Attribution–
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the publication date (see http://www.rupress.org/terms). After six months it is
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The Journal of Experimental Medicine
HCMV US10 targets HLA-G | Park et al.
with slower kinetics, yet are quantitatively significant. These
experiments also failed to take into account the possibility that
some of the HCMV US gene products might target nonclassi-
cal class I MHC products, by analogy of the effects reported for
US2 and HFE, a class I–like molecule involved in the traf-
ficking of the transferrin receptor (Ben-Arieh et al., 2001;
Vahdati-Ben Arieh et al., 2003).
HLA-G is a particularly interesting nonclassical class I MHC
molecule. It shows restricted tissue distribution and has limited
polymorphism (Shawar et al., 1994; Carosella et al., 2000).
HLA-G has strong immunomodulatory properties with specific
relevance at immune-privileged sites such as the trophoblast or
thymus, and it inhibits proliferation of T cells (Riteau et al.,
1999; Lila et al., 2001), natural killer cells (Pazmany et al., 1996;
Rouas-Freiss et al., 1997; Khalil-Daher et al., 1999), and antigen-
specific T cell cytotoxicity (Le Gal et al., 1999; Wiendl et al.,
2002). HLA-G has aroused interest not only because of its
role in feto-maternal interactions, but also because of its expres-
sion on subsets of human dendritic cells, in particular those im-
plicated in the activation of regulatory T cells (Liang et al.,
2008; Pazmany et al., 1996).
We report that, unlike any previously described nonclassi-
cal class I product, HLA-G is sensitive to proteasomal degrada-
tion in a HCMV US10-dependent manner. The underlying
mode of degradation of HLA-G under the agency of US10
appears to be unique, despite similar subcellular localization
and structural relatedness of US10 to US2 and US11. We sug-
gest that HCMV-infected cells avail themselves of all possibili-
ties to frustrate class I MHC–restricted antigen presentation,
including the inhibition of pathways that concern nonclassical
class I MHC products in the context of an HCMV infection.
HCMV US10 down-regulates surface presentation of JEG3-
derived HLA-G by degradation
Although HCMV US10 binds to classical class I MHC mole-
cules and delays their trafficking (Furman et al., 2002), it does
not affect their steady–state cell surface levels (Ahn et al., 1997).
To test whether US10 could interfere with the synthesis and
stability of nonclassical class I MHC products, we expressed
US10 in the HLA-G–positive choriocarcinoma cell line JEG3
and examined surface levels of HLA-G by cytofluorimetry
using W6/32 or MEM-G/9, both of which recognize assembled
heterodimers of heavy chain and 2-microglobulin (2m;
Fig. 1 A). We observed a significant reduction of surface ex-
pression of HLA-G in US10-expressing cells, whereas intro-
duction of US9, used as a control, was without effect on HLA-G
levels (Fig. 1 A, right). The effect of US10 on HLA-G expres-
sion is not unique to the naturally HLA-G–expressing chorio-
carcinoma cell line JEG3. Introduction of HLA-G into HeLa
cells showed that US10 also down-regulates HLA-G in this set-
ting (Fig. 1 B, top). US10 did not affect surface display of
the classical class I MHC products endogenous to HeLa cells
(Fig. 1 B, bottom). This result suggests that properties intrinsic to
US10 are sufficient to account for down-regulation of HLA-G,
and that no choriocarcinoma-specific factors contribute.
Figure 1. US10 down-regulates HLA-G molecules, but not classical
class I MHC products, by degradation. (A) Cell surface expression of
HLA-G in HCMV US9 and US10-expressing JEG3 cells. JEG3 was transiently
transfected with empty vector, US9, or US10. After 48 h, cell surface expres-
sion of HLA-G was monitored by cytofluorimetry using mAb MEM-G/9.
(B) US10 down-regulates HLA-G products. Both US10 and HLA-G or US10
alone were transiently transfected into HeLa cells. Surface expression of
HLA-G or classical class I MHC was measured by cytofluorimetry using
MEM-G/9 or W6/32. (C) Human foreskin fibroblasts (HFF) stably expressing
HLA-G were infected with wild-type HCMV AD169 (a and c, shaded area), a
HCMV mutant US2-US11 (a–d, solid black line), or RV670 (b and d, shaded
area). Surface levels of either classical class I MHC or HLA-G by cytofluorim-
etry using W6/32 or MEM-G/9. US10 gene product is verified by RT-PCR in
a HCMV AD169 or mutant virus-infected cells. The data shown are repre-
sentative of two independent experiments with similar results.
JEM VOL. 207, August 30, 2010
US10-expressing cells (Fig. 2 C, lane 6). Inclusion of ZL3VS
prevented the loss of labeled HLA-G in US10-expressing
cells. Moreover, we observed a deglycosylated intermediate
reminiscent of what is seen in US2 or US11-expressing cells
for classical class I MHC products (Fig. 2 C, lanes 8 and 9).
We obtained similar results for HeLa cell transfectants that
express HLA-G and US10 (Fig. 2 D, lanes 8 and 9). We
conclude that inclusion of proteasome inhibitors allows the
visualization of the deglycosylated HLA-G intermediate in
cells that express US10.
To ascertain the physiological relevance of US10 activities in
down-regulation of HLA-G, we examined whether US10 af-
fects cell surface levels of HLA-G in the context of HCMV in-
fection. Human foreskin fibroblasts stably expressing HLA-G
were infected either with wild-type HCMV AD169, a HCMV
deletion mutant lacking the US2-US11 region (US2-US11),
or with a HCMV mutant virus, RV670, lacking all genes in the
US2-US11 region with the exception of US10 (Jones and
Muzithras, 1992; Jones et al., 1995). We then examined surface
levels of either classical class I MHC or HLA-G by cytofluorim-
etry using W6/32 or MEM-G/9. In AD169-infected cells,
surface levels of classical class I MHC and HLA-G were signifi-
cantly reduced, whereas in US2-US11 AD169-infected cells
(Fig. 1 C, a and c), they were comparable to levels observed in
uninfected cells (Fig. 1 C). The HCMV mutant virus, RV670,
down-regulated cell surface expression of HLA-G (Fig. 1 C, d),
but did not affect levels of classical class I MHC products
(Fig. 1 C, b). RV670-infected cells express the US10 gene pro-
duct as do wild-type HCMV AD169-infected cells (Fig. 1 C,
bottom). These findings show that US10 specifically down-
regulates HLA-G, but not classical class I MHC products.
US10-dependent degradation of HLA-G involves
a deglycosylated intermediate in the presence
of proteasome inhibitor
To explore how US10 affects expression of HLA-G, we in-
stalled an HA epitope tag on US3, US9, and US10 to assess the
levels of these HCMV products in transfectants. The use of the
HA tag allowed us to select transfectants with comparable levels
of expression for each of the viral products, as they are detected
by one and the same antibody (Fig. 2 A). We quantitated the
amount of HLA-G in US3-, US9-, and US10-expressing JEG3
cells by immunoblotting using MEM-G/1 antibody, which rec-
ognizes the denatured 1 domain of HLA-G. HLA-G was barely
detectable in the JEG3-US10 cells, but neither US3 nor US9 af-
fected the robust HLA-G levels present in these transfectants
(Fig. 2 A). We confirmed the results of immunoblotting by bio-
chemical analysis on S-methionine and cysteine-labeled cells.
In the absence of US10, significant amounts of HLA-G were
detected at the end of both the pulse and the 6-h chase. In con-
trast, in JEG3 cells transfected with US10, despite similar rates of
synthesis of HLA-G during the 30-min pulse, 90% of labeled
HLA-G was degraded after the 6-h chase (Fig. 2 B, lanes 7 and 8).
Only a small portion of endoglycosidase H (endo H)–sensitive
HLA-G complexes remains at 6 h of chase (Fig. 2 B, lanes
7 and 8). We conclude that surface expression and stability of
HLA-G are sensitive to the presence of US10, whereas the
classical class I MHC molecules are not affected by US10.
To investigate whether the proteasome is involved in
US10-mediated degradation of HLA-G, we monitored
HLA-G levels in US10-expressing JEG3 cells in the presence
of the proteasome inhibitor ZL3VS (Jones et al., 1996; Wiertz
et al., 1996a). In JEG3 cells, HLA-G is a stable type I mem-
brane glycoprotein with scant reduction in the amounts of
biosynthetically labeled HLA-G, even after 12 h of chase.
Little if any HLA-G remains after the 12-h chase period in
Figure 2. HLA-G dislocation and degradation in the presence of
US10. (A) US10 degrades HLA-G. Lysates from JEG3 cells expressing HA-
tagging US3, US9, or US10 were analyzed by immunoblotting with the
indicated antibodies. (B) US10-expressing JEG3 cells were metabolically
labeled for 20 min and chased for 6 h. Assembled HLA-G molecules were
immunoprecipitated with MEM-G/9 and treated with endo H where indi-
cated. Immunoprecipitation from NP-40 lysates of JEG3 cells (C) and HeLa
cells (D) pulse-labeled for 20 min and chased for the indicated time points
in the absence (lanes 1–6) or presence (lanes 7–9) of 50 µM ZL3VS were
performed with MEM-G/1 antibodies. A representative result of two inde-
pendent experiments is shown.
HCMV US10 targets HLA-G | Park et al.
tail or both the transmembrane domain and the cytoplasmic
tail of US10, respectively, were deleted (Fig. 3 A, top). These
mutant proteins remain endo H sensitive during chase periods
of up to 4 h (Fig. 3 B). In contrast to cells that express wild-
type US10, cells that express the US10 truncation mutants
Characterization of the cytoplasmic tail residues of US10
critical for degradation of HLA-G
To identify the regions of US10 responsible for mediating the
degradation of HLA-G, we constructed two truncation mu-
tants, US10CT and TM+CT, in which the cytoplasmic
Figure 3. Characterization of the cytoplasmic determinants of US10 responsible for the degradation of HLA-G. (A) Schematic representation
of the chimeras of US10 and HLA-G. TM, transmembrane; CT, cytoplasmic tail; filled, HLA-G; open, HLA-A2. (B) Stability and maturation of US10 and its
deletion mutants. HeLa cells were transfected with US10, US10CT or US10TM+CT. Cells were labeled for 15 min and chased for the indicated times.
Immunoprecipitated proteins were digested (+) or not digested () with endo H. (C) HLA-G surface expression in JEG3 and HeLa cells expressing US10
chimeras. HeLa and JEG3 cells were transiently transfected with the individual cDNAs encoding the US10 deletion mutants. After 48 h, cell surface ex-
pression of HLA-G was analyzed by cytofluorimetry using MEM-G/9. Dashed line, no US10; solid line, US10; bolded line, transfectants. (D and E) Stability
of HLA-G in cells expressing wild-type US10, mutants, or HA-TEV epitope–tagged constructs of US10. (F and G) The tri-leucine motif of the US10 cyto-
plasmic tail is critical for degradation of HLA-G. Expression of HLA-G and the indicated point mutants of US10 were analyzed by cytofluorimetry and
immunoblotting using MEM-G/9 and MEM-G/1 antibodies. Data are from three independent experiments.
JEM VOL. 207, August 30, 2010
The cytoplasmic tail
of HLA-G is required for US10
to exert its function
The sensitivity of HLA-G to degra-
dation facilitated by US10 is intrinsic
to HLA-G. The cytoplasmic tail of
HLA-G is much shorter (6 amino
acids; RKKSSD) than that of the classi-
cal class I MHC products (Geraghty
et al., 1987; Fig. 3 A, bottom). To
address the role of HLA-G’s short cy-
toplasmic tail, we generated not only HLA-GCT, which
lacks the cytoplasmic tail, but also HLA-A2/G tail, a version
of the classical class I MHC product HLA-A2 equipped with
the cytoplasmic tail of HLA-G (HLA-A2/G tail; Fig. 3 A,
bottom). Expression levels of HLA-A2.1 or HLA-GCT
were not affected by US10 (Fig. 4 A), whereas HLA-A2/G
tail behaved like HLA-G (Fig. 4, B and C). We examined
surface expression of these mutants in US10-expressing HeLa
cells. Deletion of the cytoplasmic tail of HLA-G (HLA-
GCT) prevented its down-regulation by US10, whereas
surface expression of HLA-A2/G tail was affected by US10
like HLA-G (Fig. 4 C). HLA-E is a human nonclassical class I
MHC molecule and is similar to HLA-G with the exception
of an extension of the cytoplasmic tail when compared with
HLA-G (32 aa instead of 6 aa). We examined whether the
level of HLA-E protein expression is affected by US10
(Fig. S1). HLA-G or HLA-E–expressing HeLa cells were
transfected with US10 and examined by immunoblotting with
anti-HLA-G or anti-HLA-E antibodies. Protein levels of
HLA-G were significantly affected by US10, whereas the
HLA-E level was only slightly reduced. We conclude that
within the context of a human class I MHC molecule the
short cytoplasmic tail sequence of HLA-G is necessary and
sufficient to allow US10-mediated degradation and that US10
specifically targets HLA-G.
showed normal levels of HLA-G at the cell surface (Fig. 3 C).
Even though the levels of expression of wild-type and mutant
proteins were comparable, the levels of HLA-G heavy chain,
assessed by immunoblotting, were similar for cells that express
the US10 truncation mutants and HLA-G transfectants pro-
duced in the absence of US10 (Fig. 3, D and E). The cytoplas-
mic tail of US10 thus functions as a signal that targets HLA-G
To narrow the determinants within the cytoplasmic tail es-
sential to the down-regulatory capacity of US10, we gener-
ated a construct that lacks residues 177–185 (US10177-185;
Fig. 3 A, top). This mutant showed a wild-type pattern of
degradation of HLA-G (Fig. 3, D and F). Thus, the motif be-
tween amino acids 171 and 176 (TRSLLL) of US10 appeared
critical for the degradation of HLA-G. We then established the
importance of the leucine residues 174–176 for degradation
of HLA-G. We substituted the first leucine residue (in bold;
TRSALL), the first and second leucine residues (TRSAAL),
and all three leucine residues (TRSAAA) with alanine (Fig. 3 A,
top). The version of US10 that lacks two leucine residues re-
stored HLA-G protein levels, and its presence is permissive for
cell surface expression of HLA-G. Replacement of all three
leucines rendered US10 completely inactive (Fig. 3, F and G).
We conclude that the tri-leucine motif in the cytoplasmic tail
of US10 is crucial to target HLA-G for degradation.
Figure 4. US10 uniquely targets the
short cytoplasmic tail of HLA-G.
(A and C) HeLa cells expressed HLA-G, HLA-
GCT, or HLA-A2.1 were transiently trans-
fected with the indicated constructs and
observed for protein expression or cell
surface expression of HLA-G by immuno-
blotting or cytofluorimetry with the in-
dicated antibodies (A: -HLA-G, -HA, or
--actin; C: -HLA-G or BB7.2). (B) HeLa
cells expressing US10 were transiently trans-
fected with wild-type HLA-A2 or HLA-A/G
tail and examined for protein expression of
HLA-A2 by immunoblotting with HLA-A2
antibody. Dashed line, control Ab; solid
line: transfectants alone; bolded line:
transfectants+US10. Data are representa-
tive of similar experiments performed at
least two times.
HCMV US10 targets HLA-G | Park et al.
Although the failure to recover
these components of the dislocation
complex is a negative result, we note
that the recovery of US10 in these
experiments is very similar to that
recorded for US11, based on compa-
rable methionine content of US11 and US10. We have thus
far been unable to positively identify interactors of US10 that
might account for its ability to dislocate HLA-G. The failure
to recover the well-documented interactors of US11, as well
as the strikingly different kinetics with which dislocation of
HLA-G occurs relative to classical class I molecules, also
compared with cells that express US2, suggests that US10
employs a mechanism distinct from that used by US2 and
US11. In much the same way that the early aspects of US2-
and US11-dependent dislocation operate through recruit-
ment of different machinery (Lilley and Ploegh, 2004;
Loureiro et al., 2006; Mueller et al., 2006), the US10 mole-
cule may well use a different pathway to execute destruction
of HLA-G molecules.
US10 prevents HLA-G-mediated NK cell inhibition
HLA-G has unique immunomodulatory properties through
its ability to inhibit NK cell–mediated cytotoxicity (Pazmany
et al., 1996; Navarro et al., 1999; Sasaki et al., 1999). We there-
fore examined the ability of US10 to interfere with HLA-G–
mediated inhibition of NK cytotoxicity. In previous studies,
killing of the class I–negative human B cell line 721.221
by peripheral NK cells is inhibited by the expression of
HLA-G (Pazmany et al., 1996; Ponte et al., 1999; Söderström
et al., 1997). Peripheral NK (pNK) cells were coincubated
with 721.221, 721.221/HLA-G, or 721.221/HLA-G that
The mechanism of degradation of HLA-G by US10
is distinct from that used by US2 and US11 for classical
class I MHC products
We next investigated the physical association of US10 and
HLA-G in digitonin lysates of metabolically labeled cells.
In anti-HLA-G immunoprecipitates, both US10 and its trun-
cation mutants were present (Fig. 5 A, lanes 2–4), as verified
by a second round of immunoprecipitation with antibodies
that recognize the tagged versions of US10 (Fig. 5 A, lanes
6–8). The luminal segment of US10 is therefore sufficient for
its interaction with HLA-G, a situation analogous to the in-
teraction of US2, US3, and US11, all of which interact with
their client proteins via their luminal domains.
We have mapped in some detail the protein complexes re-
quired for US11-dependent dislocation of classical class I mol-
ecules. US11 associates in stable fashion with Sel1L, Derlin1,
and Derlin2 (Lilley and Ploegh, 2004; Mueller et al., 2006).
A similar experiment conducted for US10 fails to recover Sel1L,
Derlin1, or Derlin2 (Fig. 5 B). We confirmed these results by
using short hairpin RNA (shRNA) for Sel1L and by transfec-
tion with a dominant-negative construct of Derlin1 or Derlin2
(Lilley and Ploegh, 2004; Mueller et al., 2006). In US11-
expressing HeLa cells cotransfected with either Derlin1GFP
or Sel1L shRNA, we observed stabilization of the class I HC
(Fig. 5 C, bottom). In contrast, HLA-G was degraded in US10-
dependent fashion under the same conditions (Fig. 5 C, top).
Figure 5. US10 selectively targets
HLA-G molecules for degradation by a
mechanism distinct from that used by
HCMV US11. (A) The luminal domain of
US10 is required for the interaction of
HLA-G. Cells transfected with indicated
cDNA were labeled with [35S] methionine
and cysteine for 1 h. The anti-HLA-G
immunoprecipitate was analyzed directly
(lanes 1–4) or reimmunoprecipitated after
dissociation of the initial immunoprecipi-
tate using anti-HA antibodies (lanes 5–8).
(B) US10 does not obviously associate with
the dislocation components recruited by
US11: Sel1L and Derlin1, 2. Immunoprecipi-
tations from digitonin lysates were per-
formed with anti-HA antibodies. The
anti-HA immunoprecipitates were either
analyzed directly or reimmunoprecipitated
using the indicated antibodies. (C) Expres-
sion of a Derlin1/2 dominant-negative
construct or shRNA Sel1L does not inhibit
US10-mediated HLA-G degradation. Data
are representative of two experiments.
JEM VOL. 207, August 30, 2010
The functional consequences of HLA-G down-regulation
include, yet may not be limited to interference with HLA-G-
mediated NK cell inhibition. This is consistent with previous
studies that documented a role of HLA-G in the control of
feto-maternal interactions (Carosella et al., 2000) and the more
recently described involvement of HLA-G in the regulation of
both DC differentiation (Romani et al., 1994) and prolifera-
tion of T cells (Le Gal et al., 1999; Lila et al., 2001). Therefore,
we must consider the possibility that HCMV targets these
aspects of immune recognition no less attentively than it per-
turbs antigen presentation via classical class I MHC molecules.
The ability to exploit US10 to achieve selective elimination of
HLA-G may be useful as a tool to establish in greater detail the
immunological function of HLA-G. Our experiments do not
address the impact of US10 on the generation of soluble HLA-G,
the function of which remains to be established at physio-
Why HCMV should use several different gene products
to target a common set of substrates—class I MHC products—
remains a mystery. Earlier work has shown that inhibition of
TAP via US6 compromises HLA-G expression (Jun et al.,
2000). Variable effects of US2, US3, and US11 on expression
of HLA-G have been reported as well, but these experiments
often rely on overexpression US gene products, mostly using
vaccinia virus vectors, and do not examine their effects in the
context of HCMV-infected cells (Jun et al., 2000; Barel et al.,
2003). Because HCMV can infect many different cell types, it
is possible that each cell type prefers a distinct mode of disloca-
tion of class I MHC products, depending on the dislocation or
ER retention machinery available. The mere presence of the
HCMV gene products is sufficient to mediate accelerated de-
struction, and consequently these small viral membrane pro-
teins must co-opt host machinery to achieve their goals.
HLA-G shows a pattern of expression markedly different from
that of classical class I MHC products. Although HLA-G was
first described as a molecule whose expression is restricted to
extravillous trophoblasts, it is now appreciated that dendritic
cells and other cell types can express HLA-G and may do so to
regulate the activation of regulatory T cells through interaction
with ILT4 (Ristich et al., 2007; Liang et al., 2008). The ability
of CMV to target this nonclassical class I molecule for degrada-
tion suggests that HCMV maintains tight control over the ex-
pression not only of classical class I molecules, but also of those
whose functions may be more specialized. What could be the
selective advantage that accrues to HCMV through expression
of US10, and hence down-modulation of HLA-G? Obviously,
relieving inhibition of NK cells and inviting cytolytic attack
on HCMV-infected, US10-positive cells would seem coun-
terintuitive, unless one assumes that cytolysis may assist in the
release of already assembled intracellular virus particles to facili-
tate further spreading of the infection. In many instances, activa-
tion of NK cells need not lead to cytolysis, but rather results in
altered cytokine production instead (Pazmany et al., 1996; Li
et al., 2009; Ponte et al., 1999). Any action that would alter the
activation status of NK cells, more specifically of those that ex-
press several inhibitory receptors, might create an environment
coexpresses US10 at a 10:1 E/T ratio for 5 h at 37°C. We ob-
served that US10 also degrades HLA-G in 721.221 cells (Fig. 6 A)
and that HLA-G–expressing 721.221 cells are resistant to cyto-
toxic activity of pNK cells compared with the parental 721.221
cell line (Fig. 6 B). US10 blocked the inhibition of NK lytic
activity by HLA-G, as did addition of mAb specific for HLA-G
(Fig. 6 B). We conclude that US10 interferes with the ability of
HLA-G to regulate NK cell activity.
A deglycosylated intermediate of HLA-G is present in US10-
expressing cells exposed to proteasome inhibitors, but is
evident only with considerable delay after synthesis. The
properties of the tails of HLA-G and US10 are essential for
dislocation to occur, yet neither the HLA-G tail nor the US10
tail resemble those of classical class I MHC molecules or those
of US2 and US11, respectively: the tri-leucine cluster in the
US10 cytoplasmic tail is without an obvious counterpart in
US2 or US11. In addition, the physical association of US10
with HLA-G through the luminal domain of US10 may have
contributed to a small extent to the retention of HLA-G com-
plexes in the ER (Fig. 2 B).
Figure 6. US10 interferes with HLA-G-mediated Natural Killer cell
cytotoxic activity. (A) Expression level of HLA-G by US10 in 721.221 cells
was analyzed by immunoblotting with MEM-G/1 antibodies. (B) Target
cells were stained with DiOC18 (3) for 30 min and then pNK cells were
coincubated with different target cells at a 10:1 ratio (Effect: Target) for
5 h. After incubation time, cells were stained with propidium iodide for
dead cells for 10 min and analyzed by cytofluorimetry. Data are represen-
tative of three experiments.
HCMV US10 targets HLA-G | Park et al.
with 5% skim milk in PBS with 0.1% Tween-20 for 2 h, and probed with
the appropriate antibodies for 4 h. Membranes were washed three times
with PBS with 0.1% Tween-20 and incubated with horseradish peroxidase–
conjugated streptavidin for 1 h. The immunoblots were visualized with ECL
Cytotoxicity assay. Target cells were stained with DiOC18 (3) (Invitrogen)
for 30 min, and then washed with PBS 3 times. Effect cells were co-incubated
with different target cells for 5 h. Effect cells alone and target cells alone were
prepared for data analysis. After the incubation period, propidium idodide was
added immediately into the co-cultured cells and analyzed by Flow cytometry.
Online supplemental material. Fig. S1 shows that expression of HLA-G
is slightly reduced by US10. Online supplemental material is available at
We are grateful to Ann E. Campbell for providing us with HCMV mutant virus.
We thank Marisa Isaacson for amplification of a HCMV mutant virus and Melanie
M. Brinkmann and Ludovico Buti for critical reading of the manuscript.
The authors declare no competing financial interests.
Submitted: 17 August 2009
Accepted: 19 July 2010
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more favorable for HCMV, and herein might provide a ratio-
nale for why HCMV has retained the US10 gene as a pos-
sible immunoevasin. The ability of HCMV to be passed from
mother to child in the course of pregnancy may well be related
to the unique mechanisms by which HCMV abrogates both
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MATERIALS AND METHODS
Cells and virus. The JEG3 choriocarcinoma cell line, HeLa cells, and
human foreskin fibroblast cells were cultured in complete medium: DME
supplemented with 10% fetal bovine serum and 50 U/ml penicillin (Invitro-
gen). YTS and 721.221 cells were cultured in RPMI 1640 supplemented
with 10% fetal bovine serum. Cells were grown at 37°C in humidified air
with 5% CO2. HCMV strain AD169 was obtained from the American Type
Culture Collection. HCMV mutant virus AD169US2-US11 (deletion of
US2 to US11) and RV670 (deletion of IRS to US11, but contains only
US10) were provided by A.E. Campbell (Eastern Virginia Medical School,
Norfolk, Virginia; Jones et al., 1995).
Isolation of pNK cells. pNK cells were isolated from peripheral blood as
previously described (Koopman et al., 2003). For cytotoxicity assay, pNK
were further purified by the subsequent use of anti-CD56–coated mag-
DNA constructs and antibodies. HLA-G, HLA-E, and HLA-A2.1
cDNAs and all chimeric mutants were subcloned in the pcDNA3.1 vector
(Invitrogen, San Diego, CA). HA-US10 and the chimeras of US10 were
constructed as previously described (Lilley and Ploegh, 2004). For site-
directed mutagenesis and the truncation mutants, respective DNA fragments
were amplified by PCR with oligonucleotides that introduced the desired,
unique restriction site. Derlin1GFP, Derlin2GFP, and shRNA for Sel1L con-
structs were generated as described (Lilley and Ploegh, 2004; Mueller et al.,
2006). The monoclonal antibody W6/32 recognizes only MHC class I
heavy chains associated with 2m. The MEM-G/9, MEM-G/1, and BB7.2
recognize 2m-associated HLA-G (Abcam), denatured HLA-G (AbD Sero-
tech), and HLA-A2.1 (Abcam). Antibody against HLA-E was purchased
Flow cytometry. Surface expression of MHC class I molecules and HLA-G
were determined by flow cytometry (FACSCalibur; BD) as previously de-
scribed (Park et al., 2003).
Pulse-chase analysis and immunoprecipitation. Cells (107) were
starved for 50 min in medium lacking methionine and cysteine; labeled with
0.1 mCi/ml [35S]methionine and cysteine (TranS-label; NEN Life Science);
and chased in complete medium with or without added inhibitor for the in-
dicated times. Cells were lysed with 1% NP-40 (Sigma-Aldrich) in PBS for
30 min at 4°C. After preclearing lysates with protein G–Sepharose (Sigma-
Aldrich), primary antibodies, and protein G–Sepharose were added to super-
natants and incubated at 4°C. The protein G–Sepharose beads were washed
four times with 0.1% NP-40 in PBS. Proteins were eluted from the beads
by boiling in SDS sample buffer and separated by SDS-PAGE. For endo H
treatment, immunoprecipitates were digested with 3 mU endo H (NEB) at
37°C overnight in 50 mM sodium acetate, pH 5.6, 0.3% SDS.
Coimmunoprecipitation and Western blot analysis. Cells were lysed in
1% digitonin (Calbiochem) in buffer containing 25 mM Hepes, 100 mM
NaCl, 10 mM CaCl2, and 5 mM MgCl2, pH 7.6, supplemented with 0.5 mM
PMSF, leupeptin, and 10 mM NEM. Lysates were precleared with protein
G–Sepharose for 1 h at 4°C. For immunoprecipitation, samples were incu-
bated with the appropriate antibodies for 12 h at 4°C before protein G beads
were added. Beads were washed four times with 0.1% digitonin, and bound
proteins were eluted by boiling in SDS sample buffer. Proteins were separated
by 12% SDS-PAGE, transferred into a nitrocellulose membrane, blocked
JEM VOL. 207, August 30, 2010 Download full-text
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