By
From the * Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139; and Department of Molecular Biology, Infectious Diseases
Section, Wyeth-Ayerst Research, Pearl River, New York 10965
Human cytomegalovirus downregulates the expression of human class I major histocompatibility complex (MHC) molecules by accelerating destruction of newly synthesized class I heavy chains. The HCMV genome contains at least two genes, US11 and US2, each of which encode a product sufficient for causing the dislocation of newly synthesized class I heavy chains from the lumen of the endoplasmic reticulum to the cytosol. Based on a comparison of their abilities to degrade the murine class I molecules H-2Kb, Kd, Db, Dd, and Ld, the US11 and US2 gene products have non-identical specificities for class I molecules. Specifically, in human astrocytoma cells (U373-MG) transfected with the US11 gene, the Kb, Db, Dd, and Ld molecules expressed via recombinant vaccinia virus are rapidly degraded, whereas in US2-transfected cells, only Db and Dd are significantly destabilized. The diversity in HCMV-encoded functions that interfere with class I-restricted presentation likely evolved in response to the polymorphism of the MHC.
Class I MHC molecules present peptides derived from
proteins degraded in the cytosol to cytolytic T cells,
and as such are targets for viruses seeking to evade immune
recognition (1). The ability to downregulate the expression
of class I molecules in host cells has been documented for a
number of viruses, including adenovirus (2), herpes simplex
virus (3), and both mouse (4) and human cytomegalovirus
(5). In HCMV-infected cells, newly synthesized class I heavy
chains are rapidly degraded following their deposition in
the lumen of the ER (6). The HCMV gene product
US11 ("US11"), an ER-resident type I transmembrane glycoprotein, has been shown by transfection to be sufficient
to cause the selective degradation of endogenous class I
molecules (9). In the presence of US11, class I heavy chains
are dislocated from the lumen of the ER into the cytosol,
where they are deglycosylated by host N-glycanase, and
then degraded by the proteasome (10). A second HCMV
gene product, US2, has also been observed to cause the
premature destruction of newly synthesized class I molecules, apparently by the same pathway as described above
for US11 (9, 11). Since both US11 and US2 are expressed
at the same stage of infection, we sought to determine why
the virus might encode two separate proteins that appear to
function in the same manner.
One possibility is that US11 and US2 recognize overlapping but nonidentical subsets of class I molecules. In the
initial comparison of the US11 and US2 transfectants, only
the degradation of endogenous human class I molecules was
studied, and no differences in allelic selectivity between
US11 and US2 were apparent (9). In human fibroblasts
infected with HCMV, all endogenous class I molecules appeared to be destabilized (6). In mouse L cells (H-2k)
transfected with HLA-B27, the human, but not murine
class I molecules were degraded upon HCMV infection,
but the reason for this difference was not established (6). In
virus-infected cells, both US11 and US2 are presumably
expressed, and these experiments therefore cannot resolve
possible differences in substrate specificity between the two
viral proteins. To probe the specificities of US11 and US2,
we infected cell lines transfected with either US11 or US2 with a panel of recombinant vaccinia virus expressing different mouse class I heavy chains (H-2Kb, Kd, Db, Dd, and
Ld) to determine whether certain heavy chains would be
resistant to US11- or US2-mediated degradation. We observe that US11 and US2 differ in their abilities to degrade
murine class I heavy chains, and thus are not functionally
redundant. Specifically, we find that US11 degrades Kb,
Db, Dd, and Ld efficiently, whereas US2 is most effective in
degrading Db and Dd. We suggest that MHC polymorphism drives diversification of viral evasion strategies.
Cells and Vaccinia Virus Infections.
U373-MG astrocytoma cells
and the US11 and US2 transfectants prepared from this cell line
have been described (9). Cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% (vol/vol) FCS,
penicillin (1:1,000 dilution U/ml), streptomycin (100 µg/ml),
and puromycin (Sigma Chem. Co., St. Louis, MO) at a final concentration of 0.375 µg/ml. Recombinant vaccinia virus expressing H-2Kb (lacking the cytoplasmic tail), Kd, Db, Dd, and Ld were
obtained from Dr. J. Yewdell. Between 1 and 5 × 106 cells per
sample were detached by treatment with trypsin, resuspended in
PBS supplemented with 1% FCS, penicillin, and streptomycin, and then infected for 45 min with recombinant vaccinia virus at a
multiplicity of infection of 10, after which 10 ml of media was
added. 5 h later, cells were starved in methionine/cysteine-free medium for 45 min with or without the proteasome inhibitor
Cbz-LLL (10 µm final) (10) before labeling with 250 µCi/ml
[35S]methionine/cysteine (80:20). Labeling was terminated by addition of 1 mM cold methionine/cysteine to the labeling mix.
Aliquots of cells were spun down at each chase point, and the cell
pellets frozen before immunoprecipitation of class heavy chains.
Antibodies and Immunoprecipitations.
For immunoprecipitation
of mouse class I heavy chains, a rabbit polyclonal antiserum (RafHC)
which recognizes non-assembled or unfolded heavy chains was
used (12). Cell pellets were each lysed in 1 ml ice-cold lysis mix
(0.5% NP-40, 50 mM Tris/HCl, pH 7.4, 5 mM MgCl, 1 mM
PMSF, and 10 mM iodoacetamide), and the postnuclear supernatant precleared twice with 10% fixed Staphylococcus aureus before
specific immunoprecipitation of murine class I heavy chains with
RafHC. To enhance the visualization of degradation intermediates, the RafHC immunoprecipitates were boiled in denaturation buffer (2% SDS, 50 mM Tris/HCl, pH 7.8, 1 mM EDTA, 5 mM
DTT), and the murine class I material re-immunoprecipitated
with RafHC before gel analysis. The N-glycanase digestions in
Fig. 2 were performed according to the manufacturer instructions
(Boehringer Mannheim, Germany). SDS-PAGE and one dimensional isoelectric focusing were performed as described (13).
To probe the specificity of the
HCMV gene products US11 and US2 for class I MHC
molecules, we infected parental U373-MG cells, and transfectants expressing either US11 or US2, with a panel of recombinant vaccinia virus encoding different mouse class I heavy chains, and then performed a pulse chase experiment
(10-min labeling, 0-min and 20-min chase points) in the
presence of the proteasome inhibitor Cbz-LLL (Fig. 1). At
each chase point, aliquots of cells were pelleted and frozen
at
US11 and US2 were equally capable of degrading the
murine class I MHC heavy chains H-2Db (Fig. 1 C), and
Dd (Fig. 1 D). However, when challenged with the H-2Ld
molecule (Fig. 1 E), US11 was more efficient than US2 at
causing the breakdown of the heavy chains, as evidenced
by the nearly complete conversion in US11+ cells of fulllength Ld heavy chains into the breakdown intermediate
over the 20-min chase. In the case of H-2Kd (Fig. 1 B), the
heavy chains decay more rapidly in the presence of either
US11 or US2 than in control cells, but little degradation intermediate accumulates in either transfectant, despite the
presence of the inhibitor Cbz-LLL. This is most likely due
to the comparatively lower rate of degradation for Kd, coupled with the earlier observation that Cbz-LLL retards, but does not block, the subsequent degradation of the heavy
chain intermediate (10). Thus, while US2 can apparently
destabilize the Kd and Ld heavy chains to some extent, it
does not cause their destruction at a rate sufficient for the
intermediate to accumulate.
In the case of human class I MHC molecules, the US11-induced degradation intermediate that accumulates in the presence of Cbz-LLL is a deglycosylated
heavy chain (10). To demonstrate that the Kb degradation
intermediate observed in Fig. 1 is a deglycosylated heavy
chain, we performed a pulse chase experiment (10-min label, 0-min and 20-min chase points) on Kb vaccinia virus
infected US11 cells, and immunoprecipitated with RafHC
the Kb heavy chains from lysates of cells removed at each
chase point. Each immunoprecipitate was split in two, and
either kept on ice ( HCMV downregulates the expression of class I MHC
molecules by dislocating newly synthesized class I heavy
chains from the lumen of the ER back into the cytosol,
where they are rapidly degraded (10). The virus encodes
two proteins, US11 and US2, that are each sufficient for
causing the premature destruction of class I heavy chains
(9, 11). We show here that US11 and US2 have nonidentical specificities for a panel of murine class I heavy chains.
Over the 20-min chase period examined, US11 degraded H-2Kb, Db, Dd, and Ld with similar kinetics, while US2
only degraded Db and Dd efficiently. One possible explanation for the observed differences in class I heavy chain degradation for US11 and US2 positive cells is that each may
interact with different regions of the class I heavy chain in
the process of dislocating the latter into the cytosol. Due to
the sequence variability between different class I alleles, this
dual recognition strategy would allow the virus to downregulate the expression of a broader range of class I molecules than might be possible with either US11 or US2
alone.
Fig. 2.
The Kb degradation intermediate is a deglycosylated heavy
chain. US11+ cells were infected with vaccinia virus expressing H-2Kb
(lacking cytoplasmic tail), labeled for 10 min with [35S]methionine, and
chased as indicated. Immunoprecipitated Kb heavy chains from each chase
point were either kept on ice () or treated with recombinant N-glycanase (+) before resolution by SDS-PAGE (A) or 1D-IEF (B).
[View Larger Versions of these Images (47 + 42K GIF file)]
The Murine Class I MHC Molecule H-2Kb Is Degraded by
US11 but not by US2.
80°C before lysis and immunoprecipitation with a rabbit
anti-free heavy chain antiserum (RafHC). Immunoprecipitates were denatured by boiling in 2% SDS, and re-immunoprecipitated before analysis by SDS-PAGE. In the case
of H-2Kb (lacking cytoplasmic tail) vaccinia virus-infected
cells (Fig. 1 A), class I heavy chains synthesized in the 10min pulse are stable throughout the 20-min chase in control cells and in US2+ cells. However, in US11+ cells, the
fully intact Kb heavy chains observed immediately upon
completion of labeling (Fig. 1 A, 0-min timepoint) are rapidly converted into a faster migrating species. If the pulse
chase is performed in the absence of Cbz-LLL, then the Kb
heavy chains in US11+ cells are fully degraded by 20 min,
and no intermediates accumulate (data not shown).
Fig. 1.
Fate of murine class I MHC heavy chains in US11+ or US2+
cells. Control (nontransfected), US11+, or US2+ cells were infected with
vaccinia virus expressing H-2Kb (lacking the cytoplasmic tail; A), Kd (B),
Db (C), Dd (D), or Ld (E), labeled with [35S]methionine for 10 min and
chased as indicated. Immunoprecipitated murine class I molecules were
resolved on a 12.5% SDS-polyacrylamide gel and visualized by fluorography. The position of the breakdown intermediate is indicated with an asterisk.
[View Larger Versions of these Images (48 + 47 + 42 + 50 + 47K GIF file)]
) or treated with recombinant N-glycanase (+) before re-immunoprecipitation with RafHC.
The samples were then split again and analyzed by SDSPAGE (Fig. 2 A) or 1D-IEF (Fig. 2 B). As seen in Fig. 2 A, all of the Kb molecules at the beginning of the chase are
fully susceptible to N-glycanase treatment (Fig. 2 A, 0-min
timepoint), and no intermediate has yet accumulated. After
20 min, most of the heavy chains have been converted into
the US11-induced fragment, which migrates at the same
position as N-glycanase treated material (Fig. 2 A, 20-min
timepoint). In hydrolyzing the N-glycosidic bond of the Kb
heavy chain's glycan, N-glycanase converts the Asn sidechain to an Asp, and thus imparts an additional negative
charge upon the molecule. Isoelectric focusing of the samples in Fig. 2 Areveals that the fully intact Kb heavy chains
present at the beginning of the chase have the same pI after
N-glycanase treatment as the degradation intermediate observed after 20 min of chase (Fig. 2 B). We conclude that
the mechanism of US11-mediated class I heavy chain degradation is similar for mouse and human molecules, and
that in both cases, a deglycosylated heavy chain intermediate can be visualized when the proteasome inhibitor CbzLLL is present during the chase.
Address correspondence to Hidde Ploegh, Center for Cancer Research, MIT, 40 Ames St. E17-322, Cambridge, MA 02139.
Received for publication 20 September 1996
This work was supported by National Institutes of Health grants R01-AI33456 and R01-AI07463-17, and by Boehringer-Ingelheim.We thank Dr. J. Yewdell for the murine class I-vaccinia virus recombinants.
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