Mouse cytomegalovirus (MCMV), a β-herpesvirus that establishes latent and persistent infections in mice, is a valuable model for studying complex virus-host interactions. MCMV encodes the m145 family of putative immunoevasins with predicted MHC-I structure. Functions attributed to some family members include downregulation of host MHC-I (m152) and NKG2D ligands (m145, m152, m155) and interaction with inhibitory or activating NK receptors (m157). We present the cellular, biochemical and structural characterization of m153, which is a heavily glycosylated homodimer, that does not require β2m or peptide, and is expressed at the surface of MCMV-infected cells. Its 2.4 Å crystal structure confirms that this compact molecule preserves an MHC-I-like fold and reveals a novel mode of dimerization, confirmed by site-directed mutagenesis, and a distinctive disulfide-stabilized extended amino terminus. The structure provides a useful framework for comparative analysis of the divergent members of the m145 family

Balbo, Andrea

Eikel, Daniel

Hess, Sonja

Jonjić, Stipan

Mans, Janet

Margulies, David H.

Natarajan, Kannan

Robinson, Howard

Schuck, Peter

Tiemessen, Caroline T.

Šimić, Hrvoje

English

Caltech Authors - Main

 1 
Supplementary Experimental Procedures 
Immunoprecipitation and western blots 
NIH3T3 cells (2 x 106) were co-transfected with FLAG-m153-pcDNA (1µg) and HA-m153-
pcDNA(1µg) (Amaxa Nucleofector, solution R, program U30) and seeded in 10 cm plates. For infection 
experiments 70-80% confluent NIH3T3 cells seeded in 10 cm plates were infected with MCMV-GFP at 
an m.o.i. of two. After overnight incubation for transfections and 24h incubation for infections, the cells 
were washed twice in ice-cold PBS and lysed in the plates with 1ml of 1% NP40, 50 mM Tris-HCl pH8, 
300 mM NaCl, 5 mM EDTA, 1x protease inhibitor cocktail (Pierce), 10mM iodoacetamide, 0.02 % 
sodium azide. The cells were removed from the plate with a cell scraper and incubated for 15 min. on ice. 
All subsequent steps were performed on ice or at 4 °C. The lysate was centrifuged for 15 min. at 13,000 
rpm, and supernatants were precleared by incubating with 50 µl of anti-mouse IgG beads (eBioscience) 
for 30 min. For transfected cells the precleared cell lysates were divided into three aliquots, and either 5 
µg of FLAG M2 antibody (Sigma), anti-HA  (Sigma) or mAb153.16 were incubated with the lysates for 1 
h. For infection experiments 5 µg of mAb153.16 were incubated with infected and uninfected cell lysates 
for 1 h. Then 50 µl of anti-mouse IgG beads were added to the lysates and incubated for 1h with rotation. 
The beads were washed 5x with lysis buffer containing 0.1 % NP40. Reducing SDS-PAGE sample buffer 
(Pierce) was added to the beads and the samples heated to 100°C for ten minutes prior to electrophoresis 
on an 8-20 % gradient Tris-Glycine SDS-PAGE gel (Invitrogen). The proteins were transferred to 
polyvinylidene fluoride (PVDF) membranes using standard protocols. The membrane was blocked 
overnight with 5 % non-fat dried milk in 0.1 % Tween 20/PBS. The proteins were detected by incubation 
with either anti-FLAG M2, anti-HA (Sigma) or anti-m153 rabbit serum for 1h at room temperature. After 
washing in 0.1 % Tween 20/PBS the membranes were incubated with secondary antibody for 1h at room 
temperature (anti-mouse or anti-rabbit Trublot-eBioscience), and washed again. The blots were developed 
with the Supersignal West Dura Extended Duration substrate (Pierce) and exposed to Biomax XAR film 
(Kodak).  
 To determine whether m153 is glycosylated, immunoprecipitated samples on beads were incubated 
with 1000 U PNGaseF (New England Biolabs) in PBS at 37 °C overnight before processing for SDS-
PAGE and western blot analysis as described above.  
 
Mass spectrometry 
         For mass spectrometry infusion experiments, samples were diluted with ultrapure water (LC-MS-
Chromasolv grade, Sigma-Aldrich), heated to 65 °C for 5 min and desalted twice in ultrapure water using 
Centri•Sep™ desalting spin columns according to the manufacturers procedures (Princeton Separation). 
After further enzymatic or chemical treatment with 0.1 µg trypsin (Promega), 0.5 mU PNGaseF (QA-
Bio), 2 µg tris-(carboxyethyl)-phosphine hydrochloride (TCEP, Sigma-Aldrich) or 2 µg dithioerythritol 
(DTT, Sigma-Aldrich) and incubation at 37 °C for 1 – 48 hours, the respective samples were diluted 1:1 
in spraying buffer (40 % Isopropanol, 40 % Acetonitrile, 20 % Ultrapure Water and 0.2 % formic acid, all 
Sigma-Aldrich). Infusion nano-electrospray was performed using the TriVersa™-NanoMate chip 
technology (Advion Biosystems) coupled to an LTQ-FT™ ICR mass spectrometer (Thermo Electron).  
         5 µL solution pick-up (ca. 2-15 pmol/µL) was sufficient for ca. 45 min analysis time (1.5 kV, 0.3 
bar pressure assistance). MS and MSn fragmentation data were recorded applying collision-induced 
dissociation (CID, 35 % energy), electron capture dissociation (ECD, 5 x e5 energy, 10 ms delay, 70 ms 
duration) or infrared multi photon dissociation (IRMPD, 90 % energy, 10 ms delay, 90 ms duration) of 
indicative precursor ions. MS spectra recorded were the average of three or more FT-MS scans (max. ion 
injection time 3000 ms, resolution 100,000 to 500,000, ion accumulation values according to the 
manufacturer’s standard values). MSn spectra were recorded as the average of three FT-MS scans (5 Da 
window for precursor ion selection).  
 
 2 
Supplementary Figures 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure S1: m153 is a non-covalent dimer when expressed in S2 insect cells.  A. Size exclusion 
chromatography analysis of insect cell expressed m153 (extracellular domain – 1-314). The 
calculated molecular weight based on the column retention time is 85 kDa, whereas the 
molecular weight predicted from the amino acid sequence is 37.4 kDa for a monomer. B. SDS-
PAGE of recombinant m153 expressed in S2 insect cells under reducing (R) and non-reducing 
(NR) conditions indicates that m153 is a non-covalent dimer. The molecular weight of the 
secreted, engineered m153 expressed in insect cells, analyzed in SDS-PAGE is accounted for by 
37.4 kDa from the polypeptide and about 6 kD from the core carbohydrates. In contrast, the full 
length virus-encoded or transfected m153 chains migrate at about 80 kDa in SDS-PAGE and 45 
kDa following deglycosylation (Fig. 1B), consistent with a length of 405 amino acids and more 
complex glycosylation. 
 
 
 
 
 
 
A 
97 
66 
45 
 
30 
20 
 
14 
kDa          R         NR 
B 
Dimer 
 3 
 
 
 
 
 
 
 
 
 
 
 
 
Figure S2: FACS quantification of YFP fluorescence complementation. NIH3T3 cells were 
transfected with m153-N-YFP+m153-C-YFP or m144-N-YFP+m144-C-YFP and following 
confocal microscopy the YFP-fluorescent cells were counted by FACS. YFP fluorescence was 
visible in 18% of m153 transfected cells but only in 0.8% of m144 transfected cells, indicating 
that m153 forms dimers in NIH3T3 cells.  
 
 
 
 
 
 
 
 
 
10
0
10
1
10
2
10
3
10
4
EYFP
10
0
10
1
10
2
10
3
10
4
F
L
2
-H
18.3
10
0
10
1
10
2
10
3
10
4
EYFP
10
0
10
1
10
2
10
3
10
4
F
L
2
-H
0.79
m144-N-YFP + m144-C-YFP m153-N-YFP + m153-C-YFP 
0.79% 18.3% 
 4 
 
 
 
 
 
 
 
 
 
 
 
 
Figure S3: Mass spectrum of a disulfide-linked tryptic peptide confirms the C16-C171 disulfide 
bond. FT-MS/MS spectrum after electron capture dissociation (ECD) fragmentation of the 
quadruply charged m/z=708.097. ECD induced a fragmentation of the disulfide bond yielding 
the singly charged fragment m/z=779.375 and the doubly charged fragment m/z=1027.007, 
corresponding to the tryptic peptides TGTVDICQGPMELIFSVSR and WTEICK, respectively. 
ECD also resulted in further peptide fragmentation revealing the sequence tag FSVS.  
 
 
 
 
 
 
 
 
TGTVDIC*QGPMELIFSVSR 
 
    WTEIC*K   
 5 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure S4:  Mass spectra of C*PVPQFNC*TITHR peptide before and after reduction with TCEP 
confirmed the C101-C108 disulfide bond. Mass shift of 1.005 at the doubly charged peptide 
m/z=757.851 indicated addition of two protons after reduction of internal Cys-Cys bond in 
tryptic peptide CPVPQFNCTITHR. 
 
 
FT-MS experiments for determination of Cys-Cys bonds present in MCMV-m153 
Desalted and PNGaseF treated MCMV-m153 was tryptic digested and nano 
electrosprayed. FT-MS/MS spectra were recorded after ECD fragmentation of indicative 
peptides to selectively reduce Cys-Cys bonds between two tryptic peptides. Alternatively TCEP 
was added to the solution to obtain mass shift experiments of FT-MS survey scans for 
identification of e.g. internal Cys-Cys bonds in one and the same tryptic peptide. Figure S3 
shows exemplary the selective Cys-Cys reduction using ECD fragmentation of the quadruply 
charged m/z=708.097 (tryptic peptides TGTVDIC*QGPMELIFSVSR and WTEIC*K), whereas 
Figure S4 shows the mass shift of 1.005 at the doubly charged m/z= 757.851 caused by TCEP 
induced reduction of the internal Cys-Cys bond of tryptic peptide C*PVPQFNC*TITHR. All 
cysteines present in MCMV-m153 are engaged in bonds with C16 bound to C171, C101 to C108 
and C203 bound to C255. 
 
- TCEP + TCEP 
C*PVPQFNC*TITHR 
m/z 
 6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure S5: Mutations at the interface destabilize the m153 dimer. Size exclusion chromatography 
analysis of wild type (black), quadruple interface mutant (T128A, S131A, R225A, S241A - red) 
and control mutant (D194A - dashed blue) m153 purified from SF9 insect cells. The quadruple 
interface mutant migrates at a slower rate through the column, indicating a smaller size 
compared to wild type m153.  
 
 
 
 
 
158 66 44 kD 
 7 
 
 
Table S1: Hinge angle between the α1α2 and α3 domains of viral and MHC-Ia and MHC-
Ib molecules.  
Protein PDB code  Angle (degrees) Associated subunits 
m153  2O5N 76 m153 
m157 2NYK 81 None 
m144 1U58 99 β2m 
H-2Kb 2VAA 71 β2m, peptide 
CD1d 1ZHN 75 β2m, lipid 
T22 1C16 72 β2m 
ZAG 1TW7 90 β2m 
MICA 1B3J 116 β2m 
Hinge angles were calculated with the HINGE program as described in Experimental 
Procedures. 
 
 


Cellular expression and crystal structure of the murine cytomegalovirus MHC-Iv glycoprotein, m153

J Exp Med

Major Histocompatibility Complex (MHC) Molecules: Structure, Function, and Genetics. In:

Rheumatology (Oxford)

The PyMOL User's Manual, DeLano Scientific,

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Cellular expression and crystal structure of the murine cytomegalovirus MHC-Iv glycoprotein, m153

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