Characterization of the Interaction between the ... - Journal of Virology

Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2 Frauke Beilstein, Linda Obiang, Hélène Raux, Yves Gaudin Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, France

ABSTRACT

The matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. IMPORTANCE

The immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.

V

esicular stomatitis virus (VSV) is the prototype rhabdovirus and for years has been used as a model to study many aspects of the virus life cycle. Its negative-strand RNA genome of 11,161 nucleotides successively encodes the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and the RNAdependent RNA polymerase (L). The N, P, and L proteins are associated with the RNA molecule and compose the transcriptionally active nucleocapsid (NC). The NC is enveloped by a lipid bilayer which is derived from the host cell plasma membrane and which is acquired during the budding process. G is a transmembrane glycoprotein that is involved in virus entry. Most of the M protein is located beneath the viral membrane and bridges the NC and the lipid bilayer (1). M is a multifunctional protein involved in virus assembly and budding. In relationship with this structural role, it has been demonstrated that VSV M interacts with both artificial and cellular membranes (2–5) and that it binds to the viral nucleocapsid (6, 7). It also self-associates into large multimers at physiological salt concentrations (8–10). The flexible amino-terminal part of M has the ability to recruit cellular partners that assist with viral assembly and budding. The first 10 amino acids of M bind dynamin, and this interaction is required for efficient viral assembly (11). The amino-terminal

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part of M also contains two late domains, 24PPPY27 and 37PSAP40, which have the ability to recruit cellular partners that are involved in the ultimate step of the budding process. The 24PPPY27 domain has been shown to interact with the WW domains of Nedd4related E3 ubiquitin ligases (12–14), a feature which is essential for efficient viral budding. The 37PSAP40 domain recruits TSG101 (15), a component of the endosomal sorting complex required for transport (ESCRT) complexes that plays a key role in the biogenesis of multivesicular bodies (MVBs) (16). It has also been shown that M protein targets several cellular proteins to inhibit host gene expression at multiple levels, includ-

Received 9 July 2015 Accepted 17 August 2015 Accepted manuscript posted online 26 August 2015 Citation Beilstein F, Obiang L, Raux H, Gaudin Y. 2015. Characterization of the interaction between the matrix protein of vesicular stomatitis virus and the immunoproteasome subunit LMP2. J Virol 89:11019 –11029. doi:10.1128/JVI.01753-15. Editor: D. S. Lyles Address correspondence to Frauke Beilstein, [email protected], or Yves Gaudin, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ing transcription and nuclear cytoplasmic transport. M interacts with host proteins Nup98 (17) and Rae1 (18), which have been implicated in the regulation of mRNA nuclear-cytoplasmic transport (18) and in cellular transcription (19). Proteasomes are the major nonlysosomal machines involved in protein degradation (20, 21). They are classified into three subtypes on the basis of the nature of their catalytic subunits. The structure of all three subtypes is basically the same, consisting of the 20S core particle, which is composed of 28 subunits arranged in four heptameric rings. The upper and the lower rings are formed by ␣ subunits, while the two central rings contain the ␤ subunits (22). The standard proteasome is constitutively expressed in nearly all mammalian cells and contains the catalytic subunits ␤1 (PSMB6), ␤2 (PSMB7), and ␤5 (PSMB5). The immunoproteasome is generally expressed in very small amounts in all cells, with the exception of cells from the immune system. However, it can be significantly upregulated through factors like gamma interferon or oxidative stress (23, 24). In the immunoproteasome, the subunits ␤1, ␤2, and ␤5 are replaced by the catalytic subunits ␤1i (LMP2, PSMB9), ␤2i (MECL-1, PSMB10), and ␤5i (LMP7, PSMB8), respectively (25–27). The catalytic subunits are synthesized as inactive precursors bearing an N-terminal propeptide. Upon completion of proteasome assembly, the prosequences, which prevent premature activation, are autocatalytically cleaved. This cleavage is essential for proteasome maturation and the activation of the catalytic residues (28, 29). In the immunoproteasome, LMP2 has a chymotrypsin-like activity distinct from the caspase-like activity of PSMB6 and cleaves after hydrophobic amino acids. This shift promotes the formation of major histocompatibility complex (MHC) class I-compatible peptides that contain hydrophobic C-terminal anchors. Here, we report that VSV M protein interacts directly with LMP2, and we identified at the surfaces of both proteins residues that are critical for the interaction. Our data suggest that M is able to impede the assembly of the immunoproteasome. MATERIALS AND METHODS Cell culture, transfections, gamma interferon treatment, and virus. BHK-21, N2A, HeLa, and PANC-1 cells were grown at 37°C in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum. Cells of the mouse fibroblast cell line B8 (the parental cell line of the B27M.2 cell line) and B27M.2 cells, endogenously expressing LMP2, LMP7, and MECL-1, were kindly provided by M. Groettrup and grown as described previously (26). Cells grown to 70% confluence on 35-mm plates were transfected with 0.5 ␮g of plasmid DNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were incubated for 24 h (16 h for transfections using VSV M) in antibiotic-free medium before harvesting or infection. If indicated, HeLa cells were incubated for 24 h with 10 ng/ml recombinant human gamma interferon (BD Pharmingen). A strain of the VSV Indiana serotype (the Orsay strain) was grown and titrated on BHK-21 cells. Growth curves (multiplicities of infection [MOIs], 3 and 0.3) were performed on B8 and B27M.2 cells. Virus titers were calculated by plaque assays on BHK-21 cells. Antibodies. Antibodies against myc (9E10 antibody) and ␣-tubulin were obtained from Sigma, antibody against hemagglutinin (HA; sc-805 antibody) was from Santa Cruz, and horseradish peroxidase-conjugated IgG was from GE Healthcare. Antibodies against the VSV matrix proteins M214 and MA3 and glycoprotein G307 were described before (11). The following antibodies (all from Santa Cruz) against proteasome subunits

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were used: the sc-373689 and sc-28809 antibodies for LMP2, the sc374405 antibody for PSMB6, and the sc-1666205 antibody for PSMA3. Plasmid constructs. To obtain the plasmid constructs used in the yeast two-hybrid (Y2H) assay, cDNAs encoding premature or mature LMP2 (obtained from the Y2H screen) and PSMB6 (synthetic gene obtained from Biovalley) were constructed in pGAD-GH (Clontech), a derivative of pGAD-GE. cDNAs coding for VSV M (serotype Indiana, strain Orsay) as well as all VSV M mutants were constructed in LexA (Clontech) (11). Plasmids carrying Chandipura virus (CHAV) M and spring viremia of carp virus (SVCV) M were provided by J. Petersen, L. Her, and J. E. Dahlberg (University of Wisconsin). Mutants were generated in pLEX for M and in pGAD for LMP2. Constructs of LMP2, PSMB6, M, MI96A, and dystonin in pCDNA3.1-myc or pCDNA3.1-HA (provided by D. Pasdeloup [30, 31]) were generated by PCR. Yeast two-hybrid assay. VSV M and mutants of M were constructed in pLEX, fused to the DNA-binding domain LexBD, and used as bait to test for the interaction with LMP2 and PSMB6. cDNAs encoding these were cloned in pGAD, fused to the GAL4 activation domain AD, and used as prey. The Saccharomyces cerevisiae yeast strain L40 containing the reporter gene lacZ was transformed with combinations of the pLEX and pGAD constructs using a lithium acetate protocol. Double transformants were grown on plates containing medium lacking Trp and Leu (Trp⫺ Leu⫺) to select for the presence of both the bait and the prey. Positive clones were assayed for ␤-galactosidase activity on the basis of their ability to activate the transcription of the lacZ reporter gene. Liquid ␤-galactosidase assays were done using o-nitrophenyl-␤-D-galactopyranoside (ONPG) as the substrate as previously described (32). ␤-Galactosidase activity was expressed in arbitrary units and calculated using the following formula: (A420 · 1,000)/(A600 · T · V), where A420 is the absorbance of the reaction mixture at 420 nm, A600 is the absorbance at 600 nm as a measure of the cell density of the culture, T is the reaction time (in minutes) used for the assay, and V is the volume (in milliliters) used for the assay. Immunoprecipitation. N2A cells were transfected and subinfected with VSV at an MOI of 3 for 4 h at 37°C. To prepare extracts for immunoprecipitation, cells were lysed with cytoplasmic protein extraction buffer {CPEB; 50 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, 0.5% Igepal [octylphenoxy poly(ethyleneoxy)ethanol; catalog number CA-630; Sigma], protease inhibitors (cOmplete, EDTA free; Roche)} for 30 min on ice. Nuclei and cell debris were pelleted at 13,000 ⫻ g for 2 min. The supernatant (cytoplasmic fraction) was retained, and the pellet was resuspended in nuclear protein extraction buffer (20 mM HEPES-KOH [pH 7.9], 420 mM KCl, 20% glycerol, 1 mM EDTA, 2 mM-mercaptoethanol, protease inhibitors) for 30 min on ice. The nuclear extracts were clarified by centrifugation at 13,000 ⫻ g for 2 min, and the supernatants from the nuclear and cytoplasmic fractions were pooled to constitute the wholecell extract. All extracts were mixed with protein A for 30 min at 4°C and centrifuged at 13,000 ⫻ g for 2 min to eliminate nonspecific binding. A sample of the supernatant, constituting 6% of the total extract, was removed before the addition of antibodies for 90 min at 4°C. The immune complexes were collected on protein A-Sepharose beads (Sigma) by incubation for 1 h at 4°C and washed three times in cold CPEB. Proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and detected by Western blotting. Western blotting. Proteins were resolved on sodium dodecyl sulfatepolyacrylamide gels and transferred to a Protran nitrocellulose membrane (GE Healthcare). The blots were blocked for 1 h with 5% dried milk powder in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20 (TBS-Tween) and incubated overnight at 4°C with appropriate antibodies diluted in TBS-Tween containing 1% dried milk. Blots were developed by use of an enhanced chemiluminescence reagent (Millipore). Statistical analysis. Results are expressed as the mean ⫾ standard deviation (SD) from at last three independent experiments and were analyzed by Student’s t test for unpaired data. P values of ⬍0.05 were considered statistically significant.

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Interaction between VSV M and LMP2

Structure models. All the figures with structures were prepared using the program PyMOL (PyMOL molecular graphics system; DeLano Scientific LLC, San Carlos, CA, USA; http://www.pymol.org).

RESULTS

Identification of LMP2 as a binding partner of VSV matrix protein. To identify cellular proteins interacting with the VSV matrix protein, a cDNA library prepared from differentiated cells of the PC12 rat neuronal cell line was screened by a Y2H assay using pLEX M as the bait (11). Out of 6.4 ⫻ 106 transformants, 150 were positive for interaction, as assessed by ␤-galactosidase staining and growth on selective medium. Among these 150 clones, 40 were identified to be LMP2, making this protein the most abundant hit of the screen. LMP2, like each catalytic subunit, is expressed as a precursor, of which the N-terminal propeptide is cleaved during the maturation of the 20S core particle, a process which is necessary for the activation of the catalytic threonine residue (Thr1 in the mature protein). In this study, we refer to the LMP2 precursor harboring the N-terminal propeptide as pre-LMP2 and to the cleaved version as mature LMP2. cDNAs encoding pre-LMP2, mature LMP2, and the N-terminal propeptide were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. As shown in Fig. 1A, we could detect a strong interaction between VSV M and pre-LMP2. The interaction between mature LMP2 and VSV M, although still detected, was significantly reduced. The N-terminal propeptide of LMP2 on its own does not interact with VSV M. Next, we attempted to confirm the interaction by coimmunoprecipitation in mammalian cells. N2A cells were transfected with pmyc-pre-LMP2, ppre-LMP2-myc, and pmyc-mature-LMP2 carrying pre-LMP2 fused to an N- or C-terminal myc tag or mature LMP2 fused to an N-terminal myc tag, respectively. pmycdystonin, carrying a fragment of the unrelated dystonin protein fused to a C-terminal myc tag, was used as a control. At 24 h after transfection, cells were infected with VSV, the cells were lysed at 4 h postinfection (p.i.), and VSV M was immunoprecipitated with an M-specific antibody. The immunoprecipitates were analyzed by Western blotting using a myc-specific antibody to reveal the presence of LMP2. In the immunoprecipitates, the myc-tagged versions of pre-LMP2 and mature LMP2 could be readily detected, which was not the case for the myc-tagged version of dystonin (Fig. 1B). Coimmunoprecipitation, in which LMP2 constructs were immunoprecipitated with a myc-specific antibody before analysis by Western blotting using an M-specific antibody, also revealed the specific association between the proteins (Fig. 1B). These results validate the interaction between M and LMP2 (expressed either in its premature form or in its mature form) in infected cells. We attempted to determine if an interaction between M and LMP2 could also be detected for other members of the Vesiculovirus genus. We decided to investigate interactions between LMP2 and the M protein of Chandipura virus (CHAV), a vesiculovirus responsible for deadly encephalitis in children mainly in India (33), or spring viremia of carp virus (SVCV), a more distant member of the same genus (34). Coimmunoprecipitations were performed on lysates of N2A cells that had previously been transfected with HA-LMP2 together with either pmyc-M VSV, pmyc-M CHAV, or pmyc-M SVCV. LMP2 was immunoprecipitated from the lysates using an HA-specific antibody. The immu-


noprecipitates were analyzed by Western blotting using a mycspecific antibody to reveal the presence of M. myc-M VSV could be detected in the immunoprecipitates, whereas myc-M CHAV and myc-M SVCV were absent (Fig. 1C). VSV M specifically interacts with LMP2 but not with PSM〉6. In the immunoproteasome, LMP2 replaces PSMB6. As the genes encoding those two proteins share about 60% amino acid sequence identity, this raised the question of whether VSV M is also able to interact with PSMB6. cDNAs encoding the precursor form of PSMB6 (pre-PSMB6) as well as the mature one (mature PSMB6) were cloned in pGAD, expressing the GAL4 activation domain, and used in Y2H assays against pLEX VSV M constructs. As shown in Fig. 2A, no interaction between PSMB6 and M in the Y2H assay was observed. These results were confirmed by coimmunoprecipitation in mammalian cells. N2A cells were transfected with pmyc-preLMP2 as a positive control, pmyc-pre-PSMB6, and pmyc-maturePSMB6. pmyc-dystonin was used as a negative control. Cells were transfected and infected as described above. M proteins present in cell lysates were immunoprecipitated with an M-specific antibody. The immunoprecipitates were analyzed by Western blotting using a myc-specific antibody to reveal the presence of myc fused to PSMB6 or LMP2. Only myc-LMP2 could be readily detected (Fig. 2B). These results were confirmed by immunoprecipitation with a myc-specific antibody. Immunoprecipitates were analyzed by Western blotting using an anti-M antibody, which revealed that M was associated with myc-LMP2 and not with myc-PSMB6 (Fig. 2B). The single mutation I96A in VSV M impairs the interaction between M and LMP2. The VSV M protein is composed of two domains, a globular carboxy-terminal domain and a flexible aminoterminal domain (comprising the 57 first residues) (35, 36). In order to identify the domain involved in binding LMP2, each domain was fused to LexA and tested for LMP2 binding in the Y2H system (Fig. 3A). An interaction with pre-LMP2 could be observed with the globular domain but not with the flexible domain of M. We decided to map the regions of M which are important for LMP2 binding. According to the crystal structure of VSV M (35), residues exposed at the surface of the protein were selected and the following 27 point or double point mutations were designed: R73A, N75A, R79A/T80A, H93A/M94A, I96A, M98A, M98R, V122A/L123A, D125A/Q126A, E136A, P149A/P150A, L152D, V154Y, V154A/P155A, E156A, E156A/H157A, R159A/R160A, G165A/L166A, D181A/E182A, L183A/E184A, P187A/M188A, S199A/D200A, K214A/K215A, S217A, G218A, D223A, and V225A/S226A (Fig. 3B). First, M mutants were tested for their ability to interact with previously described M-binding partners NEDD4 and TSG101 using the Y2H system. All of them were found to interact with NEDD4 and TSG101 (data not shown). However, one of them, the I96A mutant, bound weakly to preLMP2 in the Y2H assay (Fig. 3C). This result was confirmed in mammalian cells by coimmunoprecipitation. N2A cells were transfected with pmyc-M as a positive control, pmyc-M I96A, and pHA-pre-LMP2 or ppreLMP2-HA encoding pre-LMP2 fused to an N- or C-terminal HA tag. Cells were transfected as described above, and LMP2 was immunoprecipitated with an HA-specific antibody. The immunoprecipitates were analyzed by Western blotting using a myc-specific antibody to reveal the presence of VSV M. myc-M could be

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FIG 1 The matrix protein of VSV interacts with LMP2. (A) cDNAs encoding pre-LMP2, mature LMP2, and the 20-amino-acid N-terminal propeptide were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of ␤-galactosidase activity in liquid yeast cultures as described in the Materials and Methods section. Bars represent standard deviations of the mean from three independent experiments performed in triplicate. *, P ⬍ 0.05 compared to the control. (B) Coimmunoprecipitation of M and LMP2. N2A cells were transfected or not transfected (Non TF) for 24 h with plasmids carrying pre-LMP2-myc, myc-pre-LMP2, myc-mature-LMP2, or myc-dystonin as a negative control and then infected with VSV (4 h; MOI, 3) before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody or anti-myc antibody 9E10, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting (WB) using anti-myc antibody 9E10 or anti-M antibody to reveal the presence of the different myc-LMP2 and myc-dystonin constructs and M. Non Inf, noninfected. (C) N2A cells were transfected with plasmids carrying myc-tagged versions of VSV M, CHAV M, SVCV M, and LMP2-HA, followed by immunoprecipitation with an anti-HA antibody. The immunoprecipitates were analyzed by Western blotting using a myc-specific antibody to reveal the presence of VSV M.

readily detected in the immunoprecipitates, which was not the case for myc-M I96A (Fig. 3D). The Ile residue at position 96 is not conserved among the M proteins of vesiculoviruses. We tried to introduce this mutation into the viral genome. Unfortunately, all our attempts using conditions clas-

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sically used to obtain a recombinant virus failed (11, 15). This confirmed previous results obtained by Dancho et al. (37), who were also unable to obtain a recombinant virus harboring this mutation. M binds specifically to the LMP2 precursor, which is not incorporated in the immunoproteasome. With a few exceptions,

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B6 B6 B6 B6 M M M M B6 PS n B6 PS n B6 PS n B6 PS n 2 2 2 P P P M e- ni M eM e- ni M e- ni ni PS ur to LM LM PS tur sto LM PS ur to PS ur to LM e- F re- at ys e- F ree- F re- at ys e- F re- at ys a y r r r r -p n T c-p c-m c-D c-p n T c-p c-m c-D -p T -p -m -D c-p n T c-p c-m c-D y y y y y y yc y yc n yc yc yc y y y y m m m m m m No m m No m m m No m m No m m m P2

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FIG 2 The matrix protein of VSV does not interact with PSMB6. (A) cDNAs encoding pre-LMP2 as well as pre-PSMB6 and mature PSMB6 were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of ␤-galactosidase activity in liquid yeast cultures (see the Materials and Methods section). Bars represent standard deviations of the means from three independent experiments performed in triplicate. (B) Coimmunoprecipitation of M and PSMB6. N2A cells were transfected or not transfected (Non TF) for 24 h with plasmids carrying myc-pre-LMP2 (positive control), myc-pre-PSMB6, myc-maturePSMB6, or myc-dystonin as a negative control and then infected with VSV (4 h; MOI, 3) before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody or anti-myc antibody 9E10, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 or M-specific antibody to reveal the presence of the different myc-LMP2, myc-PSMB6, and myc-dystonin constructs and M.

the immunoproteasome is expressed only in very small amounts in commonly used cell lines. Therefore, in the experiments described above, the interaction between M and LMP2 was demonstrated in transfected cells overexpressing LMP2. However, in those experiments, the immunoproteasome was not assembled and LMP2 was not mature. Therefore, in most studies the conversion from the standard proteasome to the immunoproteasome is triggered by interferon. However, infection by VSV is strongly inhibited in the presence of type I or II interferons (38–40). To examine if M also interacts with endogenous LMP2, HeLa cells were stimulated with gamma interferon. After such a treatment, the total amount of LMP2 increased and pre-LMP2 was


converted into mature LMP2, which became the most abundant form in the cell (Fig. 4, input). Interferon gamma-treated cells were transfected with pmyc-M or pmyc-M I96A. Sixteen hours later, the cells were lysed and endogenous LMP2 was immunoprecipitated using an LMP2-specific antibody. The immunoprecipitates were analyzed by Western blotting using a myc-specific antibody to reveal the presence of VSV M. This showed that M interacts with endogenously expressed LMP2, an interaction which is abolished for the M I96A mutant (Fig. 4), as previously observed using overexpressed LMP2 (Fig. 1B). More interestingly, although mature LMP2 was the major form in the cell lysate, upon immunoprecipitation of M using a

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FIG 3 Mapping of M-protein-binding domain with pre-LMP2. (A) Interaction between the flexible domain (amino acids 1 to 54) and the globular domain (amino acids 55 to 229) of M protein expressed in LexA and pre-LMP2 constructed in pGAD assayed by the Y2H assay. wt, wild type. (B) Surface representation of the crystal structure of VSV M (35). All the surface residues which were mutated are either in red or in yellow (residue I96). (C) The interaction between mutant M I96A and pre-LMP2 was assayed by the Y2H assay and quantification of ␤-galactosidase activity in liquid yeast cultures. Bars represent standard deviations of the means from three independent experiments performed in triplicate, *, P ⬍ 0.05 compared to the control. (D) N2A cells were transfected or not with plasmids carrying M, M I96A, pre-LMP2-HA, and HA-pre-LMP2 for 16 h before preparation of cell lysates. Following immunoprecipitation with an anti-HA antibody, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 to reveal the presence of M and M I96A.

myc-specific antibody, the pre-LMP2 form was found to be essentially associated with M. This indicates that M binds preferentially to this form, which is free in the cytosol (i.e., it is not incorporated in the immunoproteasome).

Mutations of residues in the S1 pocket, which are essential for the catalytic activity of LMP2, can alter the interaction with VSV M. According to the crystal structure of the murine immunoproteasome (41), mature LMP2 is tightly packed in a heptam-

Input

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FIG 4 M binds specifically to pre-LMP2. HeLa cells were stimulated with gamma interferon for 8 h before transfection for 16 h with pmyc-M or pmyc-M I96A, followed by immunoprecipitation with an LMP2- or M-specific antibody. The immunoprecipitates were analyzed by Western blotting using an LMP2- or myc-specific antibody to reveal the presence of LMP2 or VSV M, respectively.

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FIG 5 Mapping of the LMP2 binding site with M. (A) Sequence alignment of the first 60 residues of mature LMP2 and mature PSMB6. (B) The interactions of the pre-LMP2 T1A, D17A, K33A, S129A, V20T, L45R, V20T/L45R, and D17A/L45R mutants with M were assessed by the Y2H assay and quantification of ␤-galactosidase activity in liquid yeast cultures. Bars represent standard deviations of the means from three independent experiments performed in triplicate, *, P ⬍ 0.05 compared to the control. (C) N2A cells were transfected or not for 24 h with plasmids carrying myc-pre-LMP2 and myc-pre-LMP2 D17A, myc-preLMP2 L45R, or myc-pre-LMP2 D17A/L45R and infected or not with VSV for 4 h before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 or an M-specific antibody to reveal the presence of LMP2 and M, respectively.

eric ring structure. In this structure, some of its surface-exposed residues face outwards, whereas others are buried at the interface with the other immunoproteasome subunits. According to this, we designed mutants of LMP2 with mutations in regions which are exposed (E107A, K143A, R153A, and E107A/R153A) and in regions which are buried in the assembled immunoproteasome (R29A, Q39A, Y61A, Y90A, M118A, R121A, Y135A, R165A, and a deletion of the 5 last C-terminal residues). All these LMP2 mutants were used in the Y2H assay to investigate the possible binding of M. None of these 13 mutants were found to abolish the interaction with the matrix protein (not shown).


VSV M was shown by Y2H assays to interact predominantly with pre-LMP2 and to a lesser extent with the mature form of the protein (Fig. 1A). Consequently, we investigated if mutations of conserved residues essential for the catalytic activity and residues required for the structural integrity of the catalytic site of LMP2 have an effect on its interaction with M. According to the crystal structure of the mature LMP2 protein, we decided to perform an alanine scanning experiment mutating the catalytic site of LMP2 (T1A) as well as three amino acids (D17A, K33A, and S129A) facing the same site. Furthermore, as mentioned above, LMP2 and PSMB6 share about 60% amino acid sequence identity, and we

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FIG 6 Growth kinetics of VSV on cells endogenously expressing LMP2, LMP7, and MECL-1. (A) B8 cells expressing LMP2 or the empty vector and B27M.2 cells expressing LMP2, LMP7, and MECL-1 were infected with VSV at MOIs of 3 and 0.3. Samples were harvested at 1, 4, 6, 8, 10, and 12 h postinfection, and the titer of progeny virus on BHK-21 cells was determined. Bars represent standard deviations of the means from three independent experiments. (B) Infected cell lysates were separated by SDS-PAGE and analyzed by Western blotting using anti-VSV M, anti-VSV G, and antitubulin antibodies. (C) PANC-1 cells constitutively expressing high levels of endogenous immunoproteasome were infected with VSV (MOI, 3) for the indicated times. Total cell lysates were examined by Western blotting with antibodies specific for the PSMA3 and PSMB6 subunits of the proteasome, LMP2, and M. hpi, hours postinfection.

took advantage of the fact that M does not interact with PSMB6 to design new mutants. We focused on residues 20 and 45, located in the vicinity of the S1 pocket, which confer the enzymatic specificity of LMP2. In LMP2, PSMB6 Thr20 was replaced by Val and PSMB6 Arg45 was replaced by Leu (Fig. 5A). These substitutions reduce the size of the S1 pocket and change the overall charge of the local environment from positive to neutral (42). To investigate if these changes had an effect on the interaction with VSV M, the LMP2 V20T and L45R point mutants and the V20T/L45R double mutant were constructed. All the mutations were introduced into the premature form of LMP2, and the interaction of the mutant proteins with M was analyzed using the Y2H assay. As shown in Fig. 5B, the LMP2 T1A, LMP2 K33A, LMP2 S129A, and LMP2 V20T mutants interacted with M as efficiently as wild-type LMP2. On the other hand, the interaction between M and the LMP2 D17A and LMP2 L45R mutants was significantly reduced (Fig. 5B). According to these results, a double mutant combining the mutations D17A and L45R was constructed, and it was not able to interact with M (Fig. 5B). We attempted to confirm the lack of interaction between M and this double mutant in infected cells by coimmunoprecipitation. N2A cells were transfected with pmyc-pre-LMP2, pmyc-pre-LMP2 D17A, pmyc-pre-LMP2 L45R, or pmyc-pre-

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LMP2 D17A/L45R and infected by VSV, and the cell lysates were immunoprecipitated with an M-specific antibody. The immunoprecipitates were analyzed by Western blotting using a myc-specific antibody to reveal the presence of LMP2 (Fig. 5C). Pre-LMP2, pre-LMP2 D17A, and pre-LMP2 L45R were detected in the immunoprecipitates, but this was not the case for pre-LMP2 D17A/L45R, confirming that LMP2 harboring this double mutation is unable to bind M (Fig. 5C). Growth kinetics of VSV on cells endogenously expressing LMP2, LMP7, and MECL-1. As was already mentioned above, in commonly used cell lines, the immunoproteasome is expressed only in very small amounts. As we could not trigger the conversion from the standard proteasome to the immunoproteasome using interferon because infection by VSV is strongly inhibited after application of class I or II interferons (38–40), we used a cell line endogenously expressing LMP2, LMP7, and MECL-1 (26). To establish whether the presence of the immunoproteasome would influence viral growth, one-step (MOI, 3) growth curves were performed. Virus grown on the mouse fibroblast cell line B27M.2 expressing LMP2, LMP7, and MECL-1 showed no difference in virus release from that for virus grown on the control cell line B8 expressing the empty vector or a B8 cell line expressing LMP2 alone (Fig. 6A and B). Identical results were obtained at a lower MOI of 0.3.

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FIG 7 LMP2 residues which are important for the interaction with VSV M are buried within the immunoproteasome. Ribbon representation of the crystal structure of the mouse immunoproteasome (41). (Inset) Magnification of the M-binding region seen from within the immunoproteasome. Red, LMP2; blue, residues T1, D17, and L45. These residues face inwards in the immunoproteasome.

To evaluate whether an infection with VSV alters the cellular amount of proteasome or immunoproteasome subunits, PANC-1 cells, which constitutively express high levels of endogenous immunoproteasome (43), were infected with VSV for 0, 4, 6, 9, and 12 h. Total cell lysates were examined by Western blotting with antibodies specific for the constitutive proteasome subunits PSMA3 and PSMB6, the immunoproteasome subunit LMP2, and M as a marker for infection. The results in Fig. 6C show that infection with VSV does not interfere with the amount of the tested proteasome and immunoproteasome subunits present in whole-cell lysates. Furthermore, the ratio between the precursor form and the mature form of LMP2 was apparently not affected. Those results are not surprising: the in vivo half-life of the constitutive proteasome is 12 days (44), and the total replacement of the constitutive proteasome by the immunoproteasome is also a rather slow process taking several days (45), whereas the VSV life cycle in cultured cells is completed in a few hours. Finally, we repeatedly tried to inhibit LMP2 expression in PANC-1 cells using several different short hairpin RNAs. However, probably due to the lifetime of the protein, we were unable to decrease significantly the amount of LMP2 in the cell (not shown). DISCUSSION

In this study, using the yeast two-hybrid system, we have identified LMP2, a component of the immunoproteasome, to be a binding partner of the VSV matrix protein. This interaction was validated in infected cells by coimmunoprecipitation of M and a tagged version of LMP2 as well as in cells endogenously expressing LMP2. We were not able to detect an interaction between LMP2 and the matrix protein of CHAV or SVCV, suggesting that this interaction is specific of VSV. No interaction between M and PSMB6 (which is replaced by LMP2 in the immunoproteasome) was detected, despite an important amino acid sequence identity between both proteins (42).


This indicates that VSV M specifically targets the immunoproteasome and not the proteasome. Interactions of viral proteins with members of the immunoproteasome have been previously described. The HIV Tat protein interacts with LMP2 and LMP7 as well as other ␣ and ␤ subunits of the proteasome (46). Hepatitis C virus (HCV) NS3 interacts with LMP7 (47), and adenovirus E1A interacts with MECL-1 (48). The role of those interactions remains to be determined. M binding to LMP2 involves the M globular domain, and residue I96, which is located on the M surface, was found to be crucial for this interaction. It is worth noting that LMP2 is the first identified cellular partner that binds the globular domain of M. The other partners so far identified (11–15, 17, 18) bind the flexible amino-terminal part of the protein. The site of interaction comprises the LMP2 catalytic site and the S1 pocket. A double mutation of LMP2 Asp17 (which is essential for the enzymatic activity) and Leu45 (which defines the specificity of the S1 pocket), respectively, into an alanine and an arginine abolishes the interaction. This may explain the weaker interaction of M with mature LMP2 observed using the Y2H system, as the autocatalytic cleavage of the amino-terminal propeptide certainly results in a local conformational change of the catalytic site. Similar observations were made for HCV NS3, which shows a stronger interaction with pre-LMP7 and binds very weakly to the mature form of LMP7 (47). Several lines of evidence indicate that M binds LMP2 before its incorporation in the immunoproteasome. First, the M-binding site (comprising Asp17 and Leu45 of LMP2) faces inwards in the immunoproteasome after its assembly and is therefore not accessible to M in this assembly (Fig. 7). Second, the mature form of LMP2 is formed only upon completion of immunoproteasome assembly, and although the M-binding site is still present on mature LMP2 (Fig. 1A and B), we have shown that M preferentially binds to pre-LMP2 (Fig. 4), which is free in the cytosol and not

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incorporated in the immunoproteasome. This strongly suggests that M sequesters the precursor of LMP2 and, as a consequence, interferes with the assembly of the immunoproteasome. This could confer an advantage to the virus, as the immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Such an advantage is, of course, silent in cultured cell lines, which explains why VSV growth is insensitive to the presence or absence of LMP2 (or the whole immunoproteasome) in the B8 cell line. The hypothesis that the M-LMP2 interaction controls CD8 T cell activity against infected cells could have been tested by comparison of the pathogenicity of the VSV wild type with that of a mutant whose matrix protein is unable to bind LMP2. Unfortunately, introduction of the mutation I96A (which abolishes the binding between M and LMP2) in the viral genome is lethal, and, like Dancho et al. (37), we were unable to get such a recombinant virus. This is probably due to the fact that the M I96A mutant has a reduced ability to associate with nucleocapsid-M protein complexes (37). In conclusion, we suggest that VSV M, by interfering with immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy. ACKNOWLEDGMENTS We thank M. Groettrup (University of Konstanz) for the B8 and B27M.2 cell lines, I. Beau (UMR 984, Châtenay-Malabry, France) for the PANC-1 cell line, and D. Pasdeloup (UMR 984, Châtenay-Malabry, France) for the pCDNA3.1myc and the pCDNA3.1myc-dystonin plasmids. Plasmids carrying CHAV M and SVCV M were provided by J. Petersen, L. Her, and J. E. Dahlberg (University of Wisconsin). This work was supported by the Fondation pour la Recherche Médicale (grant FRM DEQ20120323711 to Y.G.).

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