INTRODUCTION

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Superantigens (Sags) constitute a group of proteins with potent effects on the immune system. Although different Sags are expressed by a wide variety of microorganisms, they share the ability to stimulate a large number of T cells through similar mechanisms. Sags are presented in the context of major histocompatibility complex (MHC) class II molecules at the cell surface and interact with subsets of T cells expressing specific variable domains in the T-cell receptor (TCR)  chain (12, 23, 31, 43). The encounter with Sag leads first to the expansion and subsequently to the deletion of reactive mature T cells (30, 42, 43). When immature T cells interact with Sag during thymic development, they undergo intrathymic deletion (22, 23, 31).

Mouse mammary tumor virus (MMTV) is a retrovirus which exists either as an infectious viral particle transmitted from mother to offspring via milk (exogenous MMTV) or as a germ line-integrated provirus stably transmitted via genetic inheritance (endogenous MMTV) (24). In addition to the classical retrovirus genes gag, pol, and env, the MMTV provirus genome contains an open reading frame within the 3' long terminal repeat which encodes the viral Sag (2, 10). The MMTV Sag plays a crucial role in the viral life cycle. MMTV preferentially binds to B cells (5), triggers their activation (3), and infects them. The Sag is then expressed at the surface of the cells in association with MHC class II molecules. Sag-reactive T cells are activated and accumulate locally, providing help to infected B cells through cognate T-cell-B-cell interactions, which lead to a large increase in the number of infected B cells (17, 27). This Sag-mediated increase in viral load has been shown elsewhere to be an essential step for the subsequent transport of the virus to the mammary gland and for its efficient transmission to the next generation of mice (16, 18).

The presentation of the Sag-MHC class II complex and its recognition by the TCR are very different from the recognition of classical antigens. The interaction of a T cell with Sags almost exclusively involves the V domain of the TCR. The other parts of the TCR do not contribute to the interaction, except for the V domain, which has a minor indirect influence (37, 40, 41). In particular, Sags are thought to interact specifically with the hypervariable region 4 (HV4) of the V domain as well as with elements of the complementarity-determining regions 1 and 2 (CDR1 and CDR2) (4, 6, 9, 11, 15, 19, 20, 35, 36). The HV4 is located on the lateral part of the molecule, far away from the central region implicated in the recognition of peptide-MHC complexes (14). In the case of bacterial Sags, the published crystal structure of a Sag-TCR complex shows a direct interaction between the Sag and the HV4, as well as with several amino acids of CDR1 and CDR2, of the TCR (13). This report, together with other studies (4, 8, 9, 15, 19, 33, 35, 36), indicates that residues of the HV4 as well as some residues outside this region are critical for the specific interaction of the TCR with Sag.

Comparisons of Sag proteins from several MMTV strains have shown a high degree of sequence identity between them (29, 46). Most sequence differences are located within two regions defined by amino acids (aa) 174 to 198 (region I) and aa 288 to the C terminus (region II). The C-terminal variability has been correlated with observations that certain Sag molecules interact with particular TCRs (46). For instance, the C3H and GR exogenous Sags reacted with T cells bearing V14 chains (10). Moreover, experiments performed by Yazdanbakhsh and colleagues showed that C-terminal replacement of endogenous mtv-1 Sag (V3 reactive) with mtv-7 Sag (V6 reactive) allowed the recombinant Sag to react with V6-expressing T cells in stable-transfection assays (46). The reciprocal experiment confirmed that a polymorphic Sag region (30 to 40 aa) at the C terminus is sufficient to specify interactions with certain TCR V chains (46). However, little is known about the precise requirements within this region for Sag function. Recently, a series of substitutions and deletions transferred into a cloned infectious MMTV provirus has been used for in vivo analysis (45). These mutant viruses induced tumors with lower incidence in mice, although all but one C-terminal amino acid substitution abolished Sag function. Interestingly, the one mutation affecting the C-terminal 3 aa that retained partial Sag function lost the ability to be transmitted through milk to susceptible offspring (45).

The aim of this work was to characterize the amino acids that determine the V specificity of the MMTV(SIM) Sag (32) and, indirectly, the mtv-8 Sag. Among the 39 sequenced viral Sags, MMTV(SIM) is the only one showing reactivity with V4 and V10^a/c TCRs (31, 32; reviewed in reference 29). Its sequence has greatest similarity with Sags interacting with V5, -11, and -12, such as mtv-8. MMTV(SIM) and mtv-8 Sags differ at only six positions and/or regions within the C-terminal 70 aa (Fig. 1). Four of them (positions 266, 273, 305, and 319-320; all amino acid positions refer to the mtv-8 sequence) can be found in Sags with V specificities other than that of mtv-8 and MMTV(SIM), whereas two (289 to 295 and 314-315) are unique to MMTV(SIM). We have addressed the importance of these two regions for SIM-specific reactivity to V4 and V10^a/c in vivo by using the recombinant vaccinia virus (RV) expression system. We have previously shown that RV can be used not only to assess the expression and posttranslational modifications of the MMTV Sag in cell cultures but also to monitor the specific Sag response in vivo (25, 26). Subsequently, we generated RVs expressing the complete Sag molecule of mtv-8 or MMTV(SIM), as well as a panel of chimeric and mutant mtv-8 Sag molecules. With this strategy, we aimed to simultaneously monitor the response in vivo to the mutant Sag in terms of gain of SIM-specific reactivity (V4 and -10^a/c) and of loss of mtv-8-specific reactivity (V5, -11, and -12). All mutants had functional Sag activity in vivo. We confirm that the C-terminal part of the viral Sag is necessary and sufficient to confer specific TCR V reactivity. In addition, we show that the exchange of 7 aa (VWGKIFH [289 to 295]) of the mtv-8 Sag with the SIM-specific 4 aa and the small deletion that they encompass (F*R---Y, designated "") established a partial SIM-specific V4 reactivity. Interestingly, only some of the original mtv-8-specific TCR V interactions were lost in parallel, thereby generating a Sag molecule with a mixed mtv-8 and MMTV(SIM) V reactivity pattern, i.e., V11 and V4. The full SIM-specific V4 reactivity was reached by the introduction in the mtv-8 Sag of two additional point mutations (NS [314-315, designated "M"]) together with the previously mentioned deletion and was paralleled by the total loss of mtv-8-specific TCR interactions. However, none of the examined mutations in the mtv-8 Sag restored the V10^a/c reactivity observed with the MMTV(SIM) Sag. In conclusion, we were able to identify residues important for the complex interactions between a viral (MMTV) Sag molecule and TCR V elements on T cells.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Comparison of amino acid sequences of mtv-8 and MMTV(SIM) Sag molecules. Asterisks indicate amino acid identity with the mtv-8 sequence. Dashes indicate gaps introduced to maximize amino acid identity. Open circles indicate the portion of the MMTV(SIM) Sag molecule used to produce the chimeric mtv-8/MMTV(SIM) Sag molecule.

	MATERIALS AND METHODS

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Mouse strains. BALB/c mice (H-2^d TCR V^b ) were purchased from Harlan OLAC Ltd. (Bicester, United Kingdom). BALB/c mice bearing the TCR V^a haplotype were kindly provided by Alexandra Livingstone (Basel Institute of Immunology, Basel, Switzerland). The mtv-free mice have been previously described (8). BALB/c TCR V^a and mtv-free mouse strains were kept as breeding pairs in our animal facilities.

Antibodies. The following antibodies were used in this study: fluorescein isothiocyanate (FITC)-labeled anti-V4 (KT4-10 [39]), FITC-labeled anti-V5 (MR9-4; Pharmingen, San Diego, Calif.), FITC-labeled anti-V11 (RR3-15; Pharmingen), FITC-labeled anti-V12 (MR11-1; Pharmingen), Cychrome-coupled anti-CD4 (RM4-5; Pharmingen), and phycoerythrin-coupled anti-CD69 (H1.2F3; Pharmingen). Anti-V10^a/c (KT10a [38]), kindly provided by K. Tomonari (Fukui Medical School, Fukui, Japan), was used as a hybridoma supernatant and was detected by an FITC-coupled goat anti-rat immunoglobulin G (IgG). A polyclonal rabbit anti-open reading frame-peptide serum (serum C [7]) raised against a 23-aa synthetic peptide of the MMTV(GR) Sag was used for immunoprecipitation of the viral Sags expressed by RVs.

Cloning and mutagenesis. The mtv-8 and the MMTV(SIM) Sag coding sequences were amplified by PCR from genomic DNA of a mouse harboring mtv-8 as a single endogenous mtv (8) and from a pUC19 backbone containing the MMTV(SIM) Sag coding sequence (32), respectively, using the following primers: 5'-TT GGA ATT CCA CCA TGC CGC GCC TGC AG-3' and 5'-CCA CGC GTT GGG AAC CGC AAG GTT GG-3'. Both PCR products were cloned into pCI-neo expression vector (Promega, Madison, Wis.) after EcoRI and AflIII digestion to generate pCIMtv-8 and pCISIM. The chimeric mtv-8-SIM Sag construct was generated by switching in the PpuMI/AflIII fragment from pCISIM into pCIMtv-8 to generate pCIMtv-8/S. These three Sag coding sequences upon digestion with EcoRI, blunted with Klenow enzyme and digested with XbaI, were shuttled into the vaccinia virus transfer vector pARO1 (25) digested with HindIII, blunted with Klenow enzyme, and digested with XbaI. The resulting plasmids, i.e., pAMtv-8, pASIM, and pAMtv-8/S, were then sequenced. To generate pAMtv-8M and pAMtv8-M, the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) was used with pAMtv-8 and pA8-M, respectively, as templates, following the manufacturer's instructions. The following primers were used: 5'-GAA CAC ATT TCA GCT GAT ACT AAT AGC ATG AGC TAT AAT GG-3' and 5'-CCA TTA TAG CTC ATG CTA TTA GTA TCA GTC GAA ATG TGT TC-3'. pAMtv-8 was generated using the ExSite PCR-based site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions and using the following primers: 5'-TCT CCA AAC GTT CAT TCC TGT TCC-3' and 5'-CCA TTA TAG CTC ATG CTA TTA GTA TCA GCT GAA ATG TGT TC-3'.

Preparation of RVs. The procedure to generate RVs has been described previously (25). To generate virus stocks, HeLa cells were infected with RV for 48 h at a multiplicity of infection of 0.1. The cells were recovered and Dounce homogenized in cold 10 mM Tris-HCl, pH 9, and cell fragments were eliminated by centrifugation for 10 min at 4°C and 750 × g The supernatant (referred to as crude stock) was incubated for 30 min at 37°C with trypsin (0.25 mg/ml). The crude stock was then laid over a 36% sucrose cushion in 10 mM Tris-HCl, pH 9, and centrifuged for 80 min at 4°C and 25,000 × g The purified virus in the pellet was resuspended in a minimal volume of 10 mM Tris-HCl, pH 9, and titrated in a plaque formation assay on huTk143B cells (ATCC CRL-8303).

Injection and sampling. Purified virus (5 × 10⁷ PFU) in a 30-µl volume was injected subcutaneously into the hind footpads of naive mice. Twenty-four hours postinjection, the mice were sacrificed; the draining popliteal lymph nodes were isolated and homogenized to a single-cell suspension. For each experiment, we used the two hind feet of two to three mice per virus tested. All experiments involving mice were repeated three times.

Flow cytometric analysis. Popliteal lymph node cells were triple stained in a single step with a mixture of a given anti-V antibody (V4, -5, -11, or -12), anti-CD4 antibody, and anti-CD69 antibody. V10^a-positive cells were labeled by incubation with KT10a antibody (hybridoma supernatant) and subsequently with FITC-coupled goat anti-rat IgG. After blocking with rat IgG (10 µg/ml), the cells were incubated with anti-CD4-Cychrome and anti-CD69-phycoerythrin. Analysis was performed on a FACScan (Becton Dickinson & Co., Mountain View, Calif.) cell analyzer using the Lysis II software for data evaluation. Dead cells were excluded on the basis of their forward and side scatter characteristics. From 30,000 to 50,000 cells were acquired, and results were analyzed with a Student t test assuming unequal variance.

Metabolic labeling and immunoprecipitation. Confluent CV-1 cells in six-well plates were infected at a multiplicity of infection of 10 for 12 h. The cells were washed with phosphate-buffered saline and starved in 1 ml of methionine- and cysteine-free medium for 1 h at 37°C. The medium was then replaced by 0.3 ml of methionine- and cysteine-free medium containing 100 µCi of ³⁵S-Easy Tag express labeling mix (NEN Life Science Products, Boston, Mass.). After 1 h of incubation at 37°C, cells were washed with cold phosphate-buffered saline and lysed for 30 min on ice with 0.5 ml of RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris-HCl, pH 8) containing protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin/ml, 10 µg of leupeptin/ml). Clarified lysates (350 µl) were precleared with 35 µl of preimmune rabbit serum and 50 µl of protein G-agarose beads (Amersham Pharmacia Biotech, Uppsala, Sweden) for 6 h at 4°C. The resulting lysates were further incubated overnight at 4°C with 10 µl of anti-MMTV(GR) Sag serum C and 50 µl of protein G-agarose beads. After two washes in cold RIPA buffer, the protein G beads were resuspended in loading buffer (3% -mercaptoethanol, 3% SDS, 0.3% bromophenol blue, 10% glycerol) and heated for 5 min at 95°C, and the samples were resolved on an SDS-8 to 12% polyacrylamide gel. The gel was fixed, soaked in Amplify (Amersham Pharmacia Biotech) for 30 min, dried, and exposed at 80°C with amplifying screens.

Statistical treatment of the data. All data were analyzed for statistical significance with a t test (two-sample test assuming unequal variance). For stimulation data, we have compared the values obtained after injection of an RV with the data obtained from mock-infected animals. For CD69 upregulation, the data obtained for CD4⁺ T cells in a given V subset were compared to data for all other V CD4⁺ subsets.

	RESULTS

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Construction of RVs expressing a wild-type or a mutated MMTV Sag molecule. Despite 90.9% sequence identity and 92% sequence homology (Fig. 1), mtv-8 and MMTV(SIM) Sags interact with different TCR V domains. The mtv-8 Sag is recognized by the V5, -11, and -12 regions of TCRs (29), whereas the MMTV(SIM) Sag has been shown previously to interact with the V4 and V10^a/c regions of TCRs (32, 33). Figure 1 shows the amino acid sequence alignment of the mtv-8 and MMTV(SIM) Sags, which differ in only 29 aa. Because previous results have shown that the C-terminal portion of a Sag dictates its reactivity with given V regions of TCRs (46) and because most of the sequence variability of viral Sags resides within these C-terminal 30 to 40 aa, we decided to examine the importance of this C-terminal domain for V reactivity. When all known Sag sequences were compared, only two regions within the C-terminal 70 aa were unique to the MMTV(SIM) and its unique V specificity. One region consists of three amino acid changes and a small deletion of 3 aa present in the MMTV(SIM) Sag molecule close to the C terminus (Fig. 1, "").The other region is characterized by the double mutation F to N and G to S compared to mtv-8 (Fig. 1, "M"). These few changes gave us an opportunity to map the amino acids responsible for the different V specificities of MMTV(SIM) and mtv-8 Sags. To do this, we constructed six RVs producing wild-type or mutant mtv-8/SIM Sags (Fig. 2A): RV-8 and RV-S produced the wild-type Sags of mtv-8 and MMTV(SIM), respectively, while RV-8/S expressed a chimeric Sag with the N-terminal portion of the mtv-8 Sag and the 106 C-terminal aa of the MMTV(SIM) Sag. We also produced three RVs to characterize the amino acids determining the V reactivity patterns of the MMTV(SIM) and mtv-8 Sag molecules. In all three cases, the mtv-8 Sag was used as a backbone to insert SIM-specific modifications. This strategy allowed us to diagnose the gain of the reactivity with SIM-specific V4 and V10^a/c TCRs and the loss of the mtv-8-specific interaction with TCRs V5, -11, and -12 of the newly generated mutant Sag. We inserted the deletion and the two flanking mutations present in the MMTV(SIM) Sag molecule (Fig. 1, aa 289 to 295 in mtv-8, "") to produce RV-8. We also introduced the double mutation F to N and G to S (Fig. 1, aa 314-315, "M") to produce RV-8M. We further combined both the deletion  and the mutation M to produce RV-8M. All the other mutations (T to S [aa 266], I to M [aa 273], Q to L [aa 305], N to Y [aa 319], and G to D [aa 320]) were ignored because they are present in other Sags displaying different V specificities (reviewed in reference 29)

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Scheme of the different Sag molecules constructed and their expression in mammalian cells infected with RVs. (A) The different Sag molecules constructed and used to produce RVs are shown. The amino acids that have been introduced in the mtv-8 backbone have been indicated: F*R---Y (aa 289 to 295, referred to as "") and NS (aa 314-315, referred to as "M"). The numbering refers to the mtv-8 Sag molecule (Fig. 1). (B) CV-1 cells were infected with Sag-expressing RVs and labeled with [35S]Met-Cys at 12 h postinfection as described in Materials and Methods. The upper panel shows the immunoprecipitated Sag molecules including an uninfected sample (Mock), a wild-type-infected sample (WT), and a positive control (rm1 [25]). The lower panel shows the same samples before immunoprecipitation. The numbers at left are molecular masses in kilodaltons.

Expression of the different MMTV Sag molecules in mammalian cells infected with RVs. Expression of the viral Sag molecules by the RVs was monitored in cultured CV-1 cells. Figure 2B shows the results of a representative immunoprecipitation of the different Sag molecules. We used noninfected cells (Mock) and wild-type vaccinia virus (WT)-infected cells as negative controls. Furthermore, we used the previously described RV-rm1 (25, 26), which expresses the MMTV(GR) Sag, as a positive control. Two specific bands were seen for each RV (upper panel), one with a size of 46 kDa as previously described (25) and one slightly smaller. The lower-molecular-weight band might reflect either an internal initiation or differential glycosylation. Overall, all RVs expressed a Sag molecule of the appropriate size at similar levels (maximally threefold difference as measured by densitometry) in tissue culture. The lysates before immunoprecipitation are shown in the lower panel.

Response of BALB/c mice to RVs expressing wild-type or mutant MMTV Sags: the  mutant induces weak reactivity, and the combination of  and M induces full reactivity, with TCR V4. BALB/c mice were tested for their response to the six RVs expressing wild-type or mutant mtv-8/SIM Sags. Because BALB/c mice harbor, among others, an integrated mtv-8 virus deleting V5, -11, and -12 T cells (reviewed in reference 29) and because their TCR V domains are of the b haplotype, only SIM-reactive V4 CD4⁺ T cells can be analyzed. Figure 3A shows the percentage of large CD4⁺ lymphoblasts expressing the V4 TCR in the lymph node draining the site of injection. The results indicate that RV-8/S (P = 1 × 10⁵), RV-8M (P = 6 × 10⁵), and to a lesser extent RV-8 (P = 2.6 × 10⁴) or RV-8M (P = 3.4 × 10²) induced the expansion of the V4-bearing CD4⁺ lymphoblasts like the control virus RV-S (P = 1.8 × 10³). The control virus, RV-8, did not stimulate V4 CD4⁺ T cells as expected. The expansion by RV-8M observed in this experiment was not seen in two other experiments. Furthermore, no expansion in total V4⁺ CD4⁺ T cells was observed to be statistically significant in all experiments done. In conclusion, RV-8M only very weakly and inconsistently caused some expansion of V4⁺ CD4⁺ blasts.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Activation and expansion of V4+ CD4+ T cells upon injection of Sag-expressing RVs in BALB/c mice. BALB/c mice (TCR Vb) were not injected (Mock) or were injected with 5 × 107 PFU of RV-8, RV-S, RV-8/S, RV-8M, RV-8, or RV-8M. (A) The response of V4+ cells to each RV tested is indicated as a percentage of large CD4+ blasts. *, P < 0.05; **, P > 0.05. (B) The percentage of CD69+ CD4+ T cells among V4-negative (Non-V4) or V4-positive (V4) cells is shown for the same experiment. *, P < 0.01. Data represent mean values ± standard errors of the means of four draining lymph nodes 24 h postinfection. The experiment is representative of three performed.

4

6

3

5

Response of BALB/c TCR V^a mice to RVs expressing wild-type or mutant MMTV Sags: the  and/or M modifications do not confer V10^a specificity on the mtv-8 Sag. The MMTV(SIM) Sag was reported previously to interact not only with V4-expressing T cells in mice with a TCR V^b haplotype (32) but also with V10-expressing T cells in BALB/c mice with the TCR V^a or the TCR V^c haplotype (33). We therefore injected our RVs into BALB/c mice with the TCR V^a haplotype in order to detect a Sag response in V4 and V10^a CD4⁺ T cells. As shown in Fig. 4, RV-S and RV-8/S triggered the expansion of both V4 and V10^a CD4⁺ T cells (P = 1.4 × 10², P = 5.9 × 10⁴, P = 1 × 10³, and P = 1.4 × 10⁴, respectively). RV-8 and RV-8M did not stimulate V10^a CD4⁺ T cells (Fig. 4B), although they were clearly capable of inducing the expansion of V4 CD4⁺ T cells (P = 3.5 × 10² and P = 3.7 × 10², respectively) (Fig. 4A). RV-8 and RV-8M were unable to change the levels of V4 and V10^a CD4⁺ T cells. The expression of the activation marker CD69 (data not shown) correlated with these results, demonstrating that neither , M, nor the combination of  and M residues mediates the interaction of the SIM Sag with the V10^a TCR and that other amino acids must be important for this V10^a TCR reactivity.

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Expansion of SIM-reactive V4+ and V10+ CD4+ T cells upon injection of the different RVs into BALB/c (TCR Va) mice. BALB/c mice bearing the TCR Va haplotype were not injected (Mock) or were injected with 5 × 107 PFU of RV-8, RV-S, RV-8/S, RV-8M, RV-8, or RV-8M. The response of V4+ (A) or V10+ (B) cells is indicated as a percentage of enlarged CD4+ T lymphoblasts. Data represent mean values ± standard errors of the means of four draining lymph nodes 24 h postinfection. The experiment is representative of three performed. *, P < 0.05.

Response of mtv-free mice to RVs expressing wild-type or mutant MMTV Sags: reactivity with V5, -11, and -12 TCRs. The experiments presented so far have addressed the gain of SIM-specific V4 and V10^a reactivity by the mtv-8/MMTV(SIM) Sags expressed by RVs. We further determined whether the gain of function with SIM-specific TCRs was paralleled by the loss of mtv-8-specific reactivity with V5, -11, and -12. Since the response to RV-8 cannot be measured in BALB/c mice because of the presence of endogenous mtv genes, we tested all the RVs in MMTV-free mice (8). These mice have no integrated mtv genome and therefore have an intact V repertoire, making it possible to test for any V reactivity for a given Sag molecule. mtv-free mice were injected with the six RVs, and the draining lymph nodes were analyzed for the preferential activation (data not shown) and the expansion of a given V population among the CD4⁺ T cells (Fig. 5). When the V5 TCR was analyzed, only RV-8 (P = 4.8 × 10²) and RV-8M (P = 3.0 × 10²) were found to stimulate a low but significant expansion of this CD4⁺ T-cell population (Fig. 5A). The analysis of V11 CD4⁺ lymphoblasts indicated that RV-8 (P = 3.7 × 10²), RV-8M (P = 2.2 × 10³), and, interestingly, also RV-8 (P = 1.1 × 10⁴) induced the expansion of this lymphoblast population (Fig. 5B). The stimulation of V11 CD4⁺ T cells by RV-8 was highly significant and was observed in other experiments. When we looked at the V12 CD4⁺ T-cell population, we observed that RV-8 (P = 4.4 × 10⁹), RV-8M (P = 3.3 × 10³), and, very weakly, RV-8 (P = 3.1 × 10²) triggered their expansion (Fig. 5C). In all other experiments, the stimulation of V12 CD4⁺ T cells by RV-8 was undetectable (data not shown). Interestingly, the expansion induced by RV-8M and RV-8 was very low compared to that of the control virus RV-8. This decreased stimulation cannot be due to lower Sag expression by RV-8M and RV-8 since these viruses stimulated V11 CD4⁺ T cells as well as RV-8 (Fig. 5B). The analysis of V4-bearing T cells (data not shown) revealed that RV-S, RV-8/S, RV-8M, and, very weakly, RV-8 triggered V4⁺ T cells, in agreement with the results obtained with BALB/c mice (Fig. 3 and 4). The expression of the activation marker CD69 measured in parallel confirmed the obtained V reactivity pattern of the tested RVs (data not shown).

View larger version (28K): [in this window] [in a new window]   FIG. 5.   V reactivity pattern in mtv-free mice observed after injection of Sag-expressing RVs. mtv-free mice were not injected (Mock) or were injected with 5 × 107 PFU of RV-8, RV-S, RV-8/S, RV-8M, RV-8, or RV-8M. The response of V5-, -11-, and -12-positive cells is indicated as a percentage of large CD4+ blasts in panels A, B, and C, respectively. V4+ CD4+ T cells from the same lymph nodes were analyzed in parallel (data not shown). Data represent mean values ± standard errors of the means of four draining lymph nodes 24 h postinfection. The experiment is representative of three performed. *, P < 0.05.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

MMTV/mtv Sags can be grouped into seven families based on the alignment of their 70 C-terminal aa (reviewed in reference 29). Within this C-terminal region, the highest sequence variability is found in the polymorphic region II containing the last 30 to 40 aa. Several studies have indeed implicated this part of the Sag molecule in the interaction with the V domain of the TCR (1, 44, 46).

Most of the knowledge about the interaction of the viral Sag with the TCR comes from studies analyzing TCR residues involved in Sag recognition. In these studies, the TCR polymorphism in wild-type mice and in rats, responsible for gain or loss of recognition of particular MMTV Sags, was used to map the key amino acids in TCR-Sag interactions (4, 8, 9, 15, 19, 33, 36). The few residues ascribed were mainly located in the HV4, but also in the CDR2 of the TCR. Although the same regions are also implicated in the recognition of bacterial Sags, the TCR binding sites for viral and bacterial Sags were shown elsewhere to be different (28).

Little is known about the precise amino acid requirements in the MMTV Sag for TCR interaction besides the already-mentioned importance of the 30 to 40 aa of the C terminus. This is undoubtedly due to the low abundance of the Sag and its lack of function in purified form. In this report, we sought to study the residues of the viral Sag that determine its interaction with the TCR by taking advantage of the closely related Sags of mtv-8 and MMTV(SIM). These Sags show 91% sequence identity in their C-terminal 70 aa but are recognized by different TCR V domains. We focused on two SIM-specific regions of the Sag because they are unique to MMTV(SIM) and its unique TCR V recognition pattern. We thus introduced into the mtv-8 backbone the MMTV(SIM)-specific mutations that we named M and  to be able to simultaneously monitor the gain of SIM-specific V TCR interaction and loss of mtv-8-specific V TCR interaction. RVs were used for the expression of wild-type or mutant mtv-8/SIM Sags in vivo, as we have previously shown that this system can be used to dissect the in vivo response to the retroviral MMTV(GR) Sag in the context of an unrelated viral infection (26).

By exchanging residues from two naturally occurring Sags, we obtained a panel of mutant proteins expressed by RVs which all had intact Sag function as monitored by the activation and expansion of Sag-reactive CD4⁺ T cells in vivo. This contrasts with the results of two recent studies addressing the impact of mutations and deletions-insertions on the function of Sag molecules (34, 45). In both studies, mutations not occurring in heterologous Sags were introduced in the polymorphic region II and led to the loss of Sag function, due in some cases to the abolition of transport to the cell surface.

In the present study, we provide the first evidence of a switch of V reactivity for a viral Sag when expressed in vivo. Thus, we confirm that the C-terminal portion of a given Sag is sufficient to confer its TCR V specificity on another Sag molecule when it is recognized in the mouse. Indeed, the exchange of the 106 C-terminal aa of the mtv-8 Sag with the 106 C-terminal aa of the MMTV(SIM) Sag results in a complete transfer of TCR V specificities, i.e., a switch from a V5, V11, and V12 to a V4 and V10^a reactivity pattern. This is in agreement with a previous report showing that the exchange of the polymorphic region II between mtv-1 Sag and mtv-7 Sag led to the exchange of the V specificity of the responding T cells in stable-transfection assays (46).

Here, we further report that the two small regions M and  of the MMTV(SIM) Sag, when transferred into the mtv-8 Sag, are sufficient to modify the V specificity of the parental Sag molecule. Importantly, the two regions of 2 and 6 aa, respectively, differentially affected the recognition by specific TCR V domains. The SIM-specific M mutation greatly decreased the V12 reactivity of the parental mtv-8 Sag, thereby indicating that amino acids F₃₁₄ and G₃₁₅ of the mtv-8 Sag play a role in the interaction with this TCR V domain. These mutations were, however, not sufficient for the induction of a SIM-specific V recognition pattern.

Interestingly, we also show that the Sag molecules containing the  region of MMTV(SIM) displayed a mixed phenotype with an acquired SIM-specific V4 reactivity while retaining an mtv-8-specific interaction with V11 and a strong reduction of mtv-8-specific V12 recognition. Importantly, the V12 reactivity was almost totally abolished despite the presence of the mtv-8 residues F₃₁₄ and G₃₁₅ shown above to be involved in its recognition. Structural changes induced by the  mutations may explain this apparent contradiction.

The combination of M and  conferred full SIM-specific V4 reactivity on the mtv-8 Sag molecule and completely abrogated its mtv-8-specific reactivity with V5, V11, and V12 CD4⁺ T cells. The two regions are, however, not sufficient to induce detectable SIM-specific V10^a reactivity in the mtv-8 Sag. Since the response to the MMTV(SIM) Sag of V10^a/c is normally higher than that of V4 (33), residues other than M and  must contribute to the interaction with V10^a/c. Only 5 aa are different between RV-8/S and RV-8M. Since all these amino acid changes can be found in Sags with V specificities other than that of MMTV(SIM) and mtv-8, a combination of one or several of them with  and/or M may be required for the recognition by V10^a.

Altogether, our results show that only a few Sag residues are involved in the interactions with specific TCR V. A similar situation is found with bacterial Sags, where two to three residues can change the TCR V specificity (21). The lack of a three-dimensional model for the MMTV Sag molecule precludes the possibility of ascribing precise positions to the interaction. We can therefore not distinguish whether M and  residues are in direct contact with the TCR V chain or whether they impose structural constraints on the Sag molecule, thereby modifying the exposure of the contact points.

Our work presents a model system that makes it possible to screen in vivo for amino acids of Sags involved in the interaction with a given TCR V domain. It conserves one of the principal Sag functions encountered in MMTV infection, namely, the activation and expansion of the TCR V-reactive CD4⁺ T cells. Another Sag function, the deletion of the amplified T cells, cannot be addressed with this model system because of the lack of a sustained Sag expression due to the rapid elimination of the RVs in the mouse.

In conclusion, we were able to precisely delineate eight residues relevant for molecular interactions between the SIM, or mtv-8, Sag and the TCR V domain within the 35 C-terminal aa of the viral Sag.