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1 Unidad de Inmunología Viral, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Pozuelo km 2, E-28220 Majadahonda, Madrid, Spain
2 Unidad de Proteómica, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Pozuelo km 2, E-28220 Majadahonda, Madrid, Spain
3 Unidad de Biología Viral, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Pozuelo km 2, E-28220 Majadahonda, Madrid, Spain
4 Centro de Biología Molecular Severo Ochoa, CSIC/Universidad Autónoma de Madrid, E-28049 Madrid, Spain
Correspondence
Margarita Del Val
mdval{at}isciii.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and also affects immunocompromised patients and the elderly. Studies of human RSV in a mouse model have shown that cytotoxic T-lymphocytes (CTLs) play a role in both lung pathology and virus clearance (Cannon et al., 1988; Graham et al., 1991). The fusion (F) protein of RSV is an important target for virus-specific CTLs (Pemberton et al., 1987) and neutralizing antibodies (Olmsted et al., 1986). Immunization of mice with recombinant vaccinia virus (rVACV) (Olmsted et al., 1986; Stott et al., 1987) or plasmid DNA encoding the F protein (Li et al., 1998; Bembridge et al., 2000), which induce F-specific antibodies and CTLs, confers protection against challenge with RSV. Human RSV F glycoprotein is a type I integral membrane protein synthesized as a polypeptide precursor, F0, which is cleaved at residues 109 and 136 by furin-like proteases (González-Reyes et al., 2001; Zimmer et al., 2001). Cleavage yields two chains, F1 and F2, which remain linked by a disulfide bond. Furin cleavage at sites I and II is necessary for the F protein to become fusion active (González-Reyes et al., 2001; Zimmer et al., 2001). Upon activation, the F protein experiences conformational changes that allow insertion of the hydrophobic fusion peptide, located at the N terminus of the F1 chain, into the target membrane for virus-mediated membrane fusion.
CTLs are able to specifically recognize and eliminate virus-infected cells. Recognition takes place through a specific interaction between a T-cell receptor of the CTL and a major histocompatibility complex (MHC) class I molecule presenting a viral peptide on the surface of the virus-infected cell. In general, peptides presented on MHC class I molecules are 8–10 residues long and derived from products of the processing of intracellular viral proteins by the proteasome multi-catalytic complex in the cytosol. Correct C termini must be generated by endoproteases such as the proteasome and tripeptidyl peptidase II (Reits et al., 2004; Guil et al., 2006), as carboxypeptidase activity is limited in this classical antigen-processing pathway (Powis et al., 1996). Peptides of 8–16 residues that survive extensive cytosolic peptidase activity (Reits et al., 2003, 2004) are efficiently translocated by the transporters associated with antigen processing (TAP) to the lumen of the endoplasmic reticulum (ER), where N-terminal residues can be further trimmed by the ER aminopeptidases ERAAP/ERAP (Serwold et al., 2002; York et al., 2002; Tanioka et al., 2003; Saveanu et al., 2005). Of all the ER peptides, only a small number is selected by their affinity to the peptide-binding groove of nascent MHC class I heavy chains. Chaperones assist in the assembly of a stable heterotrimeric complex formed by the nascent MHC class I heavy chain, β2-microglobulin and the peptide. The assembled MHC/peptide complex then exits the ER and is transported through the Golgi to the cell surface following the constitutive secretory route.
Alternative antigen-processing and -presentation pathways have been described (Del Val & López, 2002; Johnstone & Del Val, 2007). For instance, in TAP-deficient cells, different routes have been reported that are able to deliver peptides to receptive MHC class I molecules for their presentation to CTLs. Some of these peptides are processed and presented independently of TAP after liberation of leader peptides by a signal peptidase in the ER (Henderson et al., 1992; Wei & Cresswell, 1992), release of peptides by furin from proteins that mature in the trans-Golgi network (Gil-Torregrosa et al., 1998, 2000), processing of transmembrane proteins by endosomal cathepsins (Tiwari et al., 2007) or liberation of epitopes belonging to proteins associated or not with membranes by other undefined endoproteases in the secretory route (Hammond et al., 1993, 1995; Elliott et al., 1995; Snyder et al., 1998). Entry from the cytosol to the ER by unknown mechanisms has also been reported for plasmid- or virus-encoded peptides (Zweerink et al., 1993; Hammond et al., 1995; Snyder et al., 1997; Lautscham et al., 2001). Furthermore, although peptides presented on MHC class I molecules are usually processed from endogenous antigens, peptides processed from exogenous antigens can also be presented (Liu et al., 1995). TAP-independent processing and loading of endogenous or exogenous antigen-derived peptides onto recycling MHC class I molecules can occur in endolysosomal acidic compartments (Grommé et al., 1999; Schirmbeck & Reimann, 2002).
In general, an antigen presented to CTLs in cells infected with a virus is also recognized by CTLs when expressed from rVACV. Given the number of described classical and alternative pathways, there is remarkably little information concerning the proteolytic processing pathways of these antigens in their natural viral context compared with infection with rVACV.
As TAP-independent presentation has been reported for paramyxovirus proteins (Zhou et al., 1993; Grommé et al., 1999; Neumeister et al., 2001) and proteins that mature by furin allow TAP-independent presentation (Gil-Torregrosa et al., 1998, 2000), we sought to investigate the TAP requirements for RSV F antigen presentation. Specifically, we assessed processing and presentation of two epitopes, F85–93 (Chang et al., 2001) and F249–258 (Johnstone et al., 2004), presented by the same murine MHC class I molecule, Kd. Epitope F85–93 is located in the smaller N-terminal F2 chain, whereas epitope F249–258 is located in the larger C-terminal F1 chain hosting the fusion peptide, the transmembrane region and several neutralizing-antibody antigenic sites (López et al., 1998). In this study, TAP-independent presentation was found for both epitopes. Interestingly, major differences were found in RSV F protein epitopes F85–93 and F249–258 processing and presentation pathways, depending on the epitope, the protein context and the viral context. This underscores both the diversity of pathways and the influence of virus infection on presentation of epitopes to CTLs.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Construction of rVACV vvF containing the F gene of the Long strain of RSV has been described previously (Bembridge et al., 1998). rVACV vvFsig– encodes a cytosolic F protein in which the first 21 aa corresponding to the signal peptide have been deleted, introducing a new AUG codon before the sequence encoding residue 22 (Bembridge et al., 1999). rVACV NPM147–155 (Bacik et al., 1994) and SNPA147–155 (Eisenlohr et al., 1992a) express influenza nucleoprotein epitope NP147–155 from a cytosolic or a secretory minigene, respectively. All viruses were based on the Western Reserve (WR) wild-type (wt) vaccinia strain.
Cell lines.
T2/Kd is a human lymphoblastoid T2 cell line deficient in TAP and transfected with Kd; T2/Kd/TAP cells are also transfected with rat TAP1 and TAP2 genes (Zhou et al., 1994). Both cell lines were provided by Dr G. Hämmerling (German Cancer Research Centre, Heidelberg, Germany). All T2 cells were cultured in RPMI 1640 supplemented with 10 % fetal bovine serum and 5x10–5 M 2-mercaptoethanol in a 5 % CO2 atmosphere and at 37 °C.
Polyclonal CTL lines.
CTL lines were named to indicate the virus used for priming in vivo and the agent used for restimulation in vitro. Generation of CTL lines has been described previously (Johnstone et al., 2004). Briefly, splenocytes were obtained from vvF-infected BALB/c mice (H-2d haplotype). The CTL F/F85–93 and CTL F/F249–258 lines were generated by stimulation with 10–10 M F85–93 peptide or 10–9 M F249–258 peptide, respectively. Peptide sequences are KYKNAVTEL for F85–93 and TYMLTNSELL for F249–258. Peptide synthesis has been described previously (Johnstone et al., 2004). Generation of CTL NP147–155 has been described previously (Guil et al., 2006). All animal studies were reviewed and approved by an appropriate institutional review committee.
Virus infection and intracellular cytokine staining (ICS) assay.
ICS assays to detect recognition by CTLs of antigens presented on infected cells were performed as described previously (Chen et al., 2000) with modifications (Johnstone et al., 2004). CTL lines were stimulated in the presence of 10 µg brefeldin A (BFA; Sigma) ml–1 with T2/Kd or T2/Kd/TAP target cells infected with RSV or rVACV. Stimulation was performed for 4 h when targets were infected with RSV or overnight when targets were infected with rVACV. Infection with RSV was performed at a m.o.i. of 0.3–3; the virus inoculum was removed by thorough washing after 90 min adsorption and infection was allowed to proceed for 16–40 h prior to ICS. Infection with rVACV was performed at a m.o.i. of 3 as described previously (Eisenlohr et al., 1992b); the virus inoculum was removed by thorough washing after 1 h adsorption and infection was allowed to proceed for 4–6 h, unless otherwise stated, prior to ICS. For infection at time 0 h, adsorption was performed at 4 °C and virus inoculum was removed by washing in the presence of BFA prior to the assay. Adsorption in the presence of inhibitors was performed at an inhibitor concentration of fivefold the concentration maintained during infection. BFA was used during infection at 5 µg ml–1. Infection in the presence of the proteasome inhibitor lactacystin (LC) (Dr E. J. Corey, Harvard University, MA, USA) was carried out at 10 µM LC. The percentage specific inhibition was calculated from the formula: 100x{[(X–N)–(Xi–Ni)]/(X–N)} where X represents the percentage of total CD8+ lymphocytes that were stained with monoclonal antibody (mAb) to gamma interferon (IFN-
) when targets were infected with RSV or rVACV, Xi is the value when infection was carried out in presence of the inhibitor, and N and Ni are the equivalent values when targets were mock infected with medium or infected with the rVACV parental strain WR. Exogenous peptide, when used, was added during infection and ICS assay co-culture. Co-culture was performed at an effector-to-target ratio of 1 : 5. Following stimulation during co-culture, cells were incubated with fluorescein isothiocyanate-conjugated anti-CD8
mAb (ProImmune), fixed with Intrastain kit reagent A (DakoCytomation) and incubated with phycoerythrin-conjugated mAb to IFN- (Becton Dickinson), while permeabilizing with Intrastain kit reagent B (Johnstone et al., 2004). Events were acquired using a FACSCalibur flow cytometer (Becton Dickinson) and data were analysed using CellQuest software (Becton Dickinson). A mean of 5000 CD8+ cells was analysed in each sample. Several experiments were also duplicated with the more classical 51Cr-release cytotoxicity assay and gave essentially the same results.
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RESULTS |
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, left panels). Thus, we concluded that epitope F249–258 is presented through a TAP-independent pathway when the F protein is in its natural viral context. In contrast, epitope F85–93 was not presented independently of TAP.
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). Activation of CTL F/F249–258 was inhibited when ICS was performed with targets infected for 16 h with RSV in the presence of BFA (Fig. 1a
, right panels). Therefore, we concluded that TAP-independent presentation of epitope F249–258 from RSV F protein is endogenous. It should be noted that, throughout this and all other infections performed in the presence of an inhibitor, inhibition was specific, as the inhibitor did not affect the presentation of low concentrations of exogenously added synthetic peptides (data not shown).
In order also to assess TAP-dependent processing, T2/Kd/TAP cells (T2/Kd cells transfected with TAP) were infected with RSV and ICS assays were performed with CTL F/F85–93 and CTL F/F249–258 (Fig. 1b, left panels). Activation of both CTL lines was observed, indicating that epitope F85–93 was presented through a TAP-dependent pathway when the F protein was in its natural viral context. In addition, CTL F/F249–258 were activated slightly more than with TAP-deficient cells and at a lower m.o.i., suggesting, but not demonstrating, the additional existence of a TAP-dependent pathway for presentation of epitope F249–258. Presentation was endogenous, as activation of CTL F/F85–93 and CTL F/F249–258 was inhibited when infections were carried out in the presence of BFA for the longest time period possible without BFA being toxic for the cells (Fig. 1b, right panels). Therefore, we concluded that, when epitopes are processed from the F protein in its natural viral context, RSV, presentation pathways are endogenous, dependent on TAP for epitope F85–93 and mostly TAP-independent for epitope F249–258 (see Table 1 for a summary of all results).
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) was toxic under conditions for RSV infections (data not shown). In addition, the fact that a recombinant RSV lacking the F protein is not viable precluded further controls. Furthermore, although sufficient for antigen presentation, viral protein expression by T2 cells was lower after RSV infection than after rVACV infection (data not shown). Therefore, the viral context of the antigen was changed in order to study further the processing and presentation of epitopes F85–93 and F249–258. When ICS assays were performed with TAP-deficient T2/Kd targets infected with rVACV encoding the wt sequence of human RSV F protein (vvF, depicted schematically in Fig. 2), CTL F/F85–93 and CTL F/F249–258 were both activated (Fig. 3a). These results indicated that, in the case of rVACV-expressed F protein, epitopes F85–93 and F249–258 could both be processed independently of TAP, in contrast to F249–258 TAP-independent presentation only in RSV-infected targets. Thus, by introducing the F protein in a different viral context, which did not alter its expression at the plasma membrane (Bembridge et al., 1999), a new TAP-independent pathway was available for presentation of epitope F85–93 (Table 1).
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). In general, membrane or secretory proteins have an N-terminal signal sequence, which allows co-translational translocation to the ER through the translocon and which is cleaved by the signal peptidase in the ER. Thus, the rule is that proteins without a signal sequence will not be able to access the constitutive endogenous secretory route. Accordingly, after rVACV infection, we have shown previously that TAP-deficient T2 cells do not present epitopes from cytosolic proteins, but do so from their secretory counterparts (Gil-Torregrosa et al., 1998, 2000, and unpublished results). However, when CTL F/F249–258 were co-cultured with TAP-deficient targets expressing the cytosolic form of the F protein, they were activated (Fig. 3a), indicating that epitope F249–258 was processed and presented by a TAP-independent pathway that could be accessed by the cytosol-confined Fsig– protein (Table 1). In contrast, as expected and serving as an internal control, CTLs F/F85–93 were not activated by the same vvFsig–-infected T2/Kd targets (Fig. 3a), strongly suggesting that epitope F85–93 was being processed and presented through a TAP-independent pathway following the endogenous secretory route inaccessible to the cytosolic F protein.
F protein lacking a signal sequence is known to be degraded rapidly within infected cells and thus is barely detected by immunofluorescence (Bembridge et al., 1999). Precursor or final peptides derived from processing of the cytosolic F protein might be transported to the ER by TAP-independent mechanisms. To ensure that no leakiness existed for peptides in our TAP-deficient experimental setting, T2/Kd cells were infected with rVACV encoding a cytosolic or secretory minigene of an influenza virus NP epitope presented by Kd. As expected, activation of the corresponding specific CTLs with TAP-deficient T2/Kd cells was only achieved after expression of the secretory epitope, whilst TAP-competent cells also activated CTLs when expressing the cytosolic epitope (Fig. 3b). We therefore concluded that T2/Kd cells are not leaky for peptides and consider the possibility that the cytosolic F protein or derived products may enter vesicular compartments by mechanisms independent of both signal sequence and TAP, gaining access to a compartment proficient in processing the F249–258 epitope.
To study the endogenous nature of the TAP-independent presentation of epitopes F85–93 and F249–258, infections were performed with or without BFA. Again, CTL F/F85–93 and CTL F/F249–258 behaved differently in ICS assays (Fig. 3c). TAP-independent presentation of epitope F85–93 from wt F protein expressed by rVACV was endogenous, as 100 % specific inhibition by BFA was observed. This was not the case for TAP-independent presentation of epitope F249–258 from wt or cytosolic F proteins expressed by rVACV, for which less than 50 % specific inhibition by BFA was achieved, indicating a strong BFA-resistant (BFAR) component in presentation.
TAP-independent entry to the ER of hydrophobic peptides generated by cytosolic processing has been shown for Epstein–Barr virus latent membrane protein LMP2 epitopes (Lautscham et al., 2001). Although this is an exceptional case, we next studied whether TAP-independent presentation of both epitopes was dependent on the proteasome. TAP-deficient T2/Kd targets were infected with vvF or vvFsig– in the presence of LC, and ICS assays were performed with CTLs specific for either epitope (Fig. 3d, e). LC had no effect on CTL activation, demonstrating that the proteasome was not involved in the TAP-independent presentation of epitope F85–93 from native F protein or in the TAP-independent BFAR presentation of epitope F249–258 from native or cytosolic F proteins.
We therefore concluded that, when the F protein is expressed by rVACV, the epitopes under study are processed and presented differently in the absence of TAP, each following distinct TAP-independent antigen-processing and -presentation pathways. Epitope F85–93 follows a TAP-independent endogenous BFA-sensitive (BFAS) pathway, whereas epitope F249–258 is processed through a TAP-independent BFAR pathway also accessible to a cytosolic form of the F protein expressed by rVACV. These striking results are in contrast to the absence of a TAP-independent F85–93 presentation pathway, as well as to the endogenous nature of the TAP-independent presentation of F249–258, in RSV-infected cells (Table 1).
rVACV-expressed epitope F85–93 is also processed by the proteasome in a TAP-dependent pathway, unlike epitope F249–258
As presentation of epitope F85–93 is dependent on TAP when the F protein is in its natural viral context, TAP-dependent pathways were further assessed in rVACV-infected cells. T2/Kd/TAP cells were infected with vvF or vvFsig– and ICS assays were performed with CTL F/F85–93 or CTL F/F249–258. In rVACV-infected cells, epitope F85–93 could also be processed through a pathway that was dependent on TAP, as CTLs F/F85–93, which were not activated by TAP-deficient targets expressing the cytosolic F protein (Fig. 3a), were now activated (Fig. 4a). Moreover, the increase in activation of CTL F/F85–93 by vvF-infected T2/Kd/TAP targets compared with vvF-infected T2/Kd targets indicated that TAP-dependent pathways were significantly contributing, together with TAP-independent pathways, to the overall presentation of epitope F85–93 (Table 1). These differences were not observed for CTL F/F249–258 (compare Figs 3a and 4a). In addition, these results controlled the strength of the cytosolic confinement of the Fsig– protein, as well as the integrity of the F85–93 epitope in this cytosolic protein.
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). Presentation of epitope F249–258 from the wt or cytosolic F protein in T2/Kd/TAP cells was again not fully susceptible to BFA inhibition. Specific inhibition was slightly higher than that observed in TAP-deficient T2/Kd cells, suggesting, but not demonstrating, the existence of an endogenous TAP-dependent pathway for epitope F249–258 as well in rVACV-infected cells (Table 1).
To investigate whether presentation of RSV F epitopes is proteasome-dependent, experiments were carried out with the proteasome-specific inhibitor LC. T2/Kd/TAP cells were infected with vvF or vvFsig– in the presence of LC, and ICS assays were performed with CTL F/F85–93 or CTL F/F249–258 (Fig. 4c). Again, a different behaviour for each CTL line was observed. LC had only a minor effect on activation of CTL F/F249–258. Thus, no evidence supporting the processing of epitope F249–258 through a proteasome-dependent pathway was found. In contrast, activation of CTLs F/F85–93 was highly inhibited by LC. Thus, we can conclude that epitope F85–93 can be presented through the classical TAP-dependent pathway involving degradation by the proteasome (Table 1). Although technically not possible to address, it is likely that proteasomes also process the F85–93 epitope in RSV-infected cells (Fig. 1b).
We thus showed that, when the F protein is expressed by rVACV, presentation of epitope F85–93 in TAP-competent cells is contributed by two different endogenous (BFAS) pathways: the classical presentation pathway mediated by the proteasome and a VACV-specific TAP-independent endogenous pathway. Proteasomal products of the F protein are therefore produced in infected cells. However, these proteasomal products were not involved in the endogenous TAP-independent presentation of epitope F85–93, as LC barely inhibited activation of CTL F/F85–93 by T2/Kd targets infected with vvF (Fig. 3d). This result additionally showed the absence of leakiness of proteasome-produced precursor peptides in our TAP-deficient cells.
In conclusion, major differences are found in RSV F protein epitope F85–93 and F249–258 processing and presentation pathways, depending on the epitope, the protein context and the viral context.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, there is little information comparing the processing pathways of antigens in their natural viral context and in infection with rVACV. Our results indicate that antigen-processing routes may differ more often than assumed. Differences in the capacity of inducing CTLs in vivo depending on the viral context have been reported once, as CTLs specific for a herpes simplex virus epitope were induced in TAP-deficient mice immunized with DNA or rVACV, but not when infected with the natural virus (Paliard et al., 2001). TAP-independent presentation of another paramyxovirus epitope has been observed when the antigen is expressed from the natural virus and not from rVACV (Neumeister et al., 2001). In contrast, our results indicate that VACV also allows TAP-independent presentation of both RSV F protein CTL epitopes. The fact that antigen-processing pathways differ more often than assumed depending on the viral context must be taken into account when choosing a viral vector to study CTL responses in vitro or in vivo.
In addition, the presentation pathways can also be influenced by the protein context and the subcellular localization of the antigen. Thus, epitope F85–93 could be presented by a TAP-dependent mechanism from either natural or cytosolic F protein expressed by rVACV, but was only presented from natural F by a TAP-independent pathway (Table 1). In contrast, TAP-independent presentation of F249–258 did also occur from the cytosolic F protein (Table 1). TAP-independent mechanisms involving unidentified proteases located in different compartments have been described for presentation of the influenza virus epitope NP147–155 when inserted in a chimeric cellular protein and expressed by rVACV (Snyder et al., 1997, 1998). The relevance of our results lies in showing this for the first time for a natural protein, RSV F protein, and unveiling a novel BFAR, TAP-independent route of antigen presentation.
TAP-independent presentation of only a few paramyxovirus proteins has been documented (Zhou et al., 1993; Liu et al., 1995; Grommé et al., 1999; Neumeister et al., 2001) and it is reported here for RSV for the first time. In RSV-infected cells, only epitope F249–258 was presented through a TAP-independent pathway, whereas when the F protein was expressed by rVACV, epitopes F85–93 and F249–258 could both be presented through contrasting (BFAS or BFAR) TAP-independent processing pathways (Table 1). In addition, both F protein epitopes were also very different with regard to presentation dependent on TAP, as this pathway could only be unequivocally detected for epitope F85–93 in both viral contexts (Table 1). These major qualitative differences in the processing and presentation of epitopes F85–93 and F249–258 may underlie the immunodominance pattern observed in in vitro-generated CTL lines and in vivo secondary responses (Johnstone et al., 2004). The processing by the proteasome of epitope F85–93, but not of epitope F249–258, may contribute to the immunodominance of F85–93.
Epitope F85–93 was presented through an apparently classical endogenous (BFAS) TAP-dependent pathway when the F protein was expressed in its natural viral context, RSV. This pathway was also prominent when F was expressed from rVACV in TAP-positive cells. In contrast, for F85–93 TAP-independent presentation, the viral context seemed to be critical. Thus, expression of the natural F protein from rVACV allowed the alternative processing of epitope F85–93 through an endogenous (BFAS) and proteasome-independent TAP-independent pathway, which was evident in TAP-deficient cells and which was not accessible to protein Fsig– (Table 1). Probably, TAP-independent presentation of epitope F85–93 requires the action of an endoprotease of the secretory pathway other than signal peptidase. Given the extreme sensitivity of CTLs, it is unlikely that lower protein expression levels from RSV than from rVACV in T2 cells could account for differences in the processing pathways of F85–93 depending on the viral context. These differences are more likely to be related to differences in the biology of RSV and rVACV, for example in terms of their interaction with secretory compartments, which, in the case of rVACV, might help bypass TAP requirements for presentation of epitope F85–93. In fact, dual presentation of CTL epitopes by TAP-dependent and TAP-independent pathways has also been observed in cells infected with rVACV encoding the human immunodeficiency virus (HIV) envelope glycoprotein (Hammond et al., 1995), but the effect of the viral context (rVACV or HIV) was not addressed in that study.
Epitope F249–258 was presented almost exclusively through TAP-independent pathways and not by the classical TAP-dependent antigen-processing route followed by most antigens described so far. Notably, these TAP-independent pathways for epitope F249–258 were different depending on the viral context of the F protein. In RSV-infected cells, presentation was endogenous (BFAS), whereas after infection with rVACV encoding natural or cytosolic F, presentation was mostly BFAR (Table 1). The different biology of infection by each virus might explain the fact that this access of peptides to BFAR compartments was only detected in rVACV-infected cells and not in RSV-infected cells, as rVACV strongly interacts with the intracellular membranous system. Also, rVACV induces stronger shut-off of host-cell macromolecular synthesis compared with RSV, which would favour BFAR presentation, possibly by recycling MHC class I molecules. Concerning TAP independency, rVACV-infected cells otherwise show strict TAP-dependent presentation of cytosolic proteins (Snyder et al., 1997, 1998; Gil-Torregrosa et al., 1998, 2000; Fig. 3b), as exemplified by the F85–93 epitope as an internal control. Therefore, special features of the F protein may also play a role in the TAP-independent BFAR presentation of F249–258, affecting only this epitope and not F85–93. For instance, hydrophobic regions of the F1 chain such as the fusion peptide or the transmembrane region might favour translocation of cytosolic F249–258 precursors to distal BFAR compartments in the secretory pathway by an uncharacterized mechanism, independent of both TAP and the signal sequence, as postulated previously (Zweerink et al., 1993; Hammond et al., 1995; Snyder et al., 1997; Lautscham et al., 2001). The remarkable fact that epitope F249–258 is presented to CTLs mostly through a BFAR pathway is of interest for RSV immunization studies in murine models, as it opens up the possibility currently under investigation of priming CTLs in vivo with non-infectious (i.e. UV- or heat-inactivated) virus or even purified F protein.
The relevance in vivo of the different TAP requirements for presentation of the studied epitopes is currently complicated to address, as TAP-deficient knockout mice have an H-2b haplotype. However, the recent identification of RSV epitopes in this genetic background may open up new possibilities (Lukens et al., 2006). Overall, our results emphasize the diversity of cellular mechanisms available for presentation of epitopes to CTLs, even for epitopes that are processed from the same antigen and are presented by the same MHC class I molecule. The influence of the viral context on the accessibility of processing pathways is underscored, highlighting the versatility of VACV for this type of study.
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ACKNOWLEDGEMENTS |
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Received 31 March 2008;
accepted 26 May 2008.
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) presented by Kd assessed in this study were F85–93 (KYKNAVTEL) and F249–258 (TYMLTNSELL). Virus vvFsig– encoded a cytosolic form of the F protein lacking the signal peptide.
