Institute for Virology, Johannes Gutenberg University, Hochhaus am Augustusplatz, 55101 Mainz, Germany1
Author for correspondence: Matthias Reddehase. Fax +49 6131 39 35604. e-mail Matthias.Reddehase{at}uni-mainz.de
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Abstract |
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Introduction |
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For mCMV, however, the specificity spectrum of cytolytic T lymphocytes (CTL) in pulmonary infiltrates predicted the existence of a multitude of subdominant antigenic peptides attributed to the E- or late (L)-phase (Holtappels et al., 1998 ). Specifically, infected target cells were preferentially lysed in the E-phase. A more recent report directly documented a leakiness of E-phase immune evasion functions in that the Dd-presented antigenic peptide 243YGPSLYRRF251 was identified in the m04 gene product gp34 (Holtappels et al., 2000
), an E-phase protein supposed to be involved in the surface transport of MHC-I molecules (Kleijnen et al., 1997
).
For hCMV, the CD8 T-cell response to ppUL83 (pp65), a constituent of the virion tegument and a major component of dense bodies, appeared to be at least as prevalent as the response to IE1-pp72 (Boppana & Britt, 1996 ; Wills et al., 1996
). In addition, an antigenic peptide has been described for the major virion envelope glycoprotein UL55-gB (Utz et al., 1992
). While ppUL83 is an L-phase protein according to its expression kinetics, entry of dense bodies and virion uncoating after penetration can feed the alternative class I pathway of antigen processing and presentation before any virus gene expression (McLaughlin-Taylor et al., 1994
). Therefore, processing of ppUL83 is not targeted by immune evasion mechanisms. It was thus an obvious and long overdue issue to determine an immunological role of mCMV homologues of hCMV ppUL83 and other hCMV virion proteins. It is the merit of D. H. Spectors group to have taken up that task. Two ORFs in the genome of mCMV were found to possess significant homology to hCMV UL83, namely ORFs M83 and M84. Based on percentage amino acid identity, pM84 (p65) is more related to ppUL83 than is the positional homologue ppM83 (pp105). However, only ppM83 proved to be analogous to ppUL83 by virtue of its late expression kinetics, phosphorylation and, most importantly, its virion association (Cranmer et al., 1996
; Morello et al., 1999
). In contrast, pM84 was identified as a nonstructural protein expressed in the E-phase (Morello et al., 1999
) and should hence be targeted by immune evasion mechanisms operating in the E-phase. Notably, however, besides ORF m123-encoded IE1-pp89 (Gonzales Armas et al., 1996
), only pM84 induced protective immunity in the H-2d haplotype after genetic immunization of BALB/c mice with an M84 expression plasmid. Plasmids specifying the mCMV positional homologues of hCMV virion proteins UL32-pp150 (corresponding to mCMV gene M32), UL48-p212 (M48), UL56-p130 (M56), pUL69 (M69), UL82-pp71 (M82), UL83-pp65 (M83), UL85-mCP (M85), UL86-MCP (M86) and UL99-pp28 (M99) did not confer in vivo protection (Morello et al., 2000
).
Based on these data, we started a survey for identifying an antigenic peptide processed from pM84 and presented by an MHC-I molecule of the H-2d haplotype. A set of synthetic peptides deduced from the pM84 sequence was selected according to Rammensees antigenic motif forecast (Falk et al., 1991 ; Rammensee et al., 1997
), which is essentially based on the identification of conserved anchor residues extending into hydrophobic pockets of MHC-I molecules. Specifically, the major nonameric motifs are x(Y or F)xxxxxx(I or L or V), xGPxxxxx(L or I or F) and x(P or S)xxxxxx(F or L or M) for the MHC-I molecules Kd, Dd and Ld, respectively. Based on empirical antigenic peptides, this forecast gets continuously refined and probability scores for antigenicity are assigned to the motifs. For example, antigenic peptides of mCMV defined by us previously, namely IE1 168YPHFMPTNL176 (Reddehase et al., 1989
) and m04 243YGPSLYRRF251 (Holtappels et al., 2000
) got scores of 26 and 28, respectively. By using the Internet database SYFPEITHI version 1.0 (http://www.uni-tuebingen.de/uni/kxi/) for MHC ligands and peptide motifs, a refined forecast was made for pM84. For all motifs containing the major anchor residues (see above) and for all Kd and Ld motifs with a score of
20, peptides were synthesized and tested for antigenicity in a previously described microculture CTL assay system (Holtappels et al., 2000
) involving three restimulations of memory CD8 T cells present in the spleens of BALB/c mice at 3 months after intraplantar infection (Fig. 1
). In the first screening experiment, M84 nonapeptides starting at N-terminal positions 33 (score of 31) and 297 (score of 28) were found to be antigenic at restimulation concentrations of 10-6 M and 10-10 M, respectively, and there appeared to be further but minor candidates. A second screening experiment, performed with an independent group of memory T-cell donors, confirmed only the antigenic peptide at position 297. Potential candidates from either screening experiment were then tested for their ability to support the generation of a CTL line (CTLL) in a bulk culture system (Holtappels et al., 2000
). The only peptide confirmed by the generation of a CTLL (see below) was the one starting at position 297 representing an MHC-I Kd motif. In conclusion, nonapeptide 297AYAGLFTPL305 represents an antigenic M84 peptide.
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The fact that the antigenic M84 peptide is presented by the Kd molecule bears an interesting side aspect. Under conditions when the m152-encoded E-phase immune evasion gene product gp37/40 of mCMV mediates retention of peptide-loaded MHC-I molecules in a cis-Golgi compartment (Ziegler et al., 1997 ), the m04 E-phase gene product gp34 directs MHC-I molecules to the cell surface, wherefrom gp34MHC-I complexes can be immunoprecipitated (Kleijnen et al., 1997
). It was an attractive speculation that gp34 may operate as a transporter for peptide-loaded MHC-I molecules to bypass m152-gp37/40-mediated retention and to aid presentation of E-phase peptides, including its own processing product, the m04 peptide 243YGPSLYRRF251 (Holtappels et al., 2000
). However, if gp34-mediated MHC-I surface transport were essential for peptide presentation in the E-phase, Kd-restricted peptides should not exist, because, unlike the situation seen with Dd and Ld, gp34Kd complexes were not detectable (Kleijnen et al., 1997
).
Despite the high coding capacity of CMVs, only a few antigenic proteins and peptides derived therefrom have been defined for MHC-I presentation to CD8 T cells. For a more comprehensive understanding of immunity to CMVs, there is an urgent need to identify antigenic peptides assigned to different phases of the virus replication cycle. Previous work by Morello et al. (2000) has predicted an antigenic peptide in the nonstructural E-phase protein pM84, one of the two mCMV homologues of the hCMV virion tegument protein ppUL83. We have identified the Kd-restricted pM84-derived antigenic peptide 297AYAGLFTPL305. It represents the second E-phase peptide of mCMV that apparently escapes the immunosubversive mechanisms operative in the E-phase. Its existence underscores our previous notion that mCMV is held in check by a redundancy of CD8 T cells recognizing antigenic peptides in different phases of virus gene expression. The mCMV model predicts that further antigenic peptides derived from E- or L-phase proteins will exist also for hCMV.
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Acknowledgments |
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References |
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Received 8 June 2000;
accepted 21 August 2000.