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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This smooth view of mCMV antigen presentation was called into question by the finding that CD8-positive ex vivo cytolytic T lymphocytes (CTL) isolated from pulmonary infiltrates during mCMV pneumonia lysed infected foetal fibroblasts preferentially in the E phase (Holtappels et al., 1998 ). MHC restriction of that recognition predicted the existence of antigenic E phase peptides presented by MHC class I molecules. In addition, control of mCMV in the Ld gene-deletion mutant BALB/c-H-2dm2 (Alterio de Goss et al., 1998
) indicated the existence of antigenic peptide(s) presented by Kd and/or Dd. As a fancy of nature, the immune evasion protein pORFm04 (gp34) was the first E protein of mCMV for which an antigenic peptide was identified, namely peptide 243YGPSLYRRF251 presented by Dd (Holtappels et al., 2000a
). Further E proteins instantly followed. Specifically, mCMV homologues of human CMV (hCMV) pUL83 (pp65), namely pM83 (pp105) and pM84 (p65) (Cranmer et al., 1996
; Morello et al., 1999
, 2000
), were found to account for antigenic peptides 761YPSKEPFNF769 (Holtappels et al., 2001
) and 297AYAGLFTPL305 (Holtappels et al., 2000b
, 2001
) presented by Ld and Kd, respectively. However, these three E peptides represented subdominant antigenic peptides. While CTL lines (CTLL) with the respective peptide specificities were all protective in preemptive cytoimmunotherapy of mCMV disease (Holtappels et al., 2000a
, 2001
), the quantitative contribution of these peptides to the priming of an immune response during mCMV infection was minimal to undetectable (Holtappels et al., 2000c
, 2001
). The idea that subdominance might be a characteristic feature of E phase peptides soon proved to be unfounded: very recent work identified a dominant peptide in E protein pORFm164 (Holtappels et al., 2002
). Peptide 257AGPPRYSRI265 is presented by Dd and elicits acute and memory CD8 T cells at a frequency that compares to IE1.
Naturally processed m164 peptide was found to elute in fractions 22 and 23 of a high performance liquid chromatography (HPLC) separation of peptides present in lysates of foetal fibroblasts in the late phase of infection (Holtappels et al., 2002 ). Notably, in that study, an enzyme-linked immunospot (ELISPOT) assay performed with the naturally processed peptides present in fractions 22 and 23 revealed a frequency of responding memory CD8 T cells that was higher than the frequency obtained after stimulation with a saturating dose of synthetic m164 peptide. This finding had thus indicated the existence of at least one further antigenic peptide co-eluting with the m164 peptide.
How can this hidden peptide be disclosed? We knew from previous experience that mass spectrometry is not a promising approach, because an HPLC fraction represents a mixture of different peptides, including many self peptides, all present in a low absolute amount. Support came from the analysis of MHC restriction of antigenic activity. In a previous report, we had shown already that short-term microculture CTLL raised with the fraction 22 and 23 eluates lysed only L-Dd transfectants pulsed with the same eluates, but not L-Ld or L-Kd transfectants (Holtappels et al., 2000a ). As a consequence, like the m164 peptide, the unknown peptide co-eluting must be presented by Dd. In two previous reports in which the Dd-restricted m04 (Holtappels et al., 2000a
) and m164 (Holtappels et al., 2002
) peptides were identified, we had already performed genome-wide screenings of Dd-binding motifs based on the forecast developed by Rammensee and co-workers (reviewed by Rammensee et al., 1997
). Both screenings had indicated the existence of minor activities, which we had, at that time, not followed any further. One such minor activity was visible in both previous screenings and mapped to genomic position C19158 within HindIII fragment A of the mCMV Smith strain genome (Ebeling et al., 1983
; Rawlinson et al., 1996
; GenBank accession no. MCU68299). The corresponding peptide 346SGPSRGRII254 is part of the ORFm18 protein (Fig. 1a
).
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Target cells were pulsed with the HPLC fractions obtained from lysates of foetal fibroblasts in the E phase of infection and the two CTLL were used as effector cells for the localization of the respective peptides (Fig. 1c). Both CTLL detected their cognate naturally processed peptide in fraction 22 and, to a lesser extent, fraction 23. In addition, HPLC retention was determined for the corresponding synthetic peptides and both were found to elute with a peak in fraction 22 (Fig. 1c
, dashed line).
In conclusion, the m18 and m164 peptides do indeed co-elute and the hidden fraction 22 peptide is now identified as the m18 peptide 346SGPSRGRII254.
The UL18 gene of hCMV, the positional homologue of the mCMV m18 gene, has evoked great interest, as it is a sequence and structural homologue of MHC class I molecules proposed to be involved in the silencing of natural killer cells by binding to inhibitory receptors (Reyburn et al., 1997 ). Recently, it has also been identified as a ligand of the leukocyte immunoglobulin-like receptor LIR-1 (Chapman et al., 1999
; reviewed by Cosman et al., 1999
). However, the mCMV m18 gene is not a sequence homologue of cellular MHC class I or hCMV UL18 [indicated by the lowercase letter m, according to the nomenclature used by Rawlinson et al. (1996)
].
So far, nothing is known about the biological role of the ORFm18 protein in the virus life cycle. According to structure prediction algorithms, the protein is largely coiled and does not possess membrane-spanning -helices (PredictProtein PHDhtm; Rost et al., 1996
).
The expression kinetics of the m18 gene in productively infected foetal fibroblasts was studied by RTPCR performed with purified poly(A)+ RNA using oligo(dT) priming for reverse transcription. Map positions of the m18 gene as well as PCR primers and probe for the detection of m18 cDNA are illustrated in Fig. 2(a). The absence of transcripts after infection in the presence of actinomycin D confirmed that m18 RNA is not virion RNA (Bresnahan & Shenk, 2000
). Newly synthesized mRNA was detected first at 2 h post-infection. As indicated by sensitivity to cycloheximide, this transcription required preceding IE protein synthesis, whereas insensitivity to phosphonoacetic acid showed its independence of viral DNA replication. All in all, these data clearly identified gene m18 as an E gene (Fig. 2b
).
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In conclusion, we have identified pORFm18 as a further E protein of mCMV that contributes significantly to the CD8 T cell response to infection in spite of E phase immune evasion mechanisms. Thus, it appears that the prevention of antigenic peptide presentation by the immune evasion proteins of mCMV, so far documented only for the presentation of the IE1 peptide in fibroblasts, represents an exception rather than the rule.
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Bresnahan, W. A. & Shenk, T. (2000). A subset of viral transcripts packaged within human cytomegalovirus particles. Science 288, 2373-2376.
Chapman, T. L., Heikeman, A. P. & Bjorkman, P. J. (1999). The inhibitory receptor LIR-1 uses a common binding interaction to recognize class I MHC molecules and the viral homolog UL18. Immunity 11, 603-613.[Medline]
Cosman, D., Fanger, N. & Borges, L. (1999). Human cytomegalovirus, MHC class I and inhibitory signalling receptors: more questions than answers. Immunological Reviews 168, 177-185.[Medline]
Cranmer, L. D., Clark, C. L., Morello, C. S., Farrell, H. E., Rawlinson, W. D. & Spector, D. H. (1996). Identification, analysis, and evolutionary relationships of the putative murine cytomegalovirus homologs of the human cytomegalovirus UL82 (pp71) and UL83 (pp65) matrix phosphoproteins. Journal of Virology 70, 7929-7939.[Abstract]
Del Val, M., Münch, K., Reddehase, M. J. & Koszinowski, U. H. (1989). Presentation of CMV immediate-early antigen to cytolytic T lymphocytes is selectively prevented by viral genes expressed in the early phase. Cell 58, 305-315.[Medline]
Del Val, M., Hengel, H., Hacker, H., Hartlaub, U., Ruppert, T., Lucin, P. & Koszinowski, U. H. (1992). Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment. Journal of Experimental Medicine 176, 729-738.[Abstract]
Ebeling, A., Keil, G. M., Knust, E. & Koszinowski, U. H. (1983). Molecular cloning and physical mapping of murine cytomegalovirus DNA. Journal of Virology 47, 421-433.[Medline]
Hengel, H., Brune, W. & Koszinowski, U. H. (1998). Immune evasion by cytomegalovirussurvival strategies of a highly adapted opportunist. Trends in Microbiology 6, 190-197.[Medline]
Hengel, H., Reusch, U., Gutermann, A., Ziegler, H., Jonjic, S., Lucin, P. & Koszinowski, U. H. (1999). Cytomegaloviral control of MHC class I function in the mouse. Immunological Reviews 168, 167-176.[Medline]
Holtappels, R., Podlech, J., Geginat, G., Steffens, H.-P., Thomas, D. & Reddehase, M. J. (1998). Control of murine cytomegalovirus in the lungs: relative but not absolute immunodominance of the immediate-early 1 nonapeptide during the antiviral cytolytic T-lymphocyte response in pulmonary infiltrates. Journal of Virology 72, 7201-7212.
Holtappels, R., Thomas, D., Podlech, J., Geginat, G., Steffens, H.-P. & Reddehase, M. J. (2000a). The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells. Journal of Virology 74, 1871-1884.
Holtappels, R., Thomas, D. & Reddehase, M. J. (2000b). Identification of a Kd-restricted antigenic peptide encoded by murine cytomegalovirus early gene M84. Journal of General Virology 81, 3037-3042.
Holtappels, R., Pahl-Seibert, M.-F., Thomas, D. & Reddehase, M. J. (2000c). Enrichment of immediate-early 1 (m123/pp89) peptide-specific CD8 T cells in a pulmonary CD62Llo memory-effector cell pool during latent murine cytomegalovirus infection of the lungs. Journal of Virology 74, 11495-11503.
Holtappels, R., Podlech, J., Grzimek, N. K. A., Thomas, D., Pahl-Seibert, M.-F. & Reddehase, M. J. (2001). Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologues of human cytomegalovirus tegument protein ppUL83 (pp65). Journal of Virology 75, 6584-6600.
Holtappels, R., Thomas, D., Podlech, J. & Reddehase, M. J. (2002). Two antigenic peptides from genes m123 and m164 of murine cytomegalovirus quantitatively dominate CD8 T-cell memory in the H-2d haplotype. Journal of Virology 76, 151-164.
Kleijnen, M. F., Huppa, J. B., Lucin, P., Mukherjee, S., Farrell, H. E., Campbell, A. E., Koszinowski, U. H., Hill, A. B. & Ploegh, H. L. (1997). A mouse cytomegalovirus glycoprotein, gp34, forms a complex with folded class I MHC molecules in the ER which is not retained but is transported to the cell surface. EMBO Journal 16, 685-694.
Lie, W.-R., Myers, N. B., Connolly, J. M., Gorka, J., Lee, D. R. & Hansen, T. H. (1991). The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure. Journal of Experimental Medicine 173, 449-459.[Abstract]
McNally, J. M., Zarozinski, C. C., Lin, M. Y., Brehm, M. A., Chen, H. D. & Welsh, R. M. (2001). Attrition of bystander CD8 T cells during virus-induced T-cell and interferon responses. Journal of Virology 75, 5965-5976.
Morello, C. S., Cranmer, L. D. & Spector, D. H. (1999). In vivo replication, latency, and immunogenicity of murine cytomegalovirus mutants with deletions in the M83 and M84 genes, the putative homologs of human cytomegalovirus pp65 (UL83). Journal of Virology 73, 7678-7693.
Morello, C. S., Cranmer, L. D. & Spector, D. H. (2000). Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65). Journal of Virology 74, 3696-3708.
Rammensee, H.-G., Bachmann, J. & Stevanovic, S. (1997). MHC ligands and peptide motifs. Molecular Biology Intelligence Unit, Landes Bioscience, Austin, TX, USA.
Rawlinson, W. D., Farrell, H. E. & Barrell, B. G. (1996). Analysis of the complete DNA sequence of murine cytomegalovirus. Journal of Virology 70, 8833-8849.[Abstract]
Reddehase, M. J. (2000). The immunogenicity of human and murine cytomegaloviruses. Current Opinion in Immunology 12, 390396, 738.[Medline]
Reddehase, M. J., Fibi, M. R., Keil, G. M. & Koszinowski, U. H. (1986). Late-phase expression of a murine cytomegalovirus immediate-early antigen recognized by cytolytic T lymphocytes. Journal of Virology 60, 1125-1129.[Medline]
Reddehase, M. J., Rothbard, J. B. & Koszinowski, U. H. (1989). A pentapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes. Nature 337, 651-653.[Medline]
Reddehase, M. J., Balthesen, M., Rapp, M., Jonjic, S., Pavic, I. & Koszinowski, U. H. (1994). The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease. Journal of Experimental Medicine 179, 185-193.[Abstract]
Reusch, U., Muranyi, W., Lucin, P., Burgert, H. G., Hengel, H. & Koszinowski, U. H. (1999). A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation. EMBO Journal 18, 1081-1091.
Reyburn, H. T., Mandelboim, O., Vales-Gomez, M., Davis, D. M., Pazmany, L. & Strominger, J. L. (1997). The class I MHC homologue of human cytomegalovirus inhibits attack by natural killer cells. Nature 386, 514-517.[Medline]
Rost, B., Fariselli, P. & Casadio, R. (1996). Topology prediction for helical transmembrane proteins at 86% accuracy. Protein Science 5, 1704-1718.
Tough, D. F., Borrow, P. & Sprent, J. (1996). Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272, 1947-1950.[Abstract]
Ziegler, H., Thäle, R., Lucin, P., Muranyi, W., Flohr, T., Hengel, H., Farrell, H., Rawlinson, W. & Koszinowski, U. H. (1997). A mouse cytomegalovirus glycoprotein retains MHC class I complexes in the ERGIC/cis-Golgi compartments. Immunity 6, 57-66.[Medline]
Ziegler, H., Muranyi, W., Burgert, H. G., Kremmer, E. & Koszinowski, U. H. (2000). The luminal part of the murine cytomegalovirus glycoprotein gp40 catalyses the retention of MHC class I molecules. EMBO Journal 19, 870-881.
Received 16 August 2001;
accepted 9 October 2001.