Early gene m18, a novel player in the immune response to murine cytomegalovirus

Rafaela Holtappels1, Natascha K. A. Grzimek1, Doris Thomas1 and Matthias J. Reddehase1

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


   Abstract
Top
Abstract
Main text
References
 
The identification of all antigenic peptides encoded by a pathogen, its T cell ‘immunome’, is a research aim for rational vaccine design. Screening of proteome-spanning peptide libraries or computational prediction is used to identify antigenic peptides recognized by CD8 T cells. Based on their high coding capacity, cytomegaloviruses (CMVs) could specify numerous antigenic peptides. Yet, current evidence indicates that the memory CD8 T cell response in a given haplotype is actually focused on a few viral proteins. CMVs actively interfere with antigen processing and presentation by the expression of immune evasion proteins. In the case of murine CMV (mCMV), these proteins are effectual in the early (E) phase of the virus replication cycle and should thus preclude the presentation of peptides derived from E proteins. Notably, the m18 gene is here added to a growing list of mCMV E genes that encode antigenic peptides in spite of the E phase immune evasion strategies of the virus.


   Main text
Top
Abstract
Main text
References
 
For more than a decade since its identification in 1989, the IE1 protein (pp89)-derived peptide 168YPHFMPTNL176, which is encoded by the immediate-early (IE) gene m123 exon 4 (ie1) and presented by the major histocompatibility complex (MHC) class I molecule Ld, had remained the only known antigenic peptide of murine CMV (mCMV) (Reddehase et al., 1989 ). Its immunodominance appeared to be explained conveniently by the later finding that mCMV encodes early (E) phase immune evasion proteins that interfere at various steps with the MHC class I pathway of antigen processing and presentation (reviewed by Hengel et al., 1998 , 1999 ; see also Reddehase, 2000 ). Specifically, the m152 gene product gp37/40 mediates the retention of peptide-loaded MHC class I {beta}2-microglobulin complexes in a cis-Golgi compartment (Del Val et al., 1992 ; Ziegler et al., 1997 , 2000 ), the m04 gene product gp34 serves as a chaperone for MHC class I molecules (Kleijnen et al., 1997 ) and the m06 gene product gp48 re-routes MHC class I molecules for lysosomal degradation (Reusch et al., 1999 ). Collectively, these redundant mechanisms should preclude effectively the presentation of peptides derived from E proteins, whereas presentation of the IE1 peptide can occur during the IE phase and is prevented only after expression of the immune evasion proteins in the E phase (Reddehase et al., 1986 ; Del Val et al., 1989 ).

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).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1. Identification of an antigenic peptide in the ORFm18 protein. (a) HindIII map of the mCMV Smith strain (ATCC VR-194/1981) genome showing the locations of the ORFm18 (m18) and ORFm164 (m164) proteins and the sequences of the antigenic peptides therein. Molecular masses were deduced from the amino acid sequence (Rawlinson et al., 1996 ). So far, neither of the two proteins has been characterized biochemically. (b) Mutually exclusive recognition of the m18 and m164 peptides. CTLL were generated as described previously (Holtappels et al., 2002 ). The cytolysis assay was performed with P815 (H-2d) target cells pulsed with synthetic peptide at the indicated molar concentrations. (c) Co-elution of naturally processed peptides m18 and m164. m18-CTLL and m164-CTLL were used as responder cells in a cytolysis assay performed with P815 target cells that were pulsed with aliquots of HPLC fractions. Solid line, HPLC fractions containing naturally processed peptides derived from the acidic extract of foetal fibroblasts in the E phase of the virus replication cycle. Dashed line, HPLC fractionation of acidic extract of uninfected foetal fibroblasts supplemented alternatively with synthetic peptides m18 (top) or m164 (bottom). Throughout, the effector:target cell ratio was 15:1.

 
Then, we used successfully the synthetic m18 peptide in a restimulation concentration of 10-10 M for the generation of a long-term CTLL, here referred to as m18-CTLL, from memory spleen cells, according to a protocol described recently for the generation of m164-CTLL (Holtappels et al., 2002 ). As it is documented in Fig. 1(b), recognition of the Dd-presented m18 and m164 peptides by m18-CTLL and m164-CTLL was mutually exclusive, which proved the specificity of these CTLL and excluded cross-reactive recognition of these two peptides. We emphasize this as being an important control, because cross-reactive recognition of the m164 peptide by m18-CTLL could have mimicked a false co-elution of the two peptides. Dd restriction of both CTLL was confirmed by the lysis of peptide-pulsed L-Dd transfectants and the absence of lysis with peptide-pulsed transfectants L-Kd and L-Ld as target cells (data not shown). On these firm grounds we could address finally the question of whether the m18 peptide was indeed the hidden fraction 22 peptide that we had been seeking.

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 {alpha}-helices (PredictProtein PHDhtm; Rost et al., 1996 ).

The expression kinetics of the m18 gene in productively infected foetal fibroblasts was studied by RT–PCR 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).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 2. Characterization of gene m18 as an E phase gene. (a) Location map of gene m18 and of the primers and probe for the PCR amplification and Southern blot detection of an m18 cDNA fragment. Map positions refer to the sequence of mCMV strain Smith ATCC VR-194 (Rawlinson et al., 1996 ; GenBank accession no. MCU68299). (b) Kinetics and drug sensitivity of gene m18 transcription. RT–PCR was performed with purified poly(A)+ RNA isolated from mouse foetal fibroblasts at the indicated times after infection (multiplicity of 4 IU per cell). Oligo(dT) priming was used for the RT reaction. Conditions for RT–PCR were as described previously (Holtappels et al., 2001 ), except that the annealing temperature in the PCR amplification of the cDNA fragment was 64 °C. An ethidium bromide-stained gel is shown on the left; M, 100 bp size markers. A corresponding Southern blot autoradiograph obtained after hybridization with the {gamma}-32P-end-labelled oligonucleotide probe is shown on the right. ActD, actinomycin D (5 µg/ml); CH, cycloheximide (100 µg/ml); PAA, phosphonoacetic acid (250 µg/ml).

 
Finally, we asked for the quantitative contribution of the newly identified m18 peptide to the priming of an mCMV-specific immune response and to the establishment of CD8 T cell memory (Fig. 3). As a measure of this contribution, we determined the frequencies of immunomagnetically purified CD8 T cells responding in interferon (IFN)-{gamma}-based ELISPOT assays. The total number of responsive cells was determined by polyclonal stimulation via the signal-transducing CD3{epsilon} molecule of the T cell receptor–CD3 complex (CD3{epsilon}-redirected ELISPOT assay; Holtappels et al., 2001 ). Frequencies of mCMV-specific CD8 T cells were measured by stimulation with the antigenic peptides of mCMV known currently (listed in Fig. 3a). The primary immune response to mCMV was analysed in the draining popliteal lymph node on day 8 after intraplantar infection (Fig. 3b). Memory was assessed for the CD8 T cell population in the spleen during latent infection (Reddehase et al., 1994 ) at 8 months after priming (Fig. 3c). In accordance with previous results (Holtappels et al., 2002 ), peptides IE1 and m164 co-dominated the immune response to mCMV during acute infection as well as during the memory state after clearance of productive infection. As discussed recently in greater detail (Holtappels et al., 2002 ), the memory ‘immunome’ of mCMV, which is the repertoire of antigenic peptides involved in immunological memory, is more focused than the acute response immunome. ‘Bystander activation’ of CD8 T cells (Tough et al., 1996 ; McNally et al., 2001 ) with specificities for peptides unrelated to mCMV is likely to contribute to the gap between CD3{epsilon}-reactive cells and mCMV peptide-specific cells during the acute response. The new information is that the m18 peptide takes the third rank in immunogenicity among the antigenic peptides of mCMV known currently. Notably, while CD8 T cells specific for peptides IE1 and m164 are enriched in the memory cell pool during latency (Holtappels et al., 2000c , 2002 ), this is apparently not the case for m18-specific CD8 T cells (Fig. 3c).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3. Contribution of the m18 peptide to the CD8 T cell response. (a) Compilation of antigenic peptides of mCMV known currently. IE, immediate-early; E, early; L, late. Gene M83 is transcribed in the E phase, but the protein is detected in significant amounts only in the L phase (Cranmer et al., 1996 ). (b) Frequencies of acutely sensitized CD8 T cells present in the draining popliteal lymph node on day 8 after intraplantar mCMV infection (1x106 p.f.u.). IFN-{gamma}-based ELISPOT assays were performed with immunomagnetically purified CD8 T cells. P815-B7 stimulator cells were pulsed with a saturating dose (10-8 M) of the indicated mCMV peptides. {alpha}CD3, polyclonal stimulation with 145-2C11 hybridoma cells that produce monoclonal antibody directed against mouse CD3{epsilon}; {emptyset}, P815-B7 cells with no peptide added; {bullet}, data from triplicate assay cultures. Median values are marked by vertical bars. (c) Frequencies of memory CD8 T cells present in the spleen at 8 months after intraplantar mCMV infection. Details are as in (b).

 
It was surmised previously that MHC allele preferences contribute to the immunodominance of peptides. Specifically, the finding that immunodominant peptides are frequently presented in the H-2d haplotype by the Ld molecule was explained by a constitutively low loading of Ld with self peptides providing a higher number of accessible binding sites for foreign peptides (Lie et al., 1991 ). The list of antigenic peptides of mCMV now available allows us to revisit that view. At least, there is no strict correlation between the presenting MHC molecule and peptide immunogenicity: Ld and Dd both present dominant as well as subdominant peptides of mCMV.

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.


   Acknowledgments
 
Support was provided by a grant to M.J.R. from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 490, individual project B1 ‘Immune control of latent CMV infection’.


   References
Top
Abstract
Main text
References
 
Alterio de Goss, M., Holtappels, R., Steffens, H.-P., Podlech, J., Angele, P., Dreher, L., Thomas, D. & Reddehase, M. J. (1998). Control of cytomegalovirus in bone marrow transplantation chimeras lacking the prevailing antigen-presenting molecule in recipient tissues rests primarily on recipient-derived CD8 T cells. Journal of Virology 72, 7733-7744.[Abstract/Free Full Text]

Bresnahan, W. A. & Shenk, T. (2000). A subset of viral transcripts packaged within human cytomegalovirus particles. Science 288, 2373-2376.[Abstract/Free Full Text]

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 cytomegalovirus–survival 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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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, 390–396, 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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Received 16 August 2001; accepted 9 October 2001.