Institute of Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany1
Institute of Molecular Biology, University of Zürich, Winterthurer Str. 190, 8057 Zürich, Switzerland2
Children Hospital, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany3
Institute of Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany4
Author for correspondence: Stefan Niewiesk. Fax +49 931 201 3934. e-mail niewiesk{at}vim.uni-wuerzburg.de
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Abstract |
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Introduction |
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Methods |
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Infection of mice.
For generation of CTL, BALB/c mice were infected intraperitoneally (i.p.) with 8x106 p.f.u. MV (Edmonston strain) or 2x106 p.f.u. of the vaccinia virus recombinant expressing -galactosidase (vv5259/
-gal).
Preparation of UV-inactivated MV.
MV was grown on Vero cells according to standard procedures (Niewiesk et al., 1993 ) and UV-inactivated with a UV handset (wavelength 254 nm) for 10 min; 100 µg UV-inactivated virus did not yield infectious virus on Vero cells.
Cells and peptides.
RMA-S cells stably transfected with H2-Ld were obtained from Hans-Georg Rammensee, Tübingen, Germany, C1R-Ld cells from Peter Cresswell, Yale, USA, and T2-Ld cells from Jörg Reimann, Ulm, Germany. All cell lines were cultured in RPMI10% FCS.
Peptides YPALGLHEF [MV N protein aa 281289, Ld-restricted (Beauverger et al., 1993 )] and TPHPARIGL [
-galactosidase, aa 876884, Ld-restricted (Gavin et al., 1993
)] were synthesized and purified (>90%) by Jerini Biotools, Berlin, Germany.
Generation and culture of CTL.
For generation of CD8+ T cells, spleen cells from BALB/c mice were irradiated (30 Gy) and infected with MV (Edmonston strain) at an m.o.i. of 1 or incubated with epitope peptide (50 µg/107 cells) at 37 °C for 2 h; 3x106 infected stimulators were cultivated in an upright 50 ml flask containing 15 ml RPMI10% FCS and 1·5x107 spleen cells from infected animals. After 7 days, living cells were separated on a Percoll gradient (1·083 g/ml) and plated at a density of 106cells/ml in tissue culture plates. For restimulation spleen cells (106 cells/ml) infected with virus or incubated with peptide (see above) and 2% rat spleen Concanavalin A supernatant (ConA-S) were added. T cells were tested for their cytolytic activity 5 days after restimulation.
Cytotoxicity assay.
If not otherwise indicated target cells were infected with MV (m.o.i. 1) and CDV (m.o.i. 2) for 48 h and with recombinant vaccinia viruses (m.o.i. 5) overnight. For inhibition of antigen processing cells were incubated with chloroquine (Sigma) at 50 µM for 48 h and subsequently cell viability was tested by trypan blue exclusion. 106 cells were labelled with 3·7 MBq Na51CrO4 (DuPont) for 80 min at 37 °C and washed twice. If peptide was used, it was added during the labelling step (50 µg/106 cells). 104 labelled target cells in a volume of 50 µl were added to various numbers of T cells in 100 µl volumes in U-bottomed microtitre plates. After 5 h incubation at 37 °C, 75 µl supernatant was harvested and counted. The percentage lysis was calculated as: 100x(experimental-spontaneous release)/(total-spontaneous release).
Elispot for interferon-
-producing CTL.
Elispot assays for IFN- were performed with slight modifications according to established protocols (Fujihashi et al., 1993
; Sarawar & Doherty, 1994
). Briefly, 96-well nitrocellulose-based microtitre plates (Milititre HA; Milipore) were coated overnight at 4 °C with anti-IFN-
MAb clone 1-D1-K at a concentration of 15 µg/ml in PBS (Hölzel Diagnostik, Köln, Germany). After washing with PBS, 1x105 lymphocytes were added to the wells with uninfected or MV-infected APC and incubated for 24 h at 37 °C. After removal of the cells biotinylated anti-IFN-
MAb clone 7-B61 (Hölzel Diagnostik) was added, and incubated overnight at 4 °C. Plates were washed with PBS and streptavidin-conjugated alkaline phosphatase (Hölzel Diagnostik) was added, followed by 2 h incubation at room temperature. Spots representing individual IFN-
-secreting cells were visualized using the Bio-Rad alkaline phosphatase conjugate substrate kit. Wells were photographed with a Wild Fotomikroskop M400 camera (Wild, Heebrugg, Germany). Spots were counted after slide projection. All assays were performed in triplicate.
Vaccinia virus recombinants.
Recombinant vaccinia viruses expressing epitope aa 281289 on the MV-N protein together with a signal sequence for targeting the epitope into the ER, -galactosidase (vv5259/
-gal) and MV-N protein have been described previously (Neumeister & Niewiesk, 1998
).
Construction and rescue of recombinant MV-lacZ virus.
In order to generate a recombinant MV expressing -galactosidase, the green fluorescent protein (GFP) cassette cloned into the additional transcription unit (ATU) between the P and M gene of p(+)MPlrGFPV (Singh et al., 1999
) was replaced by a lacZ expression cassette. P(+)MPlrGFPV was cut with SnaBI and BssHII and the larger cleavage product containing the MV antigenome and the plasmid backbone was treated with Klenow polymerase, and subsequently dephosphorylated using calf intestinal alkaline phosphatase (CIAP) according to published protocols. The lacZ gene was isolated from plasmid pCMV lacZ (Kupfer et al., 1994
) by cleaving with PstI and PpuMI. After treatment with Klenow polymerase, the lacZ fragment was blunt-ligated into the MV vector backbone. Transformed bacteria were then screened by colony hybridization. Correct orientation of the insert as well as proper blunt-end ligation were verified by sequencing. MV shares a particular property with other Paramyxovirinae. For efficient replication and to generate functional infectious particles, the genome length has to be a multiple of six. The remaining MV antigenome after cleavage of p(+)MPlrGFPV retained 15998 nt, and the isolated lacZ transcription unit had a size of 3196 nt which after ligation with the MV backbone gave rise to the 19194 nt MV-lacZ antigenome, which is a multiple of six.
Confocal laser microscopy.
Infected and uninfected T2-Ld and C1R-Ld cells were incubated with MAbs specific for MV-N [F227 (ter Meulen et al., 1981 )], MV-M [B117 (ter Meulen et al., 1981
)], MV-H [L77 (ter Meulen et al., 1981
)] or Ld (Pharmingen) for 1 h at room temperature and then detected with a donkey anti-mouse IgG serum conjugated with Cy3 (Dianova). Subsequently, cells were incubated with an FITC-conjugated MAb specific for human CD107a (LAMP-1) from Pharmingen (MO18753) for 1 h at room temperature. Cells were examined with a Leica DM IRB by confocal laser scanning imaging using the Leica TCS 4D system. Images of both labels were obtained simultaneously with an argonkrypton laser as the source of light and were superimposed. Crosstalk between the two fluorochromes was excluded by taking control images of both labellings separately.
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Results |
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-Galactosidase expression by recombinant MV leads to TAP-independent recognition by CTL
As MV was able to introduce both MV-N and MV-M into the endolysosomal compartment we investigated whether a bacterial protein like -galactosidase could be introduced into the TAP-independent processing pathway by MV. To this end we produced a recombinant MV expressing
-galactosidase (MV-lacZ). This protein is recognized by Ld-restricted CTL via the epitope aa 876884 (Rammensee et al., 1989
). The stable expression of
-galactosidase over 15 passages was followed by Western blot analysis (Fig. 5a
). Recognition of MV-lacZ by Ld-restricted
-galactosidase CTL was compared to recognition of the vaccinia virus recombinant vv5259/
-gal which has been described previously (Neumeister & Niewiesk, 1998
). This recombinant produces in addition to a Kk-restricted epitope high levels of
-galactosidase and is recognized in P815 cells by CTL (data not shown). T2-Ld cells infected with MV-lacZ were recognized by CTL specific for epitope 876884 (Fig. 5b
). In contrast, T2-Ld cells infected with vv52-59/
-gal were not recognized by CTL.
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Discussion |
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Most often (non-infectious) viral proteins are fed into the endolysosome by exocytosis. However, in the case of parainfluenza virus type 1 (PIV-1; Sendai virus) (Zhou et al., 1993 ), canine distemper virus (this study), MV fusion protein (Gromme et al., 1999
) and N protein (this study) infection and therefore protein synthesis in the cytoplasm led to recognition in a TAP-independent and chloroquine-sensitive manner. In contrast to a recombinant vaccinia virus, MV and CDV are able to present the N protein as well as
-galactosidase protein in a TAP-independent manner. During MV infection, the ribonucleoprotein (RNP) complex containing the viral RNA, polymerase, phosphoprotein (P) and N protein forms and is translocated to the cell surface where it is enveloped into a budding virus. It has been shown that MV-N (Narang, 1973
), MV-P and the matrix (M) protein (which links the RNP complex to the envelope) (Cathomen et al., 1998
) accumulate intracellularly in small aggregates. Some of these aggregates containing MV-M and MV-N colocalize with LAMP-1 in the endolysosomal compartment (this study). The expression of MV-N alone did not reveal targeting into the endolysosomal compartment. It therefore is likely that the transport of MV-N into this localization is only possible in the context of the RNP complex or as part of an aberrant intracellular budding process. PIV-1, CDV and MV belong to the Paramyxoviridae family, share the same mode of replication and budding and are recognized in a TAP-independent fashion. It is interesting to note that a completely unrelated protein like
-galactosidase can be presented in a TAP-independent manner only after expression by a recombinant MV. The intracellular assembly of MV-RNP and the multiple interactions of the viral proteins with cellular motor proteins could influence the uptake of
-galactosidase to the lysosome by a yet undefined mechanism. To fully understand the underlying mechanism, a precise knowledge of the stepwise assembly of paramyxoviruses in intracellular compartments is worth determining.
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Acknowledgments |
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Footnotes |
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References |
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Beauverger, P., Buckland, R. & Wild, F. T.(1993). Measles virus antigens induce both type-specific and canine distemper virus cross-reactive cytotoxic T lymphocytes in mice: localization of a common Ld-restricted nucleoprotein epitope. Journal of General Virology 74, 2357-2363.[Abstract]
Cathomen, T., Mrkic, B., Spehner, D., Drillien, R., Naef, R., Pavlovic, J., Aguzzi, A., Billeter, M. A. & Cattaneo, R.(1998). A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain. EMBO Journal 17, 3899-3908.
Fujihashi, K., McGhee, J. R., Beagley, K. W., McPherson, D. T., McPherson, S. A., Huang, C. M. & Kiyono, H.(1993). Cytokine-specific ELISPOT assay. Single cell analysis of IL-2, IL-4 and IL-6 producing cells. Journal of Immunological Methods 160, 181-189.[Medline]
Gavin, M. A., Gilbert, M. J., Riddell, S. R., Greenberg, P. D. & Bevan, M. J.(1993). Alkalis hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes. Journal of Immunology 151, 3971-3980.
Gromme, M., Uytdehaag, F. G. C. M., Janssen, H., Calafat, J., van Binnendijk, R. S., Kenter, M. J. H., Tulp, A., Verwoerd, D. & Neefjes, J.(1999). Recycling MHC class I molecules and endosomal peptide loading. Proceedings of the National Academy of Sciences, USA 96, 10326-10331.
Jondal, M., Schirmbeck, R. & Reimann, J.(1996). MHC class I-restricted CTL responses to exogenous antigens. Immunity 5, 295-302.[Medline]
Kupfer, J. M., Ruan, X. M., Liu, G., Matloff, J., Forrester, J. & Chaux, A.(1994). High-efficiency gene transfer to autologous rabbit jugular vein grafts using adenovirus-transferrin/polylysine-DNA complexes. Human Gene Therapy 5, 1437-1443.[Medline]
Liu, T., Zhou, X., Örvell, C., Lederer, E., Ljunggren, H.-G. & Jondal, M.(1995). Heat-inactivated Sendai virus can enter multiple MHC class I processing pathways and generate cytotoxic T lymphocyte responses in vivo. Journal of Immunology 154, 3147-3155.
Nanan, R., Carstens, C. & Kreth, H. W.(1995). Demonstration of virus-specific CD8+ memory T cells in measles-seropositive individuals by in vitro peptide stimulation. Clinical and Experimental Immunology 102, 40-45.[Medline]
Narang, H. K.(1973). Electron-microscopic study of measles virus in lymphocytes of affected children. Journal of Hygiene 71, 447-451.[Medline]
Neumeister, C. & Niewiesk, S.(1998). Recognition of measles virus-infected cells by CD8+ T cells depends on the H-2 molecule. Journal of General Virology 79, 2583-2591.[Abstract]
Niewiesk, S., Brinckmann, U., Bankamp, B., Sirak, S., Liebert, U. G. & ter Meulen, V.(1993). Susceptibility to measles virus-induced encephalitis in mice correlates with impaired antigen presentation to cytotoxic T lymphocytes. Journal of Virology 67, 75-81.[Abstract]
Rammensee, H. G., Schild, H. S. & Theopold, U.(1989). Protein-specific cytotoxic T lymphocytes. Recognition of transfectants expressing intracellular, membrane-associated or secreted forms of beta-galactosidase. Immunogenetics 30, 296-302.[Medline]
Sarawar, S. R. & Doherty, P. C.(1994). Concurrent production of interleukin-2, interleukin-10, and gamma interferon in the regional lymph nodes of mice with influenza pneumonia. Journal of Virology 68, 3112-3119.[Abstract]
Schirmbeck, R. & Reimann, J.(1996). 'Empty Ld molecules capture peptides from endocytosed hepatitis B surface antigen particles for major histocompatibility complex class I restricted presentation. European Journal of Immunology 26, 2812-2822.[Medline]
Schirmbeck, R., Melber, K. & Reimann, J.(1995). Hepatitis B virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I-restricted epitope presentation. European Journal of Immunology 25, 1063-1070.[Medline]
Schirmbeck, T., Thoma, S. & Reimann, J.(1997). Processing of exogenous hepatitis B surface antigen particles for Ld-restricted epitope presentation depends on exogenous 2-microglobulin. European Journal of Immunology 27, 3471-3484.[Medline]
Sigal, L. J., Crotty, S., Andino, R. & Rock, K. L.(1999). Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen. Nature 398, 77-80.[Medline]
Singh, M., Cattaneo, R. & Billeter, M. A.(1999). A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice. Journal of Virology 73, 4823-4828.
ter Meulen, V., Löffler, S., Carter, M. J. & Stephenson, J. R.(1981). Antigenic characterization of measles and SSPE virus haemagglutinin by monoclonal antibodies. Journal of General Virology 57, 357-364.[Abstract]
Zhou, X., Glass, R., Liu, T., Ljunggren, H.-G. & Jondal, M.(1993). Antigen processing mutant T2 cells present viral antigen restricted through H-2Kb. European Journal of Immunology 23, 1802-1808.[Medline]
Received 2 August 2000;
accepted 10 October 2000.