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. 1996 Oct 1;184(4):1319–1329. doi: 10.1084/jem.184.4.1319

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

PMCID: PMC2192835  PMID: 8879204

Abstract

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules.

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Selected References

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  1. Abastado J. P., Miller P. F., Jackson B. M., Hinnebusch A. G. Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol Cell Biol. 1991 Jan;11(1):486–496. doi: 10.1128/mcb.11.1.486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bjorkman P. J., Saper M. A., Samraoui B., Bennett W. S., Strominger J. L., Wiley D. C. Structure of the human class I histocompatibility antigen, HLA-A2. Nature. 1987 Oct 8;329(6139):506–512. doi: 10.1038/329506a0. [DOI] [PubMed] [Google Scholar]
  3. Bjorkman P. J., Saper M. A., Samraoui B., Bennett W. S., Strominger J. L., Wiley D. C. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature. 1987 Oct 8;329(6139):512–518. doi: 10.1038/329512a0. [DOI] [PubMed] [Google Scholar]
  4. Bonetti B., Fu L., Moon J., Bedwell D. M. The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. J Mol Biol. 1995 Aug 18;251(3):334–345. doi: 10.1006/jmbi.1995.0438. [DOI] [PubMed] [Google Scholar]
  5. Boon T., Van Pel A. T cell-recognized antigenic peptides derived from the cellular genome are not protein degradation products but can be generated directly by transcription and translation of short subgenic regions. A hypothesis. Immunogenetics. 1989;29(2):75–79. doi: 10.1007/BF00395854. [DOI] [PubMed] [Google Scholar]
  6. Cohen T., Muller R. M., Tomita Y., Shibahara S. Nucleotide sequence of the cDNA encoding human tyrosinase-related protein. Nucleic Acids Res. 1990 May 11;18(9):2807–2808. doi: 10.1093/nar/18.9.2807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cossins J., Gould K., Brownlee G. G. Peptides shorter than a minimal CTL epitope may have a higher binding affinity than the epitope for the class I Kk molecule. Virology. 1993 Aug;195(2):851–854. doi: 10.1006/viro.1993.1443. [DOI] [PubMed] [Google Scholar]
  8. Coulie P. G., Lehmann F., Lethé B., Herman J., Lurquin C., Andrawiss M., Boon T. A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7976–7980. doi: 10.1073/pnas.92.17.7976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cox J. H., Galardy P., Bennink J. R., Yewdell J. W. Presentation of endogenous and exogenous antigens is not affected by inactivation of E1 ubiquitin-activating enzyme in temperature-sensitive cell lines. J Immunol. 1995 Jan 15;154(2):511–519. [PubMed] [Google Scholar]
  10. Donzé O., Damay P., Spahr P. F. The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging. Nucleic Acids Res. 1995 Mar 11;23(5):861–868. doi: 10.1093/nar/23.5.861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eisenlohr L. C., Bacik I., Bennink J. R., Bernstein K., Yewdell J. W. Expression of a membrane protease enhances presentation of endogenous antigens to MHC class I-restricted T lymphocytes. Cell. 1992 Dec 11;71(6):963–972. doi: 10.1016/0092-8674(92)90392-p. [DOI] [PubMed] [Google Scholar]
  12. Eisenlohr L. C., Yewdell J. W., Bennink J. R. Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes. J Exp Med. 1992 Feb 1;175(2):481–487. doi: 10.1084/jem.175.2.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elliott T., Bodmer H., Townsend A. Recognition of out-of-frame major histocompatibility complex class I-restricted epitopes in vivo. Eur J Immunol. 1996 May;26(5):1175–1179. doi: 10.1002/eji.1830260532. [DOI] [PubMed] [Google Scholar]
  14. Engelhard V. H. Structure of peptides associated with class I and class II MHC molecules. Annu Rev Immunol. 1994;12:181–207. doi: 10.1146/annurev.iy.12.040194.001145. [DOI] [PubMed] [Google Scholar]
  15. Falk K., Rötzschke O., Stevanović S., Jung G., Rammensee H. G. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature. 1991 May 23;351(6324):290–296. doi: 10.1038/351290a0. [DOI] [PubMed] [Google Scholar]
  16. Fearon K., McClendon V., Bonetti B., Bedwell D. M. Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family. J Biol Chem. 1994 Jul 8;269(27):17802–17808. [PubMed] [Google Scholar]
  17. Fetten J. V., Roy N., Gilboa E. A frameshift mutation at the NH2 terminus of the nucleoprotein gene does not affect generation of cytotoxic T lymphocyte epitopes. J Immunol. 1991 Oct 15;147(8):2697–2705. [PubMed] [Google Scholar]
  18. Frank J., Zhu J., Penczek P., Li Y., Srivastava S., Verschoor A., Radermacher M., Grassucci R., Lata R. K., Agrawal R. K. A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature. 1995 Aug 3;376(6539):441–444. doi: 10.1038/376441a0. [DOI] [PubMed] [Google Scholar]
  19. Germain R. N., Margulies D. H. The biochemistry and cell biology of antigen processing and presentation. Annu Rev Immunol. 1993;11:403–450. doi: 10.1146/annurev.iy.11.040193.002155. [DOI] [PubMed] [Google Scholar]
  20. Grant C. M., Miller P. F., Hinnebusch A. G. Requirements for intercistronic distance and level of eukaryotic initiation factor 2 activity in reinitiation on GCN4 mRNA vary with the downstream cistron. Mol Cell Biol. 1994 Apr;14(4):2616–2628. doi: 10.1128/mcb.14.4.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hahn Y. S., Braciale V. L., Braciale T. J. Presentation of viral antigen to class I major histocompatibility complex-restricted cytotoxic T lymphocyte. Recognition of an immunodominant influenza hemagglutinin site by cytotoxic T lymphocyte is independent of the position of the site in the hemagglutinin translation product. J Exp Med. 1991 Sep 1;174(3):733–736. doi: 10.1084/jem.174.3.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hatfield D., Oroszlan S. The where, what and how of ribosomal frameshifting in retroviral protein synthesis. Trends Biochem Sci. 1990 May;15(5):186–190. doi: 10.1016/0968-0004(90)90159-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Heemels M. T., Ploegh H. Generation, translocation, and presentation of MHC class I-restricted peptides. Annu Rev Biochem. 1995;64:463–491. doi: 10.1146/annurev.bi.64.070195.002335. [DOI] [PubMed] [Google Scholar]
  24. Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol. 1987 Aug 20;196(4):947–950. doi: 10.1016/0022-2836(87)90418-9. [DOI] [PubMed] [Google Scholar]
  25. Kozak M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989 Nov;9(11):5134–5142. doi: 10.1128/mcb.9.11.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kozak M. Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8301–8305. doi: 10.1073/pnas.87.21.8301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  29. Kozak M. Regulation of translation in eukaryotic systems. Annu Rev Cell Biol. 1992;8:197–225. doi: 10.1146/annurev.cb.08.110192.001213. [DOI] [PubMed] [Google Scholar]
  30. Lovett P. S., Rogers E. J. Ribosome regulation by the nascent peptide. Microbiol Rev. 1996 Jun;60(2):366–385. doi: 10.1128/mr.60.2.366-385.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luukkonen B. G., Tan W., Schwartz S. Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance. J Virol. 1995 Jul;69(7):4086–4094. doi: 10.1128/jvi.69.7.4086-4094.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Macejak D. G., Sarnow P. Internal initiation of translation mediated by the 5' leader of a cellular mRNA. Nature. 1991 Sep 5;353(6339):90–94. doi: 10.1038/353090a0. [DOI] [PubMed] [Google Scholar]
  33. Mackett M., Smith G. L., Moss B. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol. 1984 Mar;49(3):857–864. doi: 10.1128/jvi.49.3.857-864.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Malarkannan S., Afkarian M., Shastri N. A rare cryptic translation product is presented by Kb major histocompatibility complex class I molecule to alloreactive T cells. J Exp Med. 1995 Dec 1;182(6):1739–1750. doi: 10.1084/jem.182.6.1739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McBratney S., Chen C. Y., Sarnow P. Internal initiation of translation. Curr Opin Cell Biol. 1993 Dec;5(6):961–965. doi: 10.1016/0955-0674(93)90077-4. [DOI] [PubMed] [Google Scholar]
  36. Oukka M., Riché N., Kosmatopoulos K. A nonimmunodominant nucleoprotein-derived peptide is presented by influenza A virus-infected H-2b cells. J Immunol. 1994 May 15;152(10):4843–4851. [PubMed] [Google Scholar]
  37. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  38. Roelse J., Grommé M., Momburg F., Hämmerling G., Neefjes J. Trimming of TAP-translocated peptides in the endoplasmic reticulum and in the cytosol during recycling. J Exp Med. 1994 Nov 1;180(5):1591–1597. doi: 10.1084/jem.180.5.1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rötzschke O., Falk K., Deres K., Schild H., Norda M., Metzger J., Jung G., Rammensee H. G. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature. 1990 Nov 15;348(6298):252–254. doi: 10.1038/348252a0. [DOI] [PubMed] [Google Scholar]
  40. Shastri N., Gonzalez F. Endogenous generation and presentation of the ovalbumin peptide/Kb complex to T cells. J Immunol. 1993 Apr 1;150(7):2724–2736. [PubMed] [Google Scholar]
  41. Shastri N., Nguyen V., Gonzalez F. Major histocompatibility class I molecules can present cryptic translation products to T-cells. J Biol Chem. 1995 Jan 20;270(3):1088–1091. doi: 10.1074/jbc.270.3.1088. [DOI] [PubMed] [Google Scholar]
  42. Sibille C., Chomez P., Wildmann C., Van Pel A., De Plaen E., Maryanski J. L., de Bergeyck V., Boon T. Structure of the gene of tum- transplantation antigen P198: a point mutation generates a new antigenic peptide. J Exp Med. 1990 Jul 1;172(1):35–45. doi: 10.1084/jem.172.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stark H., Mueller F., Orlova E. V., Schatz M., Dube P., Erdemir T., Zemlin F., Brimacombe R., van Heel M. The 70S Escherichia coli ribosome at 23 A resolution: fitting the ribosomal RNA. Structure. 1995 Aug 15;3(8):815–821. doi: 10.1016/s0969-2126(01)00216-7. [DOI] [PubMed] [Google Scholar]
  44. Sweetser M. T., Morrison L. A., Braciale V. L., Braciale T. J. Recognition of pre-processed endogenous antigen by class I but not class II MHC-restricted T cells. Nature. 1989 Nov 9;342(6246):180–182. doi: 10.1038/342180a0. [DOI] [PubMed] [Google Scholar]
  45. Townsend A. R., Bastin J., Gould K., Brownlee G. G. Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence. Nature. 1986 Dec 11;324(6097):575–577. doi: 10.1038/324575a0. [DOI] [PubMed] [Google Scholar]
  46. Townsend A. R., Gotch F. M., Davey J. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell. 1985 Sep;42(2):457–467. doi: 10.1016/0092-8674(85)90103-5. [DOI] [PubMed] [Google Scholar]
  47. Townsend A., Bodmer H. Antigen recognition by class I-restricted T lymphocytes. Annu Rev Immunol. 1989;7:601–624. doi: 10.1146/annurev.iy.07.040189.003125. [DOI] [PubMed] [Google Scholar]
  48. Uenaka A., Ono T., Akisawa T., Wada H., Yasuda T., Nakayama E. Identification of a unique antigen peptide pRL1 on BALB/c RL male 1 leukemia recognized by cytotoxic T lymphocytes and its relation to the Akt oncogene. J Exp Med. 1994 Nov 1;180(5):1599–1607. doi: 10.1084/jem.180.5.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vijayasaradhi S., Bouchard B., Houghton A. N. The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product. J Exp Med. 1990 Apr 1;171(4):1375–1380. doi: 10.1084/jem.171.4.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wang R. F., Parkhurst M. R., Kawakami Y., Robbins P. F., Rosenberg S. A. Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen. J Exp Med. 1996 Mar 1;183(3):1131–1140. doi: 10.1084/jem.183.3.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Weiss R. B., Dunn D. M., Atkins J. F., Gesteland R. F. Slippery runs, shifty stops, backward steps, and forward hops: -2, -1, +1, +2, +5, and +6 ribosomal frameshifting. Cold Spring Harb Symp Quant Biol. 1987;52:687–693. doi: 10.1101/sqb.1987.052.01.078. [DOI] [PubMed] [Google Scholar]
  52. Weiss R. B., Huang W. M., Dunn D. M. A nascent peptide is required for ribosomal bypass of the coding gap in bacteriophage T4 gene 60. Cell. 1990 Jul 13;62(1):117–126. doi: 10.1016/0092-8674(90)90245-A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wiertz E. J., Jones T. R., Sun L., Bogyo M., Geuze H. J., Ploegh H. L. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell. 1996 Mar 8;84(5):769–779. doi: 10.1016/s0092-8674(00)81054-5. [DOI] [PubMed] [Google Scholar]
  54. Yewdell J. W., Bennink J. R. Cell biology of antigen processing and presentation to major histocompatibility complex class I molecule-restricted T lymphocytes. Adv Immunol. 1992;52:1–123. doi: 10.1016/s0065-2776(08)60875-5. [DOI] [PubMed] [Google Scholar]
  55. Yewdell J. W., Bennink J. R., Smith G. L., Moss B. Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1785–1789. doi: 10.1073/pnas.82.6.1785. [DOI] [PMC free article] [PubMed] [Google Scholar]

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