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. 1987 Oct;61(10):3227–3234. doi: 10.1128/jvi.61.10.3227-3234.1987

Acylation of the 176R (19-kilodalton) early region 1B protein of human adenovirus type 5.

C J McGlade, M L Tremblay, S P Yee, R Ross, P E Branton
PMCID: PMC255902  PMID: 2957509

Abstract

Antipeptide sera were prepared in rabbits against synthetic peptides corresponding to the predicted amino and carboxy termini of the early region 1B 176R (19-kilodalton [kDa]) protein of human adenovirus type 5. Both antisera specifically immunoprecipitated the 19- and 18.5-kDa forms of the 176R protein observed previously with antitumor sera. These data suggested that both species are full-length molecules of 176 residues. To identify posttranslational modifications that could explain the formation of these multiple species and possibly their known association with membranes, studies were carried out to determine whether they are glycosylated or acylated. Neither the 19- nor the 18.5-kDa species appeared to be a glycoprotein, however, they were labeled with [3H]palmitate and [3H]myristate, indicating that both species are acylated. Thus, whereas acylation does not appear to be the cause of the multiple species, it could play a role in the membrane association of these viral proteins. The acylation of 176R was found to be unusual. The fatty acid linkage was resistant to treatment with hydroxylamine or methanol-KOH, suggesting that acylation was through an amide bond. In addition, both palmitate and myristate were present in 176R, suggesting either a lack of specificity in the acylation reaction or the existence of more than one acylation site.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson C. W., Schmitt R. C., Smart J. E., Lewis J. B. Early region 1B of adenovirus 2 encodes two coterminal proteins of 495 and 155 amino acid residues. J Virol. 1984 May;50(2):387–396. doi: 10.1128/jvi.50.2.387-396.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babiss L. E., Fisher P. B., Ginsberg H. S. Effect on transformation of mutations in the early region 1b-encoded 21- and 55-kilodalton proteins of adenovirus 5. J Virol. 1984 Nov;52(2):389–395. doi: 10.1128/jvi.52.2.389-395.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bos J. L., Polder L. J., Bernards R., Schrier P. I., van den Elsen P. J., van der Eb A. J., van Ormondt H. The 2.2 kb E1b mRNA of human Ad12 and Ad5 codes for two tumor antigens starting at different AUG triplets. Cell. 1981 Nov;27(1 Pt 2):121–131. doi: 10.1016/0092-8674(81)90366-4. [DOI] [PubMed] [Google Scholar]
  4. Branton P. E., Bayley S. T., Graham F. L. Transformation by human adenoviruses. Biochim Biophys Acta. 1985;780(1):67–94. doi: 10.1016/0304-419x(84)90007-6. [DOI] [PubMed] [Google Scholar]
  5. Buss J. E., Sefton B. M. Direct identification of palmitic acid as the lipid attached to p21ras. Mol Cell Biol. 1986 Jan;6(1):116–122. doi: 10.1128/mcb.6.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buss J. E., Sefton B. M. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog. J Virol. 1985 Jan;53(1):7–12. doi: 10.1128/jvi.53.1.7-12.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen Z. Q., Ulsh L. S., DuBois G., Shih T. Y. Posttranslational processing of p21 ras proteins involves palmitylation of the C-terminal tetrapeptide containing cysteine-186. J Virol. 1985 Nov;56(2):607–612. doi: 10.1128/jvi.56.2.607-612.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chinnadurai G. Adenovirus 2 Ip+ locus codes for a 19 kd tumor antigen that plays an essential role in cell transformation. Cell. 1983 Jul;33(3):759–766. doi: 10.1016/0092-8674(83)90018-1. [DOI] [PubMed] [Google Scholar]
  9. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  10. Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Föhring B., Gallimore P. H., Mellow G. H., Raska K., Jr Adenovirus type 12 specific cell surface antigen in transformed cells is a product of the E1b early region. Virology. 1983 Dec;131(2):463–472. doi: 10.1016/0042-6822(83)90512-3. [DOI] [PubMed] [Google Scholar]
  12. Gallimore P. H., McDougall J. K., Chen L. B. In vitro traits of adenovirus-transformed cell lines and their relevance to tumorigenicity in nude mice. Cell. 1977 Apr;10(4):669–678. doi: 10.1016/0092-8674(77)90100-3. [DOI] [PubMed] [Google Scholar]
  13. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  14. Graham F. L., van der Eb A. J., Heijneker H. L. Size and location of the transforming region in human adenovirus type 5 DNA. Nature. 1974 Oct 25;251(5477):687–691. doi: 10.1038/251687a0. [DOI] [PubMed] [Google Scholar]
  15. Grand R. J., Roberts C., Gallimore P. H. Acylation of adenovirus type 12 early region 1b 18-kDa protein. Further evidence for its localisation in the cell membrane. FEBS Lett. 1985 Feb 25;181(2):229–235. doi: 10.1016/0014-5793(85)80265-9. [DOI] [PubMed] [Google Scholar]
  16. Green M., Brackmann K. H., Cartas M. A., Matsuo T. Identification and purification of a protein encoded by the human adenovirus type 2 transforming region. J Virol. 1982 Apr;42(1):30–41. doi: 10.1128/jvi.42.1.30-41.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Green M., Wold W. S., Brackmann K., Cartas M. A. Studies on early proteins and transformation proteins of human adenoviruses. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):457–469. doi: 10.1101/sqb.1980.044.01.049. [DOI] [PubMed] [Google Scholar]
  18. Hedo J. A., Collier E., Watkinson A. Myristyl and palmityl acylation of the insulin receptor. J Biol Chem. 1987 Jan 25;262(3):954–957. [PubMed] [Google Scholar]
  19. Henderson L. E., Krutzsch H. C., Oroszlan S. Myristyl amino-terminal acylation of murine retrovirus proteins: an unusual post-translational proteins modification. Proc Natl Acad Sci U S A. 1983 Jan;80(2):339–343. doi: 10.1073/pnas.80.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hopp T. P. A synthetic peptide with hepatitis B surface antigen reactivity. Mol Immunol. 1981 Sep;18(9):869–872. doi: 10.1016/0161-5890(81)90009-2. [DOI] [PubMed] [Google Scholar]
  21. Houweling A., van den Elsen P. J., van der Eb A. J. Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA. Virology. 1980 Sep;105(2):537–550. doi: 10.1016/0042-6822(80)90054-9. [DOI] [PubMed] [Google Scholar]
  22. Kamps M. P., Buss J. E., Sefton B. M. Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4625–4628. doi: 10.1073/pnas.82.14.4625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leavitt R., Schlesinger S., Kornfeld S. Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. J Virol. 1977 Jan;21(1):375–385. doi: 10.1128/jvi.21.1.375-385.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maat J., van Beveren C. P., van Ormondt H. The nucleotide sequence of adenovirus type 5 early region E1: the region between map positions 8.0 (HindIII site) and 11.8 (SmaI site). Gene. 1980 Jun;10(1):27–38. doi: 10.1016/0378-1119(80)90140-7. [DOI] [PubMed] [Google Scholar]
  25. Magee A. I., Courtneidge S. A. Two classes of fatty acid acylated proteins exist in eukaryotic cells. EMBO J. 1985 May;4(5):1137–1144. doi: 10.1002/j.1460-2075.1985.tb03751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Magee A. I., Schlesinger M. J. Fatty acid acylation of eucaryotic cell membrane proteins. Biochim Biophys Acta. 1982 Nov 30;694(3):279–289. doi: 10.1016/0304-4157(82)90008-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McIlhinney R. A., Pelly S. J., Chadwick J. K., Cowley G. P. Studies on the attachment of myristic and palmitic acid to cell proteins in human squamous carcinoma cell lines: evidence for two pathways. EMBO J. 1985 May;4(5):1145–1152. doi: 10.1002/j.1460-2075.1985.tb03752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Olson E. N., Glaser L., Merlie J. P. Alpha and beta subunits of the nicotinic acetylcholine receptor contain covalently bound lipid. J Biol Chem. 1984 May 10;259(9):5364–5367. [PubMed] [Google Scholar]
  29. Olson E. N., Towler D. A., Glaser L. Specificity of fatty acid acylation of cellular proteins. J Biol Chem. 1985 Mar 25;260(6):3784–3790. [PubMed] [Google Scholar]
  30. Omary M. B., Trowbridge I. S. Covalent binding of fatty acid to the transferrin receptor in cultured human cells. J Biol Chem. 1981 May 25;256(10):4715–4718. [PubMed] [Google Scholar]
  31. Perricaudet M., Akusjärvi G., Virtanen A., Pettersson U. Structure of two spliced mRNAs from the transforming region of human subgroup C adenoviruses. Nature. 1979 Oct 25;281(5733):694–696. doi: 10.1038/281694a0. [DOI] [PubMed] [Google Scholar]
  32. Persson H., Katze M. G., Philipson L. Purification of a native membrane-associated adenovirus tumor antigen. J Virol. 1982 Jun;42(3):905–917. doi: 10.1128/jvi.42.3.905-917.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Purchio A. F., Wells S. K., Collett M. S. Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+. Mol Cell Biol. 1983 Sep;3(9):1589–1597. doi: 10.1128/mcb.3.9.1589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rose J. K., Adams G. A., Gallione C. J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2050–2054. doi: 10.1073/pnas.81.7.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rowe D. T., Branton P. E., Yee S. P., Bacchetti S., Graham F. L. Establishment and characterization of hamster cell lines transformed by restriction endonuclease fragments of adenovirus 5. J Virol. 1984 Jan;49(1):162–170. doi: 10.1128/jvi.49.1.162-170.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rowe D. T., Graham F. L., Branton P. E. Intracellular localization of adenovirus type 5 tumor antigens in productively infected cells. Virology. 1983 Sep;129(2):456–468. doi: 10.1016/0042-6822(83)90183-6. [DOI] [PubMed] [Google Scholar]
  37. Schlesinger M. J., Magee A. I., Schmidt M. F. Fatty acid acylation of proteins in cultured cells. J Biol Chem. 1980 Nov 10;255(21):10021–10024. [PubMed] [Google Scholar]
  38. Schmidt M. F., Bracha M., Schlesinger M. J. Evidence for covalent attachment of fatty acids to Sindbis virus glycoproteins. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1687–1691. doi: 10.1073/pnas.76.4.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schmidt M. F., Schlesinger M. J. Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein. Cell. 1979 Aug;17(4):813–819. doi: 10.1016/0092-8674(79)90321-0. [DOI] [PubMed] [Google Scholar]
  40. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  41. Shih T. Y., Weeks M. O., Gruss P., Dhar R., Oroszlan S., Scolnick E. M. Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus. J Virol. 1982 Apr;42(1):253–261. doi: 10.1128/jvi.42.1.253-261.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Subramanian T., Kuppuswamy M., Gysbers J., Mak S., Chinnadurai G. 19-kDa tumor antigen coded by early region E1b of adenovirus 2 is required for efficient synthesis and for protection of viral DNA. J Biol Chem. 1984 Oct 10;259(19):11777–11783. [PubMed] [Google Scholar]
  43. Takemori N., Cladaras C., Bhat B., Conley A. J., Wold W. S. cyt gene of adenoviruses 2 and 5 is an oncogene for transforming function in early region E1B and encodes the E1B 19,000-molecular-weight polypeptide. J Virol. 1984 Dec;52(3):793–805. doi: 10.1128/jvi.52.3.793-805.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tkacz J. S., Lampen O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun. 1975 Jul 8;65(1):248–257. doi: 10.1016/s0006-291x(75)80086-6. [DOI] [PubMed] [Google Scholar]
  45. Towler D., Glaser L. Acylation of cellular proteins with endogenously synthesized fatty acids. Biochemistry. 1986 Feb 25;25(4):878–884. doi: 10.1021/bi00352a021. [DOI] [PubMed] [Google Scholar]
  46. Virtanen A., Pettersson U. Organization of early region 1B of human adenovirus type 2: identification of four differentially spliced mRNAs. J Virol. 1985 May;54(2):383–391. doi: 10.1128/jvi.54.2.383-391.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Voronova A. F., Buss J. E., Patschinsky T., Hunter T., Sefton B. M. Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells. Mol Cell Biol. 1984 Dec;4(12):2705–2713. doi: 10.1128/mcb.4.12.2705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. White E., Blose S. H., Stillman B. W. Nuclear envelope localization of an adenovirus tumor antigen maintains the integrity of cellular DNA. Mol Cell Biol. 1984 Dec;4(12):2865–2875. doi: 10.1128/mcb.4.12.2865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. White E., Faha B., Stillman B. Regulation of adenovirus gene expression in human WI38 cells by an E1B-encoded tumor antigen. Mol Cell Biol. 1986 Nov;6(11):3763–3773. doi: 10.1128/mcb.6.11.3763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. White E., Grodzicker T., Stillman B. W. Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA. J Virol. 1984 Nov;52(2):410–419. doi: 10.1128/jvi.52.2.410-419.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Willumsen B. M., Christensen A., Hubbert N. L., Papageorge A. G., Lowy D. R. The p21 ras C-terminus is required for transformation and membrane association. Nature. 1984 Aug 16;310(5978):583–586. doi: 10.1038/310583a0. [DOI] [PubMed] [Google Scholar]
  52. Willumsen B. M., Norris K., Papageorge A. G., Hubbert N. L., Lowy D. R. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus. EMBO J. 1984 Nov;3(11):2581–2585. doi: 10.1002/j.1460-2075.1984.tb02177.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yee S. P., Branton P. E. Analysis of multiple forms of human adenovirus type 5 E1A polypeptides using an anti-peptide antiserum specific for the amino terminus. Virology. 1985 Oct 30;146(2):315–322. doi: 10.1016/0042-6822(85)90015-7. [DOI] [PubMed] [Google Scholar]
  54. Yee S. P., Rowe D. T., Tremblay M. L., McDermott M., Branton P. E. Identification of human adenovirus early region 1 products by using antisera against synthetic peptides corresponding to the predicted carboxy termini. J Virol. 1983 Jun;46(3):1003–1013. doi: 10.1128/jvi.46.3.1003-1013.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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