Purification and Characterization of an Early Protein (E14K) from Adenovirus Type 2-infected Cells

Overview

Journal J Virol

Publisher American Society for Microbiology

Date 1978 Oct 1

PMID 702645

Citations 11

Authors

H Persson

B Oberg

L Philipson

Affiliations

Soon will be listed here.

Abstract

One adenovirus type 2 (Ad2) early protein, with an apparent molecular weight of 14,000 in sodium dodecyl sulfate-polyacrylamide gels (E14K), was purified to homogeneity. Purification involved fractionation of cytoplasmic extracts, precipitation at low pH, and DEAE-cellulose, phosphocellulose, and hydroxylapatite chromatography. The yield was around 12 microgram of purified protein per 10(9) HeLa cells. The two Ad2 DNA binding proteins with molecular weights of 75,000 and 45,000 (E75K and E45K) were purified by the same procedure. Tryptic peptide analyses indicated that the E14K protein is unrelated to the DNA binding proteins. The purified E14K protein has a high content of basic amino acids and a sedimentation coefficient of 5.5S in the native state, corresponding to a molecular weight of around 95,000. Pulse-chase experiments suggest that the E14K polypeptide is a primary translation product. Immunoprecipitation with a monospecific antiserum against the E14K protein revealed that it is exclusively localized in the cytoplasm of infected cells. E14K started to be synthesized at 2 hpostinfection, with a maximal rate of synthesis at 4 to 6 h postinfection. Immunoprecipitation of cell extracts from four different Ad2-transformed hamster embryo cell lines revealed that only one (Ad2HE4) of them expresses this protein. The adenovirus-simian virus 40 hybrid virus (Ad2ND1) does not express this protein, suggesting that the gene for the E14K protein is located in the part of the Ad2 genome which is deleted in this hybrid virus.

Citing Articles

Characterization of Ad5 E3-14.7K, an adenoviral inhibitor of apoptosis: structure, oligomeric state, and metal binding.

Kim H, Foster M Protein Sci. 2002; 11(5):1117-28.

PMID: 11967368 PMC: 2373546. DOI: 10.1110/ps.4180102.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters.

Lee D, Roeder R Mol Cell Biol. 1981; 1(7):635-51.

PMID: 9279377 PMC: 369711. DOI: 10.1128/mcb.1.7.635-651.1981.

The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane.

Stewart A, Tollefson A, Krajcsi P, Yei S, Wold W J Virol. 1995; 69(1):172-81.

PMID: 7983708 PMC: 188561. DOI: 10.1128/JVI.69.1.172-181.1995.

Purification of a native membrane-associated adenovirus tumor antigen.

Persson H, Katze M, Philipson L J Virol. 1982; 42(3):905-17.

PMID: 7097863 PMC: 256925. DOI: 10.1128/JVI.42.3.905-917.1982.

Mapping a new gene that encodes an 11,600-molecular-weight protein in the E3 transcription unit of adenovirus 2.

Wold W, Cladaras C, Magie S, Yacoub N J Virol. 1984; 52(2):307-13.

PMID: 6492252 PMC: 254527. DOI: 10.1128/JVI.52.2.307-313.1984.

References

Martin R, Ames B . A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961; 236:1372-9. View

LOWRY O, ROSEBROUGH N, FARR A, RANDALL R . Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265-75. View

Toneguzzo F, GHOSH H . Synthesis and glycosylation in vitro of glycoprotein of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1977; 74(4):1516-20. PMC: 430820. DOI: 10.1073/pnas.74.4.1516. View

Sugawara K, Gilead Z, Green M . Purification and molecular characterization of adenovirus type 2 DNA-binding protein. J Virol. 1977; 21(1):338-46. PMC: 353820. DOI: 10.1128/JVI.21.1.338-346.1977. View

Sugawara K, Gilead Z, Wold W, Green M . Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells. J Virol. 1977; 22(2):527-39. PMC: 515743. DOI: 10.1128/JVI.22.2.527-539.1977. View

Linne T, Jornvall H, Philipson L . Purification and characterization of the phosphorylated DNA-binding protein from adenovirus-type-2-infected cells. Eur J Biochem. 1977; 76(2):481-90. DOI: 10.1111/j.1432-1033.1977.tb11618.x. View

Klessig D . Isolation of a variant of human adenovirus serotype 2 that multiplies efficiently on monkey cells. J Virol. 1977; 21(3):1243-6. PMC: 515670. DOI: 10.1128/JVI.21.3.1243-1246.1977. View

Cleveland D, Fischer S, Kirschner M, Laemmli U . Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977; 252(3):1102-6. View

Johansson K, Persson H, Lewis A, Pettersson U, Tibbetts C, Philipson L . Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2. J Virol. 1978; 27(3):628-39. PMC: 525851. DOI: 10.1128/JVI.27.3.628-639.1978. View

10.

Harter M, Lewis J . Adenovirus type 2 early proteins synthesized in vitro and in vivo: identification in infected cells of the 38,000- to 50,000- molecular-weight protein encoded by the left end of the adenovirus type 2 genome. J Virol. 1978; 26(3):736-49. PMC: 525899. DOI: 10.1128/JVI.26.3.736-749.1978. View

11.

Jeng Y, Wold W, Sugawara K, Gilead Z, Green M . Adenovirus type 2 coded single-stranded DNA binding protein: in vivo phosphorylation and modification. J Virol. 1977; 22(2):402-11. PMC: 515731. DOI: 10.1128/JVI.22.2.402-411.1977. View

12.

Neuwald P, Meyer J, Maizel Jr J, Westpahl H . Early gene expression of adenovirus type 2: R-loop mapping of mRNA and time course of viral DNA, mRNA, and protein synthesis. J Virol. 1977; 21(3):1019-30. PMC: 515642. DOI: 10.1128/JVI.21.3.1019-1030.1977. View

13.

Sen A, Sherr C, Todaro G . Phosphorylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA. Cell. 1977; 10(3):489-96. DOI: 10.1016/0092-8674(77)90036-8. View

14.

Flint J . The topography and transcription of the adenovirus genome. Cell. 1977; 10(2):153-66. DOI: 10.1016/0092-8674(77)90211-2. View

15.

Chin W, Maizel Jr J . The polypeptides of adenovirus. VIII. The enrichment of E3 (11,000) in the nuclear matrix fraction. Virology. 1977; 76(1):79-89. DOI: 10.1016/0042-6822(77)90284-7. View

16.

Levinson A, Levine A . The isolation and identification of the adenovirus group C tumor antigens. Virology. 1977; 76(1):1-11. DOI: 10.1016/0042-6822(77)90275-6. View

17.

Gilead Z, Jeng Y, Wold W, Sugawara K, Rho H, Harter M . Immunological identification of two adenovirus 2-induced early proteins possibly involved in cell transformation. Nature. 1976; 264(5583):263-6. DOI: 10.1038/264263a0. View

18.

SCHREIER M, Staehelin T . Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems. J Mol Biol. 1973; 73(3):329-49. DOI: 10.1016/0022-2836(73)90346-x. View

19.

van der Vliet P, Levine A . DNA-binding proteins specific for cells infected by adenovirus. Nat New Biol. 1973; 246(154):170-4. DOI: 10.1038/newbio246170a0. View

20.

Fox R, Baum S . Posttranscriptional block to adenovirus replication in nonpermissive monkey cells. Virology. 1974; 60(1):45-53. DOI: 10.1016/0042-6822(74)90364-x. View