Home LiteratureArticle Details
PMID: 8139016 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity.

Journal of virology ·Vol. 68 ·No. 4 ·1994-04-00 ·Pages 2313-9

Köppe B, Menéndez-Arias L, Oroszlan S

Abstract

The mouse mammary tumor virus gag-pro transframe protein (p30) contains the nucleocapsid protein domain derived from the 3' end of gag, fused to 154 residues encoded by the 5' region of the pro open reading frame. The DNA coding for p30 was cloned into the plasmid pALTER-1, and an additional nucleotide was inserted by site-directed mutagenesis to allow the read-through from the gag into the pro open reading frame. The obtained insert was then cloned into pGEX-2T, a plasmid containing the glutathione S-transferase gene of Schistosoma japonicum and a nucleotide sequence encoding for a thrombin cleavage site. The chimeric protein (GST-p30) was isolated by affinity chromatography on a glutathione-Sepharose 4B column, and after thrombin treatment, the excised p30 was further purified on a single-stranded DNA-agarose column. This protein showed dUTPase activity, with only negligible cleavage of dATP, dGTP, dCTP, dTTP, or UTP. Its apparent Km for dUTP was 28 microM. The enzyme was inhibited by EDTA, but its effect could be reversed by Mg2+ and other divalent cations. dUTPase activity was also detected in purified mouse mammary tumor virus, and p30 was the only protein recognized by antibodies directed towards the carboxyl-terminal sequence of the dUTPase coding region.

Related Genes
gag pol
MeSH Terms
Amino Acid Sequence Base Sequence Cations, Divalent/pharmacology Chelating Agents/pharmacology Cloning, Molecular Escherichia coli/genetics Gene Expression Gene Products, gag/genetics,isolation & purification,metabolism Gene Products, pol/genetics,isolation & purification,metabolism Mammary Tumor Virus, Mouse/enzymology Molecular Sequence Data Pyrophosphatases/drug effects,genetics,isolation & purification,metabolism Reading Frames Recombinant Proteins/isolation & purification,metabolism Sequence Analysis Substrate Specificity Viral Core Proteins/drug effects,genetics,isolation & purification,metabolism
Chemicals
Cations, Divalent Chelating Agents Gene Products, gag Gene Products, pol Recombinant Proteins Viral Core Proteins Pyrophosphatases dUTP pyrophosphatase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Köppe B
Laboratory of Molecular Virology and Carcinogenesis, NCI-Frederick Cancer Research and Development Center, Maryland 21702-1201.
Menéndez-Arias L
Oroszlan S
References (42)
42 references, click to expand
  1. Isolation and characterization of alpha 2-macroglobulin-protease complexes from purified mouse mammary tumor virus and culture supernatants from virus-infected cell lines.
    J Biol Chem. 1992 Jun 5;267(16):11392-8 PMID: 1375941
  2. Drosophila deoxyuridine triphosphatase. Purification and characterization.
    J Biol Chem. 1987 Jan 5;262(1):130-4 PMID: 3025197
  3. Herpes simplex virus type 1 dUTPase mutants are attenuated for neurovirulence, neuroinvasiveness, and reactivation from latency.
    J Virol. 1992 Nov;66(11):6706-13 PMID: 1328686
  4. Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli.
    J Biol Chem. 1992 Nov 25;267(33):24134-9 PMID: 1331110
  5. Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant.
    J Virol. 1993 May;67(5):2592-600 PMID: 8386267
  6. Vaccinia virus encodes a functional dUTPase.
    Virology. 1993 Aug;195(2):863-5 PMID: 8393252
  7. Characterization of a herpes simplex virus type 2 deoxyuridine triphosphate nucleotidohydrolase and mapping of a gene conferring type specificity for the enzyme.
    Virology. 1987 Feb;156(2):282-92 PMID: 3027979
  8. Overproduction and large-scale preparation of deoxyuridine triphosphate nucleotidohydrolase from Escherichia coli.
    Eur J Biochem. 1987 Apr 1;164(1):45-51 PMID: 3030754
  9. Purification and characterization of deoxyuridine triphosphate nucleotidohydrolase from anemic rat spleen: a trimer composition of the enzyme protein.
    Arch Biochem Biophys. 1987 Mar;253(2):350-6 PMID: 3032103
  10. Relocation of a protease-like gene segment between two retroviruses.
    Proc Natl Acad Sci U S A. 1987 May;84(9):2693-7 PMID: 3106974
  11. Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins.
    Proc Natl Acad Sci U S A. 1987 Jun;84(12):4298-302 PMID: 3035577
  12. Characterization of mouse mammary tumor virus gag-pro gene products and the ribosomal frameshift site by protein sequencing.
    Proc Natl Acad Sci U S A. 1987 Oct;84(20):7041-5 PMID: 2823251
  13. Antigenic variation and lentivirus persistence: variations in envelope gene sequences during EIAV infection resemble changes reported for sequential isolates of HIV.
    Virology. 1987 Dec;161(2):321-31 PMID: 2825406
  14. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.
    Gene. 1988 Jul 15;67(1):31-40 PMID: 3047011
  15. Fidelity of HIV-1 reverse transcriptase.
    Science. 1988 Nov 25;242(4882):1168-71 PMID: 2460924
  16. The accuracy of reverse transcriptase from HIV-1.
    Science. 1988 Nov 25;242(4882):1171-3 PMID: 2460925
  17. The preparation and biochemical characterization of intact capsids of equine infectious anemia virus.
    Biochem Biophys Res Commun. 1989 Apr 28;160(2):486-94 PMID: 2541703
  18. Analysis of gag proteins from mouse mammary tumor virus.
    J Virol. 1989 Jun;63(6):2543-9 PMID: 2542570
  19. Retroviral protease-like gene in the vaccinia virus genome.
    Proc Natl Acad Sci U S A. 1989 Jun;86(11):4152-5 PMID: 2657744
  20. Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity.
    Infect Immun. 1989 Sep;57(9):2878-85 PMID: 2668192
  21. Reverse transcriptases and genomic variability: the accuracy of DNA replication is enzyme specific and sequence dependent.
    EMBO J. 1990 May;9(5):1583-93 PMID: 1691709
  22. Protein sequence comparisons show that the 'pseudoproteases' encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family.
    Nucleic Acids Res. 1990 Jul 25;18(14):4105-10 PMID: 2165588
  23. Purification and partial characterization of equine infectious anemia virus reverse transcriptase.
    Virology. 1991 Nov;185(1):387-94 PMID: 1718086
  24. Distinct subsets of retroviruses encode dUTPase.
    J Virol. 1992 Mar;66(3):1791-4 PMID: 1310783
  25. Crystal structure of a dUTPase.
    Nature. 1992 Feb 20;355(6362):740-3 PMID: 1311056
  26. Translational suppression in retroviral gene expression.
    Adv Virus Res. 1992;41:193-239 PMID: 1575083
  27. Molecular cloning and characterization of deoxyuridine triphosphatase from feline immunodeficiency virus (FIV).
    Virology. 1993 Oct;196(2):451-7 PMID: 8396797
  28. Moloney murine leukemia virus protease: bacterial expression and characterization of the purified enzyme.
    Virology. 1993 Oct;196(2):557-63 PMID: 8372434
  29. DNA sequencing with chain-terminating inhibitors.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
  30. Uracil incorporation: a source of pulse-labeled DNA fragments in the replication of the Escherichia coli chromosome.
    Proc Natl Acad Sci U S A. 1978 Jan;75(1):233-7 PMID: 203931
  31. Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia.
    J Biol Chem. 1979 Apr 25;254(8):2897-901 PMID: 285077
  32. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
    Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 PMID: 388439
  33. Methotrexate-induced misincorporation of uracil into DNA.
    Proc Natl Acad Sci U S A. 1980 Apr;77(4):1956-60 PMID: 6929529
  34. The effect of methotrexate on levels of dUTP in animal cells.
    J Biol Chem. 1980 Nov 25;255(22):10630-7 PMID: 7430142
  35. Nucleotide sequences at host-proviral junctions for mouse mammary tumour virus.
    Nature. 1981 Jan 22;289(5795):253-8 PMID: 6256658
  36. A thin-layer chromatographic method for separation of thymidine and deoxyuridine nucleotides.
    Anal Biochem. 1980 Jul 1;105(2):311-8 PMID: 7457837
  37. Chemically synthesized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope protein of Dane particles.
    Proc Natl Acad Sci U S A. 1981 Jun;78(6):3403-7 PMID: 6167985
  38. Deoxyuridine triphosphate nucleotidohydrolase induced by herpes simplex virus type 1. Purification and characterization of induced enzyme.
    J Biol Chem. 1984 Aug 25;259(16):10080-4 PMID: 6147344
  39. Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.
    J Virol. 1985 Sep;55(3):778-87 PMID: 3927012
  40. Nucleotide sequence of SRV-1, a type D simian acquired immune deficiency syndrome retrovirus.
    Science. 1986 Mar 28;231(4745):1567-72 PMID: 3006247
  41. Nucleotide sequence of Mason-Pfizer monkey virus: an immunosuppressive D-type retrovirus.
    Cell. 1986 May 9;45(3):375-85 PMID: 2421920
  42. A meristem-related gene from tomato encodes a dUTPase: analysis of expression in vegetative and floral meristems.
    Plant Cell. 1992 Feb;4(2):149-59 PMID: 1321683
Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.68.4.2313-2319.1994
Published
1994-04-00
Pages
2313-9
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC236707
Subset
IM
Grants
NCI NIH HHS · N01-CO-74101 · United States
External Links
PubMed source DOI: 10.1128/JVI.68.4.2313-2319.1994 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2