Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1992 Sep;66(9):5615–5620. doi: 10.1128/jvi.66.9.5615-5620.1992

Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains.

L Menéndez-Arias 1, C Risco 1, P Pinto da Silva 1, S Oroszlan 1
PMCID: PMC289125  PMID: 1380097

Abstract

The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.

Full text

PDF
5615

Images in this article

5616Image
on p.5616
5617Image
on p.5617
5618Image
on p.5618
5619Image
on p.5619

Selected References

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

  1. Bolognesi D. P., Montelaro R. C., Frank H., Schäfer W. Assembly of type C oncornaviruses: a model. Science. 1978 Jan 13;199(4325):183–186. doi: 10.1126/science.202022. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Cardiff R. D., Puentes M. J., Young L. J., Smith G. H., Teramoto Y. A., Altrock B. W., Pratt T. S. Serological and biochemical characterization of the mouse mammary tumor virus with localization of p10. Virology. 1978 Mar;85(1):157–167. doi: 10.1016/0042-6822(78)90420-8. [DOI] [PubMed] [Google Scholar]
  4. Chamberlain N. R., DeOgny L., Slaughter C., Radolf J. D., Norgard M. V. Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity. Infect Immun. 1989 Sep;57(9):2878–2885. doi: 10.1128/iai.57.9.2878-2885.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fine D. L., Arthur L. O., PLOWMAN J. K., Hillman E. A., Klein F. In vitro system for production of mouse mammary tumor virus. Appl Microbiol. 1974 Dec;28(6):1040–1046. doi: 10.1128/am.28.6.1040-1046.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gelderblom H. R., Hausmann E. H., Ozel M., Pauli G., Koch M. A. Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins. Virology. 1987 Jan;156(1):171–176. doi: 10.1016/0042-6822(87)90449-1. [DOI] [PubMed] [Google Scholar]
  7. Hizi A., Henderson L. E., Copeland T. D., Sowder R. C., Hixson C. V., Oroszlan S. 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):7041–7045. doi: 10.1073/pnas.84.20.7041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hizi A., Henderson L. E., Copeland T. D., Sowder R. C., Krutzsch H. C., Oroszlan S. Analysis of gag proteins from mouse mammary tumor virus. J Virol. 1989 Jun;63(6):2543–2549. doi: 10.1128/jvi.63.6.2543-2549.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jacks T., Townsley K., Varmus H. E., Majors J. 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):4298–4302. doi: 10.1073/pnas.84.12.4298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lerner R. A., Green N., Alexander H., Liu F. T., Sutcliffe J. G., Shinnick T. M. 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):3403–3407. doi: 10.1073/pnas.78.6.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Menéndez-Arias L., Risco C., Oroszlan S. 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):11392–11398. [PubMed] [Google Scholar]
  12. Moore R., Dixon M., Smith R., Peters G., Dickson C. Complete nucleotide sequence of a milk-transmitted mouse mammary tumor virus: two frameshift suppression events are required for translation of gag and pol. J Virol. 1987 Feb;61(2):480–490. doi: 10.1128/jvi.61.2.480-490.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ogawa H., Tanaka H. Immunocolloidal gold electron microscopy of viral antigens and cellular actin in C3H/He mouse mammary tumors. Cancer Res. 1986 Nov;46(11):5851–5857. [PubMed] [Google Scholar]
  14. Oroszlan S., Copeland T. D., Gilden R. V., Todaro G. J. Structural homology of the major internal proteins of endogeneous type C viruses of two distantly related species of Old World monkeys: Macaca arctoides and Colobus polykomos. Virology. 1981 Dec;115(2):262–271. doi: 10.1016/0042-6822(81)90109-4. [DOI] [PubMed] [Google Scholar]
  15. Oroszlan S., Henderson L. E., Stephenson J. R., Copeland T. D., Long C. W., Ihle J. N., Gilden R. V. Amino- and carboxyl-terminal amino acid sequences of proteins coded by gag gene of murine leukemia virus. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1404–1408. doi: 10.1073/pnas.75.3.1404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Parks W. P., Scolnick E. M., Kozikowski E. H. Dexamethasone stimulation of murine mammary tumor virus expression: a tissue culture source of virus. Science. 1974 Apr 12;184(4133):158–160. doi: 10.1126/science.184.4133.158. [DOI] [PubMed] [Google Scholar]
  17. Racevskis J., Karande K. A., Sarkar N. H. Precursor product relationship between intracytoplasmic A particle and murine mammary tumor virus core proteins established by tryptic peptide analysis. Virology. 1981 Feb;109(1):201–204. doi: 10.1016/0042-6822(81)90488-8. [DOI] [PubMed] [Google Scholar]
  18. Rhee S. S., Hunter E. A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus. Cell. 1990 Oct 5;63(1):77–86. doi: 10.1016/0092-8674(90)90289-q. [DOI] [PubMed] [Google Scholar]
  19. Rhee S. S., Hunter E. Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid. EMBO J. 1991 Mar;10(3):535–546. doi: 10.1002/j.1460-2075.1991.tb07980.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sarkar N. H., Whittington E. S. Identification of the structural proteins of the murine mammary tumor virus that are serologically related to the antigens of intracytoplasmic type-A particles. Virology. 1977 Aug;81(1):91–106. doi: 10.1016/0042-6822(77)90061-7. [DOI] [PubMed] [Google Scholar]
  21. Schultz A. M., Oroszlan S. In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid. J Virol. 1983 May;46(2):355–361. doi: 10.1128/jvi.46.2.355-361.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smith G. H. Evidence for a precursor-product relationship between intracytoplasmic A particles and mouse mammary tumour virus cores. J Gen Virol. 1978 Oct;41(1):193–200. doi: 10.1099/0022-1317-41-1-193. [DOI] [PubMed] [Google Scholar]
  23. Smith G. H., Wivel N. A. Intracytoplasmic A particles: mouse mammary tumor virus nucleoprotein cores? J Virol. 1973 Apr;11(4):575–584. doi: 10.1128/jvi.11.4.575-584.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stewart L., Schatz G., Vogt V. M. Properties of avian retrovirus particles defective in viral protease. J Virol. 1990 Oct;64(10):5076–5092. doi: 10.1128/jvi.64.10.5076-5092.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tanaka H. Precursor-product relationship between nonglycosylated polypeptides of A and B particles of mouse mammary tumor virus. Virology. 1977 Feb;76(2):835–850. doi: 10.1016/0042-6822(77)90263-x. [DOI] [PubMed] [Google Scholar]
  26. Tanaka H., Tamura A., Tsujimura D. Properties of the intracytoplasmic A particles purified from mouse tumors. Virology. 1972 Jul;49(1):61–78. doi: 10.1016/s0042-6822(72)80007-2. [DOI] [PubMed] [Google Scholar]
  27. Teramoto Y. A., Cardiff R. D., Lund J. K. The structure of the mouse mammary tumor virus: isolation and characterization of the core. Virology. 1977 Mar;77(1):135–148. doi: 10.1016/0042-6822(77)90413-5. [DOI] [PubMed] [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. 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):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yoshinaka Y., Luftig R. B. Murine leukemia virus morphogenesis: cleavage of P70 in vitro can be accompanied by a shift from a concentrically coiled internal strand ("immature") to a collapsed ("mature") form of the virus core. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3446–3450. doi: 10.1073/pnas.74.8.3446. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES