Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Feb;70(2):745–752. doi: 10.1128/jvi.70.2.745-752.1996

The block of adipocyte differentiation by a C-terminally truncated, but not by full-length, simian virus 40 large tumor antigen is dependent on an intact retinoblastoma susceptibility protein family binding domain.

C Higgins 1, S Chatterjee 1, V Cherington 1
PMCID: PMC189875  PMID: 8551611

Abstract

Simian virus 40 (SV40) can promote cell transformation and suppress differentiation. It does this partly by targeting tumor suppressors such as p53 and members of the retinoblastoma susceptibility protein (Rb) family. This work concentrates on mechanisms by which SV40 large tumor antigen (SVLT) suppresses adipocyte differentiation. We created cell lines derived from murine 3T3-L1 preadipocytes expressing different versions of SV40 early-region sequences. SVLT-expressing cells failed to exhibit adipocyte morphology, to induce glycerophosphate dehydrogenase activity, and to induce differentiation-dependent mRNA for adipocyte P2. SVLT alone was sufficient, in the absence of SV40 small tumor antigen, to inhibit differentiation. A truncated SVLT containing only the N-terminal 121 amino acids (SVLT1-121) blocked differentiation, thus mapping at least one differentiation blocking function to the N-terminal region. K1 (Glu-107-->Lys) point mutants of SVLT, which are unable to bind to the Rb protein family or induce neoplastic transformation, are defective for blocking differentiation in the case of SVLT1-121 but retain the ability to block differentiation in the case of full-length SVLT. This finding demonstrates that Rb family proteins are important in regulating adipocyte differentiation but that other functions of full-length SVLT can block adipocyte differentiation independently of RB family binding and transformation.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Armelin H. A., Armelin M. C., Kelly K., Stewart T., Leder P., Cochran B. H., Stiles C. D. Functional role for c-myc in mitogenic response to platelet-derived growth factor. Nature. 1984 Aug 23;310(5979):655–660. doi: 10.1038/310655a0. [DOI] [PubMed] [Google Scholar]
  2. Brown M., McCormack M., Zinn K. G., Farrell M. P., Bikel I., Livingston D. M. A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth. J Virol. 1986 Oct;60(1):290–293. doi: 10.1128/jvi.60.1.290-293.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  4. Chen J., Tobin G. J., Pipas J. M., Van Dyke T. T-antigen mutant activities in vivo: roles of p53 and pRB binding in tumorigenesis of the choroid plexus. Oncogene. 1992 Jun;7(6):1167–1175. [PubMed] [Google Scholar]
  5. Cherington V., Brown M., Paucha E., St Louis J., Spiegelman B. M., Roberts T. M. Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation. Mol Cell Biol. 1988 Mar;8(3):1380–1384. doi: 10.1128/mcb.8.3.1380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Cornelius P., MacDougald O. A., Lane M. D. Regulation of adipocyte development. Annu Rev Nutr. 1994;14:99–129. doi: 10.1146/annurev.nu.14.070194.000531. [DOI] [PubMed] [Google Scholar]
  8. DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [PubMed] [Google Scholar]
  9. Dickmanns A., Zeitvogel A., Simmersbach F., Weber R., Arthur A. K., Dehde S., Wildeman A. G., Fanning E. The kinetics of simian virus 40-induced progression of quiescent cells into S phase depend on four independent functions of large T antigen. J Virol. 1994 Sep;68(9):5496–5508. doi: 10.1128/jvi.68.9.5496-5508.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dyson N., Buchkovich K., Whyte P., Harlow E. The cellular 107K protein that binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus. Cell. 1989 Jul 28;58(2):249–255. doi: 10.1016/0092-8674(89)90839-8. [DOI] [PubMed] [Google Scholar]
  11. Endo T. SV40 large T inhibits myogenic differentiation partially through inducing c-jun. J Biochem. 1992 Sep;112(3):321–329. doi: 10.1093/oxfordjournals.jbchem.a123899. [DOI] [PubMed] [Google Scholar]
  12. Estervig D. N., Minoo P., Tzen C. Y., Scott R. E. Three distinct effects of SV40 T-antigen gene transfection on cellular differentiation. J Cell Physiol. 1990 Mar;142(3):552–558. doi: 10.1002/jcp.1041420314. [DOI] [PubMed] [Google Scholar]
  13. Ewen M. E., Ludlow J. W., Marsilio E., DeCaprio J. A., Millikan R. C., Cheng S. H., Paucha E., Livingston D. M. An N-terminal transformation-governing sequence of SV40 large T antigen contributes to the binding of both p110Rb and a second cellular protein, p120. Cell. 1989 Jul 28;58(2):257–267. doi: 10.1016/0092-8674(89)90840-4. [DOI] [PubMed] [Google Scholar]
  14. Ewen M. E., Xing Y. G., Lawrence J. B., Livingston D. M. Molecular cloning, chromosomal mapping, and expression of the cDNA for p107, a retinoblastoma gene product-related protein. Cell. 1991 Sep 20;66(6):1155–1164. doi: 10.1016/0092-8674(91)90038-z. [DOI] [PubMed] [Google Scholar]
  15. Fanning E., Knippers R. Structure and function of simian virus 40 large tumor antigen. Annu Rev Biochem. 1992;61:55–85. doi: 10.1146/annurev.bi.61.070192.000415. [DOI] [PubMed] [Google Scholar]
  16. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  17. Freytag S. O. Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G0/G1. Mol Cell Biol. 1988 Apr;8(4):1614–1624. doi: 10.1128/mcb.8.4.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Green H., Kehinde O. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell. 1975 May;5(1):19–27. doi: 10.1016/0092-8674(75)90087-2. [DOI] [PubMed] [Google Scholar]
  19. Green H., Meuth M. An established pre-adipose cell line and its differentiation in culture. Cell. 1974 Oct;3(2):127–133. doi: 10.1016/0092-8674(74)90116-0. [DOI] [PubMed] [Google Scholar]
  20. Gruda M. C., Zabolotny J. M., Xiao J. H., Davidson I., Alwine J. C. Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex. Mol Cell Biol. 1993 Feb;13(2):961–969. doi: 10.1128/mcb.13.2.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gu W., Schneider J. W., Condorelli G., Kaushal S., Mahdavi V., Nadal-Ginard B. Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation. Cell. 1993 Feb 12;72(3):309–324. doi: 10.1016/0092-8674(93)90110-c. [DOI] [PubMed] [Google Scholar]
  22. Gurney E. G., Tamowski S., Deppert W. Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T antigen. J Virol. 1986 Mar;57(3):1168–1172. doi: 10.1128/jvi.57.3.1168-1172.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hermeking H., Wolf D. A., Kohlhuber F., Dickmanns A., Billaud M., Fanning E., Eick D. Role of c-myc in simian virus 40 large tumor antigen-induced DNA synthesis in quiescent 3T3-L1 mouse fibroblasts. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10412–10416. doi: 10.1073/pnas.91.22.10412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ignotz R. A., Massagué J. Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8530–8534. doi: 10.1073/pnas.82.24.8530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kalderon D., Smith A. E. In vitro mutagenesis of a putative DNA binding domain of SV40 large-T. Virology. 1984 Nov;139(1):109–137. doi: 10.1016/0042-6822(84)90334-9. [DOI] [PubMed] [Google Scholar]
  26. Phillips B., Rundell K. Failure of simian virus 40 small t antigen to disorganize actin cables in nonpermissive cell lines. J Virol. 1988 Mar;62(3):768–775. doi: 10.1128/jvi.62.3.768-775.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pipas J. M., Peden K. W., Nathans D. Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene. Mol Cell Biol. 1983 Feb;3(2):203–213. doi: 10.1128/mcb.3.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Scott R. E., Florine D. L., Wille J. J., Jr, Yun K. Coupling of growth arrest and differentiation at a distinct state in the G1 phase of the cell cycle: GD. Proc Natl Acad Sci U S A. 1982 Feb;79(3):845–849. doi: 10.1073/pnas.79.3.845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shin E. K., Shin A., Paulding C., Schaffhausen B., Yee A. S. Multiple change in E2F function and regulation occur upon muscle differentiation. Mol Cell Biol. 1995 Apr;15(4):2252–2262. doi: 10.1128/mcb.15.4.2252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sompayrac L., Danna K. J. An amino-terminal fragment of SV40 T antigen induces cellular DNA synthesis in quiescent rat cells. Virology. 1994 May 1;200(2):849–853. doi: 10.1006/viro.1994.1255. [DOI] [PubMed] [Google Scholar]
  31. Sompayrac L., Danna K. J. An amino-terminal fragment of SV40 T antigen transforms REF52 cells. Virology. 1992 Nov;191(1):439–442. doi: 10.1016/0042-6822(92)90206-5. [DOI] [PubMed] [Google Scholar]
  32. Spiegelman B. M., Frank M., Green H. Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development. J Biol Chem. 1983 Aug 25;258(16):10083–10089. [PubMed] [Google Scholar]
  33. Srinivasan A., Peden K. W., Pipas J. M. The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions. J Virol. 1989 Dec;63(12):5459–5463. doi: 10.1128/jvi.63.12.5459-5463.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Symonds H., Krall L., Remington L., Saenz-Robles M., Lowe S., Jacks T., Van Dyke T. p53-dependent apoptosis suppresses tumor growth and progression in vivo. Cell. 1994 Aug 26;78(4):703–711. doi: 10.1016/0092-8674(94)90534-7. [DOI] [PubMed] [Google Scholar]
  35. Thompson D. L., Kalderon D., Smith A. E., Tevethia M. J. Dissociation of Rb-binding and anchorage-independent growth from immortalization and tumorigenicity using SV40 mutants producing N-terminally truncated large T antigens. Virology. 1990 Sep;178(1):15–34. doi: 10.1016/0042-6822(90)90375-2. [DOI] [PubMed] [Google Scholar]
  36. Tischler A. S., Ruzicka L. A., Riseberg J. C. Immunocytochemical analysis of chromaffin cell proliferation in vitro. J Histochem Cytochem. 1992 Jul;40(7):1043–1045. doi: 10.1177/40.7.1351491. [DOI] [PubMed] [Google Scholar]
  37. Tobe K., Kasuga M., Kitasato H., Takaku F., Takano T., Segawa K. Differential effects of DNA tumor virus nuclear oncogene products on adipocyte differentiation. FEBS Lett. 1987 May 11;215(2):345–349. doi: 10.1016/0014-5793(87)80175-8. [DOI] [PubMed] [Google Scholar]
  38. Wise L. S., Green H. Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells. J Biol Chem. 1979 Jan 25;254(2):273–275. [PubMed] [Google Scholar]
  39. Wolf D. A., Hermeking H., Albert T., Herzinger T., Kind P., Eick D. A complex between E2F and the pRb-related protein p130 is specifically targeted by the simian virus 40 large T antigen during cell transformation. Oncogene. 1995 Jun 1;10(11):2067–2078. [PubMed] [Google Scholar]
  40. Yaciuk P., Carter M. C., Pipas J. M., Moran E. Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products. Mol Cell Biol. 1991 Apr;11(4):2116–2124. doi: 10.1128/mcb.11.4.2116. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES