Polyomavirus large T mutants affected in retinoblastoma protein binding are defective in immortalization

Abstract

To clarify the relationship between the various activities of the polyomavirus large T antigen and the contribution of this oncogene to neoplastic transformation, we constructed a series of mutants with small deletions or single-amino-acid substitutions in two separate regions of the protein. These sequences were targeted because they showed considerable similarity to conserved regions 1 and 2 of adenovirus ElA which are thought to be binding sites for the retinoblastoma gene product (pRB). The pRB-binding properties of the large T mutants were assessed with an in vitro coimmunoprecipitation assay. pRB binding was readily detected with wild-type large T, but coprecipitation was completely abolished by as little as a single amino acid substitution (Asp-141-*Glu or Glu-146-*Asp) in region 2 of the polyomavirus large T antigen. Mutants defective in pRB binding were unable to immortalize primary rat embryo fibroblasts, suggesting that association with pRB is an important component of immortalization mediated by polyomavirus large T. The mutations in region 1 affected pRB binding only marginally, yet some of them severely impaired immortalization, indicating that pRB binding may be essential but not sufficient for immortalization. * Corresponding author. blastomas and has subsequently been found to be similarly altered in a variety of tumors and tumor cell lines (for

Testicular adenoma and seminal vesicle engorgement in polyomavirus large‐T antigen transgenic mice

“…In vivo PVLT expression in testes and heart [ 171 confers an immortal phenotype, similar to its ability to confer an immortal but not transformed phenotype in vitro. Transgenic mice expressed simian virus 40 large-T antigen, adenovirus ElA, PVLT, and PVMT in testes without consequence even while the oncogene elicitedtumorselsewhere in thesameanimal [1,2, [19][20][21].The expression of c-myc and human papillomavirus type 16 E6 and E7 proteins in transgenic mice did result in a Sertoli cell neoplasm and a seminoma, respectively [31,32]. Like our results in three lines of MT-PVLT transgenic mice, no effect of PVLT expression in testes was found in a separate study [I].…”

“…The occurrence of adenomas in both testes indicates that activating and accessory factors are either age dependent or common events or both. PVLT has been shown to bind and presumably inactivate the anti proliferation retinoblastoma protein (Rb) [39] and the ability of PVLT to bind Rb has been linked to the ability to immortalize in vitro [40]. Rb has been shown to negatively regulate the expression of the proto-oncogene c-fos, a component of activator protein-I, and has been speculated to function as a regulator of a set of genes important for cell cycling [41-431. Human fibroblasts irnmortalized by PVLT have been shown to contain elevated levels of proliferating cell nuclear antigen [44].…”

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter

“…p105-RB, p107, and p130 all interact primarily with a conserved motif located in transforming domain 2 of adenovirus type 5 (Ad5), which is required for growth activation (26,39), whereas sequences within CR1 and the amino terminus are capable of weak interactions and appear to function cooperatively with CR2 to bind these proteins (2). So, the binding of p105-RB requires a region containing the first 7 amino acid residues in CR2, which comprise the conserved (Asp)-Leu-X-Cys-X-Glu binding motif and, to some extent, the remainder of CR2 (35). In addition, mutants lacking the first 20 amino acids of CR1 (residues 36 to 60) are considerably impaired in complex formation (2,61).…”

J Domain-Independent Regulation of the Rb Family by Polyomavirus Large T Antigen

“…Much of what LT does to cells depends upon its interactions with the Rb tumor suppressor family through an LXCXE motif starting at residue 141 (17,32). Immortalization (22,34,41), induction of cell DNA synthesis (70), overcoming p53-induced cell cycle arrest (16), and prevention of differentiation (52) can all depend on Rb family binding. Transactivation of the thymidine kinase, polymerase ␣, PCNA, dihydrofolate reductase, and thymidylate synthase genes (54) as well as regulation of interferon-inducible gene expression (80) also requires an intact pRb/p107 binding site on LT and is mediated via the cellular transcription factor E2F.…”