Complex of simian virus 40 large tumor antigen and 48,000-dalton host tumor antigen.

Abstract

Simian virus 40 largetumor antigen (T Ag) can be separated by sucrose gradient sedimentation into a rapidly sedimenting, maximally phosphorylated fraction and a slowly sedimenting, less phosphorylated fraction. The Mr 48,000 host tumor antigen (48,000 HTA, also called nonviral T Ag) is preferentially complexed with the maximally phosphorylated T Ag. Pulse-labeled T Ag sediments as a 5-6S monomer, whereas T Ag radiolabeled for progressively longer periods slowly increases in sedimentation coefficient to give a, broad distribution between 5 S and greater than 28 S. Mutation in the viral A locus causes a decrease in T Ag phosphorylation and a marked decrease in 48,000 HTA binding, shifting the sedimentation coefficient of T Ag to the monomer value. The more highly phosphorylated T Ag also has the highest affinity for chromatin.Simian virus 40 (SV40) large tumor antigen (T Ag) is encoded by the viral A locus (1, 2), which governs initiation of DNA synthesis and the establishment of viral transformation (3, 4). Found in the nuclei of cells infected (5) and transformed (6) by SV40, T Ag is a DNA-binding protein (7-11) that binds DNA with heterogeneous affinities (7,12). Mutation in the viral A locus has been shown to decrease the DNA-binding affinity of T Ag (13,14). T Ag also sediments heterogeneously with sedimentation coefficients from 5S to greater than 22S (7,(15)(16)(17)(18)(19)(20). Mutation of the A locus causes T Ag to sediment as a 5-6S species (17, 18), corresponding to the 94,000 dalton monomer (13). Furthermore, T Ag, a phosphoprotein (21), is heterogeneously phosphorylated (22), generating multiple isoelectric focusing species with differing contents of phosphate. Differential phosphorylation may play a physiological role in generating the heterogeneous aggregating and DNA-binding forms of T Ag. Similarly, the addition and removal of the phosphate groups of acidic chromatin-associated proteins of eukaryotic cells has been suggested as a control mechanism for the regulation of their DNA-binding properties (23). In addition, differential phosphorylation states can affect the binding affinities of subunits for each other, as in glycogen phosphorylase (24).Two recently discovered host gene products, the Mr 48,000 and 55,000 host tumor antigens (HTA), also termed nonviral T Ags, have been shown to complex with T Ag in vitro (19,20,25,26 McCormick and Harlow (20) have proposed that the "53K NVT"-associated form oflarge T Ag becomes more highly phosphorylated than the free form. The 48,000 and 55,000 HTAs have been shown to be unrelated immunologically and structurally (19). The broad physiological significance of these nuclear phosphoproteins has been established by the fact that they are induced in mouse, rat, hamster, and human cells transformed by SV40 (19). They are expressed, in the absence of SV40, in cells transformed by a variety ofcarcinogenic agentsincluding the RNA tumor virus, murine sarcoma virus, and the chemical carcinogen methyicholanthrene (27)-and in spontaneously transformed fibro...

A mutant herpesvirus protein leads to a block in nuclear localization of other viral proteins.

“…The simian virus 40 cT mutation causes a dominant defect in large-T antigen localization to the nucleus, but transport of other viral proteins is not affected (22). This is most likely the result of the coaggregation of mutant and wild-type T S * antigen (8,13,31). The wild-type ICP4 is believed to form a homodimer in infected cells (24), and the mutant ICP4 defect for nuclear localization is dominant over the wild-type phenotype (data not shown).…”

Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

“…How these different functions are exerted by a single protein is not understood, but it is possible that large T can assume different functional forms which are interconvertible by phosphor-ylation. Compatible with this possibility are the following findings: large T is phosphorylated at multiple sites (52) in a reversible fashion (9,38); subclasses of large T can be separated by isoelectric focusing (15); and correlations exist between the degree of phosphorylation of large T on one hand and its degree of oligomerization (10,16) or its affinity for DNA (31) on the other. However, the functional significance of these quantitative differences in the phosphorylation state is not yet clear.…”

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

“…The functional significance of these phosphorylations is unclear. However, serine 389 may be important for transformation by SV40, since a reduction in phosphorylation of this residue at the nonpermissive temperature in SV40-tsA58-transformed cells correlates with a failure to form the large T/p53 complex (2, 8,32 chymotryptic activity. Phosphopeptides were separated in two dimensions by electrophoresis at pH 1.9 (1 kV for 40 min) or at pH 8.9 (1 kV for 15 min) and chromatography (11).…”