Transrepression of RNA polymerase II promoters by the simian virus 40 small t antigen.

Simian virus 40 (SV40) small t antigen (t) can activate transcription from certain RNA polymerase II and III promoters (M. Loeken, I. Bikel, D. M. Livingston, and J. Brady, Cell 55:1171-1177, 1988). Here we report a new function of t, its ability to repress human c-fos promoter and AP-1 transcriptional activity in CV-1P cells. This function is the product of a discrete N-terminal domain of t, because the large T antigen (T)/t-common polypeptide, which contains only the first 82 amino acids common to both T and t of SV40, was, like the intact protein, an active repressor. The data further suggest that the t- and T/t-common-mediated repression of c-fos expression was most likely manifest at the level of transcription. In keeping with the possibility that t affects the expression of the genomic c-fos promoter, it also led to repression of AP-1 formation. Thus, SV40 is both an activator and a repressor of transcription. Its ability to inhibit c-fos expression should be considered in light of the natural history of SV40 in its natural host.

Involvement of simian virus 40 (SV40) small t antigen in trans activation of SV40 early and late promoters.

We have previously found that simian virus 40 (SV40) small t antigen (small t) can trans activate the E2A and VA-I genes of adenovirus in plasmid DNA-transfected cells (M. R. Loeken, I. Bikel, D. M. Livingston, and J. Brady, Cell 55:1171-1177, 1988). To determine whether trans activation by small t might be involved in the SV40 productive infection cycle, we examined the effects of cotransfecting plasmids encoding small t with plasmids containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early or late promoter. Small t increased three- to fivefold the expression of a CAT plasmid linked to the SV40 early promoter and enhancer. Small t expression had no effect by itself on CAT activity directed by the SV40 late promoter, but small t enhanced the effect of a suboptimal concentration of a plasmid expressing large T up to 10-fold. When the concentration of the plasmid expressing large T was increased to a level at which large T alone stimulated the late promoter ninefold, the enhancement by small t was only twofold. The effects of small t on both the SV40 early and late promoters depended on sequences within the small t-unique domain, since a plasmid expressing only the first 82 amino acids common to both large T and small t was inactive. The effects of small t on early- and late-promoter-directed CAT enzyme activity was reflected in increased CAT mRNA as measured by S1 analysis. These results suggest that SV40 small t may play a role in viral infection by increasing transcription from the early promoter and from the late promoter at times when large T levels are low.

Simian virus 40 small tumor antigen and an amino-terminal domain of large tumor antigen share a common transforming function.

The 82-residue amino-terminal sequences of simian virus 40 large tumor antigen (TAg) and small tumor antigen (tAg) are identical. Genetic analysis of TAg lacking amino acids 1-82 revealed that it was transformation-defective, as revealed by the agar growth assay, except when introduced in the presence of tAg. Since the latter, alone, lacks overt transforming activity, it would appear that the function of the sequence common to TAg and tAg is necessary, but not sufficient, for TAg transforming activity and that tAg can provide that function or its equivalent in trans. Thus, tAg may, in part, be viewed as a "portable" copy of a TAg functional domain.

trans-activation of RNA polymerase II and III promoters by SV40 small t antigen.

A biochemical role for SV40 small t antigen (t) in the viral infectious cycle that would explain the strong conservation of t structure among papovaviruses and its role as a helper of SV40 large T antigen function in the viral transforming process is not understood. Here, we report an intracellular biochemical function of the protein--the capacity to trans-activate selected RNA polymerase II and III-requiring promoters. Since t has failed in the past to bind to DNA and did not stimulate all polymerase II-requiring promoters tested, it likely trans-activates, at least in part, by modifying the activity of selected transcription factors.

Purification and functional properties of simian virus 40 large and small T antigens overproduced in insect cells.

The insect baculovirus Autographa californica nuclear polyhedrosis virus was used as an expression vector for the simian virus 40 (SV40) small t (t) and large T (T) antigens. Spodoptera frugiperda (SF9) cells infected with recombinant viruses encoding these proteins produced approximately 1 to 2 micrograms of t and up to 30 micrograms of T per 3 X 10(6) cells. The former was highly soluble after Nonidet P-40 extraction of the infected cells, unlike its Escherichia coli-produced counterpart. Both SF9-produced proteins were of authentic size and could be readily immunoprecipitated by specific antibodies. Single-step immunoaffinity chromatography was used to purify the two proteins to near homogeneity, with yields averaging 70% in each case. Experiments to test the biological activity of the baculovirus SV40 proteins showed that SF9 t was capable of associating with two of the cellular proteins reported to bind to t in SV40-infected mammalian cells. Moreover, SF9 T had ATPase activity comparable to that of T produced in monkey cells, exhibited helicase activity and SV40 origin-specific DNA binding, and was active in the SV40 DNA replication assay in vitro. Thus, the SV40 T antigens produced in insect cells can be used in future studies of their biochemical roles in vitro and in vivo.

SV40 small t antigen enhances the transformation activity of limiting concentrations of SV40 large T antigen.

A murine recombinant Neo(r) retrovirus encoding the SV40 small t antigen was used to infect Balb/c 3T3 CIA31 cells. From analyses of G418-resistant clones containing at least as much intact t as Cos-1 cells, we found that t, alone, had no detectable A31 transforming activity. In contrast, we noted that SV40 large T promoted A31 agar colony formation when present over a 5- to 7.5-fold concentration range. However, at the low end of the spectrum, its transforming effect was manifest inefficiently except in the presence of t. Thus a major role for t in the SV40 transforming mechanism is to enhance directly or indirectly the transforming function of T.

A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth.

A recombinant murine retrovirus containing the intact cDNA sequence for the simian virus 40 (SV40) large T antigen (T) was constructed by using the pZIPNeo SV(X)1 vector. Psi 2 packaging cells were then transfected, and G418-resistant clones were used to generate helper-free viral stocks. NIH 3T3 mouse fibroblasts infected by the recombinant T cDNA retrovirus were selected fro G418 resistance. Such cultures synthesized authentic SV40 T and were transformed to anchorage-independent growth at high efficiency. Therefore, this vector has allowed the study of the transformation properties of T under conditions of neutral drug selection and in the absence of SV40 small t antigen.

Cellular proteins which can specifically associate with simian virus 40 small t antigen.

When crude, radiolabeled extracts of various cells were applied to homogeneous simian virus 40 small t antigen-Sepharose adsorbents, three cell proteins (57, 32, and 20 kilodaltons [kDa]) bound specifically. Each also bound to an insoluble, truncated t derivative composed of the COOH-terminal 123 residues of the protein. The binding of these proteins was greatly inhibited after reduction and alkylation of the t ligand. Therefore, some element of native conformation, but not all of the primary structure of t, is necessary for this binding property, which may constitute a discrete, in vitro biochemical function of this protein. Results of cell fractionation experiments suggested that the 57- and 32-kDa proteins are nonnuclear cell constituents, whereas the 20-kDa protein was closely associated with a detergent-washed nuclear fraction. Specific immunoblotting and comparative partial proteolytic digestion analyses indicated that the 57-kDa protein is tubulin, a major component of the cytoskeleton. In this regard, t and tubulin were observed to coimmunoprecipitate from crude cell extracts after incubation with monospecific anti-t antibody. Therefore, it is possible that t and tubulin interact in vivo.

The t-unique coding domain is important to the transformation maintenance function of the simian virus 40 small t antigen.

The small t antigen (t) of simian virus 40, a 174-amino-acid-containing protein, when present together with the other early viral protein, large T antigen (T), plays an important role in the maintenance of simian virus 40-induced neoplastic phenotype in certain cells. Indeed, each protein functions in a complementary manner in this process. The t coding unit is composed of two segments, a 5' region of 246 nucleotides which is identical to that of the corresponding 5' region of the T coding unit and a 3' segment of 276 nucleotides which is unique. Two mutant, t-encoding genomes, one bearing a missense and the other a nonsense mutation at the same point in the t-unique coding region were constructed in vitro and found to be defective in their ability to dissolve the actin cytoskeleton of rat fibroblasts and to complement T in the growth of mouse fibroblasts in soft agar. Therefore, the unique segment of the t gene encodes a portion of the t molecule which is essential to its transformation maintenance function.

Localization of the simian virus 40 small t antigen in the nucleus and cytoplasm of monkey and mouse cells.

Monkey and mouse cells producing simian virus 40 small t antigen in the absence of clearly detectable intact or truncated large T antigens were subjected to indirect immunofluorescence and biochemical cell compartment analyses. Results revealed specific immunofluorescence and small t polypeptide in both the nucleus and cytoplasm of these cells.

Purification of biologically active simian virus 40 small tumor antigen.

The simian virus 40 small tumor antigen (t antigen) gene has been cloned downstream from a hybrid Escherichia coli trp-lac promoter and a suitable ribosome binding site. A bacterial clone (865i) transformed by such a plasmid (pTR865) expresses this gene and, under optimal conditions, can produce greater than or equal to 5% of its total protein as t antigen. Soluble extracts of such a clone were relatively depleted in t antigen, which was found in the initial pellet fraction. The protein was recovered from this fraction in a significantly purified form by extraction with urea-containing buffer. After gel filtration of such t antigen-enriched solutions, highly purified protein was obtained. When either this fraction (freed of urea) or NaDodSO4 gel-purified 865i t antigen (rendered free of detergent) was injected into untransformed rat cells, dissolution of intracellular actin cable networks was observed.

Role of small t antigen in the acute transforming activity of SV40.

A plasmid, pHR402, containing SV40 sequences that include a truncated early region bearing an intact t-coding sequence and a functionally intact late region, was introduced into thymidine kinase deficient (tk-) mouse L cells by cotransformation with a cloned tk gene. tk+ cotransformants synthesized SV40 t but not T antigen, and no truncated T-coding sequence products were detected. The viral sequences of pHR402 were reconstituted as a virus in COS1 cells, and acute infection of untransformed mouse cells with this viral stock (SV402) also led to the appearance of t but not T or a truncated T. Abortive transformation assays of such infected cells were negative, as were those performed on the same cells infected with either of two viral mutants (dl883 and dl884), each of which leads to T but not t synthesis. However, mixed infection with SV402 and either dl883 or dl884 led to a clear abortive and permanent transformation response. Thus, at least in part, t and T appear to function in a complementary fashion in eliciting transformation expression by SV40-infected cells.

Synthesis of simian virus 40 t antigen in Escherichia coli.

Plasmids are constructed by using recombination in vitro according to Roberts, T.M., Kacich, R. & Ptashne, M. (1979) Proc. Natl. Acad. Sci. USA 76, 760-764 in which the t antigen gene of simian virus 40 is fused to a promoter of the Escherichia coli lac operon. In the fusions, transcription commences at the lac promoter, and, in some of the fusions, translation begins at the ATG initiator codon of the t gene. This translation is directed most efficiently by those plasmids in which the lac sequences abut the t gene such that a hybrid ribosome binding is encoded. In this case, the Shine-Dalgarno sequence is of lac origin but the ATG derives from the t gene. translation from this initiator codon is greatly decreased if the lac sequences are separated from the ATG by 17 base pairs and is abolished if the AT of this triplet is deleted. Cells bearing the productive fusions synthesize a 20,000-dalton protein with t antigenic determinants. This protein has an isoelectric point(s) indistinguishable from that of t antigen isolated from simian virus 40-transformed cells. Moreover, a partial sequence of the amino-terminal region of the bacterial product is that predicted for authentic t antigen. We conclude that these bacteria are for authentic t antigen. We conclude that these bacteria are producing a protein, the sequence of which is identical to that of authentic t antigen unfused to other polypeptides.

Proteins of Newcastle disease virus and of the viral nucleocapsid.

Newcastle disease virus was found to contain three major proteins. The structure unit of the viral nucleocapsid appears to be monomeric and to consist of a single large protein of an approximate molecular weight of 62,000.