Simian virus 40 functions required for the establishment and maintenance of malignant transformation.

Members of the five classes of temperature-sensitive simian virus 40 mutants were tested for their ability to transform Chinese hamster lung cells. Two criteria for transformation were used: the ability to form clones in medium with low serum concentrations and the ability to overgrow a monolayer. Only A mutants failed to transform at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. However, both A and D mutants failed to transform at the restrictive temperature when confluent monolayers and depleted medium were used. When transformed clones were selected, purified by recloning, and examined at both temperatures, only cell lines induced by A mutants lost the transformed phenotype at the higher temperature. Thus, A function is required for maintenance of the transformed phenotype in Chinese hamster lung cells. A function is known to be required for the initiation of viral DNA synthesis in permissive cells. Therefore, transformation may be a consequence of the introduction into a cell of the capacity for aberrant initiation of DNA replication.     

Simian virus 40 (SV40) small t antigen inhibits SV40 DNA replication in vitro.

We describe a biochemical function of simian virus 40 small t antigen, the inhibition of simian virus 40 large T antigen-mediated viral DNA replication in an in vitro replication system. Our results suggest that in this system, small t antigen prevents protein phosphatase 2A-mediated activation of large T antigen.     

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.     

Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells.

The ability of the two early simian virus 40 (SV40) coded proteins, the large and small T-antigens, to abortively induce the disappearance of cytoplasmic actin-containing networks in cultured cells has been studied in rat embryo fibroblasts after microinjection of intact SV40 DNA, DNA fragments from the early region of SV40, and a purified SV40 large T-antigen related protein (the D2 hybrid protein) isolated from cells infected with the adenovirus-SV40 hybrid virus Ad2+D2. Injection of either the 107,000-dalton D2 hybrid protein or SV40 DNA from the deletion mutant dl 884 SV40, which lacks part of the region (0.54 to 0.59) encoding small t-antigen, failed to cause any detectable change in the structure of actin cables in recipient cells over a period of 72 h. By contrast, injection of wild-type SV40 DNA or a DNA fragment containing the entire region coding for a small-t antigen leads to the disruption of actin cable networks within 24 h of injection. It appears likely that the SV40 small-t protein is necessary for the abortive loss of actin cables in injected cells. Epidermal growth factor also causes loss of actin cables in rat embryo fibroblasts or Rat 1 cells (an established rat embryo line), but only after exposure of the cells to epidermal growth factor in the culture medium and not after injection of epidermal growth factor into the cells.     

Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity.

The simian virus 40 small tumor antigen (small t) specifically interacts with protein phosphatase type 2A (PP2A) in vivo and alters its catalytic activity in vitro. Among the substrates for PP2A in vitro are the activated forms of MEK and ERK kinases. Dephosphorylation of the activating phosphorylation sites on MEK and ERKs by PP2A in vitro results in a decrease in their respective kinase activities. Recently, it has been shown that overexpression of small t in CV-1 cells results in an inhibition of PP2A activity toward MEK and ERK2 and a constitutive upregulation of MEK and ERK2 activity. Previously, we have observed that overexpression of either ERK1, MEK1, or a constitutively active truncated form of c-Raf-1 (BXB) is insufficient to activate AP-1 in REF52 fibroblasts. We therefore examined whether overexpression of small t either alone or in conjunction with ERK1, MEK1, or BXB could activate AP-1. We found that coexpression of small t and either ERK1, MEK1, or BXB resulted in an increase in AP-1 activity, whereas expression of either small t or any of the kinases alone did not have any effect. Similarly, coexpression of small t and ERK1 activated serum response element-regulated promoters. Coexpression of kinase-deficient mutants of ERK1 and ERK2 inhibited the activation of AP-1 caused by expression of small t and either MEK1 or BXB. Coexpression of an interfering MEK, which inhibited AP-1 activation by small t and BXB, did not inhibit the activation of AP-1 caused by small t and ERK1. In contrast to REF52 cells, we observed that overexpression of either small or ERK1 alone in CV-1 cells was sufficient to stimulate AP-1 activity and that this stimulation was not enhanced by expression of small t and ERK1 together. These results show that the effects of small t on immediate-early gene expression depend on the cell type examined and suggest that the mitogen-activated protein kinase activation pathway is distinctly regulated in different cell types.     

Simian virus 40 small t antigen activates the carboxyl-terminal transforming p53-binding domain of large T antigen.

Expression of the simian virus 40 large T antigen (large T) in F111 rat fibroblasts generated only minimal transformants (e.g., F5 cells). Interestingly, F111-derived cells expressing only an amino-terminal fragment of large T spanning amino acids 1 to 147 (e.g., FR3 cells), revealed the same minimal transformed phenotype as F111 cells expressing full-length large T. This suggested that in F5 cells the transforming domain of large T contained within the C-terminal half of the large T molecule, and spanning the p53 binding domain, was not active. Progression to a more transformed phenotype by coexpression of small t antigen (small t) could be achieved in F5 cells but not in FR3 cells. Small-t-induced progression of F5 cells correlated with metabolic stabilization of p53 in complex with large T: whereas in F5 cells the half-life of p53 in complex with large T was only slightly elevated compared with that of (uncomplexed) p53 in parental F111 cells or that in FR3 cells, coexpression of small t in F5 cells led to metabolic stabilization and to high-level accumulation of p53 complexed to large T. In contrast, coexpression of small t had no effect on p53 stabilization or accumulation in FR3 cells. This finding strongly supports the assumption that the mere physical interaction of large T with p53, and thus p53 inactivation, in F5 cells expressing large T only does not reflect the main transforming activity of the C-terminal transforming domain of large T. In contrast, we assume that the transforming potential of this domain requires activation by a cellular function(s) which is mediated by small t and correlates with metabolic stabilization of p53.     

Stabilization of the 53,000-dalton nonviral tumor antigen is not required for transformation by simian virus 40.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent about one-half of the coding region for large T antigen. We present evidence here that F8dl is able to transform mouse cells in a focus assay and that cell lines derived from these foci exhibit fully transformed phenotypes, have integrated mutant genomes, and express mutant-encoded proteins. This result implies that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for the maintenance of transformation. In addition, we have found that cells fully transformed by F8dl produce a 53,000-dalton nonviral tumor antigen (p53) that is as unstable as the p53 of untransformed cells. From this result we infer that transformation by simian virus 40 does not require the stabilization of p53.     

Simian virus 40 sequences between 0.168 and 0.424 map units are not required for abortive transformation.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent over 60% of the coding region for large T antigen. Despite this deletion, F8dl abortively transformed rat cells as efficiently as wild-type simian virus 40. From this result, we conclude that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for abortive transformation. Since abortive transformation requires the expression of the simian virus 40 maintenance functions, we also infer that the sequences deleted from F8dl are not required to maintain transformation.     

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.     

Simian virus 40 large T-antigen function is required for induction of tetraploid DNA content during lytic infection.

Infection of quiescent CV-1 cells with simian virus 40 mutant tsA30 at 37 degrees C resulted in the induction of two rounds of cellular DNA synthesis in T-antigen-positive cells, as previously described for wild-type simian virus 40. Following infection with tsA30 at 40.5 degrees C, T-antigen-positive cells were induced into S phase and reached a diploid G2 DNA content; however, a second S phase was not initiated. The failure of tsA30-infected CV-1 cells to enter tetraploid S phase at 40.5 degrees C identifies a T-antigen function, distinct from T-antigen functions responsible for stimulation of cell DNA synthesis, which is required for initiation of a second round of DNA synthesis without mitosis.     

Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences.

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.     

Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen.

Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T.     

Lectin activity of the major surfactant protein (SP-A) may participate in, but is not required for, binding to rat type II cells.

Pulmonary surfactant is a complex mixture of lipids and proteins, of which surfactant protein A (SP-A) is the most abundant glycoprotein. The SP-A molecule has several distinct structural features that include a lectin-like domain, sharing structural features with other mammalian lectins. We have tested the hypothesis that lectin activity of the SP-A molecule is required for the binding to its receptor on the surface of alveolar Type II cells. By using colloidal gold immunocytochemistry in conjunction with electron microscopy, we evaluated the ability of mannosylated proteins to inhibit canine SP-A binding to rat Type II cells in vitro. After preincubation of SP-A with the mannosylated protein horse-radish peroxidase (HRP), SP-A was incubated with isolated filter-grown Type II cells. HRP did not alter the binding of SP-A to the Type II cell surface. Evidence that SP-A did bind to HRP was shown by coincident observation of gold-labeled SP-A and HRP precipitates. These results provide visual evidence that the lectin activity associated with SP-A is not required for its binding to receptor on rat alveolar Type II epithelial cells.     

A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays.

At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays.     

Human papillomavirus type 6b DNA required for initiation but not maintenance of transformation of C127 mouse cells.

We describe the transformation of C127 mouse fibroblasts with human papillomavirus type 6b (HPV-6b) DNA, which is associated primarily with benign tumors of the human genital tract. The major transformed phenotype of the HPV-6b-transfected cells lines, which had been G418 selected, pooled, and maintained without subsequent selection, was tumorigenicity in nude mice. We found that, unlike that reported for other HPVs or papovaviruses, the transformed phenotype was expressed after a delay, in which the cells had undergone extensive culture passages (about 20 passages or 100 generations). Interestingly, the HPV-6b DNA had become reduced or nondetectable in copy number in the cells by the time the transformed phenotype was expressed and in most of the tumors induced by the cells in nude mice, indicating that high levels of HPV-6b DNA were not required for maintenance of the transformed phenotype. Clonal cell lines gave similar results. When continued G418 selection was used to maintain high-copy-number HPV-6b DNA, the cells were tumorigenic, indicating that high levels of HPV-6b DNA did not suppress tumorigenesis. These studies suggest that HPV-6b DNA initiates transformation of C127 cells but is dispensable for expression or maintenance of the transformed phenotype. Transformation by HPV-6b DNA in vitro may provide insights into the HPV type-specific association with benign versus malignant lesions in vivo and may elucidate some of the oncogenic processes involved in tumor progression.     

Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease.

The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases.     

Small t protein of simian virus 40 is required for dense focus formation in a rat cell line.

Incomplete foci were formed in a Fischer rat cell line infected with simian virus 40 detection mutants encoding defective small t antigen. The foci were small and rarely piled up. The fraction of DNA-synthesizing cells in the foci was about half of that induced by wild-type virus.