Transcriptional and post-translational regulation of the quiescence factor and putative tumor suppressor p150(Sal2).

The evolutionarily conserved SALL genes encode transcription factors with roles in embryonic development. The product of the SALL2 gene was first identified as a binding partner of the mouse polyoma virus large T antigen and later shown to possess tumor suppressor-like functions. Independent studies identified SALL2 as a factor regulating the quiescent state in human fibroblasts. Here, we investigate factors that regulate the expression of SALL2 and turnover of p150(Sal2) in growing vs. resting cells. The transcription factor AP4 increases along with SALL2 in quiescent cells and positively regulates SALL2 expression. TGFß effectively inhibits expression of SALL2 and its regulator AP4 when added to quiescent fibroblasts. TGFß repression of SALL2 and AP4 is independent of the induction of connective tissue growth factor (CTGF) by TGFß. p150(Sal2) disappears rapidly on restoration of serum. In both growing fibroblasts and established ovarian surface epithelial cells, p150(Sal2) undergoes polyubiquitination and proteosomal degradation. A CUL4/DDB1 E3 ligase containing RBBP7 as the p150(Sal2) receptor has been identified as mediating the destruction of p150(Sal2) as cells transition from a quiescent to an actively growing state.

Shp2 suppresses PyMT-induced transformation in mouse fibroblasts by inhibiting Stat3 activity.

We have examined the effect of expression of the protein tyrosine phosphatase Shp2 on transformation by the mouse polyoma virus middle T antigen (PyMT). Gain-of-function mutations in Shp2 indicate that it may serve as an oncogene in several types of human leukemia. Paradoxically, however, some catalytically dominant-negative mutations of Shp2 have also been identified in leukemia and neuroblastomas. In this study, we show that Shp2 suppresses transformation induced by PyMT, the major polyoma viral oncoprotein known to act through binding and activation of pp60(c-src). Over-expression of a catalytically inactive Shp2 mutant in NIH3T3 cells significantly enhanced PyMT-induced transformation. Conversely, re-introduction of Shp2 into Shp2-deficient cells strongly inhibited PyMT-induced transformation and tumorigenesis. Short hairpin RNA (shRNA)-mediated Shp2 knockdown potentiated PyMT-induced transformation. Finally, we present evidence that the transformation-suppressive effects of Shp2 are mediated at least partially through the inhibition of signal transducers and activators of transcription 3.

Polyoma virus-induced osteosarcomas in inbred strains of mice: host determinants of metastasis.

The mouse polyoma virus induces a broad array of solid tumors in mice of many inbred strains. In most strains tumors grow rapidly but fail to metastasize. An exception has been found in the Czech-II/Ei mouse in which bone tumors metastasize regularly to the lung. These tumors resemble human osteosarcoma in their propensity for pulmonary metastasis. Cell lines established from these metastatic tumors have been compared with ones from non-metastatic osteosarcomas arising in C3H/BiDa mice. Osteopontin, a chemokine implicated in migration and metastasis, is known to be transcriptionally induced by the viral middle T antigen. Czech-II/Ei and C3H/BiDa tumor cells expressed middle T and secreted osteopontin at comparable levels as the major chemoattractant. The tumor cell lines migrated equally well in response to recombinant osteopontin as the sole attractant. An important difference emerged in assays for invasion in which tumor cells from Czech-II/Ei mice were able to invade across an extracellular matrix barrier while those from C3H/BiDa mice were unable to invade. Invasive behavior was linked to elevated levels of the metalloproteinase MMP-2 and of the transcription factor NFAT. Inhibition of either MMP-2 or NFAT inhibited invasion by Czech-II/Ei osteosarcoma cells. The metastatic phenotype is dominant in F1 mice. Osteosarcoma cell lines from F1 mice expressed intermediate levels of MMP-2 and NFAT and were invasive. Osteosarcomas in Czech-II/Ei mice retain functional p53. This virus-host model of metastasis differs from engineered models targeting p53 or pRb and provides a system for investigating the genetic and molecular basis of bone tumor metastasis in the absence of p53 loss.

Receptor-binding and oncogenic properties of polyoma viruses isolated from feral mice.

Laboratory strains of the mouse polyoma virus differ markedly in their abilities to replicate and induce tumors in newborn mice. Major determinants of pathogenicity lie in the sialic binding pocket of the major capsid protein Vp1 and dictate receptor-binding properties of the virus. Substitutions at two sites in Vp1 define three prototype strains, which vary greatly in pathogenicity. These strains replicate in a limited fashion and induce few or no tumors, cause a disseminated infection leading to the development of multiple solid tumors, or replicate and spread acutely causing early death. This investigation was undertaken to determine the Vp1 type(s) of new virus isolates from naturally infected mice. Compared with laboratory strains, truly wild-type viruses are constrained with respect to their selectivity and avidity of binding to cell receptors. Fifteen of 15 new isolates carried the Vp1 type identical to that of highly tumorigenic laboratory strains. Upon injection into newborn laboratory mice, the new isolates induced a broad spectrum of tumors, including ones of epithelial as well as mesenchymal origin. Though invariant in their Vp1 coding sequences, these isolates showed considerable variation in their regulatory sequences. The common Vp1 type has two essential features: 1) failure to recognize "pseudoreceptors" with branched chain sialic acids binding to which would attenuate virus spread, and 2) maintenance of a hydrophobic contact with true receptors bearing a single sialic acid, which retards virus spread and avoids acute and potentially lethal infection of the host. Conservation of these receptor-binding properties under natural selection preserves the oncogenic potential of the virus. These findings emphasize the importance of immune protection of neonates under conditions of natural transmission.

Polyomavirus small T antigen controls viral chromatin modifications through effects on kinetics of virus growth and cell cycle progression.

Minichromosomes of wild-type polyomavirus were previously shown to be highly acetylated on histones H3 and H4 compared either to bulk cell chromatin or to viral chromatin of nontransforming hr-t mutants, which are defective in both the small T and middle T antigens. A series of site-directed virus mutants have been used along with antibodies to sites of histone modifications to further investigate the state of viral chromatin and its dependence on the T antigens. Small T but not middle T was important in hyperacetylation at major sites in H3 and H4. Mutants blocked in middle T signaling pathways but encoding normal small T showed a hyperacetylated pattern similar to that of wild-type virus. The hyperacetylation defect of hr-t mutant NG59 was partially complemented by growth of the mutant in cells expressing wild-type small T. In contrast to the hypoacetylated state of NG59, NG59 minichromosomes were hypermethylated at specific lysines in H3 and also showed a higher level of phosphorylation at H3ser10, a modification associated with the late G(2) and M phases of the cell cycle. Comparisons of virus growth kinetics and cell cycle progression in wild-type- and NG59-infected cells showed a correlation between the phase of the cell cycle at which virus assembly occurred and histone modifications in the progeny virus. Replication and assembly of wild-type virus were completed largely during S phase. Growth of NG59 was delayed by about 12 h with assembly occurring predominantly in G(2). These results suggest that small T affects modifications of viral chromatin by altering the temporal coordination of virus growth and the cell cycle.

Induction and utilization of an ATM signaling pathway by polyomavirus.

Progression from G(1) to S is essential for polyomavirus DNA replication and depends on the interaction of large T with the retinoblastoma gene product pRb. This virus-induced replication pathway is accompanied by p53 activation resembling a DNA damage response (12). We sought to determine whether this pathway depends in part on activation of the ATM (ataxia telangiectasia mutated) kinase and whether the virus gains advantages from this pathway beyond that of entry into S. We show that polyomavirus infection activates the S- and G(2)-phase checkpoints in primary as well as established mouse cells. Infected cells undergo a prolonged S phase compared to uninfected serum-stimulated cells and show no evidence of a G(2)-->M transition before lytic death ensues. Infection is accompanied by increases in ATM activity in vitro and in the level of ATM-S1981-P in vivo. The incubation of infected cells with caffeine, a known ATM inhibitor, did not block entry into S but reduced the rate of viral compared to cellular DNA synthesis. Importantly, caffeine lowered the yields of viral DNA an average of 3- to 6-fold and those of infectious virus by as much as 10-fold. Virus yields were 10-fold lower in ATM (-/-) p53(-/-) than in ATM(+/+) p53(-/-) mouse embryo fibroblasts, indicating a p53-independent role of ATM in productive infection. Replacement of the normal SMC1 (structural maintenance of chromosomes, or cohesin) protein, a critical ATM substrate in the DNA repair pathway, with its phosphorylation mutant SMC1(S957AS966A) also lowered virus yields by roughly 90%. We suggest that polyomavirus activates and utilizes a component(s) of an ATM pathway of DNA repair to prolong S phase and aid its own replication.

Ganglioside GD1a restores infectibility to mouse cells lacking functional receptors for polyomavirus.

Recent investigations on the pathway of cell entry by polyomavirus (Py) and simian virus 40 (SV40) have defined specific gangliosides as functional receptors mediating virus binding and transport from the plasma membrane to the endoplasmic reticulum (B. Tsai et al., EMBO J. 22:4346-4355, 2003; Gilbert and Benjamin, in press). These studies were carried out with C6 rat glioma cells, a heterologous host chosen for its known deficiency in ganglioside biosynthesis. Here, a cell genetic approach was undertaken to identify components required for the early steps of infection using mouse cells as the natural host for Py. Receptor-negative (R-) mouse cells, screened based on resistance to Py infection, were shown to bind Py but failed to allow entry of the virus. R- cells were also found to be resistant to SV40. Infectibility was restored or enhanced by the addition of the same specific gangliosides found in earlier studies with C6 cells. In one R- line, overexpression of caveolin-1 also increased infectibility. These results support and extend findings on gangliosides in lipid rafts as functional receptors and mediators of internalization for Py and SV40.

Identification of TAZ as a binding partner of the polyomavirus T antigens.

A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Delta2-4) in the common N terminus of the T antigens. To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4. TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner. TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen [lT]:middle T antigen [mT]:sT). The Delta2-4 mutant T antigens showed no detectable binding. The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D-->NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection. The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding. The Delta2-4 mutant was defective in viral DNA replication. Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2alpha. Wild-type polyomavirus T antigens effectively block transactivation by TAZ. The functional significance of TAZ interactions with polyomavirus T antigens is discussed.

Induction and bypass of p53 during productive infection by polyomavirus.

Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G(1)-to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.