Binding of p300/CBP co-activators by polyoma large T antigen.

Small DNA tumor viruses such as simian virus 40 (SV40) and polyomavirus (Py) take advantage of host cell proteins to transcribe and replicate their DNA. Interactions between the viral T antigens and host proteins result in cell transformation and tumor induction. Large T antigen of SV40 interacts with p53, pRb/p107/p130 family members, and the cyclic AMP-responsive element-binding protein (CREB)-binding protein (CBP)/p300. Py large T antigen is known to interact only with pRb and p300 among these proteins. Here we report that Py large T binds to CBP in vivo and in vitro. In co-transfection assays, Py large T inhibits the co-activation functions of CBP/p300 in CREB-mediated transactivation but not in NF-kappa B-mediated transactivation. p53 appears not to be involved in the functions of CREB-mediated transactivation and is not essential for large T:CBP interaction. Mutations introduced into a region of Py large T with homology to adenovirus E1A and SV40 large T prevent binding to the co-activators. These mutant large T antigens fail to inhibit CREB-mediated transactivation. The CBP/p300-binding Py mutants are able to transform established rat embryo fibroblasts but are restricted in their ability to induce tumors in the newborn mouse, indicating that interaction of large T with the co-activators may be essential for virus replication and spread in the intact host.     

Cell-specific modulation of papovavirus replication by tumor suppressor protein p53

Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication.     

A cell regulatory agent, CeReS-18, inhibits mouse 3T6 cell proliferation but not polyomavirus replication

We have purified a cell regulatory sialoglycopeptide, CeReS-18, from intact bovine cerebral cortex cells. This is an 18-kDa molecule that reversibly inhibits cellular DNA synthesis and the proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on mouse 3T6 host cell proliferation and polyomavirus replication was investigated. The results showed that CeReS-18 was able to inhibit 3T6 cell cycling in a concentration-dependent, calcium-sensitive, and reversible manner. Despite the inhibition of cell proliferation, CeReS-18 did not influence polyomavirus infection of 3T6 cells. Indirect immunofluorescent assays revealed that CeReS-18-treated, and cell cycle-arrested, 3T6 cells remained permissive to polyomavirus replication. Electron microscopy and immunogold labeling showed that new viral particles were assembled inside the nuclei of infected cells in the presence of CeReS-18 and during cell cycle arrest. The cellular requirements for the replication of polyomavirus DNA and the synthesis of viral proteins, as well as for the assembly of viral particles, therefore, remained available in CeReS-18-inhibited 3T6 cells. In addition, although polyomavirus infection can be mitogenic, infection of CeReS-18-treated 3T6 cells did not reverse the cell cycle arrest mediated by this cell cycle inhibitor.     

Polyoma virus middle T and small t antigens cooperate to antagonize p53-induced cell cycle arrest and apoptosis.

Wild-type p53 triggers two distinct biological responses, cell cycle arrest and apoptosis. Several small DNA tumor viruses encode proteins that bind p53 and thus block the function of p53. This probably reflects the need of these viruses to prevent p53-induced cell cycle arrest and apoptosis to allow viral DNA replication. Unlike SV40 large T, polyoma virus large T does not bind p53, and it is still unclear how polyoma virus blocks p53 function. To address this question, we transfected polyoma virus middle T or small t alone or middle T and small t together into J3D mouse T-lymphoma cells carrying temperature-sensitive p53 (ts p53). Induction of wild-type p53 by temperature shift to 32 degrees C triggered both G1 cell cycle arrest and apoptosis in parental J3D-ts p53 cells. In contrast, J3D-ts p53 cells coexpressing middle T and small t showed only a weak G1 cell cycle arrest response after induction of wild-type p53 at 32 degrees C. Fluorescence-activated cell sorter analysis revealed that nearly half of the middle T-expressing cells, 30% of the small t-expressing cells, and a majority of the cells coexpressing middle T and small t were resistant to p53-induced apoptosis. The phosphatidylinositol 3-kinase inhibitor wortmannin partially abrogated the protective effect of middle T but not small t on p53-induced apoptosis, indicating that middle T prevents p53-induced apoptosis through the phosphatidylinositol 3-kinase signal transduction pathway. Our results thus establish a mechanism for polyoma virus-mediated inhibition of p53 function.     

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.     

Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication protein A

Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase alpha-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase alpha-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia coli as a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.     

Requirements for species-specific papovavirus DNA replication.

Replication of papovavirus DNA requires a functional replication origin, a virus-encoded protein, large T antigen, and species-specific permissive factors. How these components interact to initiate and sustain viral DNA replication is not known. Toward that end, we have attempted to identify the viral target(s) of permissive factors. The functionally defined replication origins of polyomavirus and simian virus 40, two papovaviruses that replicate in different species (mice and monkeys, respectively), are composed of two functionally distinct domains: a core domain and an auxiliary domain. The origin cores of the two viruses are remarkably similar in primary structure and have common binding sites for large T antigen. By contrast, their auxiliary domains share few sequences and serve as binding sites for cellular proteins. It seemed plausible, therefore, that if cellular permissive factors interacted with the replication origin, their targets were likely to be in the auxiliary domain. To test this hypothesis we constructed hybrid origins for DNA replication that were composed of the auxiliary domain of one virus and the origin core of the other and assessed their capacity to replicate in a number of mouse and monkey cell lines, which express the large T antigen of one or the other virus. The results of this analysis showed that the auxiliary domains of the viral replication origins could substitute for one another in DNA replication, provided that the viral origin core and its cognate large T antigen were present in a permissive cellular milieu. Surprisingly, the large T antigens of the viruses could not substitute for one another, regardless of the species of origin of the host cell, even though the two large T antigens bind to the same sequence motif in vitro. These results suggest that species-specific permissive factors do not interact with the origin-auxiliary domains but, rather, with either the origin core or the large T antigen or with both components to effect DNA replication.     

Human cytomegalovirus disrupts both ataxia telangiectasia mutated protein (ATM)- and ATM-Rad3-related kinase-mediated DNA damage responses during lytic infection

Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.     

Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication.

We have investigated the species-specific replication of polyomavirus DNA in the cell-free system that was established previously (Y. Murakami, T. Eki, M. Yamada, C. Prives, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 83:6347-6351, 1986). Extracts from various species of cells supported polyomavirus DNA replication in a species-specific manner that was consistent with the host range specificity of polyomavirus; extracts prepared from mouse and hamster cells were active, whereas extracts prepared from human, monkey, and insect cells were inactive. The addition of DNA polymerase alpha-primase purified from mouse cells induced the replication of polyomavirus DNA in a cell-free system containing polyomavirus large tumor antigen and nonpermissive cell extracts, such as human and insect cell extracts. Isolated mouse DNA primase alone also induced polyomavirus DNA replication in human cell extracts but not in insect cell extracts, indicating that mouse DNA primase plays the principal role in determining permissiveness for polyomavirus DNA replication.     

Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication.

Geminiviruses are plant DNA viruses with small genomes whose replication, except for the viral replication protein (Rep), depends on host proteins and, in this respect, are analogous to animal DNA tumor viruses, e.g. SV40. The mechanism by which these animal viruses create a cellular environment permissive for viral DNA replication involves the binding of a virally encoded oncoprotein, through its LXCXE motif, to the retinoblastoma protein (Rb). We have identified such a LXCXE motif in the Rep protein of wheat dwarf geminivirus (WDV) and we show its functional importance during viral DNA replication. Using a yeast two-hybrid system we have demonstrated that WDV Rep forms stable complexes with p130Rbr2, a member of the Rb family of proteins, and single amino acid changes within the LXCXE motif abolish the ability of WDV Rep to bind to p130Rbr2. The LXCXE motif is conserved in other members of the same geminivirus subgroup. The presence of an intact Rb binding motif is required for efficient WDV DNA replication in cultured wheat cells, strongly suggesting that one of the functions of WDV Rep may be the linking between viral and cellular DNA replication cycles. Our results point to the existence of a Rb-like protein(s) in plant cells playing regulatory roles during the cell cycle.     

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts.

The induction of an S phase in the host cell is a prerequisite for the lytic replication cycle of polyomavirus. This function was attributed to proteins coded for by the early region of the viral DNA, the T antigens. A consideration of the role of the T antigens in the initiation of a mitogenic response of the host cell has to take into account the recent discovery that virus adsorption is sufficient to induce the synthesis of proteins which are known to appear early after quiescent cells are stimulated by the addition of serum, namely fos, jun, and myc (J. Zullo, C.D. Stiles, and R.L. Garcea, Proc. Natl. Acad. Sci. USA 84:1210-1214, 1987; G. M. Glenn and W. Eckhart, J. Virol. 64:2193-2201, 1990). This induction is followed by an initiation of DNA synthesis. It is therefore important to dissociate the effects of the T antigens on the host cell from those of virus adsorption. To do so, we used dexamethasone-regulated versions of the large and small T antigens of polyomavirus stably integrated into the genome of Swiss 3T3 cells to study their function in S-phase induction. When the production of the large or small T antigen in serum-starved 3T3 mouse fibroblasts was activated, only a small fraction of cells was able to leave G0/G1 despite the synthesis of considerable amounts of the respective T antigen. Activation of both T antigens within the same cell, on the other hand, resulted in S-phase induction in a notable percentage of cells, suggesting that the two proteins cooperate in this activity. Polyomavirus T antigens appear to bypass the pathway of growth regulation involving the activation of c-fos. These results are discussed in relation to other known functions of the two virally coded proteins.     

Tumor suppressor genes

The retinoblastoma sensitivity protein (Rb) and the p53 gene product both appear to function as negative regulators of cell division or abnormal cellular growth in some differentiated cell types. Several types of cancers have been shown to be derived from cells that have extensively mutated both alleles of one or both of these genes, resulting in a loss-of-function mutation. In the case of the p53 gene, this mutational process appears to occur in two steps, with the first mutation at the p53 locus resulting in a trans-dominant phenotype. The mutant p53 gene product enters into an oligomeric protein complex with the wild-type p53 protein derived from the other normal allele and such a complex is inactive or less efficient in its negative regulation of growth control. This intermediate stage of carcinogenesis selects for the proliferation of cells with one mutant allele, enhancing the probability of obtaining a cancer cell with both alleles damaged. The DNA tumor viruses have evolved mechanisms to interact with the Rb and p53 negative regulators of cellular growth in order to enhance their own replication in growing cells. SV40 and adenovirus type 5 produce viral encoded proteins that also form oligomeric protein complexes with p53 and Rb, presumably inactivating their functions. These viral proteins are also the oncogene products of these viruses. Thus, the mechanisms by which cancer may arise in a host, via mutations or virus infections, have fundamental common pathways effecting the same cellular genes and gene products; Rb and p53.     

Sequences flanking the pentanucleotide T-antigen binding sites in the polyomavirus core origin help determine selectivity of DNA replication.

Replication of the genomes of the polyomaviruses requires two virus-specified elements, the cis-acting origin of DNA replication, with its auxiliary DNA elements, and the trans-acting viral large tumor antigen (T antigen). Appropriate interactions between them initiate the assembly of a replication complex which, together with cellular proteins, is responsible for primer synthesis and DNA chain elongation. The organization of cis-acting elements within the origins of the polyomaviruses which replicate in mammalian cells is conserved; however, these origins are sufficiently distinct that the T antigen of one virus may function inefficiently or not at all to initiate replication at the origin of another virus. We have studied the basis for such replication selectivity between the murine polyomavirus T antigen and the primate lymphotropic polyomavirus origin. The murine polyomavirus T antigen is capable of carrying out the early steps of the assembly of an initiation complex at the lymphotropic papovavirus origin, including binding to and deformation of origin sequences in vitro. However, the T antigen inefficiently unwinds the origin, and unwinding is influenced by sequences flanking the T antigen pentanucleotide binding sites on the late side of the viral core origin. These same sequences contribute to the replication selectivity observed in vivo and in vitro, suggesting that the inefficient unwinding is the cause of the replication defect. These observations suggest a mechanism by which origins of DNA replication can evolve replication selectivity and by which the function of diverse cellular origins might be temporally activated during the S phase of the eukaryotic cell cycle.     

Wild-type p53 is not a negative regulator of simian virus 40 DNA replication in infected monkey cells.

To analyze the proposed growth-inhibitory function of wild-type p53, we compared simian virus 40 (SV40) DNA replication in primary rhesus monkey kidney (PRK) cells, which express wild-type p53, and in the established rhesus monkey kidney cell line LLC-MK2, which expresses a mutated p53 that does not complex with large T antigen. SV40 DNA replication proceeded identically in both cell types during the course of infection. Endogenously expressed wild-type p53 thus does not negatively modulate SV40 DNA replication in vivo. We suggest that inhibition of SV40 DNA replication by wild-type p53 in in vitro replication assays is due to grossly elevated ratios of p53 to large T antigen, thus depleting the replication-competent free large T antigen in the assay mixtures by complex formation. In contrast, the ratio of p53 to large T antigen in in vivo replication is low, leaving the majority of large T antigen in a free, replication-competent state.