Early Mutants of Polyoma Virus (dl8 and dl23) with Altered Transformation Properties: Is Polyoma Virus Middle T Antigen a Transforming Gene Product?

Griffin, B. E.; Ito, Yoshiaki; Novak, Ulrike; Spurr, Nigel K.; Dilworth, S. J.; Smolar, Nina; Pollack, Ross A.; Smith, Kevin M.; Rifkin, Daniel B.

doi:10.1101/sqb.1980.044.01.031

Cited by 41 publications

(46 citation statements)

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“…Interestingly, 257 mutants were not defective in transformation in vitro, and therefore the 14-3-3 proteins have been proposed to be part of a structural multimerization of MT rather than being involved in functional activity (70). This was further supported by the observation that dl8 mutants of Py, which lack the sequences in MT responsible for binding the 14-3-3 proteins, were not transformation deficient but were actually more efficient at transformation than was wild-type virus (136). The multimerization of MT, at most a dimer based on in vitro data, may be more important in vivo since serine 257 mutants are unusually low in induction of salivary gland and subcutaneous tissue tumors (70,307).…”

Section: -3-3 Proteinsmentioning

confidence: 78%

Natural Biology of Polyomavirus Middle T Antigen

Gottlieb

Villarreal

2001

Microbiol Mol Biol Rev

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“It has been commented by someone that ‘polyoma’ is an adjective composed of a prefix and suffix, with no root between—a meatless linguistic sandwich” (C. J. Dawe). The very name “polyomavirus” is a vague mantel: a name given before our understanding of these viral agents was clear but implying a clear tumor life-style, as noted by the late C. J. Dawe. However, polyomavirus are not by nature tumor-inducing agents. Since it is the purpose of this review to consider the natural function of middle T antigen (MT), encoded by one of the seemingly crucial transforming genes of polyomavirus, we will reconsider and redefine the virus and its MT gene in the context of its natural biology and function. This review was motivated by our recent in vivo analysis of MT function. Using intranasal inoculation of adult SCID mice, we have shown that polyomavirus can replicate with an MT lacking all functions associated with transformation to similar levels to wild-type virus. These observations, along with an almost indistinguishable replication of all MT mutants with respect to wild-type viruses in adult competent mice, illustrate that MT can have a play subtle role in acute replication and persistence. The most notable effect of MT mutants was in infections of newborns, indicating that polyomavirus may be highly adapted to replication in newborn lungs. It is from this context that our current understanding of this well-studied virus and gene is presented

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Section: -3-3 Proteinsmentioning

confidence: 78%

Natural Biology of Polyomavirus Middle T Antigen

Gottlieb

Villarreal

2001

Microbiol Mol Biol Rev

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“…It is known to bind and thereby inhibit protein phosphatase 2A (PP2A) (39, 52). Middle T antigen (MT), a 421-amino-acid protein associated with membranes and underlying cytoskeletal elements, has been shown to be essential for transformation of rodent fibroblasts in tissue cultures (20,34,47). Mammary epithelial cell-specific expression of MT in transgenic mice results in the induction of multifocal mammary tumors with 100% penetrance (21).…”

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confidence: 99%

The p110α Isoform of Phosphatidylinositol 3-Kinase Is Essential for Polyomavirus Middle T Antigen-Mediated Transformation

Utermark

Schaffhausen

Roberts

et al. 2007

J Virol

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Middle T antigen (MT) of polyomavirus is known to play an important role in virus-mediated cellular transformation. While MT has been extensively examined in spontaneously immortalized rodent fibroblasts, its interactions with cells of other types and species are less well understood. We have undertaken a cross-species and cross-cell-type comparison of MT-induced transformation in cells with genetically defined backgrounds. We tested the transforming abilities of a panel of MT mutants, Y250F, Y315F, and Y322F, that are selectively mutated in the binding sites for the principal effectors of MT-Src homology 2 domain-containing transforming protein, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-␥, respectively-in fibroblasts and epithelial cells of murine or human origin. We found that the Y315F mutation disabled the ability of MT to induce transformation in all cell types and species tested. While Y315F also failed to activate the PI3K pathway in these cells, genetic evidence has indicated Y315 may make other contributions to transformation. To confirm the role of PI3K, the PIK3CA gene, encoding p110␣, the prime effector of PI3K signaling downstream from activated growth factor receptors, was genetically ablated. This abolished the transforming activity of MT, demonstrating the essential role for this PI3K isoform in MT-mediated transformation. The Y250F mutant was able to transform the human, but not the murine, cells that were examined. Interestingly, this mutant fully activates the PI3K pathway in human cells but activated PI3K signaling poorly in the murine cells used in the study. This again points to the importance of PI3K activation for transformation and suggests that the mechanism by which MT activates the PI3K pathway differs in different species.Polyomavirus (PyV) is a small, double-stranded, closed-circular-DNA virus with an approximately 5-kb genome divided into two roughly equal regions. The late transcripts produce the viral capsid proteins, whereas the early region encodes three so-called tumor (T) antigens (46) that are important for both productive infection and transformation. The 785-aminoacid large T antigen (LT) is a nuclear protein with originspecific DNA binding properties and an ATPase activity essential for viral replication (46). LT cannot transform cells in culture but has the ability to immortalize primary cells (16,32). Small t antigen (st) is a 195-amino-acid protein found both in the nucleus and in the cytoplasm (46). It is known to bind and thereby inhibit protein phosphatase 2A (PP2A) (39, 52). Middle T antigen (MT), a 421-amino-acid protein associated with membranes and underlying cytoskeletal elements, has been shown to be essential for transformation of rodent fibroblasts in tissue cultures (20,34,47). Mammary epithelial cell-specific expression of MT in transgenic mice results in the induction of multifocal mammary tumors with 100% penetrance (21).MT functions by providing a platform for the assembly of cellular signaling proteins (11,26). Like st, it binds to the ...

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“…Although they have many similarities, the two papova viruses, simian virus 40 and polyoma virus may differ with respect to transformation (12 (28).…”

Section: Discussionmentioning

confidence: 99%

“…(iii) A new class of polyoma virus early mutants (dl-8 and dl-23), which map between 89 and 95 map units, has been shown to have altered transformation properties (11,12). These mutants code for a small T antigen of normal size and middle and large T antigens of reduced size.…”

mentioning

confidence: 99%

“…In a 90-mm dish, slightly subconfluent Rat-I cells were transfected with either the purified Bcl I/EcoRI 35% fragment (0.2 ,gg) or whole viral DNA (1 ,g) by using the calcium technique (18). DNA was absorbed at 370C for 1 hr, 5 ml of growth medium was added, and the cells were incubated for another 4 hr at 370C, trypsinized, resuspended in 0.35% agar, plated onto 10 50-mm dishes, and fed once weekly as described (12). Three weeks later the colonies were counted.…”

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confidence: 99%

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Coding capacity of a 35 percent fragment of the polyoma virus genome is sufficient to initiate and maintain cellular transformation.

Novak¹,

Dilworth²,

Griffin³

1980

Proc. Natl. Acad. Sci. U.S.A.

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Rat-I cells were transfected with the restriction enzyme fragment of polyoma virus DNA that extends clockwise from the BelI site (65.4 map units) to the EcoRI site (0/100 map units). Six transformed cell lines were obtained and one of them (BE-1) has been investigated in detail. The viral DNA that is integrated into host DNA in this line appeared to consist of two fragments arranged in a "head-to-tail" tandem with no detectable intervening host sequences. BE-I cells contained polyoma virus small and middle tumor antigens that were indistinguishable from the corresponding tumor antigens from lytically infected cells. No The early region of polyoma vinrs DNA (67-26 map units) ( Fig. 1) has been shown to code for at least three proteins (2-4) that have been designated small, middle, and large tumor antigens (T antigens). These proteins appear in susceptible cells shortly after infection with the virus and are identical to the T antigens found in cells transformed by polyoma virus. The functions of these early proteins have been defined primarily by the study of three different classes of viral mutants.(i) The host-range-and transformation-defective (hr-t) mutants in general have deletions that lie between 78 and 86 units on the physical map of the viral genome (5). They do not transform rat or hamster cells in vitro and are not tumorigenic. Infected cells produce a normal large T antigen and may also produce small amounts of truncated versions of the middle and small T antigens (6).(ii) Temperature-sensitive (tsa) mutants, which map in the distal part of the early region (between 0 and 26 map units) (7), transform cells at the permissive temperature and under certain circumstances the cells remain transformed at the nonpermissive temperature (8). tsa mutants code for normal-sized small and middle T antigens; large T antigen usually also is of normal size and is always temperature sensitive in its virus growth function. hr-t and tsa mutants can complement each other for transformation of rat or hamster cells (9, 10).(iii) A new class of polyoma virus early mutants (dl-8 and dl-23), which map between 89 and 95 map units, has been shown to have altered transformation properties (11, 12). These mutants code for a small T antigen of normal size and middle and large T antigens of reduced size.The properties of the various classes of early mutants indicate that a functional middle T antigen may be essential for cellular transformation. In contrast, large T antigen does not appear to be necessary for the maintenance of transformation and, on the basis of data presented here, may not even be necessary for the initiation of transformation. A function for small T antigen in transformation has not been defined.We have carried out experiments to define the sequences of polyoma virus DNA that are essential for cellular transformation and to correlate data on transformation with viral gene products. MATERIAL AND METHODSPreparation of Viral DNA and DNA Fragments. The large plaque A2-strain of polyoma virus was used as ...

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Early Mutants of Polyoma Virus (dl8 and dl23) with Altered Transformation Properties: Is Polyoma Virus Middle T Antigen a Transforming Gene Product?

Cited by 41 publications

References 0 publications

Natural Biology of Polyomavirus Middle T Antigen

Natural Biology of Polyomavirus Middle T Antigen

The p110α Isoform of Phosphatidylinositol 3-Kinase Is Essential for Polyomavirus Middle T Antigen-Mediated Transformation

Coding capacity of a 35 percent fragment of the polyoma virus genome is sufficient to initiate and maintain cellular transformation.

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