The members of the Polyomavirinae are small, nonenveloped, double-stranded DNA viruses with icosahedral capsids. Following its discovery in 1953 by Gross, the ecology of Py in the wild was first explored by Rowe in the early 1960s. From 1959 to 1960, Rowe and colleagues captured mice throughout New York City and in rural farms in Maryland. Collections from both locations were considered to be wild mice, as opposed to inbred laboratory strains (288, 290). They found that not only was Py infection clinically inapparent and focal, but also it remained persistent in the population at roughly the same frequency. Although virus was found in various organs of the mice and in the excreta and environment (floor sweepings), urine appeared to be the source of persistence and spread of the virus in a population (upwards of 10³ 50% infective doses /0.2 ml excreted in the urine for months from infected newborns) (289). Additional studies in farms and grain and feed mills of rural Maryland revealed that only areas with sustained breeding conditions had significant Py-positive mice, consistent with infection of newborns (154). Thus, the life cycle of mouse and other mammalian polyomaviruses involves lifelong persistent infections. All mice in these experiments were Mus musculus, but this nomenclature would correspond to Mus musculus domesticus or the more current name of Mus domesticus. Natural Py infection was not found in other rodents when the hemagglutination inhibition assay was used to detect Py in previously collected serum, including a limited survey of wild mice (Mus [rural, not associated with human dwellings], Peromyscus, Microtus, Perognathus, and Reithrodontomus) and rats (Rattus, Neotoma, and Dipodomys), although the level of Py infection in the local mice was unknown at the time (275). However, the more recent discovery of avian polyomaviruses displayed a different life cycle, which appeared to be limited to acute (not persistent) infection (355).

The studies by Rowe also suggested that the virus is most likely to be passed from mother to offspring at birth, which is consistent with subsequent laboratory observations establishing that newborns were much more permissive for Py infection. No tumors were observed in such Py-infected mice. One of the conclusions was that under natural conditions, Py does not induce tumors. Rowe suggested four main reasons to explain this conclusion: the protection from maternally transmitted antibodies, the low probability of infection during the small window of neonatal susceptibility, the low dosage of virus likely to be acquired by inhalation (or ingestion), and the effectiveness of the cell-mediated immunity in adult mice. These findings were also consistent with evaluations of tumor incidence rates in large breeding colonies containing Py-infected and uninfected mice which showed no increase in tumor rates in Py-infected colonies (288, 290). Later studies by Gardner also supported this view (124). He surveyed wild-mouse colonies in the Los Angeles area and found only one colony to be highly infected with Py (numerous separate collections were made). His original theory was that Rowe had not waited long enough in his studies for tumors to develop and that mice may show the higher incidence of Py-induced tumors if kept alive for over 2 years. He observed that Py infection did not increase the number of tumors in old age by comparing a heavily Py-infected colony (40%) with a lightly Py-infected colony (0.6%) for an extended period. In addition, even in immunosuppressed mice, tumors were not observed. His conclusion in the end was the same as Rowe's, namely, that Py does not cause cancer in the natural host under natural conditions.

Similar conclusions would apply to the other members of Polyomavirinae, such as the primate simian virus 40 (SV40), and the human BKV and JCV. Although highly prevalent, persistent, and even productive in their natural host and containing a large T antigen (LT) that binds p53 and Rb, these viruses are not associated with tumors in their natural hosts, although there have been reports of a possible connection between the presence of primate SV40 sequences and human malignant mesotheliomas (221). Considering how many people worldwide (including many with immunosuppressive disease) are infected with these agents and able to produce virus and express viral proteins, the lack of observed transformation is compelling and may suggest that our animal tumor studies may not be evaluating a biologically likely phenomena. In other words, not all mechanisms of transformation may be equally valid or biologically relevant. The Py tumor studies may therefore not identify mechanisms that are prevalent causes of human cancer. Therefore the linguistically "meatless" name "polyomavirus" appears not to describe the life-style of any of these agents but reflects phenomena (tumors) that can be induced in specific laboratory settings, such as the inoculation of specific inbred mouse strains within 24 h of birth (76).

The natural life cycles of the mammalian polyomaviruses, however, do seem similar in many respects. The best studied of these viruses (SV40, BKV, JCV, and Py) all appear to establish primary (and generally inapparent) infections via the respiratory tract or associated organs in the newborn host and then establish persistent inapparent infections in the kidneys, with periodic or episodic shedding of infectious virus from adults into the urine. Only in Py have the specific cell types that support primary and secondary infection been identified. Here, primary infection is almost completely restricted to a very specific cell type, the nonciliated epithelial cells (Clara cells) of the bronchi and bronchioles (131). Newborn immature Clara or Clara-like cells are especially permissive for Py infection, and essentially every Clara cell in the newborn lung will support Py replication. However, 4 days after birth, these cells become considerably less permissive and this reduced permissiveness is maintained in the adult lung. As this period of permissiveness corresponds to active Clara cell division and differentiation (the transition to mature Clara cells and ciliated epithelial cells), Py would appear to replicate best in differentiating cells.

The close connection between Py replication and differentiation was first proposed by us to explain why mice have certain tissues (lung and kidney tissues) which will support efficient Py replication only in newborns, not adults, whereas other tissues which continue to differentiate (bone, skin, and mammary gland) can efficiently replicate Py in adults (381). Subsequent in vivo experiments by us, in which adult kidneys were damaged and induced to differentiate, showing that these differentiating tubular epithelial cells better support Py replication, were consistent with this view (13). In addition, we showed that mice with a genetic form of polycystic kidney disease in which kidney epithelia proliferate but do not differentiate into tubular epithelia were not permissive for Py replication, suggesting that terminal differentiation, not proliferation, is needed in permissive cells (14). The general conclusion from all these studies is that Py resembles the papillomaviruses in its requirement for host cell terminal differentiation for productive infection. If this is so, the prevalent view that a role of the products of the viral early genes is to induce quiescent cells into S phase and the full cell cycle in preparation for viral DNA synthesis must necessarily be wrong in natural settings. It seems more likely that the natural role of these proteins would instead be to alter the programming that normally occurs during terminal differentiation of host cells, in order to maintain in these infected cells the capacity for virus DNA synthesis and replication, which would otherwise normally be suppressed. Given this renewed view of Py biology, we can now consider the specific characteristics of MT in relationship to host cell differentiation.

Polyomavirus evolution. Although Polyomavirinae family members establish prevalent persistent infections in their best-studied host (mouse, human, or primate), it is curious that related virus types (small icosahedral, nonenveloped double-stranded DNA viruses) are not found in nonvertebrate species, given the relative simplicity of this virus family and its parasitic dependence on host replication proteins. In addition, mammalian and avian species of polyomavirus seem to differ significantly in their biology, as noted above, in that the avian versions can infect multiple species and do not appear to establish persistent infections (262, 286, 355). Furthermore, the mammalian but not the avian polyomaviruses appear to be phylogenetically congruent with the evolution of their host, which suggests that polyomaviruses have been infecting their current mammalian host since their divergence from common ancestors (1, 139, 310, 323). Of particular interest to this review, however, is that it appears that only the rodent polyomaviruses code for the production of a MT. All the other polyomaviruses seem able to code for LT and small T antigen (ST) (although the ST for bovine polyomavirus is still questionable) but not MT. Why might this viral gene have evolved in the rodent lineage?

Is middle T antigen needed to offset a lost p53-large T-antigen interaction? One correlate that may suggest a distinct strategic difference between mouse (rodent) and other mammalian polyomaviruses is that, unlike nonrodent polyomaviruses, mouse LT lacks a p53 binding site, although Rb binding activity is conserved. This could suggest that MT may have evolved after the divergence of common ancestors and that lost p53 binding function was offset by the creation of MT. The bovine LT appears similar (and may encode a protein that appears to be ST) to the primate version. As bovines diverged very early from other mammals (including rodents), it seems likely that the early version of Py had an LT that bound p53 and pRb and had a functional ST, but no MT, and that MT was established only relatively late in the rodent lineage. Although it is apparent that the polyomaviruses of primates and rodents evolved from the same ancestor about 80 million years ago, it seems almost impossible, conceptually, that a mere LT reading frame shift could have fortuitously resulted in the complex, multitask MT protein we know today (316). Since there are no known host analogues of MT, we cannot identify a possible source of MT sequences and currently have no explanation for this conundrum.

Implications of p53 within polyomavirus infections. The different interactions between mouse and primate LT and p53 might account for the need for mouse MT and the elimination of the p53 binding ability of Py LT. The mammalian polyomaviruses are highly species specific, and it was originally established that the species-specific DNA polymerase -primase complex was the only factor that determined the tropism of SV40 or Py (240, 241). However, later evidence showed that the murine p53 blocked the ability of SV40 to grow in primate-specific extracts while Py, lacking a p53 binding site, was able to replicate in vitro within mouse-specific extracts in the presence of murine p53 (360). It was concluded that lack of p53 binding of Py LT may be due to an evolutionary need to avoid this repression (360). One example of this need that may have influenced Py evolution is the ability of p53 to repress or transactivate the proliferating-cell nuclear antigen (PCNA) (a requirement for Py DNA replication) promoter depending on the species and the cell type (78, 161, 227, 299, 313, 385). Further experiments demonstrated that human p53 did not prevent SV40 replication in Cos cells (SV40-transformed fibroblast-like cells from CV-1 cells derived from adult African green monkey kidneys) whereas murine p53 did prevent this replication (37). Specifically, murine p53 interferes with the presynthesis stages, namely, the helicase activity of SV40 LT, but not the ATPase activity (360). Furthermore, SV40 LT cannot bind DNA polymerase  when associated with murine p53 (121, 315). The mouse p53 displaces the polymerase  from SV40 T antigen, preventing replication from occurring, which may suggest a role of p53 in initiating or maintaining replicative DNA synthesis (360). This clearly indicates a difference between the mouse and human/simian forms of p53 and perhaps even a role that was acquired specifically in the primate (and bovine, possibly others) lineage but is not found in the mouse lineage. Although the human and mouse p53 proteins are only 78% homologous (with ranges from 37 to 92% among the codons), there are few if any indications that the mouse p53 and its related cell cycle, apoptosis, and other functions are effectively different from the human p53 (31, 249, 318). Thus, it is not obvious why the LT would differ in its ability to bind p53.

Middle T antigen and p53. Most DNA tumor viruses, including nonrodent polyomaviruses, papillomavirus, and adenovirus, have a mechanism for inactivating p53 (for a review, see reference 338). However, since Py LT does not directly interact with p53, the most obvious possibility, which has frequently been noted in the literature, is that MT function evolved to offset the lost interaction of mouse LT with p53. This would suggest that MT must act on the host p53 regulatory pathway someplace downstream of LT-p53 binding, such as p53 transactivation during cell cycle arrest and/or p53-induced apoptosis. However, there is no clear evidence that this is the case for Py, although there is recent support for the notion that the downstream functions of src may be to counter the negative effect of p53 on growth regulation (41, 89, 107, 232). The expression of MT (or LT) does not coincide with the activity of p53 or the levels of its expression during Py-induced tumorigenesis (within the limited number of tumors analyzed) (266). No conclusion was reached on whether anti-apoptotic functions of p53 were being manipulated by downstream signal transduction targets of MT, but it was clear that p53 transactivation abilities were not changed by the levels of MT (83, 266). Some of the tumors analyzed (sarcomas) did have mutated forms of p53 with affected transactivation abilities, but the number was similar to that of non-virus-associated tumors and could not be accounted for by Py-selected pressure. Furthermore, Py infection of wild-type or p53 knockout (/) mice (of those mice susceptible to Py-induced tumors) resulted only in a change in the time of tumorigenesis rather in the type or frequency of tumor found (83). A similar study done with cell cultures showed that the activities of p53 were not affected in the Py transformation of established rat embryo fibroblasts (REF52). Nevertheless, MT alone, as opposed to the entire early region required to transform these cells, could transform the cells in the absence of p53 (dominant negative p53 transfected into the REF52 cells) (232). Although these studies conclude that MT does not affect p53 directly, they do not rule out the possibility that MT is interfering with downstream targets of p53.

"... since its initial discovery the role of middle T-antigen in transformation has been studied almost to the exclusion of any function it might play in the life cycle of the virus" (87)

The relationship between the natural and transforming activities of MT are not at all well known. The early view that MT is important to stimulate quiescent cells into S phase seemed to also explain how these genes would be able to transform cells. However, with our better understanding of the Py life cycle, in which quiescent-cell stimulation is not observed, the relationship to transformation has become less clear. A more recent view consistent with the link between Py replication and host cell differentiation would be that the early genes are needed during terminal differentiation to alter cell programming and allow terminal cells to synthesize viral DNA and make virus. The question then becomes how such an activity would relate to transformation. Although we cannot now answer this question, we can consider established transforming activities of MT from this context.

The cellular transformation function of Py MT is well established and has been reviewed (159, 280, 342, 343). MT is sufficient to transform established cell lines but requires portions of LT or even ST as well to induce transformation in primary cultures of fibroblasts (72, 191, 280). In addition, increasing the expression of MT over levels normally associated with virus production is the only way to ultimately achieve the full characteristics associated with the transformed state (273, 275). There is a higher stringency in establishing tumorigenesis in vivo, with the large array of tumors associated with Py developing only with the expression of MT in combination with the N-terminal region of LT and the coding region for ST (11). In particular, the MT-associated tyrosine kinase activity (src) has been linked to the ability of Py to transform cells in culture or form tumors in animals (35, 277). The portions of the early region of Py required and the array of tumors that develop also depend on the type of rodent used and the strain of virus, factors that are missed in vitro (12, 77, 94, 109).

Inbred strains, however, have been frequently selected for tumor-producing phenotypes. The 30-year debate over what allows Py to establish tumors in mice is rooted in the very fact that inbred mice have different degrees of tumor induction susceptibility. The earliest experiments maintained that it was the immune response itself that was responsible for the differing effects of Py in different mouse strains (7). Some of these experiments involved removal of the thymus, irradiation of the mice, or the use of knockout mice (double CD4/CD8) and showed that an unsusceptible mouse strain could now be made susceptible to Py-induced tumors (8, 29, 193-195). One of these susceptibility factors relied on the H-2 locus of the mouse. Some mice process portions of Py that are more immunogenic than others, with H-2^k (found in AKR and C3H strains of mice) being highly prone to Py-induced tumors (110, 205). Benjamin's group has demonstrated that H-2^k mice have a superantigen (sag) derived from Mtv-7 mouse mammary tumor provirus, which eliminates V6-expressing thymocytes, particularly those that recognize amino acids (aa) 389 to 397 of MT (206, 207). Most adult inbred mice can develop Py-induced tumors following radiation, indicating that the block was due to immunological protection against the virus, namely, the particular H-2 locus (49, 110, 192). Some, however, are radiation resistant and appear to block dissemination of the virus throughout the host and thus prevent the establishment of tumors even in newborn inoculated mice (49). This form of resistance may be due to an ability of the mouse to thwart the antiapoptotic mechanisms of MT (49, 74, 225). Others demonstrated that the virus strain itself was largely responsible for the tumor profile and that large-plaque variants, often derived from tumors, were high-tumor strains due to differences in both coding (early or late proteins) and noncoding (enhancer and LT binding sites) sequences of the virus (112). It was later determined that the noncoding sequences are most responsible for the tumor profile, although changes in VP-1, the major capsid protein, significantly after the tumor profile (94, 108, 109, 111). The specific interaction between VP-1 and the sialic acid residues associated with Py cellular tropism has a profound effect on dissemination of the virus and the Py induction of tumors in mice (19, 20, 94, 108, 111). However, the very fact that most, if not all, experiments in which Py was used to study tumorigenesis were done with inbred strains of mice creates and perpetuates the misconceptions about the natural life cycle of Py that we believe should be separated from the tumor biology. As indicated below, immunosuppressed mice are not necessarily prone to Py-induced tumors. It instead seems more likely that other characteristics of inbred mice, such as genomic instability, along with failed immune clearance, are the main determinants of Py tumorigenicity.

IN VIVO VIRUS STUDIES OF MIDDLE T ANTIGEN

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Middle T-Antigen Viral Mutants in Newborn and Adult Immunocompetent Mice

Intranasal inoculation of wild-type Py into newborn mice (BALB/c) results in acute replication of the virus in the nonciliated epithelial cells of the bronchioles, with maximum replication during 3 to 6 days postinoculation (p.i.) (131). Interestingly, there is a transition from proliferation to a combination of proliferation and differentiation (based on the decline of peak bromodeoxyuridine (BrdU) incorporation and PCNA expression) in the newborn bronchiolar epithelium at around 12 to 24 h prior to the peak of virus production (5 to 6 days p.i.) (9, 69, 339). Under the same conditions, MT mutants of Py (PTA-1387T, PTA-1178, PTA-250YS, and A3-MOP1033) replicate little or not at all in the bronchiolar epithelium compared to wild-type virus of the same background strain (K. A. Gottlieb and L. P. Villarreal, unpublished data). These mutants either eliminate the key tyrosine phosphorylation sites (PTA-1178 eliminates tyrosine 315, and PTA-250YS eliminates tyrosine 250 [the importance of these is described below]) or truncate MT (PTA-1387T and A3-MOP1033). Although A3-MOP1033 is of a different strain (A3 being a small-plaque variant of Py, while PTA is a large-plaque variant), it is comparable to PTA-1387T in being a truncation mutant (opal termination codon at nucleotides 1033 to 1035 in MT and a consequential proline-to-leucine change in LT) of MT, eliminating the ability of the protein to bind the plasma membrane (335). These results indicate that MT is particularly adapted to function in the nonciliated epithelium of the newborn mouse lung since no other tissue in the mouse (including adult mouse lung) displayed this sensitivity to MT mutants (see below). In addition, since the replication levels of the mutant viruses (measured by in situ hybridization) correlated well with levels of T-antigen expression (as measured by immunofluorescence using an antibody to the shared domain of all three T antigens), this supported the idea that MT plays an autocatalytic role in Py replication in that MT function was associated with levels of early gene expression. MT function is also probably required in maintaining persistence, as indicated by the finding that the MT mutant (PTA-1387T) is unable to persist in the kidneys of mice (113). However, in contrast to their behavior in newborn lungs, the point mutants (PTA-1178 and PTA-250YS) associated with elimination of key phosphorylation sites have minor effects on virus replication in lungs of adult mice, producing subtle, often indistinguishable phenotypes from those produced by wild-type virus. The main difference in the adult is that the overall numbers of cells replicating Py are reduced, but those that are infected show a similar quantity of virus to wild-type infected cells. The lungs of adult mice given an intranasal inoculation of the truncation mutants (PTA-1387T and A3-MOP1033), on the other hand, gave similar phenotypes to the newborn lungs, with only a very few cells showing any infection at all. Since the signal transduction pathways described in the next section are an amalgamation of Py MT effects on many cell types in vitro, some of which are not even part of the life cycle of the virus, it is not clear how these identified interactions might relate to Py replication in vivo or the differences we see in newborn and adult lung infection. Our results indicate that depending on the specific cell type, whether nonciliated epithelium of the newborn or adult lung or tubular epithelium of the kidney, a different but at times redundant subset of signal transduction is required for efficient replication of the virus. On the other hand, the subtle differences in replication of the Py with MT point mutants used in our experiments could indicate either that the associated pathways (Shc/PI 3-kinase) are not required in the bronchiolar epithelium or that one pathway can substitute for the other if necessary.

A few studies have examined Py infection in SCID or SCID-beige mice, but our study was the first to study Py mutants (MT and LT) in this context (28, 326, 327). The initial studies of others used intraperitoneal inoculation of the virus into adult SCID mice, with or without NK cells, and evaluated the associated myeloproliferative disease that resulted. Mice succumbed to the virus within 16 days after infection after a period of inactivity and severe weight loss. Similar studies by Dalianis and coworkers, also using intraperitoneal inoculation, established the kinetics of viral replication and dissemination in the organs of the mice over an 8-week period. Unlike the previous study, mice did not succumb to a virus-associated disease until after the 8-week period (28). In neither experiment were tumors observed, unlike reports of Py inoculation into adult athymic nude mice (mostly mammary adenocarcinomas in females and osteosarcomas in males) or susceptible strains of newborn mice associated with the depletion of specific thymocytes that recognize MT (94, 381). Although the replication of Py was restricted to a subset of organs in nude mice, namely, skin, bone, and mammary and salivary glands, the tumors were rapid and led to death before other, slow-growing tumors could appear (326, 381). The difference in SCID and nude mouse susceptibility is curious and could possibly be associated with the strain differences, although this remains to be determined.

The myeloproliferative disease observed in histopathological studies of the bone marrow and spleen was characterized as left-shift maturation of myeloid precursors resulting in myeloid hyperplasia and megakaryocyte and erythroid degeneration. Similar to a study in which adult mice were inoculated with a recombinant Moloney leukemia virus expressing MT, the early target for T-antigen expression in SCID mice was found to be megakaryocytes (120). In the SCID mouse study, though, the megakaryocytes were depleted at later time points, perhaps as a result of lytic infection of these cells.

Our studies used intranasally inoculated SCID-bg mice over 6 weeks old with wild-type virus, strain A3. These mice began to show limited replication of the virus in the lungs by 3 weeks after infection, ranging from 5 to 15% of the epithelial cells in any given bronchiole (Gottlieb and Villarreal, unpublished). This replication increased substantially over the next few weeks, eventually encompassing a majority (over 70%) of the epithelium of the bronchioles. The notable delay in Py lung replication relative to that seen in newborns is probably due to the much lower rate of cellular differentiation occurring in the adult lung and suggests that virus replication must await the accumulation of cellular differentiation to generate permissive cells. The mice eventually succumbed to an acute myeloproliferative disease induced by Py about 35 days after infection (reviewed in reference 326). Contrary to the expectations of those who study Py, the MT mutant A3-MOP1033 (MOP), which should lack all MT-associated functions, replicated well in SCID-bg mice. Although the overall numbers of cells infected were decreased, the replication levels on a per-cell basis appeared identical to those in wild-type infection. Surprisingly, dissemination of MOP throughout the mice, including establishing infection and persistence in the kidneys, was indistinguishable from that of wild-type virus, although the numbers of kidney cells replicating Py were also lower. Although MT may still have some immune-related functions which would not be observed in SCID mice, MT function in Py replication in permissive adult tissue is subtle at best. These subtle differences include a slight decrease in virus-infected bronchioles throughout the lungs and, of particular interest, an alteration in the transition time between T-antigen expression and viral DNA replication compared to wild-type virus. The MOP-infected lungs showed a higher percentage of cells only expressing early proteins among those expressing viral proteins (early, late, or both) than did wild-type-infected lungs, indicating a delay in the onset of Py DNA replication. This latter observation indicates that MT acts as a booster to establish a cell capable of virus replication but is not an absolute requirement. However, we have not yet eliminated the possibility that the expression of the truncated MT may retain some functional capabilities that have yet to be described.

The finding of others that a similar MT mutant (PTA-1387T) eventually loses its kidney persistence also suggests a "booster" role for MT (113). Given that MT mutants which do not replicate efficiently also do not express T antigen efficiently, a likely scenario is that an array of cellular transcription factors are induced by MT to establish an environment suitable for both Py early-gene transcription and Py replication but that this permissive state is also dependent on the natural differentiation of infected cells. Over time, permissive adult SCID lung cells accumulate as they enter this window of differentiation, which allows MT to affect Py early expression and replication. Such a delayed and cumulative effect on virus replication would not be observed in a mouse with a functional and normally responsive immune system. Alternatively, since identical levels of MOP replication (with respect to wild type) in the lungs are found only in a subset of cells, MT and its associated signaling may be required to maintain virus replication as the progenitor cells proliferate and differentiate to replace cells lost due to terminal differentiation and virus lytic infection and exfoliation (132). Although we cannot eliminate the possibility that even a truncated MT has remaining activity, these results clearly illustrate that all of the signal transduction pathways associated with transformation (as described below) play a relatively minor role in Py replication in quiescent permissive adult tissue in vivo.

MIDDLE T-ANTIGEN-ACTIVATED SIGNAL TRANSDUCTION PATHWAYS

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Contextual Considerations

MT is 421 aa long (nucleotides 175 to 748 [aa 1 to 191] spliced to 811 to 1499 [aa 192 to 421]) and is encoded by a splice variant of the early region of Py, which also encodes LT and ST antigens (a general diagram of MT is shown in Fig. 1). MT has a common amino-terminal region (nucleotides 175 to 411) with LT and ST and a further common region with ST (nucleotides 412 to 748). The second exon of MT (nucleotides 811 to 1499, aa 192 to 421) is shared with the LT nucleotide sequence but has a frameshift in the amino acid sequence, astonishingly creating the tyrosines which are phosphorylated through the MT-pp60^c-src and result in activation of multiple signal transduction pathways. It has a molecular mass of 55 kDa and contains a tail of 22 hydrophobic amino acids (aa 394 to 415) at its carboxy terminus, bound on either side by clusters rich in basic residues (47). There are multiple phosphorylation sites throughout MT, including the well-studied tyrosines at aa 250, 315, and 322, serines at aa 257 and 283, and threonines at aa 160 and 291 (155, 259). In addition, MT has a protein phosphatase 2A (PP2A) binding domain (aa 90 to 120) and a region associated with pp60^c-src (aa 185 to 210). While pp60^c-src (and other members of the src family, as mentioned below) serve as the tyrosine kinases, protein kinase C and another, unnamed kinase provide the serine phosphorylation (223). However, in Py-infected cells, only a small portion of MT is phosphorylated, and among this fraction most of the phosphorylation is of serine while a significantly lesser portion is tyrosine or threonine (304).

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Schematic of MT. MT is 421 aa long and is encoded by nucleotides 175 to 748 (aa 1 to 191) spliced to 811 to 1499 (aa 192 to 421) based upon the sequence numbering of the A2 wild-type strain. The membrane-spanning domain consists of hydrophobic aa 394 to 415. On interaction with the plasma membrane, MT associates with (although does not directly bind to) src, a tyrosine kinase, resulting in the phosphorylation of tyrosines at aa 250, 315, and 322 and in the binding of Shc, PI 3-kinase, and PLC-1. In addition, MT contains serine (aa 257 and 283) and threonine (aa 160 and 291) phosphorylation sites. The phosphorylation of serine 257 results in the interaction with the 14-3-3 proteins. Finally, MT associates with both the catalytic subunit C and the regulatory domain A of PP2A at aa 90 to 120.

MT was first found associated with the plasma membrane by using antibodies against tumor antigens from Py-induced tumors (158). The insertion of MT into the plasma membrane and the sequences N terminal (RHLRRLGR) to the membrane binding sequence are functionally important for the MT association with pp60^c-src (src) and the association with the cytoskeleton needed for the transformation morphology (47, 75, 213, 337). Only a fraction of MT is associated with the plasma membrane; the majority is found in perinuclear compartments that do not colocalize with marker proteins for either the endoplasmic reticulum or other subcellular compartments of the secretory pathway (87, 158, 297, 390). Nocodazole, a drug affecting cytoskeletal structures and vesicular transport, prevents perinuclear localization of MT (228). The localization of MT at these perinuclear locations has been associated with the rearrangement of the actin cytoskeleton seen in transformed cells and the dismantling of tubular endosomes (390). Subfractionation studies show that MT in the perinuclear compartments are complexed with src and Shc and that much of the src from the plasma membrane has been relocalized by MT to these compartments (390). One theory about the discrepancies in localization of MT postulates that MT associates with the membrane skeleton and that this accounts for both the plasma membrane and perinuclear MT fractions (10). There has yet to be a study to determine whether MT complexes not associated with the plasma membrane are functional in the lytic life cycle or in transformation (87). Therefore, since such a small portion of MT is associated with the plasma membrane, it is logical to assume that functions of perinuclear MT currently unclear or speculative must be important for the virus life cycle, but these may be more closely associated with persistence than acute replication.

The importance of various domains within MT was largely determined by the ability of MT mutants to alter or abolish MT-induced transformation (46, 47, 93, 200, 214; for a review, see reference 215). The information below will show, however, that the ability to transform a specific cell line requires not only a certain threshold level of src activity but also an often undetermined subset of MT-activated signal transduction (214). Analysis of a number of MT mutants indicated that the complex with src was necessary but insufficient for cellular transformation (36, 57, 63, 137, 213). src (and to a lesser extent other tyrosine kinases, pp62^c-yes [yes] and p59^fyn [fyn]) coimmunoprecipitates with MT (56, 58, 67, 68, 138, 152, 184, 188), is associated with and activated by the amino-terminal end (aa 185 to 210) of MT, and phosphorylates three tyrosines in MT, at aa 250, 315, and 322 (143, 155, 296). However, src does not directly bind MT (38), nor does it increase the overall levels of tyrosine phosphorylation in Py-infected cells (97). Although the src family of protein tyrosine kinases includes src, yes, fyn, fgr, lck, hck, lyn, blk, and tkl, as well as other alternatively spliced forms, MT has evolved to only interact with src, fyn, and yes (59, 100, 174, 219, 243, 268, 306, 324, 329, 357, 386, 391). The selection of only this subset may indicate a specialized function of these kinases or a cell type specificity in the evolution of MT. src consists of various domains, including an N-terminal membrane localization SH4 region, a stretch of amino acids unique to each src, an SH3 domain, an SH2 domain, a kinase domain, and a conserved C-terminal tail (42). The exact function (targets) of the tyrosine kinase activity of src is unknown, although src, fyn, and yes have been found in all cell types at approximately the same levels with terminally differentiated cells apparently having higher levels (63, 341). One of the virus associated functions of src activation may be to phosphorylate the threonines found on VP1, one of the capsid proteins of Py, an apparent requirement for efficient viral encapsidation in vivo, although separable from the portions of MT required for transformation (122, 199). In addition, besides using src to phosphorylate important signal transduction activating tyrosines on MT, Py may use the cell cycle progression and/or chemotaxis functions of src, although this relationship has yet to be determined. Unfortunately, as with most of these MT domain studies, the focus of these studies has been on only the aberrant and altered regulation of src by MT in transformation.

As mentioned above, the complex between MT and pp60^c-src increases the tyrosine kinase activity of src, but complexes with fyn do not elevate kinase activity, which may be due to the low abundance of MT-fyn complexes formed (56, 188). MT complexes with a population of src that is not phosphorylated at tyrosine 527 (but is phosphorylated at tyrosine 416) (50) and locks it into a configuration in which it cannot be inactivated by the phosphorylation of tyrosine 527 (63). However, already inactivated forms of src (those with intramolecular binding of the SH2 domain to phosphorylated tyrosine 527) cannot be readily bound by MT (98). Specifically, the region bordered by aa 518 and 525 of src are essential for the MT complex and therefore are likely to mask tyrosine 527 (58). This masking of tyrosine 527 removes the normally cell cycle-specific regulation of src (active during mitosis, repressed during interphase), thereby keeping high tyrosine kinase activity throughout the cell cycle (166). Although src contains both a myristylation signal (needed to associate with membranes) and a membrane localization signal in its first 14 aa, its association with MT seems to disable these functions since truncated forms of MT which localize to the cytoplasm instead of the plasma membrane still colocalize with src (66, 383). At any one time, though, only 10 to 15% of MT is associated with the tyrosine kinases of proto-oncogenes, src, yes, or fyn (34, 55), since MT needs the less common open/activated conformation of src to bind successfully (98). Py infections in mice with knockout of any one of the above-mentioned tyrosine kinases are still able to induce tumors, although src is better able to induce the Py-associated tumor profile than is fyn (142, 340). Similar experiments determined that even when one or two of these tyrosine kinases are knocked out, the remaining ones do not compensate for the loss and increase their association with MT or elevate their kinase activity (177). This indicates that the specific stoichiometry of the individual MT and src associations may be important for different cell types, i.e., Clara cells of the lung bronchioles during acute replication, tubular epithelial cells of the kidney during persistence, or other unidentified cells that transport or harbor the virus. Although it seems clear that the MT-src complex is important for the virus life cycle, its in vivo relevance needs to be elucidated further. The shift from the predominant MT-fyn complex in hamsters to the MT-src complex in mice would indicate that pertinent changes were made during evolution, but their nature cannot be fully understood until the evolutionary tree of the MT of polyomaviruses in other species along the rodent lineage is evaluated. However, as mentioned for MT localization, the bulk of MT is not associated with the plasma membrane or with src, which means that although the MT-src complex may be important for transformation or even some facet of the normal virus life cycle, the total activities of MT cannot be simply explained by this minor complex found in both lytically infected and transformed cells (85, 297, 304).

Middle T-antigen tyrosine phosphorylation. Early experiments established that the phosphorylation of tyrosine 315, long considered the predominant tyrosine-phosphorylated site of MT, is essential for transformation, since without it there is a drastic reduction (20% of wild type) in transforming ability in rat fibroblasts (F-111 cells) (46). However, others demonstrated that tyrosine 315 was not essential for transformation of another rat cell line, Rat 1 (248). Although the conditions were somewhat different, this discrepancy indicated that studying Py in culture can be misleading. However, these experiments produced the first important mutant (Py-1178-T), which replaced tyrosine 315 with a phenylalanine to preserve the character of the wild-type MT (AT at nucleotide 1178). The residual transforming ability of this mutant was theorized to be in the phosphorylation of the tyrosine at position 322 or other phosphorylation sites but was later shown to remain even after mutation of tyrosine 322 (298) and tyrosine 297 (214). Subsequently, it was shown that the phosphorylation of tyrosine 250 was needed for transformation and that its mutation not only reduced the level of phosphorylation on MT but also weakened the interaction of MT with src (214). These results were supported by the complete lack of transformation by an MT mutant (Py-1387-T, CT at position 1387) which lacks the ability to bind the plasma membrane by eliminating the last 37 aa of the carboxy terminus and therefore is not phosphorylated by the associated src kinase activity. However, 1387T is able to replicate to almost wild-type levels in culture, although this has been attributed to the ability of the truncated MT to substitute for the ST function (122, 376).

The phosphorylated tyrosine 315 was then found to bind and result in the phosphorylation and activation of PI 3-kinase (65, 376). MT-src and MT-fyn complexes have an indistinguishable ability to phosphorylate tyrosine 315 and bind PI 3-kinase (55). PI 3-kinase is made of two subunits, the 85-kDa protein, which binds to the phosphorylated tyrosine 315 of MT, and the 110-kDa protein, with both PI 3-kinase and protein serine/threonine kinase activities, which binds the 85-kDa protein and phosphorylates the inositol ring at the D-3 position (84, 169, 250, 375, 376). Specifically, MT associates with one of two SH2 (src homology 2) domains of the p85 subunit of PI 3-kinase (317). The activation of PI 3-kinase has been associated with prevention of apoptosis, promotion of cell division, production of novel lipids which regulate Akt and atypical forms of protein kinase C (PKC), induction of protein synthesis, actin rearrangement, regulation of Ras-dependent signal transduction pathways, vesicle trafficking, and many others (for reviews, see references 16, 196, and 270). Since all of these functions must be well regulated, the cell maintains less than 0.25% of its inositol-containing lipids phosphorylated at the 3 position (270). The association and activation of PI 3-kinase by MT is necessary but not sufficient for transformation (169, 170, 376), another contradiction from the previously reported result that the Py-1178-T was not transformation deficient (215). Py-1178-T is unable to form mammary tumors except in older female mice that had experienced pregnancies, which touches on a potential in vivo role for the MT-PI 3-kinase association (332). The association of PI 3-kinase with the MT-pp60^c-src complex results in the phosphorylation of PI at the D-3 position of the inositol ring to produce PI-3-phosphate [PI(3)P], a pathway independent of inositol-1,4,5-triphosphate production (308). Additionally, PI 3-kinase phosphorylates PI(4)P and PI(4,5)P₂ to produce PI(3,4)P₂, and PIP₃, respectively, both novel polyphosphoinositides which are not hydrolyzed by PLC (170, 204, 308, 309). There is also recent evidence that a further phosphatidylinositol, PI(3,5)P₂, is formed by the phosphorylation of PI(3)P by PI 5-kinase (Fab1p) and may play roles in vesicle trafficking and stress regulation due to osmotic pressure (61, 125, 151). The production of these phosphatidylinositides has been directly linked to MT-induced transformation (65, 130, 170, 347). PI(3,4)P₂ and PIP₃ are specifically associated with cell growth since they are found only in subconfluent cultures or MT-transformed cells, not cells at high density or those transfected with a transformation-defective MT (308). Besides its association with the MT-src complexes (src, fyn, and yes), the production of PI(3)P and related phosphatidylinositides is also linked to other protein-tyrosine kinase-activated systems, including platelet-derived growth factor (PDGF)  receptor, epidermal growth factor (EGF) receptor (ErbB2R), colony-stimulating factor 1 receptor, and insulin receptors, and to the oncogenic forms of pp68^gag-ros (ros), pp130^gag-fps (fps), and pp160^gag-abl (abl) (15, 32, 117, 118, 169, 208, 212, 263, 291, 351, 352). Although most of these tyrosine kinase systems are associated with cellular growth, they can also induce the production of PI(3,4)P₂ and PI(3,4,5)P₃ while stimulating cells to differentiate or even stimulating already terminally differentiated cells, a cellular activation not associated with growth (reviewed in reference 317).

Phosphoinositides. The plethora of potential functions, including regulation of cell proliferation and survival, glucose metabolism, cytoskeletal reorganization, and vesicle trafficking, of the phosphoinositides PI(3)P, PI(3,4)P₂, and PI(3,4,5)P₃ has been reviewed extensively, but there is no consensus about how specificity of the signals is established (48, 196, 270, 317). PI(3)P has recently been shown to interact with the FYVE-containing domains, cysteine-rich zinc finger-like motifs named for the proteins Fab1p, YOTB, Vac1p, and EEA1, on numerous proteins which appear to be important for vesicle trafficking including targeting and secretion (reviewed in reference 16). There are few, if any, reports discussing the importance of vesicle trafficking in Py-infected systems, but our own observations in lung tissue show an exfoliation of virus-infected cells from the bronchiolar epithelium without apoptosis or necrosis, indicating that these mechanisms may be utilized. Furthermore, vesicle trafficking may be important during transport of the virus or movement from cell to cell without interference from the immune response. PI(3,4)P₂ and to a lesser extent PIP₃ bind and activate Akt, a serine/threonine protein kinase, which may account for the ability of the MT-PI 3-kinase complex to inhibit apoptosis, regulate glycolysis, induce protein synthesis through activation of p70^S6-K, and numerous other functions outlined below (105, 106).

Phosphatidylinositol 3-kinase and Akt. The discovery of the activation of Akt (PKB or RAC-PK) through its phosphorylation by the 110-kDa subunit of PI 3-kinase has resolved some of the apoptotic features associated with MT, as illustrated in Fig. 2 (225). The normal functions of Akt are still unclear, although there are connections with various metabolic events including lipogenesis, glycogen synthesis, and stimulation of glucose transporter GLUT4 translocation, induction of protein synthesis through S6 kinase activation, protection from apoptosis through bcl2 induction and bad phosphorylation, and, finally, cell cycle progression through c-myc induction (for reviews, see references 168 and 325). Early evidence that PI 3-kinase was responsible for the antiapoptotic signal came from an experiment which showed that rat fibroblasts transformed by Py underwent apoptosis when PI 3-kinase was inactivated by wortmannin or 1d-3-deoxy-3-fluoro-myo-inositol, blockers of both the lipid kinase and protein kinase activity of the 110-kDa subunit of PI 3-kinase (74). In addition, mutant 315YF was unable to protect the rat fibroblasts from undergoing apoptosis (74). Akt, a downstream component of the PI 3-kinase pathway, prevents apoptosis by inhibiting Ced3/ICE-like activity which is directly downstream of both the p53 and fas pathways of apoptosis (175). Currently, this is one of the few connections between MT signaling and p53 activities. Other substrates for Akt phosphorylation include bad, which loses its ability to promote apoptosis; forkhead transcription factor, a transcription factor which on phosphorylation is sequestered in the cytoplasm and is unable to activate proapoptotic proteins; glycogen synthase kinase 3, which on phosphorylation results in the maintenance of a dephosphorylated and activated glycogen synthase and cyclin D1; heart 6-phosphofructo-2-kinase, which upon phosphorylation is activated and stimulates glycolysis; endothelial NO synthase, which upon phosphorylation results in activation and production of a high level of nitric oxide; and other proteins mainly associated with the regulation of glucose metabolism, protein synthesis, or apoptosis (for a review, see reference 168). Akt activation in MT-induced transformation was found to be dependent on the MT-PI 3-kinase complex, although it is still unclear whether PI 3-kinase is sufficient to stimulate Akt since there have been reports of PI 3-kinase-independent activation of Akt kinase activity (104, 182, 225, 294, 325). The products of PI 3-kinase, particularly PIP₃ and PI(3,4)P₂, bind to the pleckstrin homology domain of Akt, resulting in a conformation change and localization to the plasma membrane (23, 105, 162, 180, 321). PDK1, a 3-PI-dependent kinase activated by PI 3-kinase, then phosphorylates threonine 308 of Akt, while a second, less well-defined kinase, tentatively called PDK2, phosphorylates at serine 473; phosphorylation of both threonine 308 and serine 473 results in the full activation of Akt (5, 6, 320, 321). PDK1, though, has the ability to not only protect against apoptosis via its activation of Akt but also induce apoptosis via its dephosphorylation by the MT-PP2A complex and the subsequent activation of the intermediary c-Jun N-terminal kinase (JNK), although JNK can mediate both pro- and anti-apoptotic signals (90, 157, 237, 287, 350). The specific signal seems to depend on the four different phosphorylation sites on PDK1. PDK1 is responsible for the phosphorylation and activation of Akt, pp70^S6K (see below), and atypical isoforms of PKC, including PKC and PKC (4, 5, 265, 320).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   MT and apoptosis. A schematic diagram of the potential antiapoptotic effects of the PI 3-kinase activation of Akt/PKB is shown. See the text for details. Akt can prevent apoptosis by inhibiting Ced3/ICE-like activity and phosphorylating and deactivating bad and glycogen synthase kinase-3 (GSK-3). In addition, Akt induces the expression of bcl2 and c-myc, which also helps protect against apoptosis. This pathway resembles those induced by many cellular growth factors, including EGF and PDGF.

40S ribosomal protein S6 kinase. The ribosomal protein S6 kinase is highly phosphorylated in growing cells and dephosphorylated in growth-arrested cells in culture. Presumably, the 40S ribosomal protein S6 (S6) is also dephosphorylated in terminally differentiated cells in vivo, although this is not well studied. S6 phosphorylation is controlled by pp70^S6K (S6K). Genetic studies have shown that S6K controls cell size, growth, and proliferation without affecting any particular phase of the cell cycle (95, 230). S6 is induced by a number of mitogens and oncogene products and results in the translation of a family of mRNA transcripts containing a polypyrimidine tract at the 5' end (5'TOP family) (73). The 5'TOP family is composed of 100 to 200 genes that encode proteins essential for translation. MT elevates the levels of S6 phosphorylation through two pathways, one of which is activated by src and S6 kinase while the other requires neither kinase (73). The src-dependent activation of S6 kinase requires the phosphorylation of tyrosine 315 (331), while the other pathway may use the binding and inactivation of PP2A by MT to stabilize the phosphates on S6. Activated PI 3-kinase results in the activation of both the cytoplasmic (70-kDa) and nuclear (85-kDa) forms of S6 kinase through the intermediary kinase PDK1 and direct phosphorylation by PI 3-kinase (73). PKC and PKC, atypical isoforms of PKC induced by the PI 3-kinase/PDK1 pathway, may be responsible for the phosphorylation of S6K in its carboxy terminus necessary for its activation, while mTOR (mammalian Target of Rapamycin), a kinase activated through the PI 3-kinase/Akt pathway, may regulate phosphatase activity (similar to the MT-PP2A complex), act as a kinase upon S6K, or act as a sensor for amino acid availability (3, 95, 305). The transcription and replication of Py would appear to benefit from the activation of S6K by the MT-PI 3-kinase complex, which may result in the upregulation of protein synthesis. However, it remains clear that neither cellular proliferation nor transformation is the ultimate goal but, rather, that the purpose of the S6 activation is only part of the viral program to establish a cell designed specifically to transcribe viral proteins and replicate the viral genome under conditions in which cellular protein synthesis has either been severely curtailed or reprogrammed to express only necessary factors.

A mutation (proline 248 to leucine) which was transformation defective but still had the ability to associate with pp60^c-src and PI 3-kinase led to the discovery of the association of the tyrosine 250 phosphorylation site of MT with the Shc protein (44, 86). A motif in MT, Asn-Pro-Thr-Tyr (NPTY), was found to be important for the ability of Py to transform cells. Shc (src homology 2 domain-containing protein) is made of three overlapping polypeptides of approximately 46, 52, and 66 kDa. The 52-kDa form contains a phosphotyrosine binding domain (PTB), a central collagen homology domain (CH1), and a carboxyl-terminal SH2 domain (33). Shc has three tyrosine phosphorylation sites, Tyr²³⁹, Tyr²⁴⁰, and Tyr³¹⁷ (considered the principal site) (257). On phosphorylation by the MT-pp60^c-src complex, the PTB domain of Shc (specifically, Tyr^239/240 is important) associates with the SH2 domain of Grb2 (growth factor receptor-bound protein 2), which leads to the Shc-Grb2-Sos complex, which activates Ras (33, 44, 86). Sos (son of sevenless) is a GDP-GTP exchange factor for Ras. Less than 1% of the 46- and 52-kDa forms are associated with MT, while there is no evidence of association with the least abundant 66-kDa form of Shc (44). Growth factors (EGF, PDGF, etc.) can stimulate the Ras pathway through Shc or directly through Grb2, but it has been shown that the association of MT with Shc and the resulting phosphorylation are needed for full activation of Grb2 and the Ras pathway, although a mimicked binding site for Grb2, similar to that found on Shc, placed into MT can replace the need for Shc. Further discoveries connected the binding of MT to Shc with the transcriptional activation of c-jun (including AP-1 jun-jun homodimers and jun-fos heterodimers) by stimulating the phosphorylation of sites in the transactivation domain of c-jun (serines 63 and 73) through Ras/Raf-1 activation (319). On activation of Ras, a cascade effect results in activation of Raf and MEK which results in nuclear translocation of members of the mitogen-activated protein (MAP) kinase family (ERK1/ERK2/JNK) and subsequently in transcriptional activation of fos, jun, or myc (319, 348). In addition, the activation of PKC by MT-expressing cells may lead to dephosphorylation of sites in the DNA binding domain of c-jun which then leads to an increase in the transcriptional activation of c-jun (319). Activated extracellular signal-regulated kinases (ERKs) also lead to the activation and phosphorylation of transcription factor Elk-1 (ternary complex factor), a member of the Ets family of transcription factors, which then forms a complex with a homodimer of the serum response factor (SRF), activates the transcription of c-fos through its serum response element (SRE), and ultimately leads to increased levels of AP-1 (126). Shc and PI 3-kinase combined Ras- and Raf-dependent signaling results in the binding of Elk-1 to the SRE of the c-fos promoter, while a Raf-independent signal through PI 3-kinase alone results in transduction via Rac1 of a signal to the SRF to bind the SRE of the c-fos promoter (349). c-fos was found to be necessary for MT-induced transformation of rat F111 fibroblasts and can be inhibited by retinoic acid (333). The induction of the Ras signaling pathway and the resulting activation of transcription factors has been associated with cell growth and differentiation, but most, if not all, of the above studies have focused on only the transformation of cells or the tumor profile in mice. Clearly, though, these pathways can result in the regulated expression of AP-1 and members of the Ets-domain (E twenty six, based on homology to v-ets of E26 avian erythroblastosis virus) family of transcription factors. Both of these transcription factors are required for activation of the Py enhancer, as discussed below.

There is some evidence that these interactions may address the LT-p53 interaction. Recent evidence shows a direct connection between Raf activation and cell cycle regulation and mitogenic control, another way in which MT may circumvent the inability of LT to bind p53. Raf1 binds to and regulates the function of cdc25A, a dual-specificity phosphatase involved in cell cycle regulation, and can physically interact with and inhibit the antiapoptotic functions of bcl-2 (176). Furthermore, on activation, Raf1 binds to the functional (hypophosphorylated) form of Rb and, through its intrinsic kinase activity or the action of cdc25A inducing the expression of cyclin D1, cdk4, and cdk6, may lead to the phosphorylation and inhibition of Rb, resulting in activation of E2F complexes and cell proliferation (362). These findings were all established through cell culture, but they illustrate one of the key steps in understanding the complex interaction between the early proteins of Py and host cell machinery. It remains to be determined if such interactions are applicable in vivo.