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Transforming Activities of JC Virus Early Proteins

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Polyomaviruses, as their name indicates, are viruses capable of inducing a variety of tumors in vivo. Members of this family, including the human JC and BK viruses (JCV, BKV), and the better characterized mouse polyomavirus and simian virus 40 (SV40), are small DNA viruses that commandeer a cell's molecular machinery to reproduce themselves. Studies of these virus-host interactions have greatly enhanced our understanding of a wide range of phenomena from cellular processes (e.g., DNA replication and transcription) to viral oncogenesis.

The current chapter will focus upon the five known JCV early proteins and the contributions each makes to the oncogenic process (transformation) when expressed in cultured cells. Where appropriate, gaps in our understanding of JCV protein function will be supplanted with information obtained from the study of SV40 and BKV.


Human Polyomaviruses

The first isolations of JCV and BKV from humans were reported in 1971, and subsequent studies have failed to identify additional animal hosts. Serologic data indicate that asymptomatic infections often occur in young children, with 80-90% of the population eventually becoming seropositive for one or both viruses. Given stable anti-viral antibody levels and the frequent occurrence of viruria in infected individuals, it is likely that JCV and BKV persist for the lifetime of their host (reviewed by Frisque and White, 1992).1

The natural host of SV40 is the rhesus monkey; however, humans were exposed to this virus by contaminated lots of poliovirus and adenovirus vaccines administered during the mid-1950s to early 1960s. It is estimated that at least 30 million of the 98 million people in the United States receiving these vaccines were given preparations containing live SV40. Prior to the advent of highly sensitive polymerase chain reaction (PCR) technologies, little evidence was available to support the suggestion that SV40 circulates in the human population. However, during the last decade, numerous reports have surfaced that require the field to reconsider this possibility (reviewed by Vilchez and Butel, 2003; Carbone et al, 2003; Garcea and Imperiale, 2003).2-4

Association of JCV with Its Human Host

JCV may establish latent, persistent or active infections in vivo depending upon the immune status of the host, the tissue infected, and the JCV variant involved. JCV has routinely been detected in kidney tissue and urine, and it has been suggested that the virus replicates at relatively low levels (persistent infection) in renal proximal tubule cells. The form of virus usually detected in urine is called archetype JCV, a variant that contains a single copy of the viral promoter-enhancer sequences within its transcriptional control region (TCR). A second variant, called rearranged JCV, has been associated with active infections in brain that lead to disease in severely immunocompromised individuals (see below). Several laboratories have reported the detection of these rearranged TCR sequences in healthy brain as well. Few amplicons are amplified from this tissue, and it is possible that a latent infection is established at this site. Relative to the archetype TCR, the promoter-enhancer sequences of a rearranged TCR have undergone deletion and duplication events that result in a virus with altered tissue tropism and pathogenic potential. In addition to the brain and kidney, one or both JCV TCR variants have been detected in tonsils, a possible primary site of infection, in peripheral blood mononuclear cells, which may seed virus to secondary sites of infection, and in colon tissue (reviewed by Atwood, 2003; Seth et al, 2003).5,6

JCV Infection and Human Disease

JCV actively infects oligodendrocytes, the myelin-producing cells of the central nervous system (CNS), and abortively infects astrocytes, a second glial cell type. The infected astrocytes take on a transformed appearance, whereas the oligodendrocytes are destroyed, leading to multiple foci of demyelination in the CNS (progressive multifocal leukoencephalopathy [PML]) and a variety of debilitating symptoms with the type and severity depending upon the region of the brain affected. Death usually occurs within a year of the onset of symptoms. PML patients nearly always have an underlying immune deficiency. Prior to the AIDS epidemic, PML was considered rare, and it occurred mostly in the elderly with lymphoproliferative diseases. PML is now common in younger individuals with AIDS, and it is the cause of death in ~5% of these patients (reviewed by Frisque and White, 1992).1 It is unclear whether oligodendrocyte death results from an apoptotic or necrotic event, although two groups have recently reported evidence of apoptosis within and near PML lesions (Yang and Prayson, 2000; Richardson-Burns et al, 2002).7,8 JCV has also been linked to polyomavirus-associated nephropathy (PAN); however, BKV is considered to be the more likely cause of this emerging disease in renal allograft recipients (reviewed by Hirsch and Steiger, 2003).9

Polyomaviruses go through their entire life cycle during an active infection (e.g., PML), with the outcome being the release of large numbers of infectious virions from the lysed cell. However, these viruses may enter certain cell types that do not support replication (nonpermissive cells) and the infection is aborted. A subset of the viral proteins (the early/tumor/regulatory proteins) may be expressed in these cells, thereby altering cell survival and proliferation responses and resulting in tumor induction in vivo or cellular transformation in vitro. The ability of viral proteins to induce proliferation by effecting transcriptional activation of critical cellular genes and through interactions with key cell cycle regulators and cell survival factors has become an area of intense interest.

The possibility that primate polyomaviruses might cause human cancer has been considered ever since SV40 was found as a contaminant in the early poliovirus vaccines; however, subsequent studies generally failed to identify adverse outcomes associated with this accidental exposure. It wasn't until the development of sensitive nucleic acid and protein detection methods that evide-nce was uncovered linking SV40, as well as JCV and BKV, to human oncogenesis. In the past decade, SV40 has been associated routinely with four types of cancer: brain tumors (ependymomas, medulloblastomas and glioblastomas), osteosarcoma, mesothelioma and non-Hodgkin lymphoma (reviewed in Klein et al, 2002; Vilchez et al, 2002).10,11 Similarly, JCV sequences have been found in tumors of the CNS (medulloblastomas, glioblastomas, oligodendrogliomas, astrocytomas, ependymomas and B cell lymphomas), colon and prostate (Del Valle et al, 2001; Laghi et al, 1999; Zambrano et al, 2002).12-14

Lytic and Oncogenic Potential of JCV in Vivo

An animal model has not been developed to study productive JCV infections. Under some conditions, transgenic mice containing JCV sequences exhibit neurological problems associated with dysmyelination of their CNS. Such mice developed a shaking disorder that resembles that of myelin-deficient strains of quaking or jimpy mice (Small et al, 1986).15 However, this phenotype does not involve cytolytic destruction of oligodendrocytes, but rather a down-regulation of myelin-specific genes by the JCV large T antigen (TAg) (Trapp et al, 1988; Haas et al, 1994).16,17 In addition, PML-like disease has been reported in SV40-infected rhesus monkeys immunosuppressed by simian immunodeficiency virus (Horvath et al, 1992).18

SV40 was first identified as a tumor virus following the appearance of sarcomas in Syrian golden hamsters injected subcutaneously with the virus (Eddy et al, 1961).19 Later studies involving injection of virus or creation of transgenic animals expressing viral tumor protein(s) revealed that several rodent hosts are susceptible to tumor induction by SV40, JCV and BKV. These studies indicated that JCV displays a predilection for inducing tumors in neural tissues, including medulloblastomas, primitive neuroectodermal tumors, glioblastomas, pineocytomas, retinoblastomas, neuroblastomas and meningiomas. Particularly relevant to the question of polyomaviruses and human cancer, was the observation that JCV inoculation of primates (owl and squirrel monkeys) results in the appearance of astrocytomas. Compilation of data from several animal systems indicate that tumor type and incidence depend upon a number of parameters, including the viral variant employed, inoculum amount, route of injection, and immune status of the host (reviewed in Frisque and White, 1992).1

Lytic and Oncogenic Potential of JCV in Vitro

A major obstacle to the study of JCV, in contrast to that of SV40 and BKV, is its highly restricted host range. JCV replicates efficiently only in primary human fetal glial (PHFG) cells and in transformed cells derived from these heterogeneous primary cultures (e.g., POJ and SVG cells). Much effort has been expended in attempts to identify a more convenient cell system (reviewed by Jensen and Major, 1999).20 Varying success has been achieved in propagating virus in human tonsillar stromal cells, astrocytes, Schwann cells, B lymphocytes, embryonic kidney cells and brain cell lines, but none of these cells support infection as efficiently as PHFG cells. Productive JCV infections involve early and late gene expression (see below), phases of the lytic cycle that are separated temporally by the process of viral DNA replication. In contrast, transformation of cells in culture requires only early gene expression. SV40, and to a lesser extent BKV, induce oncogenic transformation in cells derived from a variety of tissues and host species. Somewhat surprisingly, given its oncogenic potential in vivo, JCV transforms only a few cultured cell types (mostly rodent fibroblasts and brain cells). The inability of JCV to efficiently convert cells to a transformed state is related to both the reduced expression and activity of the JCV tumor proteins (Bollag et al, 1989; Trowbridge and Frisque, 1993).21,22

Organization of the JCV and SV40 Genomes

The organization of the primate polyomavirus genomes is highly conserved (fig. 1). Each genome can be divided into three regions: the early and late coding regions and the regulatory region. The TCR positioned within the latter region contains viral promoters that direct transcription of the early and late genes. Alternative splicing of the precursor transcripts yields mature mRNAs that encode the early and late viral proteins. The early proteins regulate virus-cell interactions through a number of mechanisms. For example, these proteins exhibit transactivating functions that are effected through interactions with cellular transcription factors and DNA. They also bind and inactivate cellular tumor suppressor proteins, thereby influencing cell cycle progression and survival. Furthermore, the viral tumor proteins alter the activities of a number of cellular factors that regulate such diverse processes as DNA replication, intracellular signaling, protein degradation and apoptosis. It is hypothesized that these interactions prepare the cellular environment for a lytic infection; in some cells these events result in malignant transformation, a biological dead end for the virus.

Figure 1. JCV and SV40 genomes.

Figure 1

JCV and SV40 genomes. A) The circular, double-stranded DNA genomes of JCV and SV40 represented by the inner circles are slightly greater than 5 kilobases in length. The genomes are divided into early and late coding regions and a regulatory region (RR) (more...)

The JCV early precursor mRNA is alternatively spliced to yield five transcripts encoding TAg, small t antigen (tAg), T'165, T'136, or T'135 (fig. 1). All five proteins share their amino-terminal (N-terminal) 81 amino acids, whereas TAg and the three T' proteins also share the contiguous 51 amino acids. Furthermore, each protein has a unique carboxy-terminus (C-terminus), except for TAg and T'165 that have overlapping C-termini. The presence of shared sequences among the JCV early proteins suggests that functional domains identified within the N-terminus of TAg may also be active in one or more of the smaller tumor proteins. On the other hand, it is likely that removal of TAg sequences during the processing of tAg and T' mRNAs leads to the production of proteins with distinct structures, post-translational modifications and stabilities. Such alterations might modify or abolish functions that the JCV early proteins are predicted to share.

The JCV regulatory region contains numerous, overlapping cis-acting sequences, including the early and late promoters, the enhancer and the core and auxiliary origins of DNA replication. Following early gene expression in a permissive cell, TAg binds specifically to a pentanucleotide motif within the origin sequences to initiate viral DNA replication. Once replication occurs, late gene expression ensues, leading to the production of Agnoprotein (also called LP1) and three capsid proteins, VP1, VP2, and VP3. Agnoprotein influences the assembly of the SV40 virion, whereas the related JCV protein was found recently to play roles in viral gene transcription and DNA replication and to influence cell cycle progression and DNA repair mechanisms (Safak and Khalili, 2001; Darbinyan et al, 2002).23,24 Because this chapter focuses on the transforming functions of the JCV early proteins, the reader is referred to other chapters in this book for a more complete discussion of JCV replication signals and late protein functions.

JCV TAg Functions

The TAg of the primate polyomaviruses functions as the viral replication protein through (i) direct interactions with a host of cellular factors involved in regulating cell cycle progression and survival (Table 1), and (ii) its ability to transregulate viral and cellular promoters to alter gene expression. TAg is modified post-translationally in a number of ways; most work has focused upon the phosphorylation of two clusters of serine and threonine residues located in N- and C-terminal domains of the protein. Phosphorylation influences a wide range of TAg activities, including nuclear import, oligomerization and modulation of viral DNA replication (Barbaro et al, 2000; H_et al, 1997; reviewed by Prives, 1990 and Fanning, 1994).25-27

Table 1. JCV early protein interactions.

Table 1

JCV early protein interactions.

TAg-Mediated Viral DNA Replication

The multifunctional TAg is the sole JCV protein required for viral DNA replication (Nesper et al, 1997).28 Replication is initiated within a 68 base pair (bp) sequence, the core origin, that includes three domains, (i) TAg binding site II (BSII) composed of four copies of the pentanucleotide sequence GAGGC embedded within a 25 bp dyad symmetry, (ii) a 15 bp imperfect palindrome (IP) and (iii) a 15 bp AT-rich region (Lynch and Frisque, 1990; Sock et al, 1991, 1993).29-31 Sequences adjacent to the core origin that enhance, but are not required for DNA replication, include TAg BSI and a pentanucleotide repeat (AGGGA AGGGA) referred to as the lytic control element (LCE). The LCE motif is bound by the transcription factors YB-1 and Pur-α and influences viral transcription as well as replication (Lynch and Frisque 1990; Tada et al, 1991; Sock et al, 1993; Chang et al, 1994).29,31-33

Polyomavirus TAgs influence both the initiation and elongation steps of DNA replication, and our understanding of these processes relies primarily on studies with SV40 TAg. Upon binding to TAg BSII, the protein oligomerizes into a double hexamer structure that leads to the distortion and unwinding of origin sequences and the recruitment of the cellular replication machinery (Chen et al, 1997, San Martin et al, 1997; Valle et al, 2000).34-36 Upon transition from the initiation to elongation phase of DNA replication, TAg displays helicase and ATPase activities resulting in the appearance of two replication forks. Replication proceeds bidirectionally as DNA is threaded through the hexameric structures and two cellular polymerases copy the unwound template strands. The rate-limiting step in the replication process occurs at the termination stage, perhaps because steric constraints begin to interfere with replication fork movement (Ishimi et al, 1992).37

Several in vivo and in vitro approaches have been taken to compare the replication potential of the primate polyomaviruses. The viral replication components, TAg and the core origin, exhibit a high degree of sequence similarity. Combinations of mutant, chimeric or naturally occurring variant origins have been examined to identify the basis for observed differences in JCV, BKV and SV40 DNA replication behavior (reviewed in Kim et al, 2001).38 These studies reveal that JCV TAg binds to both the JCV and SV40 replication origins, but with reduced efficiency relative to its SV40 counterpart. While the BKV and SV40 TAgs interact productively with the JCV origin, the JCV TAg promotes replication only from its own origin. The inability of the JCV TAg to drive replication from the SV40 origin has been mapped to amino acids 82-411 (Lynch et al, 1994) and to 3 nucleotide differences between the AT-rich regions of the two viral core origins (Lynch and Frisque, 1990).29,39 Overall, JCV TAg mediates replication, even from its own origin, less efficiently than do the BKV and SV40 proteins (Lynch and Frisque, 1990; Sock et al, 1993).29,31

JCV-SV40 early region chimeras have been used to investigate the well-established observation that JCV exhibits a more restricted host range than SV40 or BKV. When a series of constructs containing different portions of the JCV and SV40 TAg coding regions were introduced into human and monkey cells, JCV replication was extended to the latter cells if the C-terminal host range (HR) region of JCV TAg was replaced with that of SV40 TAg (fig. 2; Lynch et al, 1994).39 It should be noted that Smith and Nasheuer (2003) recently revisited the prediction that JCV's strict species specificity reflects the inability of its TAg to interact productively with a nonhuman DNA polymerase α-primase complex.40 Previous work with SV40 and mouse polyomavirus in fact supported this hypothesis (Dornreiter et al, 1990; Murakami et al, 1986), but surprisingly, the JCV TAg, unlike the SV40 TAg, was found to promote DNA replication in an in vitro system employing DNA polymerase α-primase of either human or murine origin.41,42

Figure 2. JCV-SV40 chimeric early regions.

Figure 2

JCV-SV40 chimeric early regions. A) JCV-SV40 chimeras were constructed by exchanging the amino-terminal (N; dark shading), middle (M; horizontal lines) and carboxy-terminal (C; light shading) early coding sequences. The restriction enzymes BstXI and (more...)

TAg Transformation Functions: Interactions with Rb and p53 Tumor Suppressor Proteins

Multiple TAg functional domains contribute to viral transformation by altering cellular signaling pathways regulating proliferation and survival. At least three regions of SV40 TAg are involved in these processes, the N-terminal J domain and the Rb (LXCXE) and p53 binding domains (Srinivasan et al, 1997; reviewed by DeCaprio, 1999); these motifs have also been identified in the JCV TAg.43,44 In addition, the LXCXE and J domains are present in the T. and 17kT proteins, and the J domain is found in the small tAgs of each virus (fig. 1).

To understand the mechanisms by which polyomaviruses induce transformation, researchers have focused much of their attention on the interaction of viral tumor proteins with the cellular tumor suppressors pRB and p53. Polyomavirus infection of permissive or nonpermissive cells may induce the cell's progression into S phase, with the outcome being either a lytic infection or transformation. Sheng and coworkers (1997) proposed a model to explain the ability of TAg to initiate this event.45 Members of the Rb family of tumor suppressor proteins (pRB, p107, p130) bind to the E2F family of transcription factors to negatively regulate cell cycle progression from G0/G1 to S phase (Zhu et al, 1996).46 Under the appropriate conditions, cyclin-dependent kinases (cdks) phosphorylate Rb proteins, leading to the release of the transcription factors and promotion of G1 to S phase transition. The model suggests that TAg disrupts this regulation by first binding to Rb-E2F complexes through its LXCXE domain. Once bound, TAg, via its J domain, recruits the molecular chaperone, Hsc70, and activates this cellular protein's intrinsic ATPase function. These interactions are believed to cause the release of E2F from the complex, thereby making it available to activate genes involved in S phase progression. Sullivan et al (2000, 2001) have confirmed and extended several features of this model, especially in regards to the TAg-chaperone interaction.47,48 Relevant to the current discussion of JCV tumor proteins, these investigators have shown that a chimeric SV40-JCV TAg containing a JCV J domain supports viral replication and transformation, but with reduced efficiency relative to the intact SV40 protein (Sullivan et al, 2000).49 Furthermore, they reported that an N-terminal truncated form of SV40 TAg (TN136), that shares features with the JCV T' proteins, fails to form a stable complex with Hsc70 (Sullivan et al, 2001), suggesting that sequences C-terminal to the J and LXCXE domains might be essential for stable interaction with Hsc70.50 It should be noted that interactions between viral TAgs and Hsc70 appear to influence a wide range of activities in addition to cell cycle regulation and transformation, including viral DNA replication, transactivation of viral and cellular promoters, and virion assembly (reviewed by Sullivan and Pipas, 2002).51

By promoting unscheduled cell cycle progression through the release of active E2F, small DNA tumor viruses may provoke a counter response by a cell that leads to stabilization and elevated levels of p53. Because this transcription factor regulates a wide variety of cellular processes including cell cycle arrest, DNA repair and apoptosis, p53 is an additional key target of several viral tumor proteins. To prevent this tumor suppressor protein from interfering with critical viral functions, DNA tumor viruses have evolved mechanisms to inactivate it; polyomaviruses accomplish this feat, in part, by binding to and inactivating p53. This latter event suppresses induction of the cyclin-dependent kinase inhibitor (CKI), p21, a key downstream p53 effector that promotes arrest of cells in the G1 phase of the cell cycle. JCV TAg was first shown to bind p53 in transformed hamster brain cells using a co-immunoprecipitation approach (Frisque et al, 1980).52 As noted below, a smaller subpopulation of JCV TAg, compared to SV40 TAg, appears to bind p53. Through this binding, p53 is stabilized and its levels increase dramatically in primate and rodent transformed and tumor cells. Recently, SV40 small tAg was found to contribute to p53 stabilization by an unknown mechanism (Tiemann et al, 1995); a similar role for the JCV tAg has not yet been demonstrated.53

Several in vivo and in vitro studies have confirmed that the JCV TAg interacts with cellular pRB, p107, p130, Hsc70 and p53 (Frisque et al, 1980, Tavis et al, 1994, Dyson et al, 1989, 1990; Bollag et al, 2000, Howard et al, 2000; Kilpatrick, Bollag, and Frisque, in preparation).52,54-58 As with other comparisons of the JCV, BKV and SV40 TAgs, the JCV protein appears to be less robust in binding to and overriding the functions of these cellular factors. A limited number of mutations have been introduced into the J and LXCXE domains of the JCV TAg. Initial studies created two JCV TAg LXCXE mutants, one (RbS) in which the JCV sequence was converted to an SV40-like domain and the second (RbN) designed to disrupt the Rb binding region (Table 2). The first mutant exhibited increased DNA binding activity, wild type transforming activity and, surprisingly, decreased pRB binding. The latter mutant exhibited reduced DNA binding and was unable to bind pRB or transform Rat2 fibroblasts. Both mutants were defective for DNA replication and failed to produce infectious virions (Tavis et al, 1994).54 An additional LXCXE mutant, E109K, has been generated in the JCV sequence (Tyagarajan and Frisque, in preparation); two J domain mutants were also produced in these experiments, H42Q and H42Q/D44N (Table 2). Co-immunoprecipitation/ Western blot (IP/WB) experiments performed on extracts of cells stably expressing wild type and mutant viral proteins indicated that (i) JCV early proteins influence the phosphorylation status and stability of p107 and p130; only the faster migrating hypophosphorylated forms of p107 and p130 are detected, (ii) both hypophosphorylated and hyperphosphorylated forms p107 and p130 are present in cells expressing the LXCXE and J domain mutants, suggesting that the mutant viral proteins fail to reduce the levels of the hyperphosphorylated species and (iii) J domain, but not LXCXE domain, mutant proteins bind p107 and p130. Relevant to these findings, Howard and coworkers (2000) found that induced expression of p130 resulted in elevated levels of the CKI, p27.58 In cells derived from tumors caused by JCV, p27 and p130 were identified as direct targets of TAg. Upon detecting a physical interaction between the two cellular proteins, these investigators suggested that p130 and p27 cooperate to negatively regulate cell cycle progression, and that TAg interferes with this activity either by sequestering p27 or by altering the phosphorylation state of Rb proteins.

Table 2. JCV early protein mutants.

Table 2

JCV early protein mutants.

Earlier studies involving chimeric JCV-SV40-BKV genomes support the findings that JCV tumor proteins interact with the p53 and Rb tumor suppressor proteins, but that their ability to inactivate these key regulators is reduced relative to SV40 and BKV (Chuke et al, 1986; Bollag et al, 1989; Haggerty et al, 1989; Sullivan et al, 2000).21,49,59,60 These experiments also suggest that the JCV proteins might target other cellular factors. Characterization of two sets of JCV-SV40 chimeras, generated by swapping JCV TAg sequences containing the J domain (amino acids 1-81), LXCXE domain (amino acids 82-411) or most of the p53 bipartite binding region (amino acids 412-688) (fig. 2), led to the following observations: (i) the substitution of the J domain of JCV TAg for that in the SV40 TAg resulted in reduced dense focus formation, DNA replication and p130-E2F disruption (Sullivan et al, 2000), (ii) the presence of JCV sequences in place of SV40 sequences within any of the three exchanged regions of a chimera yielded lower transformation efficiencies (Haggerty et al, 1989), (iii) hybrid proteins containing most of the p53 binding domain of JCV TAg formed less stable complexes with p53 than did a TAg with an SV40 p53 binding motif (Bollag et al, 1989; Haggerty et al, 1989) and (iv) chimeric TAgs containing the JCV C-terminal region immortalized human fibroblasts more efficiently than did the intact SV40 protein (O' Neill et al, 1995).21,47,60,61 At the time many of these studies were conducted, it was not known that the exchange of sequences within the viral early coding regions would also affect the structures of JCV T' and SV40 17kT proteins.

TAg Transformation Functions: Disruption of Wnt and IGF-IR Signaling Pathways

While most studies have examined the outcomes of interactions between polyomavirus TAgs and p53 and Rb proteins, recent work indicates that the oncogenic behavior of JCV TAg may involve the disruption of other cell signaling networks. The Wnt signal transduction pathway plays critical roles in developmental patterning events during embryogenesis; deregulation of the pathway is associated with a number of human cancers. Greater than 90% of colorectal cancers involve a genetic lesion within the Wnt pathway that leads to stabilization and nuclear import of β-catenin, a key component of this signaling cascade (reviewed by Giles et al, 2003).62 Nuclear β-catenin partners with the transcription factors TCF-4/LEF-1 to activate promoters of genes involved in induction of cellular proliferation. Importantly, JCV is associated with tumors of the colon and prostate and with medulloblastoma, cancers in which the Wnt pathway may be aberrantly activated (reviewed by Kikuchi, 2003).63 Immunostaining of cells derived from these tumors shows colocalization of TAg and β-catenin in the nucleus (Gan et al, 2001; Enam et al, 2002).64,65 Transfection of a JCV TAg-expressing vector into a colon cancer cell line indicates that TAg and wild type β-catenin form a complex and exhibit cooperativity in upregulating expression of c-myc. This latter proto-oncoprotein, a downstream target of β-catenin, is found at elevated levels in colon cancer (Morin, 1999; Dobbie et al, 2002).66,67 These results indicate that JCV TAg may activate the Wnt pathway by altering β-catenin stability and localization, suggesting another mechanism by which TAg may exert its oncogenic potential (Gan et al, 2001; Enam et al, 2002).64,65

The insulin-like growth factor I receptor (IGF-IR), when activated by its major ligand, insulin receptor substrate 1 (IRS-1), elicits numerous cellular responses involving mitogenic, anti-apoptotic and transformation signals (reviewed by Del Valle et al, 2002).68 The Khalili laboratory has presented evidence that JCV TAg may mediate transforming activity through its influence upon this signaling pathway. Analyses of human medulloblastoma tissues and medulloblastoma cell lines derived from a transgenic mouse model revealed elevated levels of IGF-IR and IRS-1 in JCV TAg-positive cells. A direct interaction was observed between TAg and IRS-1, and the latter factor, which functions in the cytoplasm, was translocated to the nucleus in the presence of the viral protein (Lassak et al, 2002; Del Valle et al, 2002).68,69 Further, the ability of JCV TAg to induce a transformed phenotype in different murine cell lines correlated with the levels of IGF-IR present in the lines examined. Finally, cells derived from IGF-IR knockout animals, but which continue to express p53 and Rb proteins, were refractory to virally induced transformation, emphasizing the importance of a functional IGF-IR/IRS-1 pathway to JCV TAg function (Del Valle et al, 2002).68

TAg and Genomic Instability

The preceding discussion argues that polyomavirus tumor proteins induce transformation by directly interacting with critical cellular factors to deregulate signal transduction pathways controlling cellular proliferation, differentiation and death. Less attention has been given to the possibility that polyomavirus infections disrupt regulated cell growth by causing genomic instability. In support of this possibility, it has been shown that JCV, BKV and SV40 TAgs exhibit mutagenic activity (Theile and Grabowski, 1990), and that JCV- and SV40-infected cells contain chromosomal abnormalities (Ray et al, 1990; Neel et al, 1996).70-72 These and other findings support the suggestion that TAg expression sets in motion a series of mutational events that activate proto-oncogenes and/or inactivate tumor suppressor genes, thereby initiating malignant transformation. While a large body of work supports the hypothesis that continued expression of viral oncoproteins is required to maintain a transformed state, the ability of TAg to initiate a cascade of genetic defects might render the continued presence of an oncogenic virus unnecessary. Although controversial, a “hit-and-run” “mechanism” has been proposed to operate in some human cancers, including medulloblastoma and colon cancer (reviewed by Croul et al, 2003; Ricciardiello et al, 2003).73,74

Physical and Functional Interactions between TAg and Replication and Transcription Factors

Based upon studies of SV40 TAg (reviewed by Kim et al, 2001), JCV TAg is predicted to physically interact with a number of cellular replication factors, including topoisomerase I, the single-stranded DNA binding protein RPA, and DNA polymerase α however, such interactions have not yet been demonstrated.38 On the other hand, because many laboratories have focused upon the glial-specific regulation of the JCV early and late promoters, progress has been made showing TAg binding to specific components of the cell's transcriptional machinery.

The POU III domain transcription factor Tst-1 (Oct-6, SCIP) is selectively expressed in myelinating glia, the cells targeted during JCV infection. Renner and colleagues (1994) discovered that the N-terminal portion of JCV TAg binds Tst-1, leading to synergistic activation of both the early and late viral promoters.75 Further, they proposed that if JCV TAg, like its SV40 counterpart (Gruda et al, 1993), binds TATA binding protein (TBP), then it might act as a co-activator to facilitate contact between Tst-1 and the basal transcriptional apparatus.76

The cellular transcription factor, Pur-α, stimulates JCV early protein expression six fold. JCV TAg interacts with Pur-a, resulting in negative modulation of both JCV promoters (Gallia et al, 1998).77 Whereas TAg antagonizes the ability of Pur-a to enhance early gene transcription, Pur-α inhibits the ability of TAg to transactivate the late promoter (Chen and Khalili, 1995).78 TAg, through its C-terminal sequences, also binds YB-1, a Y-box binding protein that regulates gene expression at both the transcriptional and translational levels (Safak et al, 1999 and references therein).79 TAg functionally interacts with YB-1, leading to synergistic transactivation of the JCV late promoter. In addition, YB-1 reduces TAg's negative regulation of its own promoter (Chen and Khalili, 1995; Kerr et al, 1994; Safak et al, 1999).78-80 Pur-α and YB-1 modulate JCV transcription, in part, by binding to the late (A/G-rich) and early (T/C-rich) strands of the LCE motif in the viral TCR. In addition to effects on viral gene transcription, the interactions between TAg, Pur-α and YB-1 also appear to dictate the association of these factors with target DNA sequences (Chen and Khalili, 1995; Gallia et al, 1998; Safak et al, 1999).77,78,81 Chen et al (1995) have proposed a model to explain the mechanism by which TAg, Pur-α and YB-1 interaction leads to transition of early to late gene transcription.82

Members of the Jun and Fos families of proto-oncoproteins form dimers that were initially recognized as the AP-1 family of transcription factors. These complexes activate a number of cellular and viral promoters and influence a variety of cellular events, including proliferation, apoptosis and differentiation (reviewed by Shaulian and Karin, 2002).83 JCV promoter function is enhanced by c-Jun and c-Fos, and c-Jun binds sequences within the JCV TCR (Amemiya et al, 1989; Sadowska et al, 2003).84,85 Kim and coworkers (2003) have reported that in the presence of JCV TAg, however, both cellular factors down-regulate TAg-mediated viral transcription and replication.86 A physical interaction between c-Jun and the central region of JCV TAg was demonstrated, leading to speculation that positive effects of AP-1 on JCV early transcription might be important to establishing an infection, but that once TAg was expressed, these same factors might temper TAg function through binding, thereby facilitating virus maturation.

The late coding regions of primate polyomaviruses encode a small protein called Agnoprotein or LP1. Initial studies of the SV40 protein suggested that it has several functions, including roles in VP1 localization and viral capsid assembly (Carswell and Alwine, 1986; Ng et al, 1985; Resnick and Shenk, 1986).87-89 Recent experiments indicate that the JCV Agnoprotein displays additional activities. This 71 amino acid protein directly binds to TAg and to p53, and it has been hypothesized these interactions may influence cellular transformation and tumor formation (Del Valle et al, 2002).90 In addition, JCV Agnoprotein decreases the levels of JCV late gene transcription, and inhibits viral DNA replication, both TAg-mediated activities (Safak et al, 2001, 2002).91,92 Recently, Agnoprotein expression was found to enhance p21 levels in cells and to delay cell cycle progression during the G2/M phase of the cell cycle (Darbinyan et al, 2002).24

JCV tAg Functions

tAg Contributes to Viral Transformation

Investigations into the functions of JCV tAg are just beginning, however, several activities can be predicted based upon studies of the related SV40 protein. Early mutagenesis studies of JCV and SV40 indicated that tAg mutants are viable (Shenk et al, 1976; Mandl et al, 1987 and unpublished data), but exhibit some defects in infectious virion production in cultured cells (Topp, 1980).93-95 Transforming potential of the SV40 mutants was found to be variable and depended upon the experimental conditions (Martin et al, 1979; Frisque et al, 1980; Sleigh et al, 1978).96-98 TAg transforms many cell types in the absence of other viral proteins, but co-expression of tAg is required to initiate transformation of certain human cells (Bikel et al, 1987; Porras et al, 1996; Yu et al, 2001; Hahn et al, 2002).99-102 Further, tAg enhances TAg-mediated transformation when quiescent cells are employed or under conditions in which low levels of TAg are present. Both the N-terminal portion of tAg, which is shared with TAg and the 17kT protein, and the unique C-terminal portion of tAg, influence its transformation functions. Significant effort has been expended to identify the functional domains of this 174 amino acid protein, and two key interactions with cellular factors have been reported (reviewed by Rundell and Parakati, 2001).103

The J Domain of tAg

The shared N-terminal 82 amino acids of SV40 TAg and tAg represent a J domain, a conserved sequence found in the DnaJ family of molecular chaperones (Srinivasan et al, 1997).42 As discussed above, DNA tumor virus proteins, through their J domain, bind the cellular chaperone Hsc70, exhibiting a cochaperone function that contributes to viral lytic and transforming behaviors. SV40 small t protein, but not large T or truncated T (TN125 or TN136) proteins, functionally replaces the chaperone function of the E. coli DnaJ protein under limited growth conditions in vivo (Genevaux et al, 2003).104 Further, tAg stimulates the ATPase activity of DnaK from E. coli in vitro. Differences in cochaperone activity of the individual SV40 proteins may be due to the unique tAg sequences (amino acids 83-174) that substitute for a glycine-phenylalanine-rich domain of the bacterial Type I DnaJ protein.

The SV40 tAg also exhibits transregulating activities that depend upon a functional J domain. These activities involve both the activation and repression of cell cycle regulators, including cyclins A and D1 and the CKI, p27 (Porras et al, 1996, 1999; Watanabe et al, 1996).100,105 In addition, tAg transregulates other cellular promoters through its binding to protein phosphatase 2A (PP2A).

tAg Interaction with PP2A

The discovery that unique C-terminal tAg sequences interact with the cellular serine/threonine phosphatase PP2A was crucial to identifying mechanisms by which tAg contributes to viral transformation (Rundell, 1987; Mungre et al, 1994).106,107 PP2A, a modular trimeric enzyme, plays critical roles in cell signaling through the dephosphorylation of specific substrates. The large number of isoforms of the regulatory B subunit directs functional specificity when joined to the core enzyme composed of subunits A and C (reviewed in Garcia et al, 2000).108 SV40 tAg forms a complex with the AC core, in part, through a cysteine-containing motif (amino acids 97-103). This association modifies the substrate specificity and intracellular localization of PP2A (Yang et al, 1991), resulting in a multitude of responses that suggest mechanisms by which tAg cooperates with TAg to induce transformation.109

Inhibition of PP2A by tAg results in the activation of the protein kinase C ? isoform (PKC ?) in quiescent cells via a mechanism requiring phosphoinositide 3-kinase (PI 3-kinase). Activated PKC ? then upregulates the stress- and mitogen-activated protein kinase signaling cascades (SAPK and MAPK). Downstream targets of these pathways include the transcription factors, CREB, AP-1, Sp1 and NF-?B, which contribute to cellular proliferation and survival (reviewed by Garcia et al, 2000).108

Yuan and colleagues (2002) reported that transformation and immortalization of human keratinocytes require the simultaneous expression of SV40 TAg and tAg.110 In parallel with these observations, the investigators detected increased phosphorylation of the protein kinase, Akt, in cells expressing both viral proteins, but not in those producing TAg only. The authors provided data supporting the hypothesis that the interaction between tAg and PP2A activates Akt, a PKC-related kinase, and telomerase, the enzyme that regulates telomere length, contributes to a cell's extended life span and displays reactivated expression in most human tumors. These experimental results fit well with earlier reports that (i) identify Akt as a target of upstream kinases of the PI 3-kinase signaling pathway and (ii) indicate activated Akt inhibits apoptosis, stimulates cell growth and enhances telomerase activity (reviewed in Yuan et al, 2002).110

tAg Expression and Genomic Instability

Human diploid fibroblasts expressing SV40 tAg are blocked in their progression through G2/M, and Gaillard et al (2001) observed that overexpression of the viral protein in these cells prevents formation of the mitotic spindle.111 This phenotype depends on the interaction between tAg and PP2A and the resulting inhibition of centrosome maturation and duplication. Such tAg effects might contribute to the TAg-mediated alterations in ploidy and genetic stability already discussed above.

tAg Expression and Effects on the Tumor Suppressor, p53

The phosphorylation state of p53 is regulated by multiple cellular kinases and phosphatases, and this post-translational modification, in turn, modulates p53 functions such as specific DNA binding, transcriptional activation, cell cycle arrest and apoptosis induction (reviewed in Yan et al, 1997).112 Okadaic acid and SV40 tAg, two inhibitors of PP2A, reduced p53 dephosphorylation and increased its transcriptional activity, but only okadaic acid induced p53-directed programmed cell death in these experiments (Yan et al 1997).112

As noted above, an important step in SV40-induced transformation is the stabilization and functional inactivation of p53 following its binding to TAg. Members of the Deppert laboratory (Tiemann et al, 1995; Zerrahn et al, 1996) have reported that the metabolic stabilization and high-level accumulation of p53 in SV40-infected mouse and rat cells requires co-expression of TAg and tAg.53,113 Further, they suggest that tAg influences transformation efficiency through its ability to activate an unknown cellular function that results in p53 stabilization.

JCV T' Protein Functions

The JCV early precursor mRNA is alternatively spliced to yield 5 transcripts encoding TAg, tAg, T'135, T'136, and T'165 (Frisque et al, 1984; Trowbridge and Frisque, 1995).114,115 The three T' proteins are 135, 136 and 165 amino acids in length, and share their N-terminal 132 amino acids with the multifunctional TAg, of which 81 amino acids are also shared with tAg. T'165 shares its 33 amino acid C-terminus with TAg, while T'135 and T'136 have unique 3 and 4 amino acid C-termini, respectively, in a different reading frame (fig. 1). Each T' protein retains the TAg nuclear localization signal (NLS) at the end of their second coding exon, and immunofluorescence experiments confirm that the signal is functional; tAg, which lacks this NLS, is found predominately in the cytoplasm (Kilpatrick et al, in preparation). Unlike the 17kT protein of SV40 (Zerrahn et al, 1993) and tiny T protein of mouse polyomavirus (Riley et al, 1997), the JCV T' protein(s) are produced at relatively high levels in transformed and productively-infected cells (reviewed by Frisque, 2001).116-118 Genetic and biochemical studies suggest that T' proteins contribute both to viral replication and oncogenesis through their interaction with key cellular regulatory factors (reviewed by Frisque et al, 2003).119

Discovery of JCV T' Proteins

Based upon sequence analysis of the JCV genome, the early region was initially proposed to encode two proteins, TAg and tAg (Frisque et al, 1984).114 Subsequent analysis of 35S-methionine labeled extracts of JCV-transformed rodent cells (Bollag et al, 1989; Haggerty et al, 1989) revealed three bands on an SDS-polyacrylamide gel representing TAg, tAg and an unidentified 17 kDa protein named T' protein (later shown to be T'136).21,60 Although this protein was originally thought to be a degradation product of TAg, pulse-chase experiments confirmed it to be an authentic early protein (Trowbridge and Frisque, 1995).115 Similar studies in lytically infected PHFG cells revealed four T' bands of 16, 17, 22 and 23 kDa. Immunoprecipitation reactions using a monoclonal antibody directed against the N-terminus of TAg recognized each T' band, whereas an antibody directed against the C-terminus of TAg recognized only the two largest T' bands.

RNA extracted from infected PHFG cells and subjected to reverse transcription polymerase chain reaction (RT-PCR) led to the identification of three T' transcripts. Sequence analyses of cloned T' cDNAs prepared from RT-PCR experiments indicated that two introns are removed during the processing of the T' mRNAs. The first intron is removed using the TAg mRNA donor and acceptor splice sites, and the second intron, unique to T' transcripts, is processed using a common donor site and three different acceptor sites. It will be noted that four T' bands were observed on SDS-polyacrylamide gels, but only three T' cDNAs were generated by RT-PCR. This discrepancy was resolved by using lambda phosphatase to identify the 23 kDa T' band as a phosphorylated form of T'165 (Prins and Frisque, 2001). Subsequently, the other two T' proteins were also found to be phosphoproteins (Swenson and Frisque, 1995; Kilpatrick et al, in preparation).120

A single T' protein (T'136) was detected in JCV-transformed rat fibroblasts, whereas all three T' proteins were observed in JCV-infected human brain cells, suggesting that T' expression patterns either reflect differences in the species (human vs. rat), cell type (brain vs. fibroblast) or virus-cell interaction (transformed vs. infected) examined. To investigate these possibilities, Jones and Frisque (unpublished data) conducted RT-PCR on infected, transformed or tumor cells derived from different species and tissues. The results suggest that changes in T' expression patterns are the result of differences in alternative splicing levels occurring during a lytic vs. transformation event.

Replication Function of JCV T' Proteins

Biochemical and genetic approaches have been employed to understand the role of the T' proteins in JCV biology. The N-terminal 132 amino acids shared by TAg and T' proteins encompass numerous functional domains influencing both viral replication and oncogenic behavior. Early mutagenesis experiments targeted the T' common donor splice site, thereby abrogating T' expression. Transfection of this mutant genome (JCVΔT') into PHFG cells resulted in a 10- to 20-fold reduction in replication, thus confirming that one or more T' proteins contributes to TAg-mediated viral DNA replication (Trowbridge and Frisque, 1995).115 To express these T' proteins individually or in combination with one another, the three unique T' acceptor sites were mutated (without changing the TAg amino acid sequence), and viral replication was measured in PHFG cells (Prins and Frisque, 2001).121 Those mutants still capable of producing one or two T' proteins had replication activities similar to that of wild type virus, whereas the triple acceptor site mutant (no T' proteins made) had the same defective replication phenotype as the JCVΔT' donor site mutant (Prins and Frisque, 2001).121 It was observed that cells transfected with the initial T'135 acceptor site mutant DNA produced new T' mRNAs and proteins as a result of the utilization of cryptic splice sites downstream of the original T'135 acceptor site. Therefore, these cryptic sites were altered to prevent expression of any T'135-like transcripts (Prins and Frisque, 2001).121

T' Proteins Influence Virus-Cell Interactions

Attempts to transform Rat2 fibroblasts with vectors expressing individual JCV early proteins have been unsuccessful (Kilpatrick et al, in preparation). Therefore, to establish cell lines expressing each JCV tumor protein, a G418-selection protocol was employed. Constructs containing the entire JCV early coding region (JCVE) or individual proteins (TAg, T'135, T'136 or T'165) under the control of a cytomegalovirus (CMV) promoter were cotransfected into Rat2 cells with the pSV2-neo plasmid. Cells acquiring G418 resistance were single-cell cloned for stable and high-level protein expression. Lines expressing T'135, T'136 or T'165 independently or all 5 early proteins together were readily obtained, but lines containing the TAg cDNA expressed TAg plus one or more T' proteins. Because the TAg construct retains the unique T' donor and acceptor splice sites, attempts were made to create lines expressing TAg only by using a JCVΔ T' cDNA mutant (Kilpatrick et al, in preparation). Only 1 of the 30 lines screened produced TAg, and in this line the viral protein was produced at low levels. Sequence analysis confirmed that the integrated TAg gene was not mutated. One interpretation of these results is that TAg induces apoptosis in Rat2 cells and that one or more T' proteins block this effect. Others have shown that SV40 TAg and tAg may exhibit both proapoptotic or antiapoptotic functions depending on experimental conditions (Gjoerup et al, 2001 and references therein).122

Analyses of cell doubling time and saturation density in media supplemented with 1% or 10% fetal bovine serum were performed on the cloned Rat2 lines expressing individual JCV proteins. Testing of 2 independent clones showed JCVE cells had accelerated growth rates and higher saturation densities, although the values were lower than those of a control transformed line isolated from a dense focus assay (Kilpatrick et al, in preparation). Cells expressing TAg, T'135, T'136 or T'165 showed growth parameters only marginally more robust than those of the parental Rat2 line. The failure of the cloned Rat2 lines to exhibit an aggressive transformed phenotype might be attributed to expression levels of the tumor proteins that are below the threshold amounts required to induce such changes (Trowbridge and Frisque, 1993).22

T' Proteins Interact with Key Cell Cycle Regulatory Proteins

The TAg isoforms produced by members of the Polyomavirus family, JCV T', SV40 17kT and mouse polyomavirus tiny T proteins, contain two of the three TAg transformation domains (LXCXE and J) (fig. 1). In addition, several truncated versions of SV40 TAg constructed in vitro contain these motifs. One of these latter truncated forms, TN136, binds to Rb proteins but fails to form a stable complex with Hsc70 (Sullivan et al, 2001), suggesting that sequences C-terminal to the J and LXCXE domains might be essential for stable interaction with Hsc70.50 Riley et al (1997) found that the mouse polyomavirus tiny T protein stimulates ATPase activity of Hsc70, although a direct, stable interaction between the two proteins was not reported.117 The SV40 17kT protein binds to and reduces the levels of p130, promotes E2F activity, and stimulates cell-cycle progression of quiescent fibroblasts (Boyapati et al, 2003).123 Cells expressing only 17kT display a minimal transformed phenotype and the protein is underphosphorylated relative to TAg, reflecting the absence of C-terminal TAg sequences that regulate modification of the N-terminus (Zerrahn et al, 1993).124

As discussed earlier, in vivo and in vitro studies confirm that JCV TAg interacts with cellular pRB, p107 and p130 (reviewed in Frisque et al, 2003).119 Using an in vitro approach, Bollag et al (2000) investigated the possibility that T'135, T'136 and T'165 might also bind Rb family proteins.57 Viral proteins immunoaffinity-purified from insect cells infected with recombinant baculoviruses were mixed with extracts of human MOLT-4 cells containing pRB, p107 and p130. Each T' protein was shown to interact with the hypophosphorylated forms of the Rb proteins, and, importantly, each viral protein exhibited differential binding affinity depending on the specific interaction examined. Similar studies conducted in vivo using Rat2 cells expressing individual JCV tumor proteins revealed binding of the T' proteins to p107 and p130; the interaction between T'136 and p107 was especially difficult to demonstrate (Kilpatrick et al, in preparation). These experiments revealed that T' proteins not only bind to hypophosphorylated p107 and p130, but that some hyperphosphorylated species of these cellular proteins are absent in the cell lines.

Given that JCV tumor proteins interact with pRB, p107 and p130, one might predict that the five viral early proteins would also bind Hsc70 via their J domains. However, the inability to demonstrate a stable interaction between the truncated SV40 N136 peptide and this molecular chaperone raised questions about the binding potential of JCV T' proteins. Using POJ (PHFG cells transformed with Ori defective JCV; Mandl et al, 1987) cell extracts, and a co-IP/WB assay, it was determined that at least one of the JCV early proteins bound Hsc70 (Bollag and Frisque, unpublished data).94 Recent experiments utilizing Rat2 cells expressing individual JCV proteins confirm that T'135 interacts with Hsc70 (Kilpatrick et al, in preparation).

The ability of JCV TAg and T' proteins to bind Rb proteins and Hsc70 is expected to result in the release of E2F and the cell's progression to S phase. To address this possibility, Rat2 lines expressing individual JCV proteins were cotransfected with a β-galactosidase expression plasmid and a luciferase vector under the control of a promoter containing four E2F-1 binding sites. Luciferase data normalized to that of β-galactosidase activity indicates that the highest levels of free E2F-1 are induced in cells expressing T'165 or all five early proteins (Tyagarajan and Frisque, in preparation). As expected, cells expressing either an LXCXE (E109K) or J (H42Q) domain mutant had relatively low levels of released E2F-1 (Table 2). Co-IP/WB experiments performed on extracts of these cells verified that the H42Q, but not the E109K, mutant proteins were capable of binding p107 and p130. Furthermore, both hypophosphorylated and hyperphosphorylated forms of p107 and p130 were detected in these cells, indicating that the mutant viral proteins fail to alter the phosphorylation states of the cellular proteins.

Summary of JCV Early Protein Function

An extensive literature details the contributions made by the SV40 multifunctional TAg and tAg to the oncogenic process. On the other hand, little is known about the17kT protein that is produced in greatly diminished amounts relative to the other SV40 early proteins. Using SV40 results as a guide, a number of studies have been conducted to examine JCV TAg functions. At the sequence level, many structural motifs (e.g., NLS, phosphorylation, zinc finger, ATP binding) and functional domains (e.g., DNA binding, transformation) have been identified that correspond to those of the SV40 TAg. Although similarities of structure and function are readily apparent, it is also clear that the JCV TAg displays less robust support of DNA replication and transformation activity in cell culture and reduced potential to interact with key cellular regulatory factors. Analyses of JCV tAg functions have only recently been initiated, and much work must be done to determine how this protein influences the transforming potential of JCV. It is expected that JCV tAg interacts with cellular proteins such as PP2A through its unique C-terminal sequences and chaperones via its J domain, thereby affecting cellular proliferation and programmed cell death. Several observations support the hypothesis that JCV T' proteins make important contributions to the biology of the virus. Their expression is differentially regulated in permissive vs. nonpermissive cells, and they are produced at significantly elevated levels relative to their SV40 and mouse polyomavirus counterparts. In addition, T' proteins enhance viral DNA replication and exhibit differential binding to critical cell cycle regulators. Finally, preliminary data suggest that one or more T. proteins block apoptosis induced by JCV TAg under certain conditions. The ability to complement or antagonize activities of the multifunctional TAg may permit T. proteins to fine-tune JCV's control over the virus-host interactions.


The authors thank Dr. Brigitte Bollag and Lisa Kilpatrick for helpful discussions. We apologize to our colleagues if we failed to cite relevant publications either because of space constraints or our own oversight. This work was supported in part by National Institutes of Health grant NS41833.


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