Large T Antigens of Polyomaviruses: Amazing Molecular Machines

JC virus (JCV) is a DNA virus causing progressive multifocal leukoencephalopathy (PML) in immunodeficient patients. In the present study, 22 genetic quasispecies with more than 1.5% variant frequency were detected in JCV genomes from six clinical samples of PML by next-generation sequencing. A mutation from A to C at nucleotide (nt) 3495 in JCV Mad1 resulting in a V-to-G amino acid substitution at amino acid (aa) position 392 of the large T antigen (TAg) was identified in all six cases of PML at 3% to 19% variant frequencies. Transfection of JCV Mad1 DNA possessing the V392G substitution in TAg into IMR-32 and human embryonic kidney 293 (HEK293) cells resulted in dramatically decreased production of JCV-encoded proteins. The virus DNA copy number was also reduced in supernatants of the mutant virus-transfected cells. Transfection of the IMR-32 and HEK293 cells with a virus genome containing a revertant mutation recovered viral production and protein expression. Cotransfection with equal amounts of wild-type genome and mutated JCV genome did not reduce the expression of viral proteins or viral replication, suggesting that the mutation did not have any dominantnegative function. Finally, immunohistochemistry demonstrated that TAg was expressed in all six pathological samples in which the quasispecies were detected. In conclusion, the V392G amino acid substitution in TAg identified frequently in PML lesions has a function in suppressing JCV replication, but the frequency of the mutation was restricted and its role in PML lesions was limited.IMPORTANCE DNA viruses generally have lower mutation frequency than RNA viruses, and the detection of quasispecies in JCV has rarely been reported. In the present study, a next-generation sequencer identified a JCV quasispecies with an amino acid substitution in the T antigen in patients with PML. In vitro studies showed that the mutation strongly repressed the expression of JC viral proteins and reduced the viral replication. However, because the frequency of the mutation was low in each case, the total expression of virus proteins was sustained in vivo. Thus, JC virus replicates in PML lesions in the presence of a mutant virus which is able to repress virus replication.

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confidence: 98%

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

Takahashi

Sekizuka

Fukumoto

et al. 2017

“…SV40 LT interacts with classic partners including heat shock protein 70 (Hsc70) through the LT DnaJ domain and also interacts with retinoblastoma "pocket protein" (Rb) family members through a classic LxCxE motif in the N-terminal region of LT. SV40 LT binding of Rb abrogates its role as a repressor of E2F transcription factors, thereby promoting transition into S phase. MCV LT is thought to interact with Hsc70 and Rb via similar mechanisms (11)(12)(13). SV40 LT is also known to interact with the tumor suppressor protein p53 through two C-terminal LT regions within the helicase domain of LT (14).…”

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confidence: 99%

Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation

Wang

Díaz

et al. 2013

103

117

bClonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers. Merkel cell polyomavirus (MCV) is the first polyomavirus to be clearly associated with cancer in humans (1). Its genome was recently found integrated into the chromosomes of a highly aggressive skin cancer, Merkel cell carcinoma (MCC) (2). Subsequent analyses of a large number of MCC tumors have revealed that this polyomavirus is associated with at least 80% of all MCC cases (2-4). Integrated MCV genome has also been detected in non-small-cell lung cancer (5). Epidemiological surveys for MCV seropositivity (6, 7) and sequencing analyses of healthy human skin (8) have indicated that MCV represents a common component of the human skin microbial flora.As with other polyomaviruses, the MCV genome contains an early region that encodes the viral tumor antigens. Differential splicing of the early mRNA produces large tumor antigen (LT), small tumor antigen (sT), and 57kT proteins (9, 10). The highly multifunctional LT protein is involved in a variety of processes, including initiation of viral genome replication, as well as manipulation of the host cell cycle through a number of protein-protein interactions. It has been shown that MCV LT interacts with at least some of the same cellular factors as simian virus 40 (SV40) LT (11). SV40 LT interacts with classic partners including heat shock protein 70 (Hsc70) through the LT DnaJ domain and also interacts with retinoblastoma "pocket protein" (Rb) family members through a classic LxCxE motif in the N-terminal region of LT. SV40 LT binding of Rb abrogates its role as a repressor of E2F transcription factors,...

“…The large tumor antigen (TAg) of polyomavirus simian virus 40 (SV40) is a multidomain oncoprotein that interacts with and disrupts the function of several host proteins, thereby interfering with specific cellular pathways (11,12). TAg expression causes transformation of several primary cell types in cell culture and also induces tumors in model organisms.…”

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confidence: 99%

“…Studies investigating the mechanism of TAg-induced proliferation and transformation have shown that TAg interacts with the Rb/E2F complexes via its LXCXE motif and induces their disruption via its J-domain, thereby upregulating E2F target genes (3,13,14). Several studies have led to the current "textbook model" in which release of activator E2Fs by TAg and subsequent upregulation of E2F target genes are required for transformation (11,15). However, in view of recent studies demonstrating cell proliferation in the absence of activator E2Fs, we investigated whether TAg-induced proliferation and transformation require E2F1-2-3.…”

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confidence: 99%

Cellular Transformation of Mouse Embryo Fibroblasts in the Absence of Activator E2Fs

Gupta

Robles

Pipas

2015

Self Cite

The E2F family of transcription factors, broadly divided into activator and repressor E2Fs, regulates cell cycle genes. Current models indicate that activator E2Fs are necessary for cell cycle progression and tumorigenesis and are also required to mediate transformation induced by DNA tumor viruses. E2Fs are negatively regulated by the retinoblastoma (RB) family of tumor suppressor proteins, and virus-encoded oncogenes disrupt the RB-E2F repressor complexes. This results in the release of activator E2Fs and induction of E2F-dependent genes. In agreement, expression of large tumor T antigens (TAg) encoded by polyomaviruses in mammalian cells results in increased transcriptional levels of E2F target genes. In addition, tumorigenesis induced by transgenic expression of simian virus 40 (SV40) TAg in choroid plexus or intestinal villi requires at least one activator E2F. In contrast, we show that SV40 TAg-induced transformation in mouse embryonic fibroblasts is independent of activator E2Fs. This work, coupled with recent studies showing that proliferation in stem and progenitor cells is independent of activator E2Fs, suggests the presence of parallel pathways governing cell proliferation and tumorigenesis. IMPORTANCEThe RB-E2F pathway is altered in many cancers and is also targeted by DNA tumor viruses. Viral oncoprotein action on RBs results in the release of activator E2Fs and upregulation of E2F target genes; thus, activator E2Fs are considered essential for normal and tumorigenic cell proliferation. However, we have observed that SV40 large T antigen can induce cell proliferation and transformation in the absence of activator E2Fs. Our results also suggest that TAg action on pRBs regulates both E2F-dependent and -independent pathways that govern proliferation. Thus, specific cell proliferation pathways affected by RB alterations in cancer may be a factor in tumor behavior and response to therapy. Cell proliferation is a highly regulated process involving four well-coordinated phases of the cell cycle. Each phase is regulated by various pro-and antiproliferation pathways, whose disruption leads to either cell death or uncontrolled cell proliferation and cancer. One of those controllers is the retinoblastoma (RB)/ E2F pathway, and mutations in members of this pathway are often found in human cancers, leading to enhanced E2F activity and upregulation of E2F target gene (1). Furthermore, various viral oncoproteins-including the large tumor (T) antigen (TAg) from polyomaviruses, E1A from adenoviruses, and E7 from papillomaviruses-have independently evolved to target this pathway, indicating its central role in cell cycle regulation (2-4).E2Fs are transcription factors that regulate cell proliferation by controlling the expression of cell cycle genes. In a growth-arrested cell, the E2Fs are bound to and negatively regulated by hypophosphorylated members of the retinoblastoma (RB) protein family (pRb, p107, p130) (1). Upon receiving mitogenic signals, pRBs become hyperphosphorylated and release E2Fs, which then...