Merkel cell polyomavirus (MCV) is a virus discovered in our laboMerkel cell carcinoma ͉ pRB interaction ͉ viral integration ͉ virus replication ͉ helicase M erkel cell carcinoma (MCC) is an aggressive skin cancer associated with sun exposure and immunosuppression (1, 2). Using digital transcriptome subtraction, we recently identified Merkel cell polyomavirus (MCV) as a novel polyomavirus integrated into the genome of MCC tumors (3, 4). The close association between MCV and MCC has been confirmed by others (5). Polyomaviruses are small circular DNA viruses encoding a T antigen oncoprotein locus. T antigens are expressed from variably spliced viral transcripts that target tumor suppressor and cell cycle regulatory proteins, including retinoblastoma tumor suppressor protein (Rb) (6), p53 (7, 8), protein phosphatase 2A (9), and Bub1 (10). Murine polyomavirus (MuPyV) middle T (MT) antigen, a membrane-bound protein, is particularly potent in initiating cell transformation through interactions with phosphatidylinositol 3-kinase, protein phosphatase 2A, Src, and Shc proteins (11,12). MCV large T (LT) antigen retains conserved domains, such as pocket Rb binding LXCXE and DnaJ motifs, present across virus species (13). LT not only encodes tumor suppressor targeting domains but also origin binding and helicase/ATPase functions required for viral genome replication. MCV integration in MCC tumors is incompatible with transmissible virus and likely represents a rare biological accident in which the tumor cell is a dead-end host. The monoclonal pattern of MCV integration into MCC tumors suggests that virus integration occurs before tumor cell expansion and that MCV is a contributing factor in a portion of MCC (3).We have isolated MCV T antigen sequences from both tumor cases and nontumor cases to characterize their abilities to act as a viral DNA replicase. Sequence analysis demonstrates that LT protein is prematurely truncated in all MCC cases, whereas the Rb-interacting domain is preserved. Viruses from nontumor sources do not possess these mutations. We also describe here an MCC cell line stably harboring MCV and an origin replication assay to assess MCV replication. We show that wild type (WT) LT from nontumorous sources activates MCV replication of integrated tumor virus, suggesting that MCV-associated MCC arises from a two-step process in which viral genome integrates into the host genome and develops T antigen mutations to prevent autonomous viral genome replication. Failure to truncate the viral T antigen may lead to DNA damage responses or immune recognition that hinders nascent tumor cell survival.

Merkel cell polyomavirus (MCV) is the recently discovered cause of most Merkel cell carcinomas (MCCs), an aggressive form of nonmelanoma skin cancer. Although MCV is known to integrate into the tumor cell genome and to undergo mutation, the molecular mechanisms used by this virus to cause cancer are unknown. Here, we show that MCV small T (sT) antigen is expressed in most MCC tumors, where it is required for tumor cell growth. Unlike the closely related SV40 sT, MCV sT transformed rodent fibroblasts to anchorageand contact-independent growth and promoted serum-free proliferation of human cells. These effects did not involve protein phosphatase 2A (PP2A) inhibition. MCV sT was found to act downstream in the mammalian target of rapamycin (mTOR) signaling pathway to preserve eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) hyperphosphorylation, resulting in dysregulated cap-dependent translation. MCV sT-associated 4E-BP1 serine 65 hyperphosphorylation was resistant to mTOR complex (mTORC1) and mTORC2 inhibitors. Steady-state phosphorylation of other downstream Akt-mTOR targets, including S6K and 4E-BP2, was also increased by MCV sT. Expression of a constitutively active 4E-BP1 that could not be phosphorylated antagonized the cell transformation activity of MCV sT. Taken together, these experiments showed that 4E-BP1 inhibition is required for MCV transformation. Thus, MCV sT is an oncoprotein, and its effects on dysregulated cap-dependent translation have clinical implications for the prevention, diagnosis, and treatment of MCV-related cancers.

Merkel cell polyomavirus (MCV) is a recently discovered human virus closely related to African green monkey lymphotropic polyomavirus. MCV DNA is integrated in 80% of Merkel cell carcinomas (MCC), a neuroendocrine skin cancer linked to lymphoid malignancies such as chronic lymphocytic leukemia (CLL). To assess MCV infection and its association with human diseases, we developed a monoclonal antibody that specifically recognizes endogenous and transfected MCV large T (LT) antigen. We show expression of MCV LT protein localized to nuclei of tumor cells from MCC having PCR quantified MCV genome at an average of 5.2 (range 0.8-14.3) T antigen DNA copies per cell. Expression of this putative viral oncoprotein in tumor cells provides the mechanistic underpinning supporting the notion that MCV causes a subset of MCC. In contrast, although 2.2% of 325 hematolymphoid malignancies surveyed also showed evidence for MCV infection by DNA PCR, none were positive at high viral copy numbers, and none of 173 lymphoid malignancies examined on tissue microarrays expressed MCV LT protein in tumor cells. As with some of the other human polyomaviruses, lymphocytes may serve as a tissue reservoir for MCV infection, but hematolymphoid malignancies associated with MCC are unlikely to be caused by MCV. ' 2009 UICC

SUMMARY Merkel cell polyomavirus (MCV) causes an aggressive human skin cancer, Merkel cell carcinoma, through expression of small T (sT) and large T (LT) viral oncoproteins. MCV sT is also required for efficient MCV DNA replication by the multifunctional MCV LT helicase protein. We find that LT is targeted for proteasomal degradation by the cellular SCFFbw7 E3 ligase, which can be inhibited by sT through its LT stabilization domain (LSD). Consequently, sT also stabilizes cellular SCFFbw7 targets, including the cell cycle regulators c-Myc and cyclin E. Mutating the sT LSD decreases LT protein levels and eliminates synergism in MCV DNA replication as well as sT-induced cell transformation. SCFFbw7 knockdown mimics sT-mediated stabilization of LT, but this knockdown is insufficient to fully reconstitute the transforming activity of a mutant LSD sT protein. Thus, MCV has evolved a regulatory system involving SCFFbw7 that controls viral replication but also contributes to host cell transformation.

Merkel cell polyomavirus (MCV) is a recently discovered human polyomavirus causing the majority of human Merkel cell carcinomas. We mapped a 71-bp minimal MCV replication core origin sufficient for initiating eukaryotic DNA replication in the presence of wild-type MCV large T protein (LT). The origin includes a poly(T)-rich tract and eight variably oriented, GAGGC-like pentanucleotide sequences (PS) that serve as LT recognition sites. Mutation analysis shows that only four of the eight PS are required for origin replication. A single point mutation in one origin PS from a naturally occurring, tumor-derived virus reduces LT assembly on the origin and eliminates viral DNA replication. Tumor-derived LT having mutations truncating either the origin-binding domain or the helicase domain also prevent LT-origin assembly. Optimal MCV replication requires coexpression of MCV small T protein (sT), together with LT. An intact DnaJ domain on the LT is required for replication but is dispensable on the sT. In contrast, PP2A targeting by sT is required for enhanced replication. The MCV origin provides a novel model for eukaryotic replication from a defined DNA element and illustrates the selective pressure within tumors to abrogate independent MCV replication.Unlike human cellular DNA replication origins, polyomavirus replication origins are discrete and well defined and yet retain many features of eukaryotic cellular origins. For this reason, polyomavirus replication origins, particularly the simian virus 40 (SV40) origin, have been used as easily tractable models to define eukaryotic replication requirements (1).Polyomaviruses are small, double-stranded DNA viruses with circular genomes functionally divided into coding and noncoding regions (10). The early coding region for all polyomaviruses encodes large tumor (LT) and small T (sT) antigens that serve as viral oncoproteins and a late coding region that produces viral structural proteins. Aside from LT and sT, other T-antigen isoforms, such as middle T antigen (MT) and 17kT/57kT, may be present and are virus specific. LT has pleiotropic functions that include initiation and maintenance of viral DNA replication, regulation of early and late genes transcription, and virion assembly (11,21,36,43,51,52,54). Expression of LT also leads to the transformation of susceptible cell lines mediated in part by functional regions such as the DnaJ, pocket protein binding, and p53 binding domains that target growth-suppressing and cell cycle regulatory proteins (53). In addition, sT has been shown to play an important role in LT mediated cell transformation in SV40 (3,6,7,24,38) and has been reported to increase virus replication efficiency in JC virus (JCV) (39).Merkel cell polyomavirus (MCV) was recently identified by digital transcriptome subtraction (23) as a new human polyomavirus present in ϳ80% of Merkel cell carcinoma (MCC) (22). Preferential detection of MCV in MCC has subsequently been confirmed in a variety of different settings (4,17,29,58). Similar to JCV and BK virus, this n...

SignificanceCancer cell proliferation is highly dependent on cap-dependent protein synthesis, which is generally assumed to be inhibited during mitosis. Using a viral oncoprotein that enforces mitosis, we show that CDK1 substitutes for mTOR interphase functions to phosphorylate eukaryotic initiation factor 4E-binding protein (4E-BP1) to a mitosis-specific δ isoform. Flow cytometric assays reveal that mitotic cells have high levels of inactivated 4E-BP1 and do not generally show specific loss of cap-dependent translation compared with interphase cells. This appears to be due to cyclin-dependent kinase 1 (CDK1) activity during mitosis. Mitotic cells typically represent less than 1% of all cells in bulk culture, and mitosis-arresting drugs, such as nocodazole, can directly inhibit mitotic protein translation, potentially explaining differences between our findings and previous studies showing reduced cap-dependent translation during mitosis.

E-cadherin is a key cell adhesion molecule implicated as a tumor suppressor, which is frequently altered in hepatocellular carcinoma, especially in hepatitis B virus (HBV)-related tumors. Here, we report that HBV X protein (HBx) represses E-cadherin expression at the transcription level. Based on the differential effects of HBx natural variants, we determined that Lys-130 in the transactivation domain of HBx is critical for the E-cadherin repression. The repression effect of HBx was abolished after treatment with DNA methyltransferase inhibitor, 5 0 -Aza-2 0 dC. In addition, methylation-specific PCR analysis revealed that the CpG island 1 of E-cadherin promoter is hypermethylated by HBx. Furthermore, HBx induces DNA methyltransferase 1 expression by stimulating its transcription. Therefore, we conclude that HBx represses E-cadherin expression by inducing methylation-mediated promoter inactivation. The reduced E-cadherin expression results in dramatic morphological changes of the HBxexpressing cells. In addition, HBx-expressing cells aggregate poorly in suspension culture, reflecting their altered intercellular interactions. The biological significance was further demonstrated by the increased collagen invasion ability of HBx-expressing cells. Therefore, the present study suggests that HBx plays a role during hepatocellular carcinogenesis by favoring cell detachment from the surrounding cells and migration outside of the primary tumor site.

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.