ABSTRACT
Merkel cell polyomavirus (MCPyV) is a double-stranded tumor virus that is the main causative agent of Merkel cell carcinoma (MCC). The MCPyV large T antigen (LT), an essential viral DNA replication protein, maintains viral persistence by interacting with host Skp1-Cullin 1-F-box (SCF) E3 ubiquitin ligase complexes, which subsequently induces LT's proteasomal degradation, restricting MCPyV DNA replication. SCF E3 ubiquitin ligases require their substrates to be phosphorylated to bind them, utilizing phosphorylated serine residues as docking sites. The MCPyV LT unique region (MUR) is highly phosphorylated and plays a role in multiple host protein interactions, including SCF E3 ubiquitin ligases. Therefore, this domain highly governs LT stability. Though much work has been conducted to identify host factors that restrict MCPyV LT protein expression, the kinase(s) that cooperates with the SCF E3 ligase remains unknown. Here, we demonstrate that casein kinase 1 alpha (CK1α) negatively regulates MCPyV LT stability and LT-mediated replication by modulating interactions with the SCF ß-TrCP. Specifically, we show that numerous CK1 isoforms (α, δ, ε) localize in close proximity to MCPyV LT through in situ proximity ligation assays (PLA) and CK1α overexpression mainly resulted in decreased MCPyV LT protein expression. Inhibition of CK1α using short hairpin RNA (shRNA) and treatment of a CK1α inhibitor or an mTOR inhibitor, TORKinib, resulted in decreased ß-TrCP interaction with LT, increased LT expression, and enhanced MCPyV replication. The expression level of the CSNK1A1 gene transcripts is higher in MCPyV-positive MCC, suggesting a vital role of CK1α in limiting MCPyV replication required for establishing persistent infection. IMPORTANCE: Merkel cell polyomavirus (MCPyV) large tumor antigen is a polyphosphoprotein and the phosphorylation event is required to modulate various functions of LT, including viral replication. Therefore, cellular kinase pathways are indispensable for governing MCPyV polyomavirus infection and life cycle in coordinating with the immunosuppression environment at disease onset. Understanding the regulation mechanisms of MCPyV replication by viral and cellular factors will guide proper prevention strategies with targeted inhibitors for MCPyV-associated Merkel cell carcinoma (MCC) patients, who currently lack therapies.
Subject(s)
Antigens, Viral, Tumor , Casein Kinase Ialpha , Merkel cell polyomavirus , beta-Transducin Repeat-Containing Proteins , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Humans , Phosphorylation , Casein Kinase Ialpha/metabolism , Casein Kinase Ialpha/genetics , beta-Transducin Repeat-Containing Proteins/metabolism , beta-Transducin Repeat-Containing Proteins/genetics , Antigens, Viral, Tumor/metabolism , Antigens, Viral, Tumor/genetics , Host-Pathogen Interactions , Proteolysis , Virus Replication , Protein Binding , Antigens, Polyomavirus Transforming/metabolism , Antigens, Polyomavirus Transforming/genetics , Polyomavirus Infections/virology , Polyomavirus Infections/metabolism , Polyomavirus Infections/geneticsABSTRACT
Merkel cell carcinoma is a skin cancer often driven by Merkel cell polyomavirus (MCPyV) with high rates of response to anti-PD-1 therapy despite low mutational burden. MCPyV-specific CD8 T cells are implicated in anti-PD-1-associated immune responses and provide a means to directly study tumor-specific T cell responses to treatment. Using mass cytometry and combinatorial tetramer staining, we find that baseline frequencies of blood MCPyV-specific cells correlated with response and survival. Frequencies of these cells decrease markedly during response to therapy. Phenotypes of MCPyV-specific CD8 T cells have distinct expression patterns of CD39, cutaneous lymphocyte-associated antigen (CLA), and CD103. Correspondingly, overall bulk CD39+CLA+ CD8 T cell frequencies in blood correlate with MCPyV-specific cell frequencies and similarly predicted favorable clinical outcomes. Conversely, frequencies of CD39+CD103+ CD8 T cells are associated with tumor burden and worse outcomes. These cell subsets can be useful as biomarkers and to isolate blood-derived tumor-specific T cells.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Oligosaccharides , Sialyl Lewis X Antigen/analogs & derivatives , Skin Neoplasms , Humans , Carcinoma, Merkel Cell/drug therapy , Carcinoma, Merkel Cell/metabolism , Carcinoma, Merkel Cell/pathology , Merkel cell polyomavirus/metabolism , Programmed Cell Death 1 Receptor/metabolism , CD8-Positive T-Lymphocytes , Skin Neoplasms/drug therapy , Skin Neoplasms/metabolism , Biomarkers/metabolismABSTRACT
Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Mice , Animals , Humans , Antigens, Polyomavirus Transforming/metabolism , Merkel cell polyomavirus/metabolism , NF-kappa B/metabolism , src-Family Kinases/metabolism , Phospholipase C gamma/metabolism , Signal Transduction , Antigens, Viral, Tumor/genetics , Carcinoma, Merkel Cell/genetics , Tyrosine/metabolismABSTRACT
BACKGROUND: Merkel cell carcinoma (MCC) is a rare, aggressive, primary neuroendocrine carcinoma of the skin whose main risk factors are immunosuppression, UV radiation exposure, and Merkel cell polyomavirus. Programmed death-1/programmed death ligand-1 (PD-L1)-based immunotherapy is currently the first choice for treating patients with metastatic MCC. METHODS: MCC biopsies (17) were evaluated for their nucleus and cytoplasm characteristics and growth patterns, as well as for intratumor lymphocytes, mitotic number, and lymphovascular invasion. Paraffin-embedded tissue samples of the biopsies were stained with MCPyV large T-antigen (LTag), RB1, p53, and PD-L1. RESULTS: We observed MCPyV LTag expression in 9 out of the 17 tumors, and all 9 cases were positive for RB1 ( P <0.000). p53 staining was not significantly correlated with MCPyV LTag. We observed no relationship between p53 expression and any other parameters, and PD-L1 expression was low in the MCC samples. We evaluated PD-L1 using both the combined positive score and tumor proportion score (TPS), and found that TPS was correlated with MCPyV LTag expression ( P =0.016). Tumors with tumor-infiltrating lymphocytes showed a better prognosis than those without these lymphocytes ( P =0.006). DISCUSSION: Our data demonstrated that RB1 was effective for immunohistochemically investigating the MCPyV status of tumors. TPS was superior to the combined positive score in evaluating PD-L1 in MCC. Tumor-infiltrating lymphocytes were the only parameters that were associated with survival. Further studies with larger series are required to confirm these results.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Humans , Carcinoma, Merkel Cell/pathology , B7-H1 Antigen/metabolism , Merkel cell polyomavirus/metabolism , Tumor Suppressor Protein p53 , Skin Neoplasms/pathology , Polyomavirus Infections/complications , Polyomavirus Infections/metabolism , Ubiquitin-Protein Ligases , Retinoblastoma Binding Proteins/metabolismABSTRACT
Senescent cells accumulate in the host during the aging process and are associated with age-related pathogeneses, including cancer. Although persistent senescence seems to contribute to many aspects of cellular pathways and homeostasis, the role of senescence in virus-induced human cancer is not well understood. Merkel cell carcinoma (MCC) is an aggressive skin cancer induced by a life-long human infection of Merkel cell polyomavirus (MCPyV). Here, we show that MCPyV large T (LT) antigen expression in human skin fibroblasts causes a novel nucleolar stress response, followed by p21-dependent senescence and senescence-associated secretory phenotypes (SASPs), which are required for MCPyV genome maintenance. Senolytic and navitoclax treatments result in decreased senescence and MCPyV genome levels, suggesting a potential therapeutic for MCC prevention. Our results uncover the mechanism of a host stress response regulating human polyomavirus genome maintenance in viral persistency, which may lead to targeted intervention for MCC.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Polyomavirus Infections/genetics , Tumor Virus Infections/genetics , Tumor Virus Infections/pathology , Antigens, Viral, Tumor/genetics , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/pathology , Skin Neoplasms/pathology , Cellular Senescence , Genome, ViralABSTRACT
BACKGROUND: Human Polyomaviruses such as MCPyV and HPyV6 are frequently found as part of healthy skin microbiota and have been associated with Merkel cell carcinoma (MCC), pruritic and dyskeratotic dermatoses, respectively. Their presence in other types of skin conditions varies greatly depending on lesion type and population. OBJECTIVE: To analyse comparatively the presence of MCPyV and HPyV6 in nonmelanoma skin cancers and healthy skin. METHODS: The authors utilized qPCR techniques to quantify these pathogens in NMSC, premalignant diseases, and healthy skin of 87 patients. RESULTS: MCPyV was detected in over 40% of samples, while HPyV6 was in 9.6%. MCPyV load was higher in squamous cell carcinomas (SCC) compared to basal cell carcinomas (BCC) (p=0.016) and HPyV6 showed a higher percentage of infected cells in areas of low solar exposure as well as normal skin (p=0.012). A fair agreement (kappa=0.301) was found between MCPyV detection in lesions and their respective perilesional skin, indicating a random process of local dissemination of the virus. STUDY LIMITATIONS: The lack of a larger sampling of different lesion types and protein expression analyses limits the correlation findings. CONCLUSION: This is the first report of HPyV6 detection in the healthy skin of a Brazilian population, but the role of both polyomaviruses in NMSC has yet to be demonstrated.
Subject(s)
Carcinoma, Basal Cell , Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus , Skin Neoplasms , Humans , Carcinoma, Merkel Cell/pathology , DNA, Viral/analysis , DNA, Viral/metabolism , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Polyomavirus/genetics , Skin Neoplasms/pathologyABSTRACT
Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of a C-terminal truncated LTag. Protein kinase A (PKA) plays a fundamental role in a variety of biological processes, including transcription by phosphorylating and thereby regulating the activity of transcription factors. As MCPyV LTag has been shown to be phosphorylated and acts as a transcription factor for the viral early and late promoter, we investigated whether LTag can be phosphorylayted by PKA, and whether this affects the transcript activity of LTag. Using a phosphorylation prediction algorithm, serine 191, 203, and 265 were identified as putative phosphorylation sites for PKA. Mass spectrometry of in vitro PKA-phosphorylated peptides confirmed phosphorylation of S203 and S265, but not S191. Full-length LTag inhibited early and late promoter activity of MCPyV, whereas the truncated MKL2 LTag variant stimulated both promoters. Single non-phosphorylable, as well as phosphomimicking mutations did not alter the inhibitory effect of full-length LTag. However, the non-phosphorylable mutations abrogated transactivation of the MCPyV promoters by MKL2 LTag, whereas phosphomimicking substitutions restored the ability of MKL2 LTag to activate the promoters. Triple LTag and MKL2 LTag mutants had the same effect as the single mutants. Activation of the PKA signaling pathway did not enhance MCPyV promoter activity, nor did it affect LTag expression levels in MCPyV-positive Merkel cell carcinoma (MCC) cells. Our results show that phosphorylation of truncated LTag stimulates viral promoter activity, which may contribute to higher levels of the viral oncoproteins LTag and sTag. Interfering with PKA-induced LTag phosphorylation/activity may be a therapeutic strategy to treat MCPyV-positive MCC patients.
Subject(s)
Antigens, Polyomavirus Transforming , Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Carcinoma, Merkel Cell/metabolism , Carcinoma, Merkel Cell/virology , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Merkel cell polyomavirus/metabolism , Phosphorylation , Polyomavirus Infections/metabolism , Polyomavirus Infections/virology , Skin Neoplasms/metabolism , Skin Neoplasms/virology , Tumor Virus Infections/metabolism , Tumor Virus Infections/virology , Antigens, Polyomavirus Transforming/metabolism , Transcription, GeneticABSTRACT
Merkel cell polyomavirus (MCV) is a small DNA tumor virus that persists in human skin and causes Merkel cell carcinoma (MCC) in immunocompromised individuals. The multi-functional protein MCV small T (sT) activates viral DNA replication by stabilizing large T (LT) and promotes cell transformation through the LT stabilization domain (LTSD). Using MCVΔsT, a mutant MCV clone that ablates sT, we investigated the role of sT in MCV genome maintenance. sT was dispensable for initiation of viral DNA replication, but essential for maintenance of the MCV genome and activation of viral early and late gene expression for progression of the viral lifecycle. Furthermore, in phenotype rescue studies, exogenous sT activated viral DNA replication and mRNA expression in MCVΔsT through the LTSD. While exogenous LT expression, which mimics LT stabilization, increased viral DNA replication, it did not activate viral mRNA expression. After cataloging transcriptional regulator proteins by proximity-based MCV sT-host protein interaction analysis, we validated LTSD-dependent sT interaction with four transcriptional regulators: Cux1, c-Jun, BRD9, and CBP. Functional studies revealed Cux1 and c-Jun as negative regulators, and CBP and BRD9 as positive regulators of MCV transcription. CBP inhibitor A-485 suppressed sT-induced viral gene activation in replicating MCVΔsT and inhibited early gene expression in MCV-integrated MCC cells. These results suggest that sT promotes viral lifecycle progression by activating mRNA expression and capsid protein production through interaction with the transcriptional regulators. This activity is essential for MCV genome maintenance, suggesting a critical role of sT in MCV persistence and MCC carcinogenesis.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Humans , Merkel cell polyomavirus/metabolism , Antigens, Viral, Tumor/genetics , Antigens, Viral, Tumor/metabolism , Viral Transcription , DNA Replication , Virus Replication , DNA, Viral/genetics , DNA, Viral/metabolism , Transcription Factors/metabolism , Skin Neoplasms/pathology , Genome, Viral , RNA, Messenger/metabolism , Polyomavirus Infections/metabolismABSTRACT
Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Antigens, Viral, Tumor/genetics , Antigens, Viral, Tumor/metabolism , Carcinoma, Merkel Cell/genetics , Carcinoma, Merkel Cell/pathology , Epigenesis, Genetic , Humans , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Polyomavirus Infections/genetics , Skin Neoplasms/pathology , Ubiquitin-Specific Peptidase 7/metabolismABSTRACT
MCC (Merkel cell carcinoma) is an aggressive neuroendocrine cutaneous neoplasm. Integration of the Merkel cell polyomavirus (MCPyV) is observed in about 80% of the cases, while the remaining 20% are related to UV exposure. Both MCPyV-positive and -negative MCCs-albeit by different mechanisms-are associated with RB1 inactivation leading to overexpression of SOX2, a major contributor to MCC biology. Moreover, although controversial, loss of RB1 expression seems to be restricted to MCPyV-negative cases.The aim of the present study was to assess the performances of RB1 loss and SOX2 expression detected by immunohistochemistry to determine MCPyV status and to diagnose MCC, respectively.Overall, 196 MCC tumors, 233 non-neuroendocrine skin neoplasms and 70 extra-cutaneous neuroendocrine carcinomas (NEC) were included. SOX2 and RB1 expressions were assessed by immunohistochemistry in a tissue micro-array. Diagnostic performances were determined using the likelihood ratio (LHR).RB1 expression loss was evidenced in 27% of the MCC cases, 12% of non-neuroendocrine skin tumors and 63% of extra-cutaneous NEC. Importantly, among MCC cases, RB1 loss was detected in all MCPyV(-) MCCs, while MCPyV( +) cases were consistently RB1-positive (p < 0.001). SOX2 diffuse expression was observed in 92% of the MCC cases and almost never observed in non-neuroendocrine skin epithelial neoplasms (2%, p < 0.0001, LHR + = 59). Furthermore, SOX2 diffuse staining was more frequently observed in MCCs than in extra-cutaneous NECs (30%, p < 0.001, LHR + = 3.1).These results confirm RB1 as a robust predictor of MCC viral status and further suggest SOX2 to be a relevant diagnostic marker of MCC.
Subject(s)
Carcinoma, Merkel Cell , Merkel cell polyomavirus , Polyomavirus Infections , Skin Neoplasms , Tumor Virus Infections , Carcinoma, Merkel Cell/diagnosis , Carcinoma, Merkel Cell/pathology , Humans , Merkel cell polyomavirus/metabolism , Polyomavirus Infections/complications , Polyomavirus Infections/metabolism , Retinoblastoma Binding Proteins , SOXB1 Transcription Factors/metabolism , Skin Neoplasms/pathology , Tumor Virus Infections/complications , Ubiquitin-Protein LigasesABSTRACT
Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, ß-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.
Subject(s)
Merkel cell polyomavirus , Polyomavirus Infections , Polyomavirus , Tumor Virus Infections , Antigens, Viral, Tumor/genetics , Humans , Kinetics , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Polyomavirus/genetics , Polyomavirus/metabolism , Recombinases/metabolism , Virus Replication/geneticsABSTRACT
BACKGROUND: Merkel cell polyomavirus (MCPyV) has been studied in several malignant and nonmalignant tissues. However, only in Merkel cell carcinoma (MCC) has the connection to tumorigenesis been established. Previously, eccrine porocarcinoma samples were shown to express MCPyV in the majority of samples. We aimed to examine MCPyV in porocarcinoma and poroma samples using MCC as the reference material. METHODS: We analyzed 17 porocarcinoma and 50 poroma samples for the presence of MCPyV using LT antigen immunostaining and DNA detection methods. In addition, 180 MCC samples served as controls. RESULTS: MCPyV LT antigen immunostaining was detected in 10% of poroma and 18% of porocarcinoma samples; on the other hand, it was present in 65% of MCC samples. MCPyV DNA was detected in only 10% of poroma and porocarcinoma samples compared with 96% of MCC samples. The viral DNA copy number in all MCPyV DNA-positive MCCs was at least 25 times higher than that in porocarcinoma or poroma samples with the highest MCPyV DNA-to-PTPRG ratio. CONCLUSIONS: The low number of viral DNA copies in poroma and porocarcinoma samples, together with the negative LT expression of MCPyV DNA-positive tumors, indicates that MCPyV is simply a passenger virus rather than an oncogenic driver of porocarcinoma.
Subject(s)
Carcinoma, Merkel Cell , Eccrine Porocarcinoma , Merkel cell polyomavirus/metabolism , Polyomavirus Infections , Sweat Gland Neoplasms , Tumor Virus Infections , Adolescent , Adult , Aged , Aged, 80 and over , Carcinoma, Merkel Cell/metabolism , Carcinoma, Merkel Cell/pathology , Carcinoma, Merkel Cell/virology , Child , Eccrine Porocarcinoma/metabolism , Eccrine Porocarcinoma/pathology , Eccrine Porocarcinoma/virology , Female , Humans , Male , Middle Aged , Polyomavirus Infections/metabolism , Polyomavirus Infections/pathology , Polyomavirus Infections/virology , Sweat Gland Neoplasms/metabolism , Sweat Gland Neoplasms/pathology , Sweat Gland Neoplasms/virology , Tumor Virus Infections/metabolism , Tumor Virus Infections/pathology , Tumor Virus Infections/virologyABSTRACT
BACKGROUND: Approximately 15% of human cancers are attributed to viruses. Numerous studies have shown that high-risk human polyomaviruses (HR-HPV) and Merkel cell polyomavirus (MCPyV) are two human tumor viruses associated with anogenetal and oropharyngeal cancers, and with Merkel cell carcinoma, respectively. MCPyV has been found in HR-HPV positive anogenetal and oropharyngeal tumors, suggesting that MCPyV can act as a co-factor in HR-HPV induced oncogenesis. This prompted us to investigate whether the oncoproteins large T-antigen (LT) and small antigen (sT) of MCPyV could affect the transcriptional activity HPV16 and HPV18 and vice versa whether HPV16 and HPV18 E6 and E7 oncoproteins affected the expression of MCPyV LT and sT. Reciprocal stimulation of these viral oncoproteinscould enhance the oncogenic processes triggered by these tumor viruses. METHODS: Transient co-transfection studies using a luciferase reporter plasmid with the long control region of HPV16 or HPV18, or the early or late promoter of MCPyV and expression plasmids for LT and sT, or E6 and E7, respectively were performed in the HPV-negative cervical cancer cell line C33A, in the keratinocyte cell line HaCaT, and in the oral squamous cell carcinoma cell line HSC-3. Transfections were also performed with deletion mutants of all these promoters and with mutants of all four oncoproteins. Finally, the effect of E6 and E7 on LT and sT expression in the MCPyV-positive Merkel cell carcinoma cell line WaGa and the effect of LT and sT on the expression of E6 and E7 was monitored by Western blotting. RESULTS: LT and sT stimulated the transcriptional activity of the HPV16 and HPV18 LCR and v.v. E6 and E7 potentiated the MCPyV early and late promoter in all cell lines. Induction by E6 and E7 was p53- and pRb-independent, and transactivation by LT did not require DNA binding, nuclear localization and HSC70/pRb interaction, whereas sT stimulated the HPV16/18 LCR activity in a PP2A- and DnaJ-independent manner. CONCLUSIONS: These results indicate that the co-infection of MCPyV may act as a co-factor in the initiation and/or progression of HPV-induced cancers.
Subject(s)
Carcinoma, Merkel Cell , Carcinoma, Squamous Cell , Merkel cell polyomavirus , Mouth Neoplasms , Oncogene Proteins, Viral , Papillomavirus Infections , Carcinoma, Merkel Cell/virology , Carcinoma, Squamous Cell/virology , Cell Line, Tumor , HaCaT Cells , Human papillomavirus 16/genetics , Human papillomavirus 18 , Humans , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Mouth Neoplasms/virology , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins/genetics , Transcriptional ActivationABSTRACT
Merkel cell polyomavirus (MCPyV) large tumor (LT) antigen is a DNA binding protein essential for viral gene transcription and genome replication. MCPyV LT interacts with multiple E3 ligases in a phosphorylation-dependent manner, limiting its own viral replication by enhancing LT protein degradation, which is a unique mechanism for MCPyV latency. Thus, identifying LT ubiquitination sites is an important step toward understanding the biological role of these virus-host interactions that can potentially result in viral oncogenesis. The ubiquitin (Ub) attachment sites in LT were predicted by using Rapid UBIquitination (RUBI), a sequence-based ubiquitination web server. Using an immunoprecipitation approach, the lysine (Lys, K) 585 residue in LT is identified as the ubiquitin conjugation site. Lysine 585 is deleted from tumor-derived truncated LTs (tLTs), resulting in stable expression of tLTs present in cancers. Substitution of lysine 585 to arginine (Arg, R) increased LT protein stability, but impaired MCPyV origin replication, due to a loss of ATP hydrolysis activity. These findings uncover a never-before-identified ubiquitination site of LT and its importance not only in the regulation of protein turnover, but also in MCPyV genome replication.
Subject(s)
Antigens, Viral, Tumor/metabolism , Merkel cell polyomavirus/immunology , Adenosine Triphosphate/metabolism , HEK293 Cells , Humans , Merkel cell polyomavirus/metabolism , Protein Stability , Ubiquitination , Virus ReplicationABSTRACT
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin with two distinct etiologies. Clonal integration of Merkel cell polyomavirus DNA into the tumor genome with persistent expression of viral T antigens causes at least 60% of all MCC. UV damage leading to highly mutated genomes causes a nonviral form of MCC. Despite these distinct etiologies, both forms of MCC are similar in presentation, prognosis, and response to therapy. At least three oncogenic transcriptional programs feature prominently in both forms of MCC driven by the virus or by mutation. Both forms of MCC have a high proliferative growth rate with increased levels of cell cycle-dependent genes due to inactivation of the tumor suppressors RB and p53, a strong MYC signature due to MYCL activation by the virus or gene amplification, and an attenuated neuroendocrine differentiation program driven by the ATOH1 transcription factor.
Subject(s)
Carcinogenesis/genetics , Carcinoma, Merkel Cell/genetics , Skin Neoplasms/genetics , Carcinoma, Merkel Cell/pathology , Carcinoma, Merkel Cell/virology , Humans , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Mutation , Polyomavirus Infections/complications , Polyomavirus Infections/genetics , Polyomavirus Infections/pathology , Skin Neoplasms/pathology , Skin Neoplasms/virology , Tumor Virus Infections/complications , Tumor Virus Infections/genetics , Tumor Virus Infections/pathologyABSTRACT
Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of â¼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses.IMPORTANCE Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.
Subject(s)
Carcinoma, Merkel Cell/virology , Gene Expression Regulation, Viral , Merkel cell polyomavirus/genetics , MicroRNAs/genetics , RNA, Circular/genetics , RNA, Viral/genetics , Antigens, Viral, Tumor/genetics , Antigens, Viral, Tumor/metabolism , Humans , Merkel cell polyomavirus/metabolism , MicroRNAs/metabolism , RNA, Circular/metabolism , RNA, Viral/metabolism , Virus ReplicationABSTRACT
Merkel cell polyomavirus (MCV) is the only known human oncogenic virus in the polyomaviridae family and the etiological agent of most Merkel cell carcinomas (MCC). MCC is an aggressive and highly metastatic skin cancer with a propensity for recurrence and poor prognosis. Large tumor antigen (LT), is an essential oncoprotein for MCV transcription, viral replication, and cancer cell proliferation. MCV LT is a short-lived protein that encodes a unique domain: MCV LT unique regions (MURs). These domains consist of phosphorylation sites that interact with multiple E3 ligases, thus limiting LT expression and consequently, viral replication. In this study, we show that MURs are necessary for regulating LT stability via multiple E3 ligase interactions, resulting in cell growth arrest. While expression of wild-type MCV LT induced a decrease in cellular proliferation, deletion of the MUR domains resulted in increased LT stability and cell proliferation. Conversely, addition of MURs to SV40 LT propagated E3 ligase interactions, which in turn, reduced SV40 LT stability and decreased cell growth activity. Our results demonstrate that compared to other human polyomaviruses (HPyVs), MCV LT has evolved to acquire the MUR domains that are essential for MCV LT autoregulation, potentially leading to viral latency and MCC.
Subject(s)
Antigens, Viral, Tumor/metabolism , Cell Cycle/physiology , Merkel cell polyomavirus/metabolism , Protein Stability , Carcinoma, Merkel Cell/virology , Cell Proliferation , HEK293 Cells , Humans , Neoplasm Recurrence, Local , Polyomavirus , Polyomavirus Infections/virology , Skin Neoplasms/virology , Tumor Virus Infections/virology , Ubiquitin-Protein Ligases , Virus Latency/physiology , Virus ReplicationABSTRACT
Polyomavirus infection is widespread in the human population. This family of viruses normally maintains latent infection within the host cell but can cause a range of human pathologies, especially in immunocompromised individuals. Among several known pathogenic human polyomaviruses, JC polyomavirus (JCPyV) has the potential to cause the demyelinating disease progressive multifocal leukoencephalopathy (PML); BK polyomavirus (BKPyV) can cause nephropathy in kidney transplant recipients, and Merkel cell polyomavirus (MCPyV) is associated with a highly aggressive form of skin cancer, Merkel cell carcinoma (MCC). While the mechanisms by which these viruses give rise to the relevant diseases are not well understood, it is clear that the control of gene expression in each polyomavirus plays an important role in determining the infectious tropism of the virus as well as their potential to promote disease progression. In this review, we discuss the mechanisms governing the transcriptional regulation of these pathogenic human polyomaviruses in addition to the best-studied simian vacuolating virus 40 (SV40). We highlight the roles of viral cis-acting DNA elements, encoded proteins and miRNAs that control the viral gene expression. We will also underline the cellular transcription factors and epigenetic modifications that regulate the gene expression of these viruses.
Subject(s)
Gene Expression Regulation, Viral/genetics , Polyomavirus Infections/pathology , Polyomavirus/genetics , Polyomavirus/metabolism , Tumor Virus Infections/pathology , BK Virus/genetics , BK Virus/metabolism , Carcinoma, Merkel Cell/pathology , Carcinoma, Merkel Cell/virology , Humans , JC Virus/genetics , JC Virus/metabolism , Latent Infection/virology , Leukoencephalopathy, Progressive Multifocal/pathology , Leukoencephalopathy, Progressive Multifocal/virology , Merkel cell polyomavirus/genetics , Merkel cell polyomavirus/metabolism , Polyomavirus Infections/virology , Simian virus 40/genetics , Simian virus 40/metabolism , Tumor Virus Infections/virologyABSTRACT
Viral infection underlies a significant share of the global cancer burden. Merkel cell polyomavirus (MCPyV) is the newest member of the human oncogenic virus family. Its discovery over a decade ago marked the beginning of an exciting era in human tumor virology. Since then, significant evidence has emerged to support the etiologic role of MCPyV in Merkel cell carcinoma (MCC), an extremely lethal form of skin cancer. MCPyV infection is widespread in the general population. MCC diagnoses have tripled over the past 20 years, but effective treatments are currently lacking. In this review, we highlight recent discoveries that have shaped our understanding of MCPyV oncogenic mechanism and host cellular tropism, as well as the molecular events occurring in the viral infectious life cycle. These insights will guide future efforts in developing novel virus-targeted therapeutic strategies for treating the devastating human cancers associated with this new tumorigenic virus.
Subject(s)
Carcinogenesis , Merkel cell polyomavirus/genetics , Viral Tropism/genetics , Virus Replication/genetics , Carcinoma, Merkel Cell/pathology , Carcinoma, Merkel Cell/virology , Genes, Viral , Humans , Merkel cell polyomavirus/metabolism , Oncogenes , Polyomavirus Infections/complications , Polyomavirus Infections/virology , Tumor Virus Infections/virologyABSTRACT
Merkel cell polyomavirus (MCPyV) is a human double-stranded DNA tumor virus. MCPyV cell entry is unique among members of the polyomavirus family as it requires the engagement of two types of glycans, sialylated oligosaccharides and sulfated glycosaminoglycans (GAGs). Here, we present crystallographic and cryo-electron microscopic structures of the icosahedral MCPyV capsid and analysis of its glycan interactions via nuclear magnetic resonance (NMR) spectroscopy. While sialic acid binding is specific for α2-3-linked sialic acid and mediated by the exposed apical loops of the major capsid protein VP1, a broad range of GAG oligosaccharides bind to recessed regions between VP1 capsomers. Individual VP1 capsomers are tethered to one another by an extensive disulfide network that differs in architecture from previously described interactions for other PyVs. An unusual C-terminal extension in MCPyV VP1 projects from the recessed capsid regions. Mutagenesis experiments show that this extension is dispensable for receptor interactions.IMPORTANCE The MCPyV genome was found to be clonally integrated in 80% of cases of Merkel cell carcinoma (MCC), a rare but aggressive form of human skin cancer, strongly suggesting that this virus is tumorigenic. In the metastasizing state, the course of the disease is often fatal, especially in immunocompromised individuals, as reflected by the high mortality rate of 33 to 46% and the low 5-year survival rate (<45%). The high seroprevalence of about 60% makes MCPyV a serious health care burden and illustrates the need for targeted treatments. In this study, we present the first high-resolution structural data for this human tumor virus and demonstrate that the full capsid is required for the essential interaction with its GAG receptor(s). Together, these data can be used as a basis for future strategies in drug development.