Large T Antigen Research Articles

Oncogene-specific T cells fail to eradicate lymphoma-initiating B cells in mice

AbstractTo date, little is known about the interaction between (pre-)malignant B cells and T cells. We generated transgenic mice that allow B cell-specific induction of the oncogene SV40 large T-antigen (TAg) to analyze the role of oncogene-specific T cells during sporadic B-cell lymphoma development. Constitutive TAg expression in CD19-Cre × LoxP-Tag mice resulted in TAg-tolerant CD8+ T cells and development of B-cell lymphomas. In contrast, CD19-CreERT2 × LoxP-Tag mice retained TAg-competent CD8+ T cells at time of oncogene induction and TAg expression in few B cells of adult mice resulted in exceptionally rare lymphoma formation late in life. Increased lymphoma incidence in the absence of TAg-specific T cells suggested T cell-mediated inhibition of lymphoma progression. However, TAg-initiated B cells were not eliminated by T cells and detected long term. Our results demonstrate a failure of the immune system to eradicate lymphoma-initiating B cells, retaining the risk of lymphoma development.

BK Polyomavirus MicroRNA Levels in Exosomes Are Modulated by Non-Coding Control Region Activity and Down-Regulate Viral Replication When Delivered to Non-Infected Cells Prior to Infection.

In immunosuppressed patients, BKPyV-variants emerge carrying rearranged non-coding control-regions (rr-NCCRs) that increase early viral gene region (EVGR) expression and replication capacity. BKPyV also encodes microRNAs, which have been reported to downregulate EVGR-encoded large T-antigen transcripts, to decrease viral replication in infected cells and to be secreted in exosomes. To investigate the interplay of NCCR and microRNAs, we compared archetype- and rr-NCCR-BKPyV infection in cell culture. We found that laboratory and clinical rr-NCCR-BKPyV-strains show higher replication rates but significantly lower microRNA levels than archetype virus intracellularly and in exosomes. To investigate whether rr-NCCR or increased EVGR activity modulated microRNA levels, we examined the (sp1-4)NCCR-BKPyV, which has an archetype NCCR-architecture but shows increased EVGR expression due to point mutations inactivating one Sp1 binding site. We found that microRNA levels following (sp1-4)NCCR-BKPyV infection were as low as in rr-NCCR-variants. Thus, NCCR rearrangements are not required for lower miRNA levels. Accordingly, Sp1 siRNA knock-down decreased microRNA levels in archetype BKPyV infection but had no effect on (sp1-4)- or rr-NCCR-BKPyV. However, rr-NCCR-BKPyV replication was downregulated by exosome preparations carrying BKPyV-microRNA prior to infection. To explore the potential relevance in humans, urine samples from 12 natalizumab-treated multiple sclerosis patients were analysed. In 7 patients, rr-NCCR-BKPyV were detected showing high urine BKPyV loads but low microRNAs levels, whereas the opposite was seen in 5 patients with archetype BKPyV. We discuss the results in a dynamic model of BKPyV replication according to NCCR activity and exosome regulation, which integrates immune selection pressure, spread to new host cells and rr-NCCR emergence.

New polyomavirus species identified in nutria, Myocastor coypus polyomavirus 1.

A novel polyomavirus (PyVs) comprising 5,422 bp was identified by high-throughput sequencing (HTS) in pooled organs of nutria (Myocastor coypus). The new genome displays the archetypal organization of PyVs, which includes open reading frames for the regulatory proteins small T antigen (sTAg) and large T antigen (LTAg), as well as for the capsid proteins VP1, VP2 and VP3. Based on the International Committee on Taxonomy of Viruses (ICTV) Polyomaviridae Study Group criteria, this genome comprises a new PyVs species for the Alphapolyomavirus genus and is putatively named "Myocastor coypus Polyomavirus 1" . The complete genome sequence of this Myocastor coypus Polyomavirus 1 (McPyV1) isolate is publically available under the GenBank accession no. MH182627.

CCL17/TARC and CCR4 expression in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare, highly aggressive neuroendocrine skin cancer. In more than 80% of the cases, Merkel cell polyomavirus (MCPyV) is a causal factor. The oncogenic potential of MCPyV is mediated through its viral oncoproteins, large T antigen (LT) and small t antigen (sT). To investigate the role of cytokines in MCC, a PCR array analysis for genes encoding inflammatory cytokines and receptors was performed on MCPyV-negative and MCPyV-positive MCC cell lines, respectively. We detected an increased expression of CCL17/TARC in the MCPyV-positive MKL2 cell line compared to the MCPyV-negative MCC13 cell line. Transfection studies in MCC13 cells with LT expression plasmid, and a luciferase reporter plasmid containing the CCL17/TARC promoter, exhibited stimulated promoter activity. Interestingly, the ectopic expression of CCL17/TARC upregulated MCPyV early and late promoter activities in MCC13 cells. Furthermore, recombinant CCL17/TARC activated both the mitogen-activated protein kinase and the NF-κB pathways. Finally, immunohistochemical staining on human MCC tissues showed a strong staining of CCL17/TARC and its receptor CCR4 in both LT-positive and -negative MCC. Taken together, CCL17/TARC and CCR4 may be a potential target in MCC therapy providing MCC patients with a better overall survival outcome.

The two-faced nature of BK polyomavirus: lytic infection or non-lytic large-T-positive carcinoma.

In immunocompromised patients, reactivation of latent BK polyomavirus (BKPyV) can cause disease with lytic infections of the kidneys and the lower urinary tract. Emerging evidence also links BKPyV to oncogenesis and high‐grade intrarenal and transitional cell carcinomas. These neoplasms strongly express polyomavirus large‐T antigen as a defining feature; that is, they are ‘large‐T‐positive carcinomas’. Such neoplasms arise in immunocompromised patients, typically in renal allograft recipients, and preferentially in tissues harbouring latent BKPyV. In recent articles in this journal, it was shown that tumour cells harbour replication‐incompetent clonal BKPyV. The virus can be truncated and randomly integrated into the genome, and/or it can be mutated in an episomal state. Truncation and/or deletions in the BKPyV non‐coding control region can hamper late viral gene expression, replication, and cell lysis, while facilitating overexpression of early genes, including that encoding large‐T. Biologically active fusion proteins or alterations in human tumour suppressor or promoter function have not been described so far, making uncontrolled large‐T gene expression in non‐lytically infected cells a prime suspect for neoplastic transformation. Current concepts of BKPyV‐induced disease, including recent reports from this journal, are discussed, and evolving paradigms of BKPyV‐associated oncogenesis are highlighted. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

PO-021 Merkel cell polyomavirus-encoded miRNA subverts the host cell immune response

IntroductionVirus-encoded miRNAs are emerging as key regulators of persistent infection and host-cell immune evasion. Merkel cell polyomavirus (MCPyV), the predominant aetiological agent of the rare but deadly skin cancer, Merkel cell carcinoma (MCC), encodes a single miRNA, MCV-miR-M1, which targets the oncogenic MCPyV large T antigen (LT). MCV-miR-M1 has previously been shown to play an important role in establishment of long-term infection, however, the underlying mechanism is not fully understood. A key unanswered question is whether, in addition to auto-regulating LT, MCV-miR-M1 also targets cellular transcripts to orchestrate an environment conducive for persistent infection, which is a prerequisite for oncovirus-mediated transformation.Material and methodsWe adopted an unbiased RNA-Seq-based approach to identify differentially-expressed (DE) targets of MCV-miR-M1 in cell expressing either the 5 p or 3 p arm of the miRNA. Bioinformatic analysis of DE transcripts was carried out using DAVID. DE targets identified by RNA-seq were confirmed via RT-qPCR in a variety of cell lines expressing either MCV-miR-M1 mimics or an MCPyV replication system based on synthetic MCPyV genomes. MCV-miR-M1 interaction with full length 3’UTR regions of DE genes was analysed via dual-luciferase assay to identify direct MCV-miR-M1 targets. IL-8 secretion was determined by ELISA and neutrophil chemotaxis assessed via transwell migration assays.Results and discussionsBioinformatic analysis of DE transcripts revealed numerous genes implicated in immune evasion. Subsequent target validation led to the identification of the innate immunity protein, SP100, as a direct target of MCV-miR-M1. Intriguingly, MCV-miR-M1-mediated modulation of SP100 was associated with a significant decrease in IL-8 secretion in cells harbouring synthetic MCPyV genomes. Moreover, the observed MCV-miR-M1-mediated reduction in IL-8 secretion was significantly increased by ectopic expression of MCV-miR-M1-resistant SP100. The attenuation of IL-8 secretion resulted in a significant reduction in neutrophil chemotaxis towards cells harbouring replicative MCPyV.ConclusionThese data represent the fist global analysis of MCPyV miRNA targets. Based on our observations, we propose that MCV-miR-M1 targets key immune response regulators in the host cell to help facilitate persistent infection, in part by attenuating neutrophil chemotaxis towards infected cells.

Abstract 4084: Identifying characteristics of orthotopic HCC mouse models to predict response to immunotherapy

Abstract Introduction Immunotherapy is a promising treatment strategy for hepatocellular carcinoma (HCC). A phase III trial for advanced HCC shows favorable results for nivolumab compared to sorafenib. In order to evaluate the outcome of different therapeutic strategies, we compared two HCC mouse models. The orthotopic transplanted HCC fragment Hep-55.1c model and the inducible HCC mouse model (iAST) were extensively characterized ex vivo and in vivo in response to immunotherapies, and were compared to the clinical situation of HCC patients. Experimental Procedures Fragments of mouse Hep-55.1c tumors were implanted in the left lateral liver lobe of C57/Bl6 mice and tumor growth kinetic was monitored by µ-CT. HCC tumor growth in iAST mice was induced via intravenous injection of the adenovirus expressing Cre recombinase. iAST mice express a loxP flanked stop cassette and the SV40 large T-antigen under control of a hepatocyte-specific albumin promoter. Both tumor models were characterized with respect to immune infiltration, cytokine release, somatic mutational load and histopathological characteristics. Mice were treated using different immunotherapeutic agents in monotherapy or in combination such as anti-PD-1, anti-CTLA-4 or the TLR7/8 agonist R848. Human HCC samples were analyzed for mutational load by sequencing, while tumor architecture and immune infiltrate were analyzed by histology. Results Hep-55.1c tumors showed high stroma content accompanied by a low and disorganized vasculature whereas multinodular iAST tumors were highly vascularized lacking stroma content. Furthermore, a high mutational load and a strong immune cell infiltrate including cytotoxic T cells, NK cells and myeloid cells were found in Hep-55.1c tumors. The iAST tumors were characterized by a relatively low immune infiltrate and a small number of mutations. Comparison of these baseline results with data obtained from immune-histopathological analysis and sequencing of human HCC samples confirmed this differentiated picture in the clinics. Following treatment, iAST mice showed no response to immune checkpoint monotherapies and only a marginal number of reactive T cells was found within the tumor. In contrast, tyrosine kinase inhibitor sorafenib led to tumor growth inhibition in iAST model. Treatment of mice bearing Hep-55.1c tumors with immunotherapy e.g. anti-PD-1 showed good response. Combination of anti-PD-1 with R848 increased therapeutic efficacy compared to monotherapies. Conclusions In this study, we have established two orthotopic HCC mouse models that reflect the diverse clinical situation of human HCC. Our findings demonstrate that the composition of the tumor microenvironment has a tremendous influence on the outcome of therapeutic strategies for HCC. Hence, thorough characterization of tumor patients is indispensable in order to predict response to immunotherapy. Citation Format: Carina Hage, Sabine Hoves, Mailin Ashoff, Leanne Strauss, Mario Perro, Frank Herting, Fabian Kiessling, Thomas Pöschinger. Identifying characteristics of orthotopic HCC mouse models to predict response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4084.

PO-024 SETD1A regulates cell proliferation by modulating miR205 in triple negative breast cancers (TNBCS)

IntroductionVirus-encoded miRNAs are emerging as key regulators of persistent infection and host-cell immune evasion. Merkel cell polyomavirus (MCPyV), the predominant aetiological agent of the rare but deadly skin cancer, Merkel cell carcinoma (MCC), encodes a single miRNA, MCV-miR-M1, which targets the oncogenic MCPyV large T antigen (LT). MCV-miR-M1 has previously been shown to play an important role in establishment of long-term infection, however, the underlying mechanism is not fully understood. A key unanswered question is whether, in addition to auto-regulating LT, MCV-miR-M1 also targets cellular transcripts to orchestrate an environment conducive for persistent infection, which is a prerequisite for oncovirus-mediated transformation.Material and methodsWe adopted an unbiased RNA-Seq-based approach to identify differentially-expressed (DE) targets of MCV-miR-M1 in cell expressing either the 5 p or 3 p arm of the miRNA. Bioinformatic analysis of DE transcripts was carried out using DAVID. DE targets identified by RNA-seq were confirmed via RT-qPCR in a variety of cell lines expressing either MCV-miR-M1 mimics or an MCPyV replication system based on synthetic MCPyV genomes. MCV-miR-M1 interaction with full length 3’UTR regions of DE genes was analysed via dual-luciferase assay to identify direct MCV-miR-M1 targets. IL-8 secretion was determined by ELISA and neutrophil chemotaxis assessed via transwell migration assays.Results and discussionsBioinformatic analysis of DE transcripts revealed numerous genes implicated in immune evasion. Subsequent target validation led to the identification of the innate immunity protein, SP100, as a direct target of MCV-miR-M1. Intriguingly, MCV-miR-M1-mediated modulation of SP100 was associated with a significant decrease in IL-8 secretion in cells harbouring synthetic MCPyV genomes. Moreover, the observed MCV-miR-M1-mediated reduction in IL-8 secretion was significantly increased by ectopic expression of MCV-miR-M1-resistant SP100. The attenuation of IL-8 secretion resulted in a significant reduction in neutrophil chemotaxis towards cells harbouring replicative MCPyV.ConclusionThese data represent the fist global analysis of MCPyV miRNA targets. Based on our observations, we propose that MCV-miR-M1 targets key immune response regulators in the host cell to help facilitate persistent infection, in part by attenuating neutrophil chemotaxis towards infected cells.

Efficient generation of MCC oncoprotein-specific CD4+ T cells for potential adoptive immunotherapy

Abstract Merkel Cell Carcinoma (MCC) is an aggressive skin tumor caused by Merkel Cell Polyomavirus (MCV) in ~80% of cases that responds poorly to standard chemotherapies. Virus positive tumors have a low mutational burden, and are driven by alternatively spliced T-antigens (TAG), small T (sT) and truncated large T antigen (LTT). These intracellular proteins are constitutively expressed in MCC tumors and are required for tumor survival. We hypothesized that MCV TAG can be targeted by cytotoxic T lymphocytes for potential use as an adoptive T cell immunotherapy to treat MCC. Peripheral blood T cells were stimulated with autologous dendritic cells pulsed with overlapping peptide libraries derived from MCV sT and LTT in the presence of a pro-inflammatory cytokine cocktail (IL-1β, IL-6, IL-7, IL-15, IL-21, IL-23, and TGFβ). This culturing method increases yields by 6 fold of antigen-specific T cells that produce TNFα and IFNγ, from both MCC subjects and healthy donors. These expanded TAG-specific cells are predominately CD4+ cells of a memory phenotype that expresses low levels of PD-1, but no expression of IL-17A. Growing evidence suggests that CD4+ Th1 and Th17 cells can have potent anti-tumor activities, especially against MHC class II positive tumors. Three virus-positive and two virus-negative MCC cell lines were tested for HLA-DR expression by flow cytometry. We found that MCC cell lines, both virus positive and virus negative, can be induced to express HLA-DR in a dose-dependent fashion in response to IFNγ in vitro. These results suggest that MCC virus positive tumors can likely be directly recognized and destroyed by adoptively transferred Th1 or Th17 CD4+ T cells and offers a promising avenue for adoptive T cell immunotherapy as a MCC treatment.

Cross-Talk between Myeloid-Derived Suppressor Cells and Mast Cells Mediates Tumor-Specific Immunosuppression in Prostate Cancer.

Immunotherapy, including the use of checkpoint inhibitors, is a potent therapeutic approach for some cancers, but has limited success with prostate tumors, in which immune suppression is instigated by the tumor. The immunosuppressive capacity of mast cells, which promote adenocarcinoma development in the prostate, prompted our investigation on whether mast cells promote tolerance to SV40 Large-T antigen, the transforming oncogene in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. The incidence of adenocarcinoma was reduced in the offspring of a cross between TRAMP mice and mast cell-deficient KitWsh mice. TRAMP mice are tolerant to the SV40 Large T antigen, which is otherwise immunogenic in normal syngeneic B6 mice. Genetic ablation of mast cells in TRAMP mice restored their ability to mount a tumor-specific cytotoxic T-cell response. In KitWsh-TRAMP mice, the restored T-cell immunity correlated with the reduced activity of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC), along with their reduced expression of Arg1, Nos2, and Stat3 Having found that CD40L-expressing mast cells can interact in vivo with CD40-expressing PMN-MDSC, we then determined that only KitWsh-TRAMP mice reconstituted with mast cells expressing CD40L could restore PMN-MDSCs suppressive functions, T-cell unresponsiveness and adenocarcinoma development. Thus, mast cells have an immunoregulatory effect on PMN-MDSCs activity through CD40L-CD40 interaction, favoring immunosuppression and tumor onset. In prostate cancer patients, in silico analyses correlated poor clinical outcomes with high expression of genes related to mast cells and PMN-MDSCs. Cancer Immunol Res; 6(5); 552-65. ©2018 AACR.

469 Usefulness of ulceration and hyperkeratosis as clinical predictors of merkel cell polyomavirus-negative and combined merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma, typically associated with Merkel cell polyomavirus (MCPyV). Combined MCC is occurred with non-neuroendocrine components, particularly squamous cell carcinoma, and thought to represent a distinct disease process from pure MCC, with absent MCPyV. Combined MCC may be occasionally difficult to distinguish histologically from pure MCC, and the diagnosis of combined MCC is not highly-reproducible based only on histologic assessment. This study analyzed the predictive clinical factors for MCPyV status and its morphological variants. We aimed to evaluate the significance of clinical parameters of ulceration/hyperkeratosis as potential novel predictors for MCPyV-negative MCC patients. In our study, 20 MCC specimens from 20 patients were examined. Patients were divided into two groups based on MCPyV status and morphological subsets among those diagnosed with MCC: MCPyV- positive/negative MCCs and pure/combined MCCs. The clinicopathological variants that predict the presence of MCPyV-negative MCC and combined MCC were evaluated. Of the 20 patients studied, 10 cases (50%) were not immunohistochemically identified as showing MCPyV using the MCPyV large T-antigen antibody (CM2B4). The presence of ulceration/hyperkeratosis was a predictive factor for detection of MCPyV-negative and combined MCC, being observed in 80%/50% of cases in which MCPyV was not identified. Among 10 MCPyV-positive MCCs, absence of ulceration and hyperkeratosis was found in 10 cases demonstrating smooth surface. Clinically, presence of ulceration/hyperkeratosis is a predictive factor for diagnosing MCPyV-negative MCC and combined MCC. Cases presenting with clinical evidence of ulceration and hyperkeratosis possibly suggest the presence of MCPyV-negative and combined MCC.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Establishment and characterization of pygmy killer whale (Feresa attenuata) dermal fibroblast cell line.

The pygmy killer whale (Feresa attenuata) (PKW) is a tropical and subtropical marine mammal commonly found in the Atlantic, Indian and Pacific oceans. Since the PKWs live in offshore protected territories, they are rarely seen onshore. Hence, PKW are one of the most poorly understood oceanic species of odontocetes. The dermal tissue comes primarily from stranding events that occur along the coast of the Shantou, Guangdong, China. The sampled tissues were immediately processed and attached on collagen-coated 6-well tissue culture plate. The complete medium (DMEM and Ham’s F12, fetal bovine serum, antibiotic and essential amino acids) was added to the culture plates. The primary culture (PKW-LWH) cells were verified as fibroblast by vimentin and karyotype analyses, which revealed 42 autosomes and two sex chromosomes X and Y. Following transfection of PKW-LWH cells with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. Using RT-PCR, western blot, immunofluorescence analysis and SV40 large T-antigen stability was confirmed. The cell proliferation rate of the fibroblast cells, PKW-LWHT was faster than the primary cells PKW-LWH with the doubling time 68.9h and 14.4h, respectively. In this study, we established PKW dermal fibroblast cell line for the first time, providing a unique opportunity for in vitro studies on the effects of environmental pollutants and pathogens that could be determined in PKW and/or Cetaceans.

Co-infection with human polyomavirus BK enhances gene expression and replication of human adenovirus

Immunocompromised patients are susceptible to multiple viral infections. Relevant interactions between co-infecting viruses might result from viral regulatory genes which trans-activate or repress the expression of host cell genes as well as the genes of any co-infecting virus. The aim of the current study was to show that the replication of human adenovirus 5 is enhanced by co-infection with BK polyomavirus and is associated with increased expression of proteins including early region 4 open reading frame 1 and both the large tumor antigen and small tumor antigen. Clinical samples of whole blood and urine from 156 hematopoietic stem cell transplant recipients were tested. We also inoculated adenocarcinomic human alveolar basal epithelial cells with both human adenovirus 5 and BK polyomavirus to evaluate if co-infection of viruses affected their replication. Data showed that adenovirus load was significantly higher in the plasma (mean 7.5 x 103 ± 8.5 x 102 copies/ml) and urine (mean 1.9 x 103 ± 8.0 x 102 copies/ml) of samples from patients with co-infections, in comparison to samples from patients with isolated adenovirus infection. In vitro co-infection led to an increased (8.6 times) expression of the adenovirus early region 4 open reading frame gene 48 hours post-inoculation. The expression of the early region 4 open reading frame gene positively correlated with the expression of BK polyomavirus large tumor antigen (r = 0.90, p < 0.0001) and small tumor antigen (r = 0.83, p < 0.001) genes. The enhanced expression of the early region 4 open reading frame gene due to co-infection with BK polyomavirus was associated with enhanced adenovirus, but not BK polyomavirus, replication. The current study provides evidence that co-infection of adenovirus and BK polyomavirus contributes to enhanced adenovirus replication. Data obtained from this study may have significant importance in the clinical setting.

Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements.

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r, 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific.IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

The Human Polyomavirus Middle and Alternative T-Antigens; Thoughts on Roles and Relevance to Cancer

Approximately 15–20% of human cancer is related to infection, which renders them potentially preventable by antimicrobial or antiviral therapy. Human polyomaviruses (PyVs) are relevant in this regard, as illustrated by the involvement of Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma. The polyomavirus Small and Large tumor antigen (ST and LT) have been extensively studied with respect to their role in oncogenesis. Recently it was shown that a number of human PyVs, including MCPyV and the trichodysplasia spinulosa polyomavirus (TSPyV), express additional T-antigens called Middle T (MT) and alternative T (ALT). ALT is encoded by ORF5, also known as the alternative T open reading frame (ALTO), which also encodes the second exon of MT, and overlaps out-of-frame with the second exon of LT. Previously, MT was considered unique for oncogenic rodent polyomaviruses, and ALT was still unknown. In this mini-review, we want to point out there are important reasons to explore the involvement of MT and ALT in human cellular transformation. First, just like their rodent equivalents, MT and ALT probably disrupt cellular pathways that control signaling and proliferation. Second, expression of the MT and ALT-encoding ORF5/ALTO characterizes a monophyletic polyomavirus clade that includes human and animal PyVs with known oncogenic potential. And third, ORF5/ALTO is subject to strong positive selection aimed specifically at a short linear motif within MT and ALT that overlaps completely with the RB-binding motif in LT. The latter suggests tight interplay between these T-antigens with possible consequences for cell transformation.

The cellular chloride channels CLIC1 and CLIC4 contribute to virus-mediated cell motility

Ion channels regulate many aspects of cell physiology, including cell proliferation, motility, and migration, and aberrant expression and activity of ion channels is associated with various stages of tumor development, with K+ and Cl− channels now being considered the most active during tumorigenesis. Accordingly, emerging in vitro and preclinical studies have revealed that pharmacological manipulation of ion channel activity offers protection against several cancers. Merkel cell polyomavirus (MCPyV) is a major cause of Merkel cell carcinoma (MCC), primarily because of the expression of two early regulatory proteins termed small and large tumor antigens (ST and LT, respectively). Several molecular mechanisms have been attributed to MCPyV-mediated cancer formation but, thus far, no studies have investigated any potential link to cellular ion channels. Here we demonstrate that Cl− channel modulation can reduce MCPyV ST-induced cell motility and invasiveness. Proteomic analysis revealed that MCPyV ST up-regulates two Cl− channels, CLIC1 and CLIC4, which when silenced, inhibit MCPyV ST-induced motility and invasiveness, implicating their function as critical to MCPyV-induced metastatic processes. Consistent with these data, we confirmed that CLIC1 and CLIC4 are up-regulated in primary MCPyV-positive MCC patient samples. We therefore, for the first time, implicate cellular ion channels as a key host cell factor contributing to virus-mediated cellular transformation. Given the intense interest in ion channel modulating drugs for human disease. This highlights CLIC1 and CLIC4 activity as potential targets for MCPyV-induced MCC.

Simian Virus 40 Large T Antigen as a Model to Test the Efficacy of Flouroquinolones against Viral Helicases.

Simian virus 40 large T-antigen (SV40 LT-Ag) is a 708 amino acid nuclear phosphoprotein. Among many functions of LT-Ag is its ability to perform as an ATPase-helicase, catalyzing the unwinding of viral genome during replication. The LT-Ag has been employed in the studies of helicase structure and function, and has served as a model helicase for the screening of antiviral drugs that target viral helicase. In this study, using in vitro enzyme assays and in silico computer modeling, we screened a batch of 18 fluoroquinolones to assess their potential as antivirals by virtue of their inhibition of the LT-Ag helicase. We found all fluoroquinolones to be inhibitory to the helicase activity of LT-Ag. In our docking analysis, most of these tested drugs showed similarity in their interactions with LT-Ag. Our study shows the potential of fluoroquinolones as antiviral drugs and of SV40 LT-Ag as a model protein for screening drugs against viral helicases.

Viral microRNA effects on persistent infection of human lymphoid cells by polyomavirus SV40.

BackgroundPolyomaviruses, including simian virus 40 (SV40), display evidence of lymphotropic properties. This study analyzed the nature of SV40–human lymphocyte interactions in established cell lines and in primary lymphocytes. The effects of viral microRNA and the structure of the viral regulatory region on SV40 persistence were examined.ResultsSV40 DNA was maintained in infected B cell and myeloid cell lines during cell growth for at least 28 days. Limiting dilution analysis showed that low amounts of SV40 DNA (~2 copies per cell) were retained over time. Infected B cells remained viable and able to proliferate. Genome copies of the SV40 microRNA-null mutant persisted at higher levels than the DNA of wild-type viruses. Complex viral regulatory regions produced modestly higher DNA levels than simple regulatory regions. Viral large T-antigen protein was detected at low frequency and at low levels in infected B cells. Following infection of primary lymphocytes, SV40 DNA was detected in CD19+ B cells and CD14+ monocytes, but not in CD3+ T cells. Rescue attempts using either lysates of SV40-infected B lymphocytes, coculture of live cells, or infectious center assays all showed that replication-competent SV40 could be recovered on rare occasions. SV40 infections altered the expression of several B cell surface markers, with more pronounced changes following infections with the microRNA-null mutant.ConclusionThese findings indicate that SV40 can establish persistent infections in human B lymphocytes. The cells retain low copy numbers of viral DNA; the infections are nonproductive and noncytolytic but can occasionally produce infectious virus. SV40 microRNA negatively regulates the degree of viral effects on B cells.SignificanceLymphocytes may serve as viral reservoirs and may function to disseminate polyomaviruses to different tissues in a host. To our knowledge, this report is the first extensive analysis of viral microRNA effects on SV40 infection of human lymphocytes.

Determination of EPAC2 function using EPAC2 null Min6 sublines generated through CRISPR-Cas9 technology.

Min6 cells, a mouse β cell line derived from transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells, are commonly utilized as an invitro cellular model for investigating targets involved in insulin secretion. Epac2, an exchange protein that can be directly activated by cyclic AMP (cAMP), is critical for pharmacologic stimuli-induced insulin secretion and has been hypothesized to be a direct target of sulfonylurea. Previous loss of function studies only specifically knocked out EPAC2 isoform A, leaving the other two isoforms intact. In this study, we investigated the function of EPAC2 in Min6 cells by generating EPAC2 knock-out sublines using CRISPR-Cas9 technology, by removing all three isoforms of EPAC2. Our results indicate that Min6 cells can be successfully cloned from a single cell after electroporation with plasmids expressing EPAC2 specific guide RNA, Cas9 and GFP, followed by sorting for GFP expressing single cells. Two clones were found to have a single nucleotide deletion in targeted site of EPAC2 gene by sequencing, therefore creating a frame shift in exon 13. The EPAC2 null clones have an unexpectedly increased secretion of insulin at basal level and an elevated total intracellular insulin content. However, EPAC2 deficiency impaires glucose and sulfonylurea induced insulin secretion without affecting sulfonylurea binding to cells. Potassium chloride induced insulin secretion remains intact. Interestingly, cAMP levels remained unchanged in EPAC2 null cells during these processes. To understand the global function of EPAC2, RNA Seq study was performed, which reveals that EPAC2 deficiency affects expression of multiple previously unrecognized genes, suggesting that EPAC2 can function through multiple pathways in addition to being a cAMP sensor.