The relation of NCCR variations and host transcription factors gene regulation in BK polyomavirus infected kidney transplant patients.

BACKGROUND: BK polyomavirus (BKPyV) infection in immunocompromised patients can led to polyomavirus-associated nephropathy (BKPyVAN) especially after kidney transplantation. The polyomavirus genome contains enhancer elements that are important transcription activators. In this study, the association between viral and host gene expression and NCCR variations was evaluated in kidney transplant recipients (KTRs) with BKPyV active, and BKPyV in-active infection. METHODS AND RESULTS: Blood samples were collected from selected KTRs who divided to patients with active and in-active BKPyV infection. Transcriptional control region (TCR) anatomy was compared to the genomic sequence of archetype BKPyV strain WW using nested PCR method and sequencing. The expression level of some transcription factor genes was evaluated using in-house Real-time PCR (SYBR Green) technique. Most changes were observed after TCR anatomy detection in the Q and P blocks. The expression level of VP1 and LT-Ag viral genes were significantly higher in patients with active infection compared with non-infected ones. Transcription factor genes SP1, NF1, SMAD, NFκB, P53, PEA3, ETS1, AP2, NFAT and AP1 were significantly higher in BKPyV active group in comparison in-active and control groups. The analyses revealed that viral load level and mutations frequency has significant correlation. CONCLUSIONS: Based on the results, increasing of NCCR variations were associated with higher viral load of BKPyV especially in Q block. Host transcriptional factors and viral genes all had higher express level in active BKPyV patients versus no in-active ones. Detection of the relation between NCCR variation and BKPyV severity in KTRs need to be confirmed in further complicated studies.

Neurocan expression associates with better survival and viral positivity in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that is frequently divided into Merkel cell polyomavirus negative and positive tumors due their distinct genomic and transcriptomic profiles, and disease outcomes. Although some prognostic factors in MCC are known, tumorigenic pathways, which that explain outcome differences in MCC are not fully understood. We investigated transcriptomes of 110 tissue samples of a formalin-fixed, paraffin-embedded MCC series by RNA sequencing to identify genes showing a bimodal expression pattern and predicting outcome in cancer and that potentially could play a role in tumorigenesis. We discovered 19 genes among which IGHM, IGKC, NCAN, OTOF, and USH2A were associated also with overall survival (all p-values < 0.05). From these genes, NCAN (neurocan) expression was detected in all 144 MCC samples by immunohistochemistry. Increased NCAN expression was associated with presence of Merkel cell polyomavirus DNA (p = 0.001) and viral large T antigen expression in tumor tissue (p = 0.004) and with improved MCC-specific survival (p = 0.027) and overall survival (p = 0.034). We conclude that NCAN expression is common in MCC, and further studies are warranted to investigate its role in MCC tumorigenesis.

Expression of Genes Associated With Epithelial-Mesenchymal Transition in Merkel Cell Polyomavirus-Negative Merkel Cell Carcinoma.

Two accepted possible pathways for Merkel cell carcinoma (MCC) pathogenesis include the clonal integration of the Merkel cell polyomavirus (MCPyV) into the neoplastic cells and by UV irradiation. We hypothesize that, in UV etiology, the expression of genes associated with epithelial-mesenchymal transition (EMT) would be higher in MCPyV-negative MCCs. We compared RNA expression in 16 MCPyV-negative with that in 14 MCPyV-positive MCCs in 30 patients using NanoString panel of 760 gene targets as an exploratory method. Subsequently, we confirmed the findings with a publicly available RNA sequencing data set. The NanoString method showed that 29 of 760 genes exhibited significant deregulation. Ten genes (CD44, COL6A3, COL11A1, CXCL8, INHBA, MMP1, NID2, SPP1, THBS1, and THY1) were part of the EMT pathway. The expression of CDH1/E-cadherin, a key EMT gene, and TWIST1, regulator gene of EMT, was higher in MCPyV-negative tumors. To further investigate the expression of EMT genes in MCPyV-negative MCCs, we analyzed publicly available RNA sequencing data of 111 primary MCCs. Differential expression and gene set enrichment analysis of 35 MCPyV-negative versus 76 MCPyV-positive MCCs demonstrated significantly higher expression of EMT-related genes and associated pathways such as Notch signaling, TGF-ß signaling, and Hedgehog signaling, and UV response pathway in MCPyV-negative MCCs. The significance of the EMT pathway in MCPyV-negative MCCs was confirmed independently by a coexpression module analysis. One of the modules (M3) was specifically activated in MCPyV-negative MCCs and showed significant enrichment for genes involved in EMT. A network analysis of module M3 revealed that CDH1/E-cadherin was among the most connected genes (hubs). E-cadherin and LEF1 immunostains demonstrated significantly more frequent expression in MCPvV-negative versus MCPyV-positive tumors (P < .0001). In summary, our study showed that the expression of EMT-associated genes is higher in MCPyV-negative MCC. Because EMT-related proteins can be targeted, the identification of EMT pathways in MCPyV-negative MCCs is of potential therapeutic relevance.

A review on the oncogenesis of Merkel cell carcinoma: Several subsets arise from different stages of differentiation of stem cell.

Merkel cell carcinoma (MCC), a rare primary cutaneous neuroendocrine neoplasm, is extremely aggressive and has a higher mortality rate than melanoma. Based on Merkel cell polyomavirus (MCPyV) status and morphology, MCCs are often divided into several distinct subsets: pure MCPyV-positive, pure MCPyV-negative, and combined MCC. MCPyV-positive MCC develops by the clonal integration of viral DNA, whereas MCPyV-negative MCC is induced by frequent ultraviolet (UV)-mediated mutations, that are characterized by a high mutational burden, UV signature mutations, and many mutations in TP53 and retinoblastoma suppressor gene (RB1). Combined MCC consists of an intimate mix of MCC and other cutaneous tumor populations, and is usually MCPyV-negative, with rare exceptions. Based on the existing subsets of MCC, it is speculated that there are at least 4 stages in the natural history of stem cell differentiation: primitive pluripotent stem cells, divergent differentiated stem cells, unidirectional stem cells, and Merkel cells (or epidermal/adnexal cells). In the first stage, MCPyV may integrate into the genome of primitive pluripotent stem cells, driving oncogenesis in pure MCPyV-positive MCC. If MCPyV integration does not occur, the stem cells enter the second stage and acquire the ability to undergo multidirectional neuroendocrine and epidermal (or adnexal) differentiation. At this stage, accumulated UV-mediated mutations may drive the development of combined MCC. In the third stage, the stem cells differentiate into unidirectional neuroendocrine stem cells, UV-mediated mutations can induce carcinogenesis in pure MCPyV-negative MCC. Therefore, it has been speculated that several subsets of MCCs arise from different stages of differentiation of common stem cells.

Merkel Cell Polyomavirus Large T Antigen Induces Cellular Senescence for Host Growth Arrest and Viral Genome Persistence through Its Unique Domain.

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.

Chromosomal Aberrations Accumulate during Metastasis of Virus-Negative Merkel Cell Carcinoma.

Merkel cell carcinoma is a rare, aggressive skin tumor initiated by polyomavirus integration or UV light DNA damage. In New Zealand, there is a propensity toward the UV-driven form (31 of 107, 29% virus positive). Using archival formalin-fixed, paraffin-embedded tissues, we report targeted DNA sequencing covering 246 cancer genes on 71 tumor tissues and 38 nonmalignant tissues from 37 individuals, with 33 of 37 being negative for the virus. Somatic variants of New Zealand virus-negative Merkel cell carcinomas partially overlapped with those reported overseas, including TP53 variants in all tumors and RB1, LRP1B, NOTCH1, and EPHA3/7 variants each found in over half of the cohort. Variants in genes not analyzed or reported in previous studies were also found. Cataloging variants in TP53 and RB1 from published datasets revealed a broad distribution across these genes. Chr 1p gain and Chr 3p loss were identified in around 50% of New Zealand virus-negative Merkel cell carcinomas, and RB1 loss of heterozygosity was found in 90% of cases. Copy number variants accumulate in most metastases. Virus-negative Merkel cell carcinomas have complex combinations of somatic DNA-sequence variants and copy number variants. They likely carry the small genomic changes permissive for metastasis from early tumor development; however, chromosomal alterations may contribute to driving metastatic progression.

Transcriptomic analyses reveal three distinct molecular subgroups of Merkel cell carcinoma with differing prognoses.

Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine malignancy with a poor prognosis and an unknown cell of origin. Proffered cells of origin include epithelial stem cells of the hair follicle or interfollicular epidermis, dermal stem cells and pro/pre- or pre-B cells. MCC has also been proposed to have more than one cell of origin and indeed to represent more than one type of carcinoma, currently grouped together due to phenotypic similarities. We explored the heterogeneous nature of MCC by studying the most variably expressed genes with the goal of identifying gene expression patterns that are either clinically relevant or have implications regarding the cell(s) of origin. We performed RNA sequencing on primary tumor samples from 102 patients and identified the top 200 most variably expressed genes. These genes and the tumor samples were hierarchically clustered based on their expression. The functions of three gene clusters exhibiting clearly divergent expression between samples were studied by cross-referencing the lists of genes with online databases. High expression of a gene cluster related to embryonic developmental processes and low expression of a gene cluster related to neuroendocrine processes distinguished Merkel cell polyomavirus (MCPyV)-negative tumors from MCPyV-positive tumors. Furthermore, two prognostically relevant subgroups of MCPyV-positive MCC were identified based on dichotomic expression of genes related to epidermal structures and processes. We identified three distinct molecular subgroups of MCC with prognostic relevance. We propose that the dichotomic expression of epidermis-related genes might reflect both an epidermal and a nonepidermal origin for MCPyV-positive MCC.

Single-Cell Transcriptome Identifies the Renal Cell Type Tropism of Human BK Polyomavirus.

BK polyomavirus (BKPyV) infection is the main factor affecting the prognosis of kidney transplant recipients, as no antiviral agent is yet available. A better understanding of the renal-cell-type tropism of BKPyV can serve to develop new treatment strategies. In this study, the single-cell transcriptomic analysis demonstrated that the ranking of BKPyV tropism for the kidney was proximal tubule cells (PT), collecting duct cells (CD), and glomerular endothelial cells (GEC) according to the signature of renal cell type and immune microenvironment. In normal kidneys, we found that BKPyV infection-related transcription factors P65 and CEBPB were PT-specific transcription factors, and PT showed higher glycolysis/gluconeogenesis activities than CD and GEC. Furthermore, in the BKPyV-infected kidneys, the percentage of late viral transcripts in PT was significantly higher than in CD and GEC. In addition, PT had the smallest cell-cell interactions with immune cells compared to CD and GEC in both normal and BKPyV-infected kidneys. Subsequently, we indirectly demonstrated the ranking of BKPyV tropism via the clinical observation of sequential biopsies. Together, our results provided in-depth insights into the renal cell-type tropism of BKPyV in vivo at single-cell resolution and proposed a novel antiviral target.

YAP1 and WWTR1 expression inversely correlates with neuroendocrine markers in Merkel cell carcinoma.

BackgroundMerkel cell carcinoma (MCC) is an aggressive neuroendocrine (NE) skin cancer caused by severe UV-induced mutations or expression of Merkel cell polyomavirus (MCPyV) large and small T antigens (LT and ST). Despite deep genetic differences between MCPyV-positive and -negative subtypes, current clinical diagnostic markers are indistinguishable, and the expression profile of MCC tumors is, to our knowledge, unexplored.MethodsHere, we leveraged bulk and single-cell RNA-Seq of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional environment of MCC.ResultsStrikingly, MCC samples could be separated into transcriptional subtypes that were independent of MCPyV status. Instead, we observed an inverse correlation between a NE gene signature and the Hippo pathway transcription factors Yes1-associated transcriptional regulator (YAP1) and WW domain-containing transcriptional regulator 1 (WWTR1). This inverse correlation was broadly present at the transcript and protein levels in the tumor biopsies as well as in established and patient-derived cell lines. Mechanistically, expression of YAP1 or WWTR1 in a MCPyV-positive MCC cell line induced cell-cycle arrest at least in part through TEA domain-dependent (TEAD-dependent) transcriptional repression of MCPyV LT.ConclusionThese findings identify what we believe to be a previously unrecognized heterogeneity in NE gene expression within MCC and support a model of YAP1/WWTR1 silencing as essential for the development of MCPyV-positive MCC.FundingUS Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.

HLA-G expression in Merkel cell carcinoma and the correlation with Merkel cell polyomavirus infection.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine cutaneous carcinoma with a high mortality rate. The MCC etiology is not fully understood. Merkel cell-associated polyomavirus (MCPyV) was found in MCC patients, indicating a risk factor for the tumor. Caucasian, elderly, and immunocompromised individuals are more likely to develop this tumor. HLA-G consists of a non-classical class I (Ib) HLA molecule with an immunoregulatory function and was associated with tumor escape in different types of tumors, nonetheless, never been studied in MCC. The purpose of this study was to evaluate the HLA-G expression and also to detect the MCPyV in MCC patients and correlate it with the clinical course of the disease. Forty-five MCC patients were included in a retrospective study. Formalin-fixed paraffin-embedded cutaneous skin biopsies were used by immunohistochemistry and RT-PCR to verify the HLA-G expression and MCPyV infection. HLA-G expression was found in 7 (15.6%), while the presence of MCPyV was detected in 28 (62.2%) of the studied patients. No significant association was found between HLA-G expression and MCPyV infection (p = 0.250). The presence of MCPyV was associated with areas of low sunlight exposure (p = 0.042) and the HLA-G expression with progression to death (p = 0.038). HLA-G expression was detected in MCC patients, as well as the MCPyV presence was confirmed. These markers could represent factors with a possible impact on patient survival; however, further studies with a greater number of patients are needed, to better elucidate the possible role in disease progression.

Prevalence of Merkel Cell Polyomavirus in Primary Eyelid Merkel Cell Carcinomas and Association With Clinicopathological Features.

PURPOSE: Merkel cell polyomavirus (MCPyV) infection is a known to be a critical risk factor for the development of Merkel cell carcinoma (MCC). Various reports on cutaneous MCC have shown that the differences in clinicohistopathological characteristics depend on the presence of MCPyV, but the situation in eyelid MCC is unknown. This study aimed to assess the prevalence of MCPyV in patients with eyelid MCC and examine the clinicohistopathological characteristics of MCPyV-associated eyelid MCC. DESIGN: Retrospective observational case series with laboratory investigations. METHODS: Ten patients treated for eyelid MCC were included. Histopathological characteristics were examined by immunohistochemical staining using 12 antibodies. MCPyV infection was evaluated by PCR using primer sets targeting large T antigens of the MCPyV genome and by immunohistochemical staining using CM2B4 and Ab3 monoclonal antibodies. The MCPyV viral load was also quantified by PCR using 3 primer sets. RESULTS: All patients (4 males and 6 females) were Japanese with mean age of 79 (range: 63 to 87) years. One patient died due to distant metastasis 8 months after surgery for MCC. Immunohistochemical studies showed typical MCC findings in all cases, including CK20 and neuroendocrine marker positivity. PCR and immunohistochemistry with CM2B4 and Ab3 detected MCPyV antigen in all tumors. Quantitative PCR using sT, LT4, and TAg primers yielded 0.94, 1.72, and 1.05 copies per cell, respectively. CONCLUSION: Clinical and histopathological characteristics of 10 patients with eyelid MCC were elucidated. MCPyV infection was detected in all eyelids. These results provide insight for understanding the tumorigenesis of eyelid MCC.

MicroRNA dysregulations in Merkel cell carcinoma: Molecular mechanisms and clinical applications.

Merkel cell carcinoma (MCC) is an aggressive skin malignancy with two distinct etiologies. The first, which accounts for the highest proportion, is caused by Merkel cell polyomavirus (MCPyV), a DNA tumor virus. A second, UV-induced, MCC form has also been identified. Few MCC diagnostic, prognostic, and therapeutic options are available. MicroRNAs (miRNAs) are small noncoding RNA molecules, which play a key role in regulating various physiologic cellular functions including cell cycling, proliferation, differentiation, and apoptosis. Numerous miRNAs are dysregulated in cancer, by acting as either tumor suppressors or oncomiRs. The aim of this review is to collect, summarize, and discuss recent findings on miRNAs whose dysregulation has been assumed to play a role in MCC. The potential clinical application of miRNAs as diagnostic and prognostic biomarkers in MCC is also described. In the future, miRNAs will potentially gain clinical significance for the improvement of MCC diagnostic, prognostic, and therapeutic options.

Correlation between CYP3A5 gene polymorphism and BK virus infection in kidney transplant recipients.

BACKGROUND: Cytochrome P450 3A5 (CYP3A5) includes two active genotypes, namely CYP3A5*1/*1 or *1/*3 with the fast metabolic activity and CYP3A5*3/*3 with slow metabolic. We retrospectively analyzed the correlation between CYP3A5 gene polymorphism and the susceptibility to the BK virus (BKV) infection in renal transplant recipients. METHODS: According to the inclusion/ exclusion criteria, we selected 134 recipients who received kidney transplantation at the Renmin Hospital of Wuhan University from January 2019 to December 2019. Based on the pre-operative CYP3A5 sequencing results, 134 recipients were divided into two groups: those expressing the fast metabolic CYP3A5*1/*1 or *1/*3 genotype; and, those expressing slow metabolic CYP3A5*3/*3 genotype. These two recipient groups were then analyzed for the BKV infection rate with different metabolic types to establish the potential relationship between CYP3A5 gene polymorphism and BKV infection. RESULTS: The overall incidence of BKV viruria was 37.3%, whereas BKV viremia was 4.5% among all 134 recipients. The fast metabolism group had 9.1% incidence of BKV viremia and 49.1% incidence of BKV viruria. In contrast, the slow metabolism group had only 1.3%incidence of BKV viremia (P = 0.031) with only 29.1% BKV viruria (P = 0.011). The incidence of low levels of urinary BKV in the fast metabolism group was higher than that in the slow metabolism group (P = 0.005), while no significant statistical difference in the incidence of high levels of urinary BKV and high and low levels of blood BKV. CONCLUSION: After kidney transplantation, CYP3A5 gene polymorphism of recipients present a certain relationship with the occurrence of BKV infection, which may be of value for the prediction and prevention of BKV infection.

Deciphering the role of predicted miRNAs of polyomaviruses in carcinogenesis.

Human polyomaviruses are relatively common in the general population. Polyomaviruses maintain a persistent infection after initial infection in childhood, acting as an opportunistic pathogen in immunocompromised populations and their association has been linked to carcinogenesis. A comprehensive understanding of the underlying molecular mechanisms of carcinogenesis in consequence of polyomavirus infection remains elusive. However, the critical role of viral miRNAs and their potential targets in modifying the transcriptome profile of the host remains largely unknown. Polyomavirus-derived miRNAs have the potential to play a substantial role in carcinogenesis. Employing computational approaches, putative viral miRNAs along with their target genes have been predicted and possible roles of the targeted genes in many significant biological processes have been obtained. Polyomaviruses have been observed to target intracellular signal transduction pathways through miRNA-mediated epigenetic regulation, which may contribute to cancer development. In addition, BKPyV-infected human renal cell microarray data was coupled with predicted target genes and analysis of the downregulated genes indicated that viruses target multiple signaling pathways (e.g. MAPK signaling pathway, PI3K-Akt signaling pathway, PPAR signaling pathway) in the host as well as turning off several tumor suppression genes (e.g. FGGY, EPHX2, CACNA2D3, CDH16) through miRNA-induced mechanisms, assuring cell transformation. This study provides a conceptual framework for the underlying molecular mechanisms involved in the course of carcinogenesis upon polyomavirus infection.

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma.

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.

BK Polyomavirus bkv-miR-B1-5p: A Stable Micro-RNA to Monitor Active Viral Replication after Kidney Transplantation.

BACKGROUND: Bkv-miR-B1-5p is a viral micro-RNA (miRNA) specifically produced during BK polyomavirus (BKPyV) replication. Recent studies have suggested using bkv-miR-B1-5p as a biomarker to monitor viral infection and predict complications in kidney transplant patients. To identify the technical limitations of this miRNA quantification in biological samples, knowledge of its stability and distribution in the extracellular compartment is necessary. Moreover, a proof of concept for using bkv-miR-B1-5p as a biomarker of active replication in chronic infection is still missing in the published literature. METHODS: The stability of bkv-miR-B1-5p was evaluated in samples derived from cell cultures and in urine from BKPyV-infected kidney transplant recipients. The miRNA was quantified in different fractions of the extracellular compartment, including exosomes, and protein binding was evaluated. Finally, we developed an in vitro model for chronic culture of BKPyV clinical isolates to observe changes in the bkv-miR-B1-5p level during persistent infections. RESULTS: Bkv-miR-B1-5p is a stable biomarker in samples from humans and in vitro experiments. Marginally associated with the exosomes, most of the circulating bkv-miR-B1-5p is bound to proteins, especially Ago2, so the miRNA quantification does not require specific exosome isolation. The bkv-miR-B1-5p level is predictable of viral infectivity, which makes it a potential specific biomarker of active BKPyV replication after kidney transplantation.

Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses.

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.

IFNL4 rs368234815 polymorphism does not predict risk of BK virus associated nephropathy after living-donor kidney transplant: A case-control study.

BACKGROUND: BK polyoma virus (BKPyV) associated nephropathy (BKPyVAN) is a major cause of kidney graft loss in renal transplant patients. Interferons (IFNs) are an important innate immune response against viral infections and genetic polymorphisms of the IFN-pathways can affect susceptibility and mortality during viral infection. Here, we investigated whether the dinucleotide polymorphism rs368234815 (ΔG/TT) in the IFNL4 gene contributed to BKPyV reactivation or BKPyVAN after living-donor kidney transplantation. METHODS: This retrospective case-control study determines the prevalence of IFNL4 variants in a Caucasian population of living-donor kidney transplant recipients and donors and explores its association with BKPyV infection and BKPyVAN development. We included 28 recipients with BKPyV reactivation, 10 of which developed BKPyVAN and 30 BKPyV negative controls. Targeted sequencing of the IFNL4 gene from both recipients and their respective donors was performed. RESULTS: We found IFNL4 rs368234815 ΔG allele frequencies of 41.7% in BKPyV negative and 39.3% in BKPyV positive recipients (P = .85), and 41.7% and 40.4% (P>.99) in their respective donors. IFNL4 rs368234815 ΔG allele frequencies in BKPyVAN developing recipients and their respective donors were 50% and 43.7% (P = .60 and P>.99). CONCLUSIONS: Our results indicate that the IFNL4 rs368234815 ΔG allele is not associated with BKPyV reactivation, nor the manifestation of BKPyVAN.

A human-derived 3D brain organoid model to study JC virus infection.

Progressive multifocal leukoencephalopathy (PML) is a frequent neurological complication in immunosuppressed patients. PML is caused by the JC virus (JCV), a neurotropic DNA polyomavirus that infects oligodendrocytes and astrocytes, causing inflammation and demyelination which lead to neurological dysfunction. The pathogenesis of PML is poorly understood due to the lack of in vitro or animal models to study mechanisms of disease as the virus most efficiently infects only human cells. We developed a human-derived brain organotypic system (also called brain organoid) to model JCV infection. The model was developed by using human-induced pluripotent stem cells (iPSC) and culturing them in 3D to generate an organotypic model containing neurons, astrocytes, and oligodendrocytes which recapitulates aspects of the environment of the human brain. We infected the brain organoids with the JCV MAD4 strain or cerebrospinal fluid of a patient with PML. The organoids were assessed for evidence of infection by qPCR, immunofluorescence, and electron microscopy at 1, 2, and 3 weeks post-exposure. JCV infection in both JCV MAD4 strain and PML CSF-exposed brain organoids was confirmed by immunocytochemical studies demonstrating viral antigens and electron microscopy showing virion particles in the nuclear compartment of oligodendrocytes and astrocytes. No evidence of neuronal infection was visualized. Infection was also demonstrated by JCV qPCR in the virus-exposed organoids and their media. In conclusion, the brain organoid model of JCV infection establishes a human model suitable for studying the mechanisms of JCV infection and pathogenesis of PML and may facilitate the exploration of therapeutic approaches.

Direct cellular reprogramming enables development of viral T antigen-driven Merkel cell carcinoma in mice.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that frequently carries an integrated Merkel cell polyomavirus (MCPyV) genome and expresses viral transforming antigens (TAgs). MCC tumor cells also express signature genes detected in skin-resident, postmitotic Merkel cells, including atonal bHLH transcription factor 1 (ATOH1), which is required for Merkel cell development from epidermal progenitors. We now report the use of in vivo cellular reprogramming, using ATOH1, to drive MCC development from murine epidermis. We generated mice that conditionally expressed MCPyV TAgs and ATOH1 in epidermal cells, yielding microscopic collections of proliferating MCC-like cells arising from hair follicles. Immunostaining of these nascent tumors revealed p53 accumulation and apoptosis, and targeted deletion of transformation related protein 53 (Trp53) led to development of gross skin tumors with classic MCC histology and marker expression. Global transcriptome analysis confirmed the close similarity of mouse and human MCCs, and hierarchical clustering showed conserved upregulation of signature genes. Our data establish that expression of MCPyV TAgs in ATOH1-reprogrammed epidermal cells and their neuroendocrine progeny initiates hair follicle-derived MCC tumorigenesis in adult mice. Moreover, progression to full-blown MCC in this model requires loss of p53, mimicking the functional inhibition of p53 reported in human MCPyV-positive MCCs.