RESUMEN
Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or alternate LT open reading frames (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-κB signaling. ALTO activates NF-κB by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1). Following activation, NF-κB dimers bind the MCPyV noncoding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-κB signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-κB- and NTAR-dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.
Asunto(s)
Regulación Viral de la Expresión Génica , FN-kappa B , Transducción de Señal , Humanos , FN-kappa B/metabolismo , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , Poliomavirus de Células de Merkel/genética , Infecciones por Polyomavirus/virología , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/metabolismo , Carcinoma de Células de Merkel/virología , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/metabolismo , Sistemas de Lectura Abierta/genética , Línea Celular Tumoral , Regulación hacia Abajo , Empalme AlternativoRESUMEN
The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world's adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.
Asunto(s)
Secuencias de Aminoácidos , Virus BK , Proteínas de la Cápside , Vesículas Extracelulares , Virión , Ensamble de Virus , Virus BK/genética , Virus BK/fisiología , Virus BK/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virología , Humanos , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/química , Virión/metabolismo , Virión/genética , Infecciones por Polyomavirus/virología , Infecciones por Polyomavirus/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HEK293RESUMEN
While Merkel cell polyomavirus (MCPyV or MCV) is an abundant virus frequently shed from healthy skin, it is one of the most lethal tumor viruses in immunocompromised individuals, highlighting the crucial role of host immunity in controlling MCPyV oncogenic potential. Despite its prevalence, very little is known about how MCPyV interfaces with the host immune response to maintain asymptomatic persistent infection and how inadequate control of MCPyV infection triggers MCC tumorigenesis. In this study, we discovered that the MCPyV protein, known as the Alternative Large Tumor Open Reading Frame (ALTO), also referred to as middle T, effectively primes and activates the STING signaling pathway. It recruits Src kinase into the complex of STING downstream kinase TBK1 to trigger its autophosphorylation, which ultimately activates the subsequent antiviral immune response. Combining single-cell analysis with both loss- and gain-of-function studies of MCPyV infection, we demonstrated that the activity of ALTO leads to a decrease in MCPyV replication. Thus, we have identified ALTO as a crucial viral factor that modulates the STING-TBK1 pathway, creating a negative feedback loop that limits viral infection and maintains a delicate balance with the host immune system. Our study reveals a novel mechanism by which a tumorigenic virus-encoded protein can link Src function in cell proliferation to the activation of innate immune signaling, thereby controlling viral spread, and sustaining persistent infection. Our previous findings suggest that STING also functions as a tumor suppressor in MCPyV-driven oncogenesis. This research provides a foundation for investigating how disruptions in the finely tuned virus-host balance, maintained by STING, could alter the fate of MCPyV infection, potentially encouraging malignancy.
Asunto(s)
Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Proteínas Serina-Treonina Quinasas , Infecciones Tumorales por Virus , Proteínas Serina-Treonina Quinasas/metabolismo , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/inmunología , Infecciones por Polyomavirus/virología , Humanos , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/virología , Carcinoma de Células de Merkel/virología , Carcinoma de Células de Merkel/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo , Replicación Viral , Neoplasias Cutáneas/virología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/inmunología , AnimalesRESUMEN
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.
Asunto(s)
Antígenos Virales de Tumores , Caseína Quinasa Ialfa , Poliomavirus de Células de Merkel , Proteínas con Repetición de beta-Transducina , Poliomavirus de Células de Merkel/genética , Poliomavirus de Células de Merkel/metabolismo , Humanos , Fosforilación , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/genética , Proteínas con Repetición de beta-Transducina/metabolismo , Proteínas con Repetición de beta-Transducina/genética , Antígenos Virales de Tumores/metabolismo , Antígenos Virales de Tumores/genética , Interacciones Huésped-Patógeno , Proteolisis , Replicación Viral , Unión Proteica , Antígenos Transformadores de Poliomavirus/metabolismo , Antígenos Transformadores de Poliomavirus/genética , Infecciones por Polyomavirus/virología , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/genéticaRESUMEN
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.
Asunto(s)
Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Humanos , Carcinoma de Células de Merkel/patología , Antígeno B7-H1/metabolismo , Poliomavirus de Células de Merkel/metabolismo , Proteína p53 Supresora de Tumor , Neoplasias Cutáneas/patología , Infecciones por Polyomavirus/complicaciones , Infecciones por Polyomavirus/metabolismo , Ubiquitina-Proteína Ligasas , Proteínas de Unión a Retinoblastoma/metabolismoRESUMEN
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.
Asunto(s)
Antígenos Transformadores de Poliomavirus , Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Infecciones Tumorales por Virus , Humanos , Carcinoma de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/virología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Poliomavirus de Células de Merkel/metabolismo , Fosforilación , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/virología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología , Antígenos Transformadores de Poliomavirus/metabolismo , Transcripción GenéticaRESUMEN
PURPOSE: BK Polyomavirus (BKPyV) infection manifests as renal inflammation and can cause kidney damage. Tumor necrosis factor-α (TNF-α) is increased in renal inflammation and injury. The aim of this study was to investigate the effect of TNF-α blockade on BKPyV infection. METHODS: Urine specimens from 22 patients with BKPyV-associated nephropathy (BKPyVN) and 35 non-BKPyVN kidney transplant recipients were analyzed. RESULTS: We demonstrated increased urinary levels of TNF-α and its receptors, TNFR1 and TNFR2, in BKPyVN patients. Treating BKPyV-infected human proximal tubular cells (HRPTECs) with TNF-α stimulated the expression of large T antigen and viral capsid protein-1 mRNA and proteins and BKPyV promoter activity. Knockdown of TNFR1 or TNFR2 expression caused a reduction in TNF-α-stimulated viral replication. NF-κB activation induced by overexpression of constitutively active IKK2 significantly increased viral replication and the activity of the BKPyV promoter containing an NF-κB binding site. The addition of a NF-κB inhibitor on BKPyV-infected cells suppressed viral replication. Blockade of TNF-α functionality by etanercept reduced BKPyV-stimulated expression of TNF-α, interleukin-1ß (IL-1ß), IL-6 and IL-8 and suppressed TNF-α-stimulated viral replication. In cultured HRPTECs and THP-1 cells, BKPyV infection led to increased expression of TNF-α, interleukin-1 ß (IL-1ß), IL-6 and TNFR1 and TNFR2 but the stimulated magnitude was far less than that induced by poly(I:C). This may suggest that BKPyV-mediated autocrine effect is not a major source of TNFα. CONCLUSION: TNF-α stimulates BKPyV replication and inhibition of its signal cascade or functionality attenuates its stimulatory effect. Our study provides a therapeutic anti-BKPyV target.
Asunto(s)
Virus BK , Infecciones por Polyomavirus , Humanos , Virus BK/genética , Factor de Necrosis Tumoral alfa , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral/genética , FN-kappa B , Interleucina-6 , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/patología , InflamaciónRESUMEN
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.
Asunto(s)
Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Infecciones Tumorales por Virus , Humanos , Poliomavirus de Células de Merkel/metabolismo , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , Transcripción Viral , Replicación del ADN , Replicación Viral , ADN Viral/genética , ADN Viral/metabolismo , Factores de Transcripción/metabolismo , Neoplasias Cutáneas/patología , Genoma Viral , ARN Mensajero/metabolismo , Infecciones por Polyomavirus/metabolismoRESUMEN
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.
Asunto(s)
Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Infecciones Tumorales por Virus , Carcinoma de Células de Merkel/diagnóstico , Carcinoma de Células de Merkel/patología , Humanos , Poliomavirus de Células de Merkel/metabolismo , Infecciones por Polyomavirus/complicaciones , Infecciones por Polyomavirus/metabolismo , Proteínas de Unión a Retinoblastoma , Factores de Transcripción SOXB1/metabolismo , Neoplasias Cutáneas/patología , Infecciones Tumorales por Virus/complicaciones , Ubiquitina-Proteína LigasasRESUMEN
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.
Asunto(s)
Carcinoma de Células de Merkel , Porocarcinoma Ecrino , Poliomavirus de Células de Merkel/metabolismo , Infecciones por Polyomavirus , Neoplasias de las Glándulas Sudoríparas , Infecciones Tumorales por Virus , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/patología , Carcinoma de Células de Merkel/virología , Niño , Porocarcinoma Ecrino/metabolismo , Porocarcinoma Ecrino/patología , Porocarcinoma Ecrino/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/patología , Infecciones por Polyomavirus/virología , Neoplasias de las Glándulas Sudoríparas/metabolismo , Neoplasias de las Glándulas Sudoríparas/patología , Neoplasias de las Glándulas Sudoríparas/virología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/patología , Infecciones Tumorales por Virus/virologíaRESUMEN
The BK polyomavirus (BKPyV) is a ubiquitous human virus that persists in the renourinary epithelium. Immunosuppression can lead to BKPyV reactivation in the first year post-transplantation in kidney transplant recipients (KTRs) and hematopoietic stem cell transplant recipients. In KTRs, persistent DNAemia has been correlated to the occurrence of polyomavirus-associated nephropathy (PVAN) that can lead to graft loss if not properly controlled. Based on recent observations that conventional dendritic cells (cDCs) specifically infiltrate PVAN lesions, we hypothesized that those cells could play a role in BKPyV infection. We first demonstrated that monocyte-derived dendritic cells (MDDCs), an in vitro model for mDCs, captured BKPyV particles through an unconventional GRAF-1 endocytic pathway. Neither BKPyV particles nor BKPyV-infected cells were shown to activate MDDCs. Endocytosed virions were efficiently transmitted to permissive cells and protected from the antibody-mediated neutralization. Finally, we demonstrated that freshly isolated CD1c+ mDCs from the blood and kidney parenchyma behaved similarly to MDDCs thus extending our results to cells of clinical relevance. This study sheds light on a potential unprecedented CD1c+ mDC involvement in the BKPyV infection as a promoter of viral spreading.
Asunto(s)
Antígenos CD1/metabolismo , Virus BK/inmunología , Células Dendríticas/inmunología , Células Epiteliales/inmunología , Glicoproteínas/metabolismo , Riñón/inmunología , Infecciones por Polyomavirus/inmunología , Infecciones Tumorales por Virus/inmunología , Anticuerpos Neutralizantes/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Humanos , Riñón/metabolismo , Riñón/virología , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/virología , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología , Replicación ViralRESUMEN
The aims were to investigate the incidence of BKV infection and the presence of HC in pediatric patients undergoing HSCT. Twenty-four children patients (M/F: 17/7) undergoing HSCT in a single center over a period of 1 year were included in the study. The presence of BKV DNA was determined by quantitative real-time PCR in plasma and urine samples at the following times: before transplantation, twice a week until engraftment time, and weekly for + 100 days. The mean age of the patients was 7.79 ± 5.03 years, the mean follow-up time was 95.6 ± 25.9 days, and the average number of samples per patient was 15.8 ± 3.2. BKV DNA was detected in at least one urine sample in 91.6% (n: 22) and at least one plasma sample in 75% (n:18) of the patients. The median time to the first BKV DNA positivity in urine and plasma samples was 11 (range: 1-80) and 32 days (range: 2-79), respectively. The median value of BKV DNA copies in urine and plasma were 1.7 × 106 (range: 2.8 × 101 -1.2 × 1014 ) and 1.9 × 103 copies/mL (range: 3-2.1 × 106 ), respectively. Thirteen patients (54.2%) had hematuria with BKV viruria; 8 (33.3%) patients had viremia. The median value of the BKV DNA copies in urine and plasma was 4.4 × 107 (range: 65-1 × 1011 ) and 2.9 × 103 (range: 7-7.8 × 104 ) copies/mL in these patients. Two (15.4%) of the 13 patients with BKV viruria and hematuria were diagnosed with BKV-related HC. BKV DNA viral load monitoring of urine and plasma in pediatric HSCT patients with a high risk for viral infections is valuable for understanding the development of BKV-related HC.
Asunto(s)
Virus BK/aislamiento & purificación , Cistitis/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Huésped Inmunocomprometido , Inmunosupresores/efectos adversos , Infecciones por Polyomavirus/inmunología , Adolescente , Niño , Preescolar , Cistitis/diagnóstico , Cistitis/epidemiología , Cistitis/virología , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Infecciones por Polyomavirus/diagnóstico , Infecciones por Polyomavirus/epidemiología , Infecciones por Polyomavirus/metabolismo , Carga Viral , Adulto JovenRESUMEN
BK virus (BKV) is a polyomavirus with high seroprevalence in the general population with an unremarkable clinical presentation in healthy people, but a potential for causing serious complications in immunosuppressed transplanted patients. Reactivation or primary infection in kidney allograft recipients may lead to allograft dysfunction and subsequent loss. Currently, there is no widely accepted specific treatment for BKV infection and reduction of immunosuppressive therapy is the mainstay therapy. Given this and the sequential appearance of viruria-viremia-nephropathy, screening and early detection are of utmost importance. There are numerous risk factors associated with BKV infection including genetic factors, among them human leukocyte antigens (HLA) and killer cell immunoglobulin-like receptors (KIR) alleles have been shown to be the strongest so far. Identification of patients at risk for BKV infection would be useful in prevention or early action to reduce morbidity and progression to frank nephropathy. Assessment of risk involving HLA ligands and KIR genotyping of recipients in the pre-transplant or early post-transplant period might be useful in clinical practice. This review summarizes current knowledge of the association between HLA, KIR and BKV infection and potential future directions of research, which might lead to optimal utilization of these genetic markers.
Asunto(s)
Virus BK/fisiología , Antígenos HLA/inmunología , Trasplante de Riñón/efectos adversos , Infecciones por Polyomavirus/etiología , Infecciones por Polyomavirus/metabolismo , Receptores KIR/metabolismo , Alelos , Biomarcadores , Susceptibilidad a Enfermedades , Antígenos HLA/genética , Humanos , Huésped Inmunocomprometido , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Pronóstico , InvestigaciónRESUMEN
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV+, 11 MCV-) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV large T antigen, CDKN2AIP, SERPINB5, and TRIM29) that discriminate MCV+ and MCV- MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be upregulated in virus-infected samples. Remarkably, 4 (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify potentially novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.
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Antígenos Virales de Tumores/metabolismo , Biomarcadores/metabolismo , Carcinoma de Células de Merkel/diagnóstico , Infecciones por Polyomavirus/complicaciones , Proteoma/metabolismo , Neoplasias Cutáneas/diagnóstico , Infecciones Tumorales por Virus/complicaciones , Carcinoma de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/patología , Carcinoma de Células de Merkel/virología , Formaldehído/química , Humanos , Poliomavirus de Células de Merkel/fisiología , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Proteoma/análisis , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virologíaRESUMEN
Polyomaviruses are a family of small, non-enveloped DNA viruses that can cause severe disease in immunosuppressed individuals. Studies with SV40, a well-studied model polyomavirus, have revealed the role of host proteins in polyomavirus entry and trafficking to the nucleus, in viral transcription and DNA replication, and in cell transformation. In contrast, little is known about host factors or cellular signaling pathways involved in the late steps of productive infection leading to release of progeny polyomaviruses. We previously showed that cytoplasmic vacuolization, a characteristic late cytopathic effect of SV40 infection, depends on the specific interaction between the major viral capsid protein VP1 and its cell surface ganglioside receptor GM1. Here, we show that, late during infection, SV40 activates a signaling cascade in permissive monkey CV-1 cells involving Ras, Rac1, MKK4, and JNK to stimulate SV40-specific cytoplasmic vacuolization and subsequent cell lysis and virus release. Inhibition of individual components of this signaling pathway inhibits vacuolization, lysis, and virus release, even though high-level intracellular virus replication occurs. Identification of this pathway for SV40-induced vacuolization and virus release provides new insights into the late steps of non-enveloped virus infection.
Asunto(s)
Muerte Celular/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Virus 40 de los Simios/metabolismo , Liberación del Virus/fisiología , Proteínas de la Cápside , Línea Celular , Humanos , MAP Quinasa Quinasa 4/metabolismo , Infecciones por Polyomavirus/metabolismo , Virus 40 de los Simios/genética , Infecciones Tumorales por Virus/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
BACKGROUND: Recent studies have confirmed the integration of the BK polyomavirus (BKPyV) gene into the cellular genome of urothelial carcinomas in transplant recipients, further confirming the correlation between BKPyV and urothelial carcinomas after transplantation. However, the role BKPyV infections play in the biological function of bladder cancer remains unclear. METHODS: We developed a BKPyV-infected bladder cancer cell model and a mice tumor model to discuss the role of BKPyV infections. RESULTS: Our research proves that BKPyV infections promote the proliferation, invasion and migration of bladder cancer cells, while the activation of ß-catenin signaling pathway is one of its mediation mechanisms. CONCLUSIONS: We first described BKPyV infection promotes the proliferation, invasion and migration of bladder cancer. We verified the role of ß-catenin signaling pathway and Epithelial-Mesenchymal Transition effect in BKPyV-infected bladder cancer. These results provide meaningful information towards the diagnosis and treatment of clinical bladder cancer.
Asunto(s)
Virus BK/patogenicidad , Infecciones por Polyomavirus/patología , Infecciones Tumorales por Virus/patología , Neoplasias de la Vejiga Urinaria/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/virología , Vía de Señalización Wnt , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
Merkel cell carcinoma (MCC) is an uncommon, lethal cancer of the skin caused by either Merkel cell polyomavirus (MCPyV) or UV-linked mutations. MCPyV is found integrated into MCC tumor genomes, accompanied by truncation mutations that render the MCPyV large T antigen replication incompetent. We used the open access HPV Detector/Cancer-virus Detector tool to determine MCPyV integration sites in whole-exome sequencing data from five MCC cases, thereby adding to the limited published MCPyV integration site junction data. We also systematically reviewed published data on integration for MCPyV in the human genome, presenting a collation of 123 MCC cases and their linked chromosomal sites. We confirmed that there were no highly recurrent specific sites of integration. We found that chromosome 5 was most frequently involved in MCPyV integration and that integration sites were significantly enriched for genes with binding sites for oncogenic transcription factors such as LEF1 and ZEB1, suggesting the possibility of increased open chromatin in these gene sets. Additionally, in one case we found, for the first time, integration involving the tumor suppressor gene KMT2D, adding to previous reports of rare MCPyV integration into host tumor suppressor genes in MCC.
Asunto(s)
Carcinoma de Células de Merkel/metabolismo , Proteínas de Unión al ADN/metabolismo , Poliomavirus de Células de Merkel/fisiología , Proteínas de Neoplasias/metabolismo , Infecciones por Polyomavirus/metabolismo , Infecciones Tumorales por Virus/metabolismo , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/virología , Línea Celular Tumoral , Cromosomas Humanos Par 5/genética , Cromosomas Humanos Par 5/metabolismo , Cromosomas Humanos Par 5/virología , Proteínas de Unión al ADN/genética , Genes Supresores de Tumor , Humanos , Poliomavirus de Células de Merkel/genética , Proteínas de Neoplasias/genética , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/virología , Integración Viral , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
SP1 binding in SV40 chromatin in vitro and in vivo was characterized in order to better understand its role during the initiation of early transcription. We observed that chromatin from disrupted virions, but not minichromosomes, was efficiently bound by HIS-tagged SP1 in vitro, while the opposite was true for the presence of endogenous SP1 introduced in vivo. Using ChIP-Seq to compare the location of SP1 to nucleosomes carrying modified histones, we found that SP1 could occupy its whole binding site in virion chromatin but only the early side of its binding site in most of the minichromosomes carrying modified histones due to the presence of overlapping nucleosomes. The results suggest that during the initiation of an SV40 infection, SP1 binds to an open region in SV40 virion chromatin but quickly triggers chromatin reorganization and its own removal.
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Cromatina/virología , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Virus 40 de los Simios/metabolismo , Factor de Transcripción Sp1/metabolismo , Virión/metabolismo , Cromatina/genética , Cromatina/metabolismo , Interacciones Huésped-Patógeno , Humanos , Nucleosomas/genética , Nucleosomas/metabolismo , Infecciones por Polyomavirus/genética , Unión Proteica , Virus 40 de los Simios/genética , Factor de Transcripción Sp1/genética , Virión/genéticaRESUMEN
The demyelinating disease progressive multifocal leukoencephalopathy (PML) is caused by the human polyomavirus, JCPyV, under conditions of prolonged immunosuppression. Initial infection is asymptomatic, and the virus establishes lifelong persistence in the host. Following the loss of immune surveillance, the virus can traffic to the central nervous system and infect oligodendrocytes to cause demyelination and PML. The mechanisms involved in glial cell infection are not completely understood. In a screen for N-glycosylated proteins that influence JCPyV pathology, we identified Adipocyte Plasma Membrane Associated Protein (APMAP) as a host cell modulator of JCPyV infection. The removal of APMAP by small interfering siRNA as well as by CRISPR-Cas9 gene editing resulted in a significant decrease in JCPyV infection. Exogenous expression of APMAP in APMAP knockout cell lines rescued susceptibility to infection. These data suggest that virus infection of glial cells is dependent on APMAP.
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Virus JC/fisiología , Neuroglía/metabolismo , Infecciones por Polyomavirus/metabolismo , Línea Celular , Interacciones Huésped-Patógeno , Humanos , Virus JC/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Neuroglía/virología , Oligodendroglía/metabolismo , Oligodendroglía/virología , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/virologíaRESUMEN
Merkel cell polyomavirus (MCV) small T antigen (sT) is the main oncoprotein for the development of Merkel cell carcinoma (MCC). MCC is a rare, clinically aggressive neuroendocrine tumor of the skin with a high propensity for local, regional, and distant spread. The dysregulation of matrix metalloproteinase-9 (MMP-9) has been implicated in multiple essential roles in the development of various malignant tumor cell invasion and metastasis. Previously, MCV sT was shown to induce the migratory and invasive phenotype of MCC cells through the transcriptional activation of the sheddase molecule, ADAM 10 (A disintegrin and metalloprotease domain-containing protein 10). In this study, we show that MCV sT protein stimulates differential expression of epithelial-mesenchymal transition (EMT)-associated genes, including MMP-9 and Snail. This effect is dependent on the presence of the large T stabilization domain (LSD), which is known to be responsible for cell transformation through targeting of promiscuous E3 ligases, including FBW7, a known MMP-9 and Snail regulator. Chemical treatments of MMP-9 markedly inhibited MCV sT-induced cell migration and invasion. These results suggest that MCV sT contributes to the activation of MMP-9 as a result of FBW7 targeting and increases the invasive potential of cells, which can be used for targeted therapeutic intervention.IMPORTANCE Merkel cell carcinoma (MCC) is the most aggressive cutaneous tumor without clearly defined treatment. Although MCC has a high propensity for metastasis, little is known about the underlying mechanisms that drive MCC invasion and metastatic progression. MMP-9 has been shown to play a detrimental role in many metastatic human cancers, including melanoma and other nonmelanoma skin cancers. Our study shows that MCV sT-mediated MMP-9 activation is driven through the LSD, a known E3 ligase-targeting domain, in MCC. MMP-9 may serve as the biochemical culprit to target and develop a novel approach for the treatment of metastatic MCC.