Human Polyomavirus Research Articles

132 Merkel cell polyomavirus small t antigen activates non-canonical nf-κb signaling to promote tumorigenesis

Multiple human polyomaviruses (HPyVs) can infect the skin, but only Merkel Cell Polyomavirus (MCPyV) has been implicated in the development of a cancer, Merkel Cell Carcinoma (MCC). While expression of HPyV6, HPyV7, and MCPyV small T antigens (ST) all induced a senescence associated secretory phenotype (SASP), MCPyV ST uniquely activated non-canonical NF-κB (ncNF-κB), instead of canonical NF-κB signaling to evade senescence. MCPyV activated NFKB2 and RELB transcription through H3K4 trimethylation and promoted the post-translational stability and processing of NFKB2 by inhibiting FBXW7.

Development of a Nucleic Acid Lateral Flow Immunoassay for the Detection of Human Polyomavirus BK.

The BK virus (BKV) is an emerging pathogen in immunocompromised individuals and widespread in the human population. Polymerase chain reaction is a simple and highly sensitive method for detecting BKV, but it is time consuming and requires expensive instruments and expert judgment. The lateral flow assay, a rapid, low-cost, minimal-labor, and easy-to-use diagnostic method, was successfully applied for pathogen detection. In this study, we used oligonucleotide probes to develop a simple and rapid sandwich-type lateral flow immunoassay for detecting BKV DNA within 45 minutes. The detection limit for the synthetic single-stranded DNA was 5 nM. The specificity study showed no cross-reactivity with other polyomaviruses, such as JC virus and simian virus 40. For the Escherichia coli containing BKV plasmid cultured samples, the sensitivity was determined to be 107 copies/mL. The approach offers great potential for BKV detection of various target analytes in point-of-care settings.

BK Polyomavirus Evades Innate Immune Sensing by Disrupting the Mitochondrial Network and Promotes Mitophagy.

SummaryImmune escape contributes to viral persistence, yet little is known about human polyomaviruses. BK-polyomavirus (BKPyV) asymptomatically infects 90% of humans but causes premature allograft failure in kidney transplant patients. Despite virus-specific T cells and neutralizing antibodies, BKPyV persists in kidneys and evades immune control as evidenced by urinary shedding in immunocompetent individuals. Here, we report that BKPyV disrupts the mitochondrial network and membrane potential when expressing the 66aa-long agnoprotein during late replication. Agnoprotein is necessary and sufficient, using its amino-terminal and central domain for mitochondrial targeting and network disruption, respectively. Agnoprotein impairs nuclear IRF3-translocation, interferon-beta expression, and promotes p62/SQSTM1-mitophagy. Agnoprotein-mutant viruses unable to disrupt mitochondria show reduced replication and increased interferon-beta expression but can be rescued by type-I interferon blockade, TBK1-inhibition, or CoCl2-treatment. Mitochondrial fragmentation and p62/SQSTM1-autophagy occur in allograft biopsies of kidney transplant patients with BKPyV nephropathy. JCPyV and SV40 infection similarly disrupt mitochondrial networks, indicating a conserved mechanism facilitating polyomavirus persistence and post-transplant disease.

Adipocyte Plasma Membrane Protein (APMAP) promotes JC Virus (JCPyV) infection in human glial cells

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.

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Evidence of the Mechanism by Which Polyomaviruses Exploit the Extracellular Vesicle Delivery System during Infection.

Increasing evidence suggests that human viruses can hijack extracellular vesicles (EVs) to deliver proteins, mRNAs, microRNAs (miRNAs) and whole viral particles during viral persistence in the host. Human polyomavirus (PyV) miRNAs, which downregulate large T-antigen expression and target host factors, help the virus escape immune elimination and may have roles in the success of viral persistence/replication and the development of diseases. In this context, several investigations have detected PyV miRNAs in EVs obtained from cell culture supernatants after viral infection, demonstrating the ability of these vesicles to deliver miRNAs to uninfected cells, potentially counteracting new viral infection. Additionally, PyV miRNAs have been identified in EVs derived from the biological fluids of clinical samples obtained from patients with or at risk of severe PyV-associated diseases and from asymptomatic control healthy subjects. Interestingly, PyV miRNAs were found to be circulating in blood, urine, cerebrospinal fluid, and saliva samples from patients despite their PyV DNA status. Recently, the association between EVs and PyV viral particles was reported, demonstrating the ability of PyV viral particles to enter the cell without natural receptor-mediated entry and evade antibody-mediated neutralization or to be neutralized at a step different from that of the neutralization of naked whole viral particles. All these data point toward a potential role of the association between PyVs with EVs in viral persistence, suggesting that further work to define the implication of this interaction in viral reactivation is warranted.

Analysis of viruses present in urine from patients with interstitial cystitis.

The question of whether some cases of interstitial cystitis may have an infectious etiology has been debated for some time. Previous studies have looked for the presence of certain specific viruses, but generally did not use the types of sensitive and unbiased approaches that are currently available. As part of the MAPP (Multidisciplinary Approach to the Study of Chronic Pelvic Pain) Research Network, we examined urine specimens from interstitial cystitis patients who provided specimens over time and also reported various symptoms at the time of urine collection. We first performed next-generation sequencing to look for the presence of viruses in urines, and detected two human polyomaviruses that are known to be excreted into urine, BKPyV and JCPyV. We were especially interested in BKPyV because it is a known cause of another bladder disease, hemorrhagic cystitis, in bone marrow transplant recipients. Further analysis of individual samples indicates a trend toward higher excretion of polyomaviruses in patients experiencing increased symptoms.

Current and Future Point-of-Care Tests for Emerging and New Respiratory Viruses and Future Perspectives.

The availability of pathogen-specific treatment options for respiratory tract infections (RTIs) increased the need for rapid diagnostic tests. Besides, retrospective studies, improved lab-based detection methods and the intensified search for new viruses since the beginning of the twenty-first century led to the discovery of several novel respiratory viruses. Among them are human bocavirus (HBoV), human coronaviruses (HCoV-HKU1, -NL63), human metapneumovirus (HMPV), rhinovirus type C (RV-C), and human polyomaviruses (KIPyV, WUPyV). Additionally, new viruses like SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), novel strains of influenza virus A and B, and (most recently) SARS coronavirus 2 (SARS-CoV-2) have emerged. Although clinical presentation may be similar among different viruses, associated symptoms may range from a mild cold to a severe respiratory illness, and thus require a fast and reliable diagnosis. The increasing number of commercially available rapid point-of-care tests (POCTs) for respiratory viruses illustrates both the need for this kind of tests but also the problem, i.e., that the majority of such assays has significant limitations. In this review, we summarize recently published characteristics of POCTs and discuss their implications for the treatment of RTIs. The second key aspect of this work is a description of new and innovative diagnostic techniques, ranging from biosensors to novel portable and current lab-based nucleic acid amplification methods with the potential future use in point-of-care settings. While prototypes for some methods already exist, other ideas are still experimental, but all of them give an outlook of what can be expected as the next generation of POCTs.

Promoter activity of Merkel cell Polyomavirus variants in human dermal fibroblasts and a Merkel cell carcinoma cell line

BackgroundMerkel cell polyomavirus (MCPyV) is a human polyomavirus that establishes a life-long harmless infection in most individuals, with dermal fibroblasts believed to be the natural host cell. However, this virus is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. Several MCPyV variants with polymorphism in their promoter region have been isolated, but it is not known whether these differences affect the biological properties of the virus.MethodsUsing transient transfection studies in human dermal fibroblasts and the MCC cell line MCC13, we compared the transcription activity of the early and late promoters of the most commonly described non-coding control region MCPyV variant and six other isolates containing specific mutation patterns.ResultsBoth the early and late promoters were significantly stronger in human dermal fibroblasts compared with MCC13 cells, and a different promoter strength between the MCPyV variants was observed. The expression of full-length large T-antigen, a viral protein that regulates early and late promoter activity, inhibited early and late promoter activities in both cell lines. Nonetheless, a truncated large T-antigen, which is expressed in virus-positive MCCs, stimulated the activity of its cognate promoter.ConclusionThe promoter activities of all MCPyV variants tested was stronger in human dermal fibroblasts, a cell line that supports viral replication, than in MCC13 cells, which are not permissive for MCPyV. Truncated large T-antigen, but not full-length large T-antigen stimulated viral promoter activity. Whether, the difference in promoter strength and regulation by large T-antigen may affect the replication and tumorigenic properties of the virus remains to be determined.

The Obesity-Related Gut Bacterial and Viral Dysbiosis Can Impact the Risk of Colon Cancer Development.

An incorrect food regimen from childhood is suggested to negatively impact the gut microbiome composition leading to obesity and perhaps to colon rectal cancer (CRC) in adults. In this study, we show that the obesity and cancer gut microbiota share a characteristic microbial profile with a high colonization by mucin degraders species, such as Hafnia alvei and Akkermansia muciniphila. In addition, the species Clostridium bolteae, a bacterium associated with insulin resistance, dyslipidemia, and inflammation, has been associated with the presence of oncogenic Human Polyomaviruses (HPyVs). Merkel cell Polyomavirus (MCPyV) and BK Polyomavirus (BKPyV) were the most frequently oncogenic viruses recovered in the gut of both obese and tumor patients. Considering the high seroprevalence of HPyVs in childhood, their association with specific bacterial species deserve to be further investigated. Data from the present study highlight the presence of a similar microbiome pattern in CRC and obese subjects, suggesting that obese microbiome may represent an opportunity for tumorigenic/driver bacteria and viruses to trigger cell transformation.

JC Virus infected choroid plexus epithelial cells produce extracellular vesicles that infect glial cells independently of the virus attachment receptor.

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial infection with JCPyV is common and the virus establishes a long-term persistent infection in the urogenital system of 50–70% of the human population worldwide. A major gap in the field is that we do not know how the virus traffics from the periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to virus infection together with reports of JCPyV infection of choroid plexus in vivo has led us to hypothesize that the choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major targets of virus infection in the central nervous system (CNS), do not express the known attachment receptors for the virus and do not bind virus in human tissue sections. In this report we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to human glial cells. Transmission of the virus by extracellular vesicles is independent of the known virus attachment receptors and is not neutralized by antisera directed at the virus. We also show that extracellular vesicles containing virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that the choroid plexus plays a fundamental role in the dissemination of virus to brain parenchyma.

Associations of Viral Seroreactivity with AIDS-Related Non-Hodgkin Lymphoma.

Infection with human immunodeficiency virus (HIV) is associated with substantially increased incidence of non-Hodgkin lymphoma (NHL). This risk may be driven, in part, by reduced immune control over viral infections in the setting of acquired immunodeficiency syndrome (AIDS), although the lymphomagenic mechanisms are not yet established. We used bead-based multiplex assays to measure antibody seroreactivity to 32 viral antigens representing 22 different viral infections (human herpesviruses 1-8, hepatitis B and C virus, human T-lymphotropic virus type-1, and human polyomaviruses) in two prospective HIV cohorts. Incident (n = 28) and prevalent (n = 38) AIDS-related NHL cases were matched by age, sex, race, and CD4 count to 67 HIV-positive control individuals without AIDS-NHL. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for associations of AIDS-NHL with the number of different viruses to which an individual was seropositive and seroreactivity to individual antigens. Seropositivity to an increasing number of viruses was inversely associated with AIDS-NHL (OR per virus = 0.84, 95% CI = 0.72-0.98). Seroreactivity to herpes simplex virus 2 2mgG unique antigen (OR = 0.47; 95% CI = 0.23-0.97) and to WU polyomavirus viral capsid protein (OR = 0.26, 95% CI = 0.10-0.65) was significantly lower in AIDS-NHL cases compared to controls. In this evaluation of antibodies to multiple viruses, we observed an inverse association between seropositivity to a larger number of viruses and AIDS-NHL. While in need of further evaluation, our data raise the novel hypothesis that insufficient exposures or impaired humoral immune responses to viral infections may be associated with AIDS-related lymphomagenesis.

Investigation of simian virus 40 (SV40) and human JC, BK, MC, KI, and WU polyomaviruses in glioma.

The gliomagenesis remains not fully established and their etiological factors still remain obscure. Polyomaviruses were detected and involved in several human tumors. Their potential implication in gliomas has been not yet surveyed in Africa and Arab World. Herein, we investigated the prevalence of six polyomaviruses (SV40, JCPyV, BKPyV, MCPyV, KIPyV, and WUPyV) in 112 gliomas from Tunisian patients. The DNA sequences of polyomaviruses were examined by PCR assays. Viral infection was confirmed by DNA in situ hybridization (ISH) and/or immunohistochemistry (IHC). The relationships between polyomavirus infection and tumor features were evaluated. Specific SV40 Tag, viral regulatory, and VP1 regions were identified in 12 GBM (10.7%). DNA ISH targeting the whole SV40 genome and SV40 Tag IHC confirmed the PCR findings. Five gliomas yielded JCPyV positivity by PCR and DNA ISH (2.7%). However, no BKPyV, KIPyV, and WUPyV DNA sequences were identified in all samples. MCPyV DNA was identified in 30 gliomas (26.8%). For GBM samples, MCPyV was significantly related to patient age (p = 0.037), tumor recurrence (p = 0.024), and SV40 (p = 0.045) infection. No further significant association was identified with the remaining tumor features (p > 0.05) and patient survival (Log Rank, p > 0.05). Our study indicates the presence of SV40, JCPyV, and MCPyV DNA in Tunisian gliomas. Further investigations are required to more elucidate the potential involvement of polyomaviruses in these destructive malignancies.

Applicability of crAssphage, pepper mild mottle virus, and tobacco mosaic virus as indicators of reduction of enteric viruses during wastewater treatment

This study was conducted to evaluate the applicability of crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV) as indicators of the reduction of human enteric viruses during wastewater treatment. Thirty-nine samples were collected from three steps at a wastewater treatment plant (raw sewage, secondary-treated sewage, and final effluent) monthly for a 13-month period. In addition to the three indicator viruses, eight human enteric viruses [human adenoviruses, JC and BK polyomaviruses, Aichi virus 1 (AiV-1), enteroviruses, and noroviruses of genogroups I, II, and IV] were tested by quantitative PCR. Indicator viruses were consistently detected in the tested samples, except for a few final effluents for crAssphage and TMV. The mean concentrations of crAssphage were significantly higher than those of most tested viruses. The concentrations of crAssphage in raw sewage were positively correlated with the concentrations of all tested human enteric viruses (p <0.05), suggesting the applicability of crAssphage as a suitable indicator to estimate the concentrations of human enteric viruses in raw sewage. The reduction ratios of AiV-1 (1.8 ± 0.7 log10) were the lowest among the tested viruses, followed by TMV (2.0 ± 0.3 log10) and PMMoV (2.0 ± 0.4 log10). Our findings suggested that the use of not only AiV-1 and PMMoV but also TMV as indicators of reductions in viral levels can be applicable during wastewater treatment.

Sirolimus and Other Mechanistic Target of Rapamycin Inhibitors Directly Activate Latent Pathogenic Human Polyomavirus Replication.

BackgroundHuman polyomaviruses can reactivate in transplant patients, causing nephropathy, progressive multifocal leukoencephalopathy, Merkel cell carcinoma, pruritic, rash or trichodysplasia spinulosa. Sirolimus and related mechanistic target of rapamycin (mTOR) inhibitors are transplant immunosuppressants. It is unknown if they directly reactivate polyomavirus replication from latency beyond their general effects on immunosuppression.MethodsIn vitro expression and turnover of large T (LT) proteins from BK virus, JC virus (JCV), Merkel cell polyomavirus (MCV), human polyomavirus 7 (HPyV7), and trichodysplasia spinulosa polyomavirus (TSV) after drug treatment were determined by immunoblotting, proximity ligation, replicon DNA replication, and whole virus immunofluorescence assays.ResultsmTOR inhibition increased LT protein expression for all 5 pathogenic polyomaviruses tested. This correlated with LT stabilization, decrease in the S-phase kinase-associated protein 2 (Skp2) E3 ligase targeting these LT proteins for degradation, and increase in virus replication for JCV, MCV, TSV, and HPyV7. Treatment with sirolimus, but not the calcineurin inhibitor tacrolimus, at levels routinely achieved in patients, resulted in a dose-dependent increase in viral DNA replication for BKV, MCV, and HPyV7.ConclusionsmTOR inhibitors, at therapeutic levels, directly activate polyomavirus replication through a Skp2-dependent mechanism, revealing a proteostatic latency mechanism common to polyomaviruses. Modifying existing drug regimens for transplant patients with polyomavirus-associated diseases may reduce symptomatic polyomavirus replication while maintaining allograft-sparing immunosuppression.

The Ubiquitin-Specific Protease Usp7, a Novel Merkel Cell Polyomavirus Large T-Antigen Interaction Partner, Modulates Viral DNA Replication.

Merkel cell polyomavirus (MCPyV) is the major cause for Merkel cell carcinoma (MCC), a rare but highly aggressive skin cancer predominantly found in elderly and immunosuppressed patients. The early viral gene products large T-antigen (LT) and small T-antigen (sT) are important for efficient viral DNA replication, and both contribute to transformation processes. These functions are executed mainly through interactions with host factors. Here, we identify the cellular ubiquitin-specific processing protease 7 (Usp7) as a new interaction partner of the MCPyV LT. Using glutathione S-transferase pulldown experiments, we show that MCPyV LT directly binds to Usp7 and that N- as well as C-terminal regions of LT bind to the TRAF (tumor necrosis factor receptor-associated) domain of Usp7. We demonstrate that endogenous Usp7 coprecipitates with MCPyV T-antigens and relocalizes to viral DNA replication centers in cells actively replicating MCPyV genomes. We show that Usp7 does not alter ubiquitination levels of the T-antigens; however, Usp7 binding increases the binding affinity of LT to the origin of replication, thereby negatively regulating viral DNA replication. Together, these data identify Usp7 as a restriction factor of MCPyV replication. In contrast to other DNA viruses, Usp7 does not affect MCPyV gene expression via its ubiquitination activity but influences MCPyV DNA replication solely via a novel mechanism that modulates binding of LT to viral DNA.IMPORTANCE MCPyV is the only human polyomavirus that is associated with cancer; the majority of Merkel cell cancers have a viral etiology. While much emphasis was placed on investigations to understand the transformation process by MCPyV oncoproteins and cellular factors, we have only limited knowledge of cellular factors participating in the MCPyV life cycle. Here, we describe Usp7, a cellular deubiquitination enzyme, as a new factor involved in MCPyV replication. Usp7 is known in the context of large DNA tumor viruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus, to restrict viral replication. Similar to EBV, where Usp7 binding to EBNA1 increases EBNA1 binding affinity to viral DNA, we find MCPyV LT binding to the origin of replication to be increased in the presence of Usp7, resulting in restriction of viral DNA replication. However, Usp7-induced restriction of MCPyV replication is independent of its enzymatic activity, thereby constituting a novel mechanism of Usp7-induced restriction of viral replication.

JCPyV NCCR analysis in PML patients with different risk factors: exploring common rearrangements as essential changes for neuropathogenesis

BackgroundDuring severe immunosuppression or treatment with specific biological drugs, human polyomavirus JC (JCPyV) may establish a lytic infection in oligodendrocytes, leading to progressive multifocal leukoencephalopathy (PML). Beyond AIDS, which represents the most common predisposing condition, several biological drugs have been associated to the development of PML, such as natalizumab, fingolimod and dimethyl fumarate, which have been showed to increase the risk of PML in the multiple sclerosis (MS) population. JCPyV non-coding control region (NCCR) can be found in two different forms: a virulent neurotropic pathogenic form and a latent non-pathogenic form. The neurotropic forms contain a rearranged NCCR and are typically found in the cerebrospinal fluid, brain or blood of PML patients.Case presentationWe sequenced and critically examined JCPyV NCCR from isolates detected in the cerebrospinal fluid of four newly diagnosed progressive multifocal leukoencephalopathy patients: two HIV-positive and two HIV-negative multiple sclerosis patients. More complex NCCR rearrangements were observed in the two HIV-positive patients compared to the HIV-negative multiple sclerosis patients with PML.ConclusionsThe comparison of HIV-positive and HIV-negative MS patients with PML, allowed us to evidence the presence of a common pattern of JCPyV NCCR rearrangement, characterized by the deletion of the D-block, which could be one of the initial rearrangements of JCPyV NCCR needed for the development of PML.

Human polyomavirus modulation of the host DNA damage response.

The human DNA damage response (DDR) is a complex signaling network constituting many factors responsible for the preservation of genomic integrity. Human polyomaviruses (HPyVs) are able to harness the DDR machinery during their infectious cycle by expressing an array of tumor (T) antigens. These molecular interactions between human polyomavirus T antigens and the DDR create conditions that promote viral replication at the expense of host genomic stability to cause disease as well as carcinogenesis in the cases of the Merkel cell polyomavirus and BK polyomavirus. This review focuses on the six HPyVs with disease association, emphasizing strain-dependent differences in their selective manipulation of the DDR. Appreciation of the HPyV-DDR interface at a molecular scale is conducive to the development of novel therapeutic approaches.

Merkel cell polyomavirus detected in head and neck carcinomas from Chile

BackgroundThe role of human polyomaviruses (HPyVs) in epithelial tumors such as head and neck carcinomas (HNSCCs) including oral and oropharyngeal carcinomas has not been established. In this study, we evaluated for the first time the presence of Merkel cell polyomavirus (MCPyV), BK human polyomavirus (BKPyV), and JC human polyomavirus (JCPyV) in HNSCCs from Chilean subjects.MethodsOne hundred and twenty HNSCCs were analyzed for the presence of MCPyV, BKPyV and JCPyV using real-time polymerase chain reaction procedures. In addition, 54 oral brushes from age- and sex-paired subjects were analyzed.ResultsOf the total of 120 HNSCCs, 15 were positive for MCPyV (12.5%). Only one case was positive for BKPyV (0.8%) and none for JCPyV (0%). In subjects without cancer, only one case (1.8%) resulted positive for MCPyV and none for JCPyV and BKPyV. MCPyV was associated with HNSCCs (p = 0.0239; OR = 7.571; 95% CI: 1.192–81.46). No association was found between age (p = 0.1996), gender (p = 0.7111) or differentiation status (p > 0.9999) and MCPyV presence in HNSCCs.ConclusionsMCPyVs were detected in HNSCCs from Chilean patients and were not detected in oral brushes from patients without cancer. More studies are warranted for defining an etiological role and clinical/molecular consequences of these viruses in HNSCCs.

Human Polyomavirus 6 with the Asian–Japanese Genotype in Cases of Kimura Disease and Angiolymphoid Hyperplasia with Eosinophilia

Human Polyomavirus 6 with the Asian–Japanese Genotype in Cases of Kimura Disease and Angiolymphoid Hyperplasia with Eosinophilia