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High expression of JC polyomavirus-encoded microRNAs in progressive multifocal leukoencephalopathy tissues and its repressive role in virus replication.

ABSTRACT: JC polyomavirus (JCPyV, JCV) causes progressive multifocal leukoencephalopathy (PML) in immunocompromised hosts. JCPyV replicates in oligodendrocytes within the brain tissue of patients with PML. The JCPyV genome encodes a microRNA (miRNA) in the region encoding the large T antigen. JCPyV-encoded miRNA (miR-J1) has been detected in the tissue and cerebrospinal fluid samples of patients with PML; however, there are no reports describing the localization of polyomavirus-encoded miRNA in histological samples of patients with virus-associated diseases. In the present study, we detected high miR-J1 expression in the nuclei of JCPyV-infected cells in PML tissue samples via in situ hybridization. Additionally, in situ hybridization also revealed the expression of BK polyomavirus (BKPyV, BKV)-encoded miRNA in lesions of BKPyV-associated nephropathy. In situ hybridization for miR-J1-5p and -3p showed positive signals in 24/25 (96%) of PML tissues that were positive for JCPyV by immunohistochemistry. Higher copy numbers of miR-J1 were detected in PML tissues than in non-PML tissues by real-time reverse transcription PCR. Next generation sequencing showed that miR-J1-5p, a mature miRNA of primary miRNA, was predominant in the lesions compared with miR-J1-3p, another mature miRNA. Deletion or mutation of miR-J1 in recombinant JCPyV promoted the production of JCPyV-encoded proteins in cells transfected with JCPyV DNA, suggesting that polyomavirus-encoded miRNA may have a repressive role in viral replication in PML tissues. In situ hybridization for viral miRNA may be a useful diagnostic tool for PML.

SUBMITTER: Takahashi K

PROVIDER: S-EPMC7200015 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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High expression of JC polyomavirus-encoded microRNAs in progressive multifocal leukoencephalopathy tissues and its repressive role in virus replication.

Takahashi Kenta K Sato Yuko Y Sekizuka Tsuyoshi T Kuroda Makoto M Suzuki Tadaki T Hasegawa Hideki H Katano Harutaka H

PLoS pathogens 20200423 4

JC polyomavirus (JCPyV, JCV) causes progressive multifocal leukoencephalopathy (PML) in immunocompromised hosts. JCPyV replicates in oligodendrocytes within the brain tissue of patients with PML. The JCPyV genome encodes a microRNA (miRNA) in the region encoding the large T antigen. JCPyV-encoded miRNA (miR-J1) has been detected in the tissue and cerebrospinal fluid samples of patients with PML; however, there are no reports describing the localization of polyomavirus-encoded miRNA in histologic ...[more]

PMID: 32324824

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Project description:The human JC polyomavirus (JCPyV) is the causative agent of the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). The Mad-1 prototype strain of JCPyV uses the glycan lactoseries tetrasaccharide c (LSTc) and serotonin receptor 5-HT2A to attach to and enter into host cells, respectively. Specific residues in the viral capsid protein VP1 are responsible for direct interactions with the ?2,6-linked sialic acid of LSTc. Viral isolates from individuals with PML often contain mutations in the sialic acid-binding pocket of VP1 that are hypothesized to arise from positive selection. We reconstituted these mutations in the Mad-1 strain of JCPyV and found that they were not capable of growth. The mutations were then introduced into recombinant VP1 and reconstituted as pentamers in order to conduct binding studies and structural analyses. VP1 pentamers carrying PML-associated mutations were not capable of binding to permissive cells. High-resolution structure determination revealed that these pentamers are well folded but no longer bind to LSTc due to steric clashes in the sialic acid-binding site. Reconstitution of the mutations into JCPyV pseudoviruses allowed us to directly quantify the infectivity of the mutants in several cell lines. The JCPyV pseudoviruses with PML-associated mutations were not infectious, nor were they able to engage sialic acid as measured by hemagglutination of human red blood cells. These results demonstrate that viruses from PML patients with single point mutations in VP1 disrupt binding to sialic acid motifs and render these viruses noninfectious. IMPORTANCE Infection with human JC polyomavirus (JCPyV) is common and asymptomatic in healthy individuals, but during immunosuppression, JCPyV can spread from the kidney to the central nervous system (CNS) and cause a fatal, demyelinating disease, progressive multifocal leukoencephalopathy (PML). Individuals infected with HIV, those who have AIDS, or those receiving immunomodulatory therapies for autoimmune diseases are at serious risk for PML. Recent reports have demonstrated that viral isolates from PML patients often have distinct changes within the major capsid protein. Our structural-functional approach highlights that these mutations result in abolished engagement of the carbohydrate receptor motif LSTc that is necessary for infection. Viruses with PML-associated mutations are not infectious in glial cells, suggesting that they may play an alternative role in PML pathogenesis.

Project description:BackgroundLytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples.MethodsA total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab.ResultsJCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV.ConclusionThis study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients.

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High expression of JC polyomavirus-encoded microRNAs in progressive multifocal leukoencephalopathy tissues and its repressive role in virus replication.

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High expression of JC polyomavirus-encoded microRNAs in progressive multifocal leukoencephalopathy tissues and its repressive role in virus replication.

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