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JC virus quasispecies analysis reveals a complex viral population underlying progressive multifocal leukoencephalopathy and supports viral dissemination via the hematogenous route.

ABSTRACT: Opportunistic infection of oligodendrocytes by human JC polyomavirus may result in the development of progressive multifocal encephalopathy in immunocompromised individuals. Neurotropic JC virus generally harbors reorganized noncoding control region (NCCR) DNA interspersed on the viral genome between early and late coding genes. By applying 454 sequencing on NCCR DNA amplified from body fluid samples (urine, plasma, and cerebrospinal fluid [CSF]) from 19 progressive multifocal leukoencephalopathy (PML) patients, we attempted to reveal the composition of the JC polyomavirus population (the quasispecies, i.e., the whole of the consensus population and minor viral variants) contained in different body compartments and to better understand intrapatient viral dissemination. Our data demonstrate that in the CSF of PML patients, the JC viral population is often a complex mixture composed of multiple viral variants that contribute to the quasispecies. In contrast, urinary JC virus highly resembled the archetype virus, and urine most often did not contain minor viral variants. It also appeared that archetype JC virus could sporadically be identified in PML patient brain, although selection of rearranged JC virus DNA was favored. Comparison of the quasispecies from different body compartments within a given patient suggested a strong correlation between the viral population in plasma and CSF, whereas the viral population shed in urine appeared to be unrelated. In conclusion, it is shown that the representation of viral DNA in the CSF following the high-level DNA replication in the brain underlying PML has hitherto been much underestimated. Our data also underscore that the hematogenous route might play a pivotal role in viral dissemination from or toward the brain.For the first time, the JC polyomavirus population contained in different body compartments of patients diagnosed with progressive multifocal encephalopathy has been studied by deep sequencing. Two main findings came out of this work. First, it became apparent that the complexity of the viral population associated with PML has been highly underestimated so far, suggestive of a highly dynamic process of reorganization of the noncoding control region of JC polyomavirus in vivo, mainly in CSF and blood. Second, evidence showing viral dissemination from and/or toward the brain via the hematogenous route was provided, confirming a hypothesis that was recently put forward in the field.

SUBMITTER: Van Loy T

PROVIDER: S-EPMC4300648 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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JC virus quasispecies analysis reveals a complex viral population underlying progressive multifocal leukoencephalopathy and supports viral dissemination via the hematogenous route.

Van Loy Tom T Thys Kim K Ryschkewitsch Caroline C Lagatie Ole O Monaco Maria C MC Major Eugene O EO Tritsmans Luc L Stuyver Lieven J LJ

Journal of virology 20141112 2

<h4>Unlabelled</h4>Opportunistic infection of oligodendrocytes by human JC polyomavirus may result in the development of progressive multifocal encephalopathy in immunocompromised individuals. Neurotropic JC virus generally harbors reorganized noncoding control region (NCCR) DNA interspersed on the viral genome between early and late coding genes. By applying 454 sequencing on NCCR DNA amplified from body fluid samples (urine, plasma, and cerebrospinal fluid [CSF]) from 19 progressive multifocal ...[more]

PMID: 25392214

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Project description:JC virus (JCV) is a DNA virus causing progressive multifocal leukoencephalopathy (PML) in immunodeficient patients. In the present study, 22 genetic quasispecies with more than 1.5% variant frequency were detected in JCV genomes from six clinical samples of PML by next-generation sequencing. A mutation from A to C at nucleotide (nt) 3495 in JCV Mad1 resulting in a V-to-G amino acid substitution at amino acid (aa) position 392 of the large T antigen (TAg) was identified in all six cases of PML at 3% to 19% variant frequencies. Transfection of JCV Mad1 DNA possessing the V392G substitution in TAg into IMR-32 and human embryonic kidney 293 (HEK293) cells resulted in dramatically decreased production of JCV-encoded proteins. The virus DNA copy number was also reduced in supernatants of the mutant virus-transfected cells. Transfection of the IMR-32 and HEK293 cells with a virus genome containing a revertant mutation recovered viral production and protein expression. Cotransfection with equal amounts of wild-type genome and mutated JCV genome did not reduce the expression of viral proteins or viral replication, suggesting that the mutation did not have any dominant-negative function. Finally, immunohistochemistry demonstrated that TAg was expressed in all six pathological samples in which the quasispecies were detected. In conclusion, the V392G amino acid substitution in TAg identified frequently in PML lesions has a function in suppressing JCV replication, but the frequency of the mutation was restricted and its role in PML lesions was limited. DNA viruses generally have lower mutation frequency than RNA viruses, and the detection of quasispecies in JCV has rarely been reported. In the present study, a next-generation sequencer identified a JCV quasispecies with an amino acid substitution in the T antigen in patients with PML. In vitro studies showed that the mutation strongly repressed the expression of JC viral proteins and reduced the viral replication. However, because the frequency of the mutation was low in each case, the total expression of virus proteins was sustained in vivo. Thus, JC virus replicates in PML lesions in the presence of a mutant virus which is able to repress virus replication.

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.

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JC virus quasispecies analysis reveals a complex viral population underlying progressive multifocal leukoencephalopathy and supports viral dissemination via the hematogenous route.

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JC virus quasispecies analysis reveals a complex viral population underlying progressive multifocal leukoencephalopathy and supports viral dissemination via the hematogenous route.

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