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Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection.

ABSTRACT: John Cunningham virus (JCPyV) is an ubiquitous human pathogen that causes disease in immunocompromised patients. The JCPyV genome is composed of an early region and a late region, which are physically separated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been very limited in their availability and robustness. Prior to this study, it was demonstrated that JCPyV archetype DNA replicates in COS-7 simian kidney cells expressing SV40 TAg and COS-7 cells expressing HIV-1 Tat. Based on these observations, the present study was conducted to reproduce an in vitro model in COS-7 cells transfected with the JCPyV archetype strain in order to study JCPyV DNA replication and analyze NCCR rearrangements during the viral life cycle. The efficiency of JCPyV replication was evaluated by quantitative PCR (Q-PCR) and by hemagglutination (HA) assay after transfection. In parallel, sequence analysis of JCPyV NCCR was performed. JCPyV efficiently replicated in kidney-derived COS-7 cells, as demonstrated by a progressive increase in viral load and virion particle production after transfection. The archetypal structure of NCCR was maintained during the viral cycle, but two characteristic point mutations were detected 28 days after transfection. This model is a useful tool for analyzing NCCR rearrangements during in vitro replication in cells that are sites of viral persistence, such as tubular epithelial cells of the kidney.

SUBMITTER: Prezioso C

PROVIDER: S-EPMC5671531 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection.

Prezioso Carla C Scribano Daniela D Bellizzi Anna A Anzivino Elena E Rodio Donatella Maria DM Trancassini Maria M Palamara Anna Teresa AT Pietropaolo Valeria V

Archives of virology 20170907 12

John Cunningham virus (JCPyV) is an ubiquitous human pathogen that causes disease in immunocompromised patients. The JCPyV genome is composed of an early region and a late region, which are physically separated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been ve ...[more]

PMID: 28884263

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Project description:BackgroundPatients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the white matter caused by human neurotropic polyomavirus, JC virus. It is now widely accepted that pathologic strains of JCV shows unique rearrangements consist of deletions and insertions within viral NCCR. While these kinds of rearrangements are related to viral tropism and pathology of the disease, their roles in molecular regulation of JCV gene expression and replication are unclear. We have previously identified SF2/ASF as a negative regulator of JCV gene expression in glial cells. This negative impact of SF2/ASF was dependent on its ability to bind a specific region mapped to the tandem repeat within viral promoter. In this report, functional role of SF2/ASF binding region in viral gene expression and replication was investigated by using deletion mutants of viral regulatory sequences.ResultsThe second 98-base-pair tandem repeat on Mad1 strain was first mutated by deletion and named Mad1-(1X98). In addition to this mutant, the CR3 region which served the binding side for SF2/ASF was also mutated and named Mad1-ΔCR3 (1X73). Both mutations were tested for SF2/ASF binding by ChIP assay. While SF2/ASF was associated with Mad1-WT and Mad1-(1X98), its interaction was completely abolished on Mad1-ΔCR3 (1X73) construct as expected. Surprisingly, reporter gene analysis of Mad1-(1X98) and Mad1-ΔCR3 (1X73) early promoter sequences showed two and three fold increase in promoter activities, respectively. The impact of "CR3" region on JCV propagation was also tested on the viral background. While replication of Mad1-(1X98) strain in glial cells was similar to Mad1-WT strain, propagation of Mad1-ΔCR3 (1X73) was less productive. Further analysis of the transcription mediated by Mad1-ΔCR3 (1X73) NCCR revealed that late gene expression was significantly affected.ConclusionsThe results of this study reveal a differential role of CR3 region within JCV NCCR in expression of JCV early and late genes.

Dataset Information

Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection.

Publications

Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection.

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