Abstract
BackgroundHuman polyomavirus JCV is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease characterized by lytic infection of glial cells in the central nervous system. PML is seen primarily in immunosuppressed patients and is mainly classified as an AIDS-defining disease. In addition to structural capsid proteins, JCV encodes multiple regulatory proteins, including T-antigen and agnoprotein, which are required for functional lytic infection. Previous studies have suggested that molecular interaction between viral proteins and host factors play an important role in reactivation of JCV and progression of the viral life cycle in glial cells. Recently, serine/arginine rich splicing factor 1 (SRSF1), a cellular alternative splicing factor, was identified as a strong negative regulator of JCV in glial cells. SRSF1 inhibits JCV gene expression and viral replication by directly interacting with viral promoter sequences. Here, we have investigated possible impact of JCV regulatory proteins, T-antigen and agnoprotein, on SRSF1-mediated suppression of JCV gene expression in glial cells.ResultsReporter gene analysis has suggested that T-antigen rescues viral transcriptional suppression mediated by SRSF1. Further analyses have revealed that T-antigen promotes viral gene expression by suppressing SRSF1 gene transcription in glial cells. A subsequent ChIP analysis revealed that T-antigen associates with the promoter region of SRSF1 to induce the transcriptional suppression.ConclusionsThese findings have revealed a molecular interplay between cellular SRSF1 and viral T-antigen in controlling JCV gene expression, and may suggest a novel mechanism of JCV reactivation in patients who are at risk of developing PML.
Highlights
JC virus (JCV) is a human polyomavirus and the etiological agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the white matter [1,2,3]
These results have revealed a novel interaction between T-antigen and serine/arginine rich splicing factor 1 (SRSF1) in controlling JCV gene transcription and replication, which may suggest a unique mechanism of JCV reactivation in patients who are at risk of developing PML
Effect of JCV regulatory proteins, T-antigen and agnoprotein, on SRSF1-mediated suppression of JCV transcription In order to investigate the role of JCV regulatory proteins T-antigen and agnoprotein in SRSF1-mediated suppression of JCV gene expression, primary human fetal astrocytes (PHFA) were transiently transfected with a luciferase reporter construct in both early and late orientations
Summary
Effect of JCV regulatory proteins, T-antigen and agnoprotein, on SRSF1-mediated suppression of JCV transcription In order to investigate the role of JCV regulatory proteins T-antigen and agnoprotein in SRSF1-mediated suppression of JCV gene expression, primary human fetal astrocytes (PHFA) were transiently transfected with a luciferase reporter construct in both early and late orientations (pLuc.JCV-Early or pLuc.JCV-Late). Luciferase assay of JCV-early gene transcription showed a strong suppression with SRSF1, which was rescued by co-expression of T-antigen, as well as a significant increase in transcription with the expression of T-antigen alone (Fig. 1a). Western blot analysis of the whole cell protein extracts were completed, demonstrating overexpression of SRSF1 and the expression of the viral proteins that were transfected in parallel to luciferase assays (Fig. 1b, d) These results suggest a novel role of viral Tantigen in suppression of cellular SRSF1 in glial cells. D. Western blot analysis of whole cell extracts prepared in parallel to the samples in panel c it was found that higher levels of T-antigen correspond to a greater decrease in the transcriptional activity of SRSF1, which accounts for the decreased expression of SRSF1 in T-antigen expressing cells. ChIP analysis of the cells demonstrated the association of T-antigen with SRSF1 promoter sequences (Fig. 3b)
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