JC Virus-Induced Changes in Cellular Gene Expression in Primary Human Astrocytes

Abstract

Cell-type-specific transcription of the JC virus (JCV) promoter in glial cells initiates a series of events leading to viral replication in the brain and the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in patients with neurologic complications due to infection with human immunodeficiency virus type 1. Here we employed an in vitro infection of primary cultures of human astrocytes to compare the transcriptional profile of cellular genes after JCV infection by using an oligonucleotide-based microarray of 12,600 genes. Transcription of nearly 355 genes was enhanced and expression of 130 genes was decreased to various degrees. Many transcripts that were increased upon JCV infection were found to encode proteins with properties that suggest their involvement in cell proliferation, including cyclin A and cyclin B1; signaling pathways, such as transforming growth factor ␤ receptor 1, platelet-derived growth factor receptor and fibroblast growth factor family receptor; and other regulatory events, such as inflammatory responses, including cyclo-oxygenase-2 (Cox-2). Microarray-based data for several cell cycle-regulatory genes were further examined by using Western blot analysis of in vitro infected astrocytes harvested early and late during the infection. Results demonstrate that protein levels of all upregulated genes were found to increase at some point during the infection time course. In parallel, immunohistochemical assessment of cell cycle proteins, including cyclins A, B1, E, and Cdk2, showed positive staining of astrocytes within PML lesions of brain tissue from patients with neuro-AIDS. Microarray analysis was found to be a useful predictor of gene expression in infected cells; however, it may not directly correlate with protein levels during infection with JCV.The human polyomavirus, JC (JCV), is the etiologic agent for the fatal demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML) (for review see references 5 and 11). Once a rare disease seen primarily in patients with impaired immune systems due to myeloproliferative and lymphoproliferative disorders, the incidence of PML has been significantly increased due to the AIDS epidemic (27). Clinical studies show that Ͼ4% of AIDS patients with neurologic problems are diagnosed with PML (1,2,6,7,10,18,19,20,26,31,32). JCV infects Ͼ65% of the human population by early childhood with no major clinical symptoms (16,23,28,30). Reactivation of the viral genome that preferentially occurs in oligodendrocytes and astrocytes leads to cytolytic destruction of the myelin-producing oligodendrocytes and degeneration of the myelin sheath in white matter. The chief pathological features of PML are the presence of altered oligodendrocytes whose nuclei are two to three times the size of normal cells and are filled with virions and viral antigens (34-36); giant, bizarre astrocytes with pleomorphic, hyperchromatic nuclei; and occasional mitotic figures. Early studies revealed that expre...

Intracellular Approach for Blocking JC Virus Gene Expression by Using RNA Interference during Viral Infection

“…4, high levels of T antigen and VP1 were expressed and were appropriately localized in the nuclei of JCV-infected primary human fetal astrocytic cells. Also, in accord with previous reports (8,19,20), high levels of agnoprotein with cytoplasmic perinuclear accumulation were observed in the JCV-infected cells. Treatment of the cells with JCV T-antigen siRNA caused a drastic decrease in the expression of both T antigen and VP1 but had a lesser effect on agnoprotein production.…”

“…The cells were subcultured and plated onto poly-L-lysine-coated chamber slides (Falcon) on day 13 and were fixed on day 15 postinfection with ice-cold acetone for 3 min. Viral proteins were detected by immunocytochemistry as described previously (20), using the same primary antibodies as for the Western blot analysis (see the legend to Fig. 1).…”

“…JCV T-antigen siRNA decreases expression of JCV proteins in transiently transfected and infected primary human astrocytes. Primary human fetal astrocytes were prepared as described previously and were seeded into six-well plates at a density of 500,000 cells/well (20). For transient transfections, cells were transfected by using Fu-GENE 6 with plasmid expressing JCV T antigen (15).…”

Human glial chimeric mice reveal astrocytic dependence of JC virus infection

“…Although PML has traditionally been viewed as a disease of oligodendrocytes, both astrocyte and GPC infection have been reported in vitro (9)(10)(11)(12)(13). Therefore, we first sought to assess the phenotypic selectivity of viral infection and propagation among the different phenotypes of human macroglia.…”

Host Cell Gene Expression during Human Immunodeficiency Virus Type 1 Latency and Reactivation and Effects of Targeting Genes That Are Differentially Expressed in Viral Latency

“…Other studies have described changes in cellular gene expression due to acute infection following the addition of infectious virus to cells (16,29,67), and additional studies have described changes in host cell gene expression that accompany infections by other viruses, offering interesting insights into viral pathogenesis (12,33,37,51,63,72). While these studies provide insights into the extensive effects of specific viral proteins or acute HIV infection (reviewed in reference 4), the global gene expression changes that may accompany reactivation of cells latently infected with HIV and completion of the lytic phase of viral replication have not been described.…”