SF2/ASF binding region within JC virus NCCR limits early gene transcription in glial cells

Abstract

Background: Patients 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.

Results: The 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.

Conclusions: The results of this study reveal a differential role of CR3 region within JCV NCCR in expression of JCV early and late genes.

Figure 1

The “CR3” region within JCV NCCR is the target for SF2/ASF. A. Sequence alignment for the NCCR region of JCV. CLUSTAL sequence alignment was performed for JCV Mad1 and Archetype strains (gene bank accession number NC_001699). The positions of replication origin (ORI), CR1, CR2, CR3, CR4 and 98-bp-tandem repeats are illustrated in Mad1 strain. CR3 region is highlighted with sequences in red. Numbering is relative to the Mad-1 strain of JCV. B. Upper panel: Schematic presentation of Mad1-WT, Mad1-(1X98), and ΔCR3-(1X73) promoter. Arrows point the position of forward (PF) and reverse (PR) primers used in ChIP experiments. Lower panel: PHFA cells were transiently transfected with pCGT7-SF2/ASF expression vector (SF2) or pCGT7 vector alone (vector) and pBLCAT3-JCV-RR-WT, pBLCAT3-JCV-RR-(1X98), and pBLCAT3-JCV-RR-ΔCR3-(1X73) reporter plasmids. Cells were cross-linked and ChiP assay was performed using antibody to T7-tagged SF2/ASF (lanes 4 to 12). In lanes 1, 2 and 3, pBLCAT3-JCV-RR-WT, pBLCAT3-JCV-RR-(1X98), and pBLCAT3-JCV-RR-ΔCR3-(1X73) reporter plasmid DNAs were used as positive controls. C. Western blot analyzes of whole cell lysates from panel A (lanes 4 to 12), using specific antibodies against SF2/ASF and Tubulin.

Figure 2

Transcriptional activities of mutant JCV promoter sequences. A. Cat enzyme activity of JCV-early promoter constructs were detected, and presented as bar graph. Schematic representation of JCV NCCR sequences cloned into CAT reporter constructs in early orientations was shown at the top of the graph. B. Effect of SF2/ASF on transcription induced by mutant JCV promoter sequences. pBLCAT3-JCVE-RR WT, pBLCAT3-JCVE-RR-(1X98), and pBLCAT3-JCVE-RR- ΔCR3-(1X73) reporter plasmids were transiently transfected into PHFA cells in the presence or absence of either pCGT7 vector alone or pCGT7-SF2/ASF expression plasmid. Cat enzyme activity of JCV promoter constructs were detected, and presented as bar graph.

Figure 3

Viral propagation of mutant JCV strains in PHFA cells. A. Schematic representation of JCV wild type and mutant genomes. B. Western blot analyzes of whole cell extracts from PHFA cells infected with JCV-Mad1-WT and mutants, JCV-Mad1-(1x98), and JCV-Mad1-ΔCR3-(1X73), using specific antibodies against LT-Ag, VP1, SF2/ASF and Agno protein. In lanes 7 and 8, whole cell extracts from uninfected cells were loaded as negative control. Western blot analyzes of same extracts with anti-Grb2 antibody was used as loading control. DPI depicts “day post-infection”. C. Southern blot analyses of replicated JCV genomic DNAs in parallel to protein samples in panel A. In lanes 1, 2 and 3, 2 ng of linearized Mad1-WT, Mad1-(1X98), and Mad1-ΔCR3-(1X73) were used as positive controls, respectively. In lane 4, DNA samples from uninfected cells were loaded as negative control. D. Q-PCR analyses of the JCV copy numbers in growth media of the infected PHFA cultures. Culture media was collected at 14 dpi, and was processed for the detection of viral particles by Q-PCR as described earlier [12,16].

Figure 4

The “CR3” region within JCV NCCR is critical for the expression of late genes. Cat enzyme activity of JCV-late promoter constructs were detected, and presented as bar graph. Schematic representation of JCV NCCR sequences cloned into CAT reporter constructs in late orientations was shown at the top of the graph.