Rearranged JC virus noncoding control regions found in progressive multifocal leukoencephalopathy patient samples increase virus early gene expression and replication rate

Abstract

Polyomavirus JC (JCV) infects ∼ 60% of the general population, followed by asymptomatic urinary shedding in ∼ 20%. In patients with pronounced immunodeficiency, including HIV/AIDS, JCV can cause progressive multifocal leukoencephalopathy (PML), a devastating brain disease of high mortality. While JCV in the urine of healthy people has a linear noncoding control region called the archetype NCCR (at-NCCR), JCV in brain and cerebrospinal fluid (CSF) of PML patients bear rearranged NCCRs (rr-NCCRs). Although JCV NCCR rearrangements are deemed pathognomonic for PML, their role as a viral determinant is unclear. We sequenced JCV NCCRs found in CSF of eight HIV/AIDS patients newly diagnosed with PML and analyzed their effect on early and late gene expression using a bidirectional reporter vector recapitulating the circular polyomavirus early and late gene organization. The rr-NCCR sequences were highly diverse, but all increased viral early reporter gene expression in progenitor-derived astrocytes, glia-derived cells, and human kidney compared to the expression levels with the at-NCCR. The expression of simian virus 40 (SV40) large T antigen or HIV Tat expression in trans was associated with a strong increase of at-NCCR-controlled early gene expression, while rr-NCCRs were less responsive. The insertion of rr-NCCRs into the JCV genome backbone revealed higher viral replication rates for rr-NCCR compared to those of the at-NCCR JCV in human progenitor-derived astrocytes or glia cells, which was abrogated in SV40 large T-expressing COS-7 cells. We conclude that naturally occurring JCV rr-NCCR variants from PML patients confer increased early gene expression and higher replication rates compared to those of at-NCCR JCV and thereby increase cytopathology.

FIG. 1.

JCV NCCR architecture and reporter gene expression in PDA cells. The at-NCCR of JCV is shown with the following arbitrarily denoted blocks (the number of base pairs is in parentheses): Ori(117)-A(36)-B(23)-C(55)-D(66)-E(18)-F(69). Also shown is the architecture of the rr-NCCRs. Pt, patient; source, origin of sample; ins, insertions corresponding to duplications of the numbered base pairs; del, deletions denoted by gaps and nucleotide number; confluence, samples showing phase contrast; early, red fluorescence; late, green fluorescence. Cells were transfected with the indicated bidirectional reporter constructs, and expression was quantified at 2 dpt. Expression level indicates the fold induction of green and red fluorescence-positive cells normalized to GFP- and RFP-positive cells bearing pHRG/urine at the at-NCCR; ratio, the number of RFP-positive cells divided by the number of GFP-positive cells; n.a., not applicable, since the NCCR has been generated by in vitro mutagenesis or represents the NCCR of reference strain Mad-4. Means from three independent experiments are shown. Error bars represent the means ± standard deviations.

FIG. 2.

Reporter gene expression in progenitor-derived astrocytes (PDA). The distribution of the red (early gene) and green (late gene) fluorescence of at-NCCR and rearranged CSF-1 (patient 1) rr-NCCR dual-reporter gene expression was examined in PDA cells by automated microscopy and MetaXpress software (see Materials and Methods). The histogram displays pixels carrying red and green signal distributed according to their relative signal intensity along the respective axes.

FIG. 3.

Replication of rr-NCCR JCV in cell culture. (A) Time course for prototypes CSF (patient 8), CSF-1 (patient 1), Mad-4, and archetype urine (patient 1) in Hs683 cells. DNase-protected JCV DNA was quantified by qPCR from cell supernatants collected at the indicated times after transfection. The data display the means from two independent experiments determined in triplicate. Error bars indicate the means ± standard deviations. (B) Immunofluorescence of JCV prototypes CSF (patient 8) (a), CSF-1 (patient 1) (b), Mad-4 (c), and urine (patient 1) (d) was performed at 7 dpt in Hs683 cells. Large T antigen (red) and agnoprotein (green) were detected using monoclonal mouse anti-large T antigen visualized with anti-mouse Cy3 and polyclonal rabbit anti-agno stained by anti-rabbit Cy2, respectively. Cell nuclei were visualized by Hoechst 33342 dye (blue). (C) Time course for prototypes in PDA cells as described for panel A. DNase-protected JCV DNA was quantified by qPCR from cell supernatants collected at the indicated times after infection. The data display the means from two independent experiments determined in triplicate. Error bars indicate the means ± standard deviations. (D) Immunofluorescence for prototypes was performed at 14 dpi in PDA cells as described for panel B.

FIG. 4.

Replication of recombinant rr-NCCR JCV in COS-7 cells. (A) Time course for prototypes CSF (patient 8), CSF-1 (patient 1), Mad-4, and urine (patient 1). DNase-protected JCV DNA was quantified by qPCR from cell supernatants collected at the indicated times after transfection. The data display the means from two independent experiments determined in triplicate. Error bars indicate the means ± standard deviations. (B) Immunofluorescence of JCV prototypes CSF (patient 8) (a), CSF-1 (patient 1) (b), Mad-4 (c), and urine (patient 1) (d) for VP1 (red) and agnoprotein (green) was performed at 6 dpt using monoclonal mouse anti-VP1 visualized with anti-mouse Cy3 and polyclonal rabbit anti-agno stained by anti-rabbit Cy2, respectively. Cell nuclei were detected by Hoechst 33342 dye (blue).

FIG. 5.

Modulation of NCCR-directed viral early and late gene expression in trans. (A) Effect of SV40 LTag on NCCR-mediated reporter gene expression. RFP-positive cells (red) were analyzed in CV-1 and COS-7 cells at 2 dpt. Early gene expression was normalized for at-NCCR-mediated expression in CV-1 cells. (B) Effect of HIV Tat on NCCR-directed reporter gene expression. At 2 dpt, RFP-positive (early) and GFP-positive (late) cells were analyzed after the transfection of pHRGNCCR reporter constructs into HeLa cells or HeLa SX cells constitutively expressing HIV Tat protein. The number of RFP-positive cells was set to 1 for at-NCCR-mediated reporter gene expression. The data display means from two independent experiments. Error bars indicate the means ± standard deviations.