Fig 1.
In situ hybridization (ISH) for miR-J1 and miR-B1 in clinical samples.
(A–D) PML. (A) Haematoxylin and eosin (HE) and (B) immunohistochemistry for JCPyV VP1, (C) ISH for miR-J1-5p, and (D) ISH for miR-J1-3p. (E–H) BKPyV-associated nephropathy. (E) HE and (F) immunohistochemistry for BKPyV VP1, (G) ISH for miR-B1-5p, and (H) ISH for miR-J1-3p.
Fig 2.
Specificity of in situ hybridization (ISH) for miR-J1 and Ago2 expression in clinical samples.
(A) ISH for miR-J1 in clinical samples. HE and ISH for miR-J1-5p, miR-J1-3p, and a scramble probe are shown. All control samples were obtained from patients with conditions unrelated to PML or BKPyV-associated nephropathy. (B) miR-J1, JCPyV-VP1, and Ago2 protein expression in a case of PML. In situ hybridization detected miR-J1 in the PML case (upper left). JCPyV VP1 (upper right) and Ago2 (lower panels) were detected with immunohistochemistry in a PML lesion (upper right and lower left) and non-PML lesion (lower right). (C) Immunofluorescence assay for JCPyV VP1, agnoprotein, and Ago2 in a case of PML. JCPyV proteins (VP1 and agnoprotein) and Ago2 were labelled with red and green, respectively.
Table 1.
In situ hybridization for polyomavirus miRNA in pathological samples.
Table 2.
Results of in situ hybridization (ISH), immunohistochemistry (IHC), and PCR in histological samples.
Fig 3.
Detection of miR-J1 expression by northern blot and real-time PCR.
(A) Northern blot analysis. Three PML and non-PML brain tissues were examined. (B) Stem-loop real-time RT-PCR for miR-J1 in clinical samples from PML (n = 10) and non-PML (n = 3) subjects. The relative ratio of miR-J1/miR21 is shown. Horizontal and vertical bars indicate the mean and standard error, respectively. (C) Correlation between miR-J1 and JCPyV-DNA copy numbers. The numbers of miR-J1 copies per ng of RNA and JCPyV DNA copies per ng of DNA in each sample were plotted on the vertical and horizontal axes, respectively. Ten samples of JCPyV-positive PML tissues were examined. The Spearman’s rank correlation is indicated at the bottom of each graph.
Fig 4.
Expression profile of miR-J1 by next generation sequencing.
(A) Read coverages of miRNA reads for pre-miR-J1 and miR-B1. Pre-miRNA sequences are shown at the bottom of the graph. Numbers under the bars of 5p and 3p indicate the start and stop nucleotides of 5p and 3p miRNA, respectively, in the reference sequences (JCPyV: J02226 and BKPyV: NC_001538). (B) Read counts of exact and non-exact mature miRNA in each sample.
Table 3.
NGS data from circularized JCPyV Mad1 DNA-transfected IMR32 cells, two PML cases, and one BKV-associated nephropathy case.
Table 4.
Polyomavirus-encoded miRNAs are indicated in red.
Fig 5.
JCPyV replication in miR-J1-mutants.
(A) Construction of JCPyV mutants. Point or deletion mutants were constructed in pre-miR-J1 of the JCPyV genome. (B) Western blot analysis on lysates of mutant-transfected IMR32 cells. Each number (1–6) corresponds to that in (A). The results from samples taken on day 2, 4, and 6 post-transfection are shown.