Detection of JC virus DNA in human tonsil tissue: evidence for site of initial viral infection

Abstract

Progressive multifocal leukoencephalopathy is a demyelinating disease of the human central nervous system that results from lytic infection of oligodendrocytes by the polyomavirus JC (JCV). Originally, JCV was thought to replicate exclusively in human glial cells, specifically oligodendrocytes. However, we have recently shown that JCV can replicate in cells of lymphoid origin such as hematopoietic precursor cells, B lymphocytes, and tonsillar stromal cells. To determine whether tonsils harbor JCV, we tested a total of 54 tonsils, 38 from children and 16 from adult donors. Nested PCRs with primer sets specific for the viral T protein and regulatory regions were used for the detection of JCV DNA. JCV DNA was detected in 21 of 54 tonsil tissues, or 39% (15 of 38 children and 6 of 16 adults) by using regulatory-region primers and in 19 of 54 tonsil tissues, or 35% (13 of 38 children and 6 of 16 adults) by using the T-protein primers. The DNA extracted from children's nondissected tonsil tissue, isolated tonsillar lymphocytes, and isolated stromal cells that demonstrated PCR amplification of the JCV regulatory region underwent cloning and nucleotide sequencing. Of the regulatory-region sequences obtained, nearly all contained tandem repeat arrangements. Clones originating from nondissected tonsil tissue and tonsillar lymphocytes were found to have sequences predominantly of the Mad-1 prototype strain, whereas the majority of clones from the DNA of tonsillar stromal cells had sequences characteristic of the Mad-8br strain of JCV. A few clones demonstrated structures other than tandem repeats but were isolated only from tonsillar lymphocytes. These data provide the first evidence of the JCV genome in tonsil tissue and suggest that tonsils may serve as an initial site of viral infection.

Figures

FIG. 1

Schematic diagram of the JCV genome, including the nucleotide positioning and 5′-to-3′ conformation of primer pairs used for n-PCR amplification of the T protein and the regulatory region (RR). Broad arrows represent early (dark shading) and late (light shading) protein coding regions roughly divided by the origin of DNA replication (ori). Dots represent the noncoding region of the large T protein. Numbering is adopted from the prototype Mad-1 sequence (16). Int., internal primer; Ext., external primer.

FIG. 2

Detection of the JCV genome by n-PCR amplification and Southern blot hybridization from tonsillar lymphocytes. The primer sets used in this experiment were specific for the T-protein region of the JCV genome. The internal primers amplified a 577-bp segment. Lanes marked 48 to 54 contained DNA extracted from tonsillar lymphocytes isolated from tonsil tissue of healthy donors. Negative control, PCR amplification without template. HaeIII-digested φX, DNA molecular weight markers. Positive control, JCV plasmid pM1TC.

FIG. 3

Detection of the JCV genome by n-PCR amplification and Southern blot analysis of tonsillar lymphocytes and stromal cells isolated from tonsil tissue. For amplification of segments of the JCV genome regulatory region, primer pairs which amplified a 388-bp internal segment were used. Lanes marked 51 through 53 contain DNA extracted from isolated and cultured tonsillar lymphocytes and DNA extracted from stromal cells from three different donors. Positive and negative controls are similar to those in Fig. 2.

FIG. 4

EcoRI restriction enzyme digests of JCV regulatory-region clones from nondissected tonsil tissue, tonsillar lymphocytes, and stromal cells isolated from tonsil 53. DNA fragments were visualized after staining with ethidium bromide and were photographed with UV light. The upper bands represent vector pCR2.1 (Invitrogen). The lower bands are excised inserts that were subsequently sequenced. The different molecular weights of the insert bands correspond to different viral regulatory-region sequences.

FIG. 5

Representative sequences of four variations in the regulatory region of JCV, derived from 59 different clones sequenced from pediatric and adult donor tonsil tissue, tonsillar lymphocytes, and stromal cells (adapted from reference 28). See Table 2.