Detection of JC virus DNA fragments but not proteins in normal brain tissue

Abstract

Objective: Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the white matter affecting immunocompromised patients that results from the cytolytic destruction of glial cells by the human neurotropic JC virus (JCV). According to one model, during the course of immunosuppression, JCV departs from its latent state in the kidney and after entering the brain, productively infects and destroys oligodendrocytes. The goal of this study was to test the hypothesis that JCV may reside in a latent state in a specific region of the brains of immunocompetent (non-PML) individuals without any neurological conditions.

Methods: Gene amplification was performed together with immunohistochemistry to examine the presence of JCV DNA sequences and expression of its genome in five distinct regions of the brain from seven immunocompetent non-PML individuals.

Results: Although no viral proteins were expressed in any of these cases, fragments of the viral DNA were present in various regions of normal brain. Laser-capture microdissection showed the presence of JCV DNA in oligodendrocytes and astrocytes, but not in neurons.

Interpretation: The detection of fragments of viral DNA in non-PML brain suggests that JCV has full access to all regions of the brain in immunocompetent individuals. Thus, should the immune system become impaired, the passing and/or the resident virus may gain the opportunity to express its genome and initiate its lytic cycle in oligodendrocytes. The brain as a site of JCV latency is a possibility.

Fig 1

Structural organization of JC virus (JCV) genome. The bidirectional control region of Mad-1 composed of a 98bp tandem repeat, the early genome with the ability to express several proteins, T-antigen family, the early leader protein (ELP), and the late region with four distinct open-reading frames for the regulatory agnoprotein, and capsid proteins VP1, VP2, and VP3 are shown. The positions of the primers and probes for amplification of the various regions of the JCV genome are shown.

Fig 2

Detection of JC virus (JCV) DNA sequence corresponding to the early genome. (A) Representative Southern blot analysis of amplified DNA from two cases using primers that are specific for the JCV early gene and the housekeeping gene, GAPDH. Negative and positive controls demonstrate results from reactions containing no DNA and DNA from brains of patients with progressive multifocal leukoencephalopathy (PML), respectively. Lane 8 represents gene amplification using pBJC, a plasmid containing JCV DNA as a template. Lane 7 in the bottom panel corresponding to GAPDH demonstrates gene amplification using DNA from the human astrocytic cell line, U-87MG. (B) Representative results from immunohistochemical labeling for detection of JCV early protein, T-antigen in the various regions of non-PML brain (Case 4) and in a section of a brain from a PML patient where T antigen is present in the nuclei of infected oligodendrocytes.

Fig 3

Detection of DNA sequence corresponding to VP1 in brain. (A) Representative results from polymerase chain reaction/Southern blot from two cases. (B) Representative immunohistochemical evaluation for detection of VP1 expression in the various regions of non-PML brain, as well as in demyelinated areas from a brain of a PML patient. VP1 was detected only in the oligodendrocytes of PML samples.

Fig 4

Detection of DNA sequence corresponding to a region of JC virus (JCV) that encodes agnoprotein. (A) Representative results from gene amplification in two cases. (B) Immunohistochemical labeling of brain from non-PML and PML showing perinuclear accumulation of agnoprotein in oligodendrocytes from PML, but not non-PML specimens.

Fig 5

Detection of DNA sequence corresponding to the JC virus (JCV) control region in non-progressive multifocal leukoencephalopathy (non-PML) (non-PML) brain. (A) Results from polymerase chain reaction (PCR) amplification of the control region of JCV in two cases. The negative control illustrates results from amplification with no template DNA, and the positive control shows gene amplification using pBJC plasmid DNA. (B) Structural organization of the JCV control region and the nucleotide composition of amplified JCV DNA in normal brain. The position of a single nucleotide mutation is shown.

Fig 6

Laser-capture microdissection of frontal cortex from normal brain. Selection of astrocytes (A), neurons (B), and oligodendrocytes (C) was guided by immunohistochemical detection of GFAP, neurofilament (left panels) and MBP. After the thermoplastic film is removed, the tissue left behind after laser capture shows punched holed in the receiving section (middle panels). Removal of the cells is verified by microscopic visualization prior to DNA preparation (right panels) D. Gene amplification followed by Southern blot by hybridization using a pair of primers that recognizes sequences of the JCV early gene.