Context.-It is generally accepted that JC virus variants in the brains of patients with progressive multifocal leukoencephalopathy are generated from archetypal strains through sequence rearrangement (deletion and duplication, or deletion alone) in the control region. This change is thought to occur during persistence of JC virus in patients.
Objective.-The present study was performed to ascertain whether amino acid substitution in the viral proteins is involved in the generation and propagation of JCV variants with rearranged control regions.
Design.-Many complete JC DNA clones were established from brain tissues (cerebellum, occipital lobe, and brainstem) autopsied in a case of progressive multifocal leukoencephalopathy in which multiple distinct control sequences were detected. Control and coding sequences were determined and compared among the JC DNA clones.
Results.-Twenty-eight control-region and 20 coding sequences of JC virus were compared. Five rearranged control sequences were detected, but they could be classified into 3 groups that shared common structural features. Viral coding sequences were identical among clones with different control regions and among clones derived from different brain regions.
Conclusion.-In the present case, nucleotide substitution in the viral coding regions (and resultant amino acid change in the viral proteins) was involved neither in the genesis of rearranged JC virus variants nor in the proliferation of demyelinated lesions in the brain.
(Arch Pathol Lab Med. 2004;128:275-278)
Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system that affects individuals with decreased immunocompetence.1 The causative agent is the human polyomavirus JC virus (JCV), first isolated in 1971 from the brain of a PML patient.2 Although PML was once a rare disease, it is now a common opportunistic infection in patients with acquired immunodeficiency syndrome.3
Most individuals are asymptomatically infected with JCV during childhood.4,5 The infecting JCV reaches the kidney, probably through viremia, and persists there throughout life.6,7 In adults, the renal JCV replicates and excretes progeny in urine.8-11 Renal JCV DNA carries the archetype control region (archetype CR)/ whereas JCV DNA in the brain of PML patients contains various CRs (PML-type CRs) that harbor deletions and duplications, or deletions alone, in reference to the archetype.12-15
To explain the correlation between archetype and PML-type JCVs, Yogo and Sugimoto15 proposed the archetype concept, which is formulated as follows: (1) JCVs with the archetype CR are circulating in the human population; (2) the archetype CR is highly conserved, in marked contrast to the hypervariable CRs (PML-type CRs) of JCVs in the brain of PML patients; (3) each PML-type CR is produced from the archetype by deletion and duplication, or by deletion alone; (4) the shift of the CR from archetype to PML type occurs during persistence in the host; and (5) PML-type JCVs never return to the human population.
The archetype concept assumes that no amino acid change in the viral proteins is involved in the generation of PML-type JCV To examine this assumption, we studied a PML case in which multiple, distinct, PML-type JCVs were detected in autopsied brain tissues."' We established and sequenced many complete JCV DNA clones. Viral coding sequences were identical among clones with different control regions and among clones derived from different brain regions. We concluded that nucleotide substitution (and resultant amino acid change) was involved neither in the genesis of rearranged JCVs nor in the proliferation of demyelinated lesions in the brain.
MATERIALS AND METHODS
A detailed case report was published previously.16 In brief, a 14-year-old boy with Wiskott-Aldrich syndrome suffered from progressive impairment of ocular movement and annrthria 6 months after allogenic bone marrow transplantation. T2-weighted magnetic resonance imaging showed high-signal areas in the right occipital lobe, cerebellum, and pons. JC viral DNA was detected in the cerebrospinal fluid by nested polymerase chain reaction. Several therapeutic approaches were not effective, and the patient died 8 months after bone marrow transplantation.
DNA was previously extracted from autopsied brain tissues (cerebellum, occipital lobe, and brainstem).16 From these DNA samples, entire JCV DNA sequences were cloned into pUC19 at the unique BamHI site, ns described elsewhere.17 The resultant recombinant plasmids containing complete JCV DNA sequences were prepared using a QIAGEN Plasmid Midi kit (QIAGEN GmbH, Hilden, Germany). Purified plasmids were sequenced as described by Sugimoto et al.18 The CR sequences were aligned by eye in reference to the archetype sequence,12 while entire DNA sequences, excluding the CR sequences, were aligned with the CLUSTAL W program (ftp://ftp-igbmc.u-strasbg.fr).19
Control Region Sequences
We established 14 clones from the cerebellum, 7 from occipital lobe, and 8 from brainstem (29 total). We first determined the CR sequences of these clones. We identified 5 rearranged CR sequences, designated CRs I to V. These sequences are diagrammatically represented in the Figure in reference to the archetype CR at the top. Deletions in rearranged sequences I through V are shown as gaps, with duplications depicted by parallel lines. Control regions I, II, and IV corresponded to TK-Ia, -Ic, and -Id, respectively, previously identified by polymerase chain reaction from the same brain tissue DNA.16 Control regions III and V were not detected in the previous study.16
The structural features of the 5 CR sequences are summarized as follows: (1) CR I had only deletions, whereas the others (II to V) had both deletions and duplications. (2) Control regions II and III had 2 common deletions, spanning nucleotides (nt) 37 to 60 and nt 201 to 247. However, duplications in CRs II and III were unique, and CR III had a small deletion (nt 138-139) not present in CR II. These features suggest that the same intermediate carrying only the common deletions generated CRs II and III by different subsequent changes. (3) Control regions IV and V shared the same deletion (nt 63-85) and the same breakpoints (nt 41 and 110). However, these CRs also had a unique deletion (IV) and unique duplications (IV and V). A common intermediate probably generated IV and V, but it was not readily inferred how this intermediate produced CRs IV and V. all in all, it was concluded that at least 3 independent CR rearrangements occurred in a JCV strain (or strains) in the present PML case.
In the present case of PML, we previously amplified various rearranged CRs of JCV in the brain using polymerase chain reaction and found that each rearranged CR showed a unique distribution pattern in the brain.1'1 These distribution patterns were confirmed in the present study, for which many complete JCV DNA clones were analyzed (see the Table). Thus, CR 1 (TK-Ia) was widespread in the brain, with the highest incidence in the cerebellum; CR II (TK-1c) mainly occurred in the occipital lobe; and CR IV (TK-1d) mainly occurred in the brainstem. In addition, CR III, which was first detected in the present study but was structurally related to CR II (see above), was detected only in the occipital lobe, where CR II was mainly detected.
JC virus DNA clones were classified into groups according to origin and CR sequences (Table). We determined the complete coding sequences of representative or all clones belonging to these groups (Table). A single complete coding sequence of JCV was mainly detected in the brain in the current case, but a minor complete coding sequence was detected in the 3 clones with CR IV. These minor clones carried T at a position (nt 824) within the VP2 (a minor capsid protein) gene, whereas the major clones carried C at this position. A clone with CR V (CR structurally related to CR IV) carried C, rather than T, at nt 824. The C/T nucleotide change at position 824 caused an amino acid difference of alanine/valine (a change between amino acids with similar properties).
The result regarding the complete coding sequences described above had 2 implications. First, the complete coding sequence was identical regardless of the structures of CRs. The CR sequences were classified into 3 groups with distinct rearrangements. Thus, the identity of the complete coding sequence among clones with distinctly rearranged CRs suggested that in the present PML case, JCV DNA sequences with distinctly rearranged CRs were generated from the same archetype strain. second, the complete coding sequence was identical among clones that carried the same CR, even if they were derived from different brain regions. For example, the same complete coding sequence (ie, the sequences harboring C at nt 824) was detected in all clones with CR I derived from all 3 brain regions. Likewise, this sequence was detected in all clones with CR II derived from 2 brain regions (cerebellum and occipital lobe).
It is now generally accepted that JCV variants in the brains of PML patients (PML-type JCVs) are generated from archetype strains through sequence rearrangement (deletion and duplication, or deletion alone) in the CR.12-14,20,21 Yogo and Sugimoto15 developed this view into the archetype concept. This concept assumes that no significant amino acid change in the viral proteins is involved in the generation of PML-type JCVs. We used a PML case in which multiple distinct PML-type JCVs were detected in the brain to examine this assumption.16 We established and sequenced many complete JCV DNA clones from autopsied brain tissues and compared CR and coding sequences among the JCV DNA clones obtained. Five CR sequences were detected in total, but they could be classified into 3 groups that we believe evolved independently from the archetype. The same complete coding sequence was detected in most clones with various CRs, excluding CR IV. Although we did not analyze the archetype strain that would have generated the various PML-type JCVs, it can be assumed that no common mutation occurred during the genesis of PML-type JCVs.22 Therefore, the detection of the same complete coding sequence in most JCV DNA clones, regardless of the structure of the CR, suggested that the detected complete coding sequence originally existed in the hypothetical archetype JCV and had been conserved during the generation of various PMLtype JCVs.
In JCV isolates with CR IV, we detected a single nucleotide change in the VPl (a minor capsid protein) gene. Because this nucleotide substitution caused only a change between amino acids (alanine and valine) with similar properties, the structure and function of VP2 would not have been influenced by this mutation. all in all, we can conclude that at least in the current case, amino acid substitution in the viral proteins played no important role in the genesis of JCVs with various rearranged CRs.
In the present PML case, the same rearranged CRs occurred in multiple brain regions.16 For instance, CR I occurred in 3 regions (cerebellum, occipital lobe, and brainstem), and CR II occurred in 2 regions (cerebellum and occipital lobe). We believe this observation reflects the proliferation of demyelinated lesions in the brain.16 We detected the same complete coding sequence in JCV DNA clones derived from different brain regions. We concluded that nucleotide substitutions and resultant amino acid changes rarely occurred in the proliferating demyelinated lesions in the brain.
JC viruses that persist in renal tissue and that are excreted in the urine carry the archetype CR and represent JCVs circulating in human populations.15 Zheng et al23 recently investigated how frequently renal/urinary JCVs undergo nucleotide substitution in their coding regions. In brief, they established 5 to 9 complete JCV DNA clones (61 in total) from the urine of 11 individuals (parents and children) belonging to 5 families. The complete sequences of these clones were determined and compared in each family. In the viral coding sequences, 1 or a few nucleotide substitutions per individual were detected in 5 individuals, but none were detected in 6 individuals. It is not easy to compare mutation rates in the coding regions between archetype and PML-type JCVs on the basis of the findings in the present and previous study,21 as only a single PML case was analyzed in the present study. Nevertheless, it is likely that mutation in the JCV genome is not very frequent in PML patients, although sequence rearrangements in the CR more frequently occur in immunosuppressed patients than in immunocompetent patients.24
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Huai-Ying Zheng, PhD; Yukiharu Yasuda, MD; Shunichi Kato, MD; Tadaichi Kitamura, MD; Yoshiaki Yogo, PhD
Accepted for publication November 4, 2003.
From the Department of Urology, The University of Tokyo, Tokyo, Japan (Drs Zheng, Kitamura, and Yogo), and the Department of Pediatrics, Tokai University of School of Medicine, lsehara, Japan (Drs Yasuda and Kato). Dr Yasuda is now with Yasuda Pediatric Clinic, Machida, Japan.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Yoshiaki Yogo, PhD, Department of Urology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (e-mail: firstname.lastname@example.org).
Copyright College of American Pathologists Mar 2004
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