New sequence polymorphisms in the outer loops of the JC polyomavirus major capsid protein (VP1) possibly associated with progressive multifocal leukoencephalopathy

Huai-Ying Zheng1,2, Tomokazu Takasaka1, Kazuyuki Noda3, Akira Kanazawa3, Hideo Mori3, Tomoyuki Kabuki4, Kohsuke Joh4, Tsutomu Oh-ishi4, Hiroshi Ikegaya5, Kazuo Nagashima6, William W. Hall7, Tadaichi Kitamura1 and Yoshiaki Yogo1

1 Department of Urology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
2 Japanese Foundation for AIDS Prevention, Tokyo 105-0001, Japan
3 Department of Neurology, Juntendo University School of Medicine, Tokyo 113-0033, Japan
4 Division of Infectious Diseases, Immunology and Allergy, Saitama Children's Medical Center, Iwatsuki 339-8551, Japan
5 Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan
6 Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo 060-8638, CREST, Japan
7 Department of Medical Microbiology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland

Correspondence
Yoshiaki Yogo
yogo-tky{at}umin.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML) in patients with decreased immune competence. To elucidate genetic changes in JCPyV associated with the pathogenesis of PML, multiple complete JCPyV DNA clones originating from the brains of three PML cases were established and sequenced. Although unique rearranged control regions occurred in all clones, a low level of nucleotide variation was also found in the coding region. In each case, a parental coding sequence was identified, from which variant coding sequences with nucleotide substitutions would have been generated. A comparison between the parental and variant coding sequences demonstrated that all 12 detected nucleotide substitutions gave rise to amino acid changes. Interestingly, seven of these changes were located in the surface loops of the major capsid protein (VP1). Finally, 16 reported VP1 sequences of PML-type JCPyV (i.e. derived from the brain or cerebrospinal fluid of PML patients) were compared with their genotypic prototypes, generated as consensus sequences of representative archetypal isolates belonging to the same genotypes; 13 VP1 proteins had amino acid changes in the surface loops. In contrast, VP1 proteins from isolates from the urine of immunocompetent and immunosuppressed patients rarely underwent mutations in the VP1 loops. The present findings suggest that PML-type JCPyV frequently undergoes amino acid substitutions in the VP1 loops. These polymorphisms should serve as a new marker for the identification of JCPyV isolates associated with PML. The biological significance of these mutations, however, remains unclear.

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB183534–AB183544 and AB190446–AB190453 (see Table 1).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
JC polyomavirus (JCPyV) is the causative agent of a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML) (Walker, 1985), and is widespread in humans. Primary infection occurs asymptomatically during childhood (Padgett & Walker, 1973). JCPyV is then disseminated throughout the body, probably through viraemia (Ikegaya et al., 2004). It is well established that JCPyV persists in renal tissue (Chesters et al., 1983; Tominaga et al., 1992; Kitamura et al., 1997; Aoki et al., 1999). Nevertheless, JCPyV also persists in other sites, including lymphoid tissues and peripheral blood lymphocytes (Gallia et al., 1997; Kato et al., 2004).

The genome of JCPyV has a non-coding control region (CR) between the origin of replication and the start site of the agnogene (Frisque et al., 1984). JCPyV CRs in the brain of PML patients (PML-type CRs) are so variable that identical PML-type CRs have never been detected in different PML patients (Yogo & Sugimoto, 2001). In contrast, JCPyV CRs detected in the urine, kidney and tonsils of immunocompetent individuals have the same basic structure, designated the archetype (Yogo & Sugimoto, 2001; Kato et al., 2004). Yogo & Sugimoto (2001) formulated a correlation between archetype and PML-type JCPyV as the archetype concept, consisting of the following five principles: (i) JCPyV with the archetype CR circulates in the human population; (ii) the archetype CR is highly conserved, in marked contrast to the hypervariable CRs (PML-type CRs) of JCPyV in the brain of PML patients; (iii) each PML-type CR is produced from the archetype by deletion and duplication or by deletion alone; (iv) the shift of the CR from archetype to PML type occurs during persistence in the host; and (v) PML-type JCPyV never returns to the human population.

The archetype concept adequately explains changes in the JCPyV CR from a molecular epidemiological standpoint. However, this concept does not address a medically important issue, i.e. whether these changes are involved in pathogenesis of PML. A few studies have challenged this issue by using in vitro expression assays (Sock et al., 1996; Ault, 1997). According to the results of these studies, there is little doubt that JCPyV with archetype CR can propagate in the human brain. Indeed, O'Neill et al. (2003) recently demonstrated successful propagation of an archetypal JCPyV strain in human fetal brain cells. Thus, the question remains open as to why JCPyV DNAs in the brains of PML patients regularly undergo sequence rearrangement in their CRs.

JCPyV DNA replicates in the nucleus by using a cellular DNA polymerase with proofreading activity. Therefore, the fidelity of JCPyV DNA replication is thought to be as high as that of cellular DNA replication. Nevertheless, by sequencing five to eight complete JCPyV DNA clones established from each family member, we found that nucleotide substitutions sometimes occur in the coding region of JCPyV (Zheng et al., 2004a). The coding region of JCPyV is about 4800 bp in size and encompasses several genes that encode at least six viral proteins (agnoprotein, capsid proteins VP1–3 and large and small T antigens) (Frisque et al., 1984). Nucleotide changes in the coding region have been used successfully as a marker for distinguishing between JCPyV variants transmitted to offspring and those not transmitted (Zheng et al., 2004a).

Furthermore, we recently analysed 29 complete JCPyV DNA sequences detected in autopsied brain tissue in a paediatric case of PML (Zheng et al., 2004b). From the results obtained, it was concluded that, in the studied case, nucleotide substitution (and resultant amino acid change) was not involved in either the genesis of rearranged JCPyV or the expansion of demyelinated lesions in the brain. However, from the autopsied brain in the same PML case noted immediately above, a single nucleotide substitution occurred in three rare clones with a rearranged CR sequence. As the studied PML case was atypical in terms of the patient's age, it could be that, in adult PML patients, the stability of the coding sequences might decrease (i.e. more nucleotide substitutions might occur in the coding region).

Thus, changes in the coding region, if combined with CR rearrangements, might offer useful information about the history of JCPyV DNAs from persistence to reactivation. In this study, an overall analysis of JCPyV DNA sequences from the brain tissue of three adult PML patients was performed. It was found that all nine detected nucleotide substitutions gave rise to amino acid changes; these changes frequently occurred in the surface loops of the major capsid protein (VP1). To confirm this finding, 16 reported VP1 sequences from PML-type isolates were compared with their closest typological prototypes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Patients.
As a detailed case report was described previously (case 1) (Hall et al., 1991) or will be described elsewhere (cases 2 and 3), cases are only described briefly here.

Case 1.
A 33-year-old homosexual male was admitted to North Shore University Hospital, New York, USA, with AIDS. He developed various opportunistic infections before and after hospitalization. On 2 May 1988, he exhibited a change in mental status, becoming extremely confused and progressively disoriented as to time and place. He continued to do poorly and developed cortical blindness. The patient became unresponsive and died on 4 June 1988. Pathological findings, including the detection of JCPyV DNA by in situ hybridization, have been documented previously (Hall et al., 1991).

Case 2.
A 59-year-old female with progressive impairment of memory, motor aphasia and right-sided weakness was admitted to Juntendo University Hospital, Tokyo, Japan, on 7 December 2000. Three years earlier, she had been diagnosed with mixed connective tissue disease based on the presence of antinuclear antibodies, anti-ribonucleoprotein positivity and clinical symptoms. She had been treated with predonisolone and azathioprine. Cerebral magnetic resonance imaging (MRI) performed upon administration showed progressive, confluent, non-enhancing lesions in the left frontal subcortical white matter. The cerebrospinal fluid (CSF) was positive for JCPyV DNA by nested PCR. The patient became unresponsive and died on 1 March 2001.

Case 3.
A 20-year-old male had suffered from chronic candidiasis of the skin and mucosa since early childhood. As he developed a motor disturbance of the right upper limb and difficulty in speech at the end of August 2001, he was admitted to Saitama Children's Medical Center, Iwatsuki, Japan. Based on a diagnosis of multiple sclerosis, he received {gamma}-globulin and steroid-pulse therapy. However, his symptoms got worse and he presented quadriplegia and pseudobulbar paralysis. T2-weighted brain MRI showed a high-intensity area in a white-matter area of the right parietal lobe. JCPyV DNA was detected by nested PCR in CSF collected on 23 October 2001. He was unresponsive to either plasma-exchange therapy or intravenous interleukin 2 and Ara-C injection. He became unconscious in January 2002 and died from septicaemia on 29 October 2003.

Molecular methods.
Autopsied brain tissue was digested with 100 µg proteinase K ml–1 at 56 °C for 1 h in the presence of 0·5 % SDS. The digest was extracted once with phenol and once with chloroform/isoamyl alcohol (24 : 1). DNA was recovered by ethanol precipitation and dissolved in water. Entire JCPyV DNAs were cloned into pUC19 at the unique BamHI site as described previously (Yogo et al., 1991a). The resultant recombinant plasmids containing complete JCPyV DNA sequences were prepared by using a Qiagen Plasmid Midi kit. Purified plasmids were sequenced as described previously (Sugimoto et al., 2002a).

Phylogenetic analysis.
The determined and reference sequences were aligned by using the program CLUSTAL W (Thompson et al., 1994). Aligned sequences were subjected to phylogenetic analysis by using the neighbour-joining (NJ) method (Saitou & Nei, 1987). Phylogenetic trees were constructed by using CLUSTAL W and divergences were estimated by the two-parameter method (Kimura, 1980). Phylogenetic trees were visualized by using TREEVIEW (Page, 1996). To assess the confidence of branching patterns of the NJ trees, 1000 bootstrap replications were performed (Felsenstein, 1985). Translation of nucleotide sequences into amino acid sequences and alignment of multiple amino acid sequences were performed with GENETYX-MAC ver. 11.10 (GENETYX).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CR sequence variations
From DNA extracted from autopsied brain tissue, complete JCPyV DNAs were cloned by using a plasmid vector. Twelve, 14 and 14 complete JCPyV DNA clones were established in cases 1, 2 and 3, respectively. The CR and coding sequences of all these clones were determined. Two (1A and 1B), three (2A, 2B and 2C) and three (3A, 3B and 3C) rearranged CR sequences were identified in cases 1, 2 and 3, respectively (Table 1). The structures of the detected CRs are shown diagrammatically in Fig. 1, with reference to the archetype at the top. Deletions in rearranged CR sequences are shown as gaps and duplications are depicted by parallel lines. CRs 1A and 1B were identical to CRs detected previously in the brain of the same patient (Yogo et al., 1994).


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Table 1. JCPyV DNA sequences determined in this study

 


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Fig. 1. Diagrammatic representation of the detected JCPyV CR sequences. The structure of the archetypal CR is shown schematically at the top. The origin of replication (Ori), TATA sequence and agnogene (Agno) are indicated (Frisque et al., 1984). Domain A indicates a sequence duplicated in many PML-derived JCPyV isolates and domain B indicates a sequence deleted in many PML-derived JCPyV isolates (Iida et al., 1993). The JCPyV CRs detected in this study (1A, 1B, 2A, 2B, 2C, 3A, 3B and 3C) are shown, with deletions relative to the archetype described as gaps. Reading from left to right, when a repeat is encountered, the linear representation is displaced to the line below and to a position corresponding to the sequence of the archetype. Numbers below each box and lines are nucleotide numbers indicating end locations [nucleotide numbers are those of the archetype (Yogo et al., 1990)].

 
Coding sequence variations
Three (1-1 to 1-3), three (2-1 to 2-3) and five (3-1 to 3-5) complete coding sequences of JCPyV were detected in cases 1, 2 and 3, respectively (Table 1). Nucleotide differences were examined among detected sequences in each case (Table 2). Sequences in cases 1 and 2 showed single nucleotide polymorphisms (SNPs) at two positions, whereas those in case 3 showed SNPs at five positions. Sequences in cases 1 and 2 were distinguished by one nucleotide difference, with the exception that 1-1 and 1-2 were distinguished by two nucleotide substitutions, whereas those in case 3 were distinguished by one to four nucleotide substitutions. Numbers of clones with individual CR and coding sequences are shown in Table 3.


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Table 2. Nucleotide variations among the coding sequences detected in each case

Nucleotides (amino acids) at positions of the Mad-1 genome (Frisque et al., 1984) are shown. See text for explanation of ancestral sequences.

 

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Table 3. Frequencies of complete JCPyV DNA clones with distinct control and coding sequences

 
Relationships among the coding sequences detected in each case
Classification of JCPyV DNAs detected in the three cases into genotypes was attempted by using NJ phylogenetic analysis (Saitou & Nei, 1987) based on complete coding sequences of JCPyV. According to the resultant phylogenetic tree (not shown), the JCPyV DNAs detected in cases 1, 2 and 3 belonged to genotypes EU-a2, CY-a and MY-b, respectively. These genotypes were recently recognized as independent genotypes based on phylogenetic analysis using complete JCPyV DNA sequences (Zheng et al., 2003, 2004c; Ikegaya et al., 2005).

To elucidate the ancestral states for polymorphic sites in the coding sequences (Table 2), the complete coding sequences detected in each case and a number of reference sequences belonging to the same genotype of JCPyV as that to which the detected sequences belonged were aligned. The latter included 10 EU-a2, 13 CY-a and 18 MY-b sequences, shown in Table 4. A consensus nucleotide identified at each polymorphic site was considered to be the ancestral state. Thus, sequences 1-1, 2-1 and 3-1, detected in cases 1, 2 and 3, respectively, were found to contain the ancestral states at all polymorphic sites (Table 2) and thus designated parental coding sequences.


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Table 4. Complete JCPyV DNA sequences used for phylogenetic analysis

Sequences belonging to EU-a2, CY-a and MY-b were used to identify ancestral states for variable sites in cases 1, 2 and 3, respectively (see text).

 
NJ phylogenetic trees were constructed from the complete coding sequences detected in cases 1, 2 and 3, together with many reported sequences grouped as EU-a1 and -a2, CY-a and -b, and MY-b, respectively (Table 4). On the resultant tree (Fig. 2a), the sequences detected in case 1 (1-1 to 1-3) clustered together with a high bootstrap probability (96 %), with 1-1 located at the node. Similarly, the sequences detected in case 2 (2-1 to 2-3) clustered together with a high bootstrap probability (98 %), with 2-1 located at the node (Fig. 2b). The sequences detected in case 3 (3-1 to 3-5), together with eight other MY-b sequences derived from unrelated individuals, formed a cluster on the phylogenetic tree (Fig. 2c), with sequence 3-1 at the node. The observation that the case 3 sequences and many other MY-b sequences clustered together (Fig. 2c) could be explained by assuming that the parental sequence (i.e. 3-1) in case 3 happened to be the ancestral sequence for the other sequences belonging to the cluster.



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Fig. 2. NJ phylogenetic trees relating complete JCPyV DNA sequences detected in cases 1, 2 and 3. NJ phylogenetic trees were constructed from: (a) three complete coding sequences (1-1, 1-2 and 1-3) detected in case 1 and 17 complete coding sequences belonging to EU-a; (b) three complete coding sequences (2-1, 2-2 and 2-3) detected in case 2 and 22 complete coding sequences belonging to CY; and (c) five complete coding sequences (3-1, 3-2, 3-3, 3-4 and 3-5) detected in case 3 and 17 complete coding sequences belonging to MY-b (see Table 4 for sequence details). Phylogenetic trees were visualized by using TREEVIEW and rooted by using a genotype Af1 isolate (GH-1) (Sugimoto et al., 2002a) as the outgroup. Numbers at nodes indicate the bootstrap confidence levels (%) obtained with 1000 replications (only values >=70 % are shown). It should be noted that 3-4 and 3-5 could not be discriminated, as gaps were excluded in the present phylogenetic analysis.

 
Amino acid variations among VP1 sequences detected in cases 1, 2 and 3
All nucleotide substitutions detected in JCPyV DNA clones in cases 1, 2 and 3 gave rise to amino acid changes (Table 2). Interestingly, seven of these substitutions caused amino acid changes in the major capsid protein, VP1. Although the crystal structure of the JCPyV VP1 has not yet been elucidated, it can be assumed that it is similar to that of the crystallized Simian virus 40 (SV-40) VP1 protein (Liddington et al., 1991), as the amino acid sequences between JCPyV and SV-40 VP1 proteins are highly similar (Shishido-Hara & Nagashima, 2001). Thus, by analogy with the SV-40 VP1 structure (Liddington et al., 1991), Chang et al. (1996) identified various elements in the JCPyV VP1. The amino acid changes detected in the VP1 sequences were mapped and, to our surprise, all seven amino acid changes were located within the possible surface loops, designated BC, DE and HI (Chang et al., 1996) (Table 5). In the BC loop, substitutions of lysine-60 with methionine in coding sequence 3-3 and serine-61 with leucine in coding sequence 2-2 were detected; in the DE loop, a substitution of serine-123 with cysteine was detected in coding sequences 3-4 and 3-5; and in the HI loop, substitutions of serine-269 with either phenylalanine in sequences 1-2 and 1-3 or tyrosine in sequence 3-2 were detected. Most of the detected amino acid substitutions, excluding the substitution of serine-123 with cysteine in the coding sequences 3-4 and 3-5, caused changes in the amino acid properties based on a Venn diagram grouping of amino acids (Betts & Russell, 2003).


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Table 5. Amino acid residue variations in VP1 sequences detected in cases 1, 2 and 3

The BC, DE and HI loops are structural elements of the JCPyV VP1 defined by amino acid sequence similarity to SV-40 VP1 (Chang et al., 1996).

 
Thus, VP1 loop mutations were detected in two of the three coding sequences in case 1, in one of the three coding sequences in case 2 and in four of the five coding sequences in case 3. In terms of clone frequencies, VP1 loop mutations were identified in three of the 12 clones in case 1, two of the 14 clones in case 2 and 12 of the 14 clones in case 3 (Table 3).

Amino acid changes in VP1 sequences identified previously in the brain or CSF of PML patients
To our knowledge, complete VP1 sequences (designated PML-type VP1 sequences for convenience) have been reported for 16 JCPyV isolates derived from the brain or CSF of different PML patients [NY-1B, which was isolated from one of the patients (case 1) investigated in this study, was excluded] (Table 6). The presence of any amino acid changes in the surface loops of these VP1 sequences was examined. Naturally, detection of such amino acid changes requires parental VP1 sequences from which PML-type VP1 sequences might have been generated. The genotypic prototype was used as a substitute for the real parental sequence for each PML-type VP1 sequence. The genotypic prototype was identified as the consensus sequence of VP1 sequences detected in representative archetypal isolates (i.e. isolates derived from the urine of healthy individuals and non-PML patients) belonging to each genotype. The JCPyV genotype designation used was that of Yogo et al. (2004) with modifications (Saruwatari et al., 2002; Ikegaya et al., 2005; Takasaka et al., 2005). Furthermore, B1-b was divided into B1-b1 and -b2 according to a recent phylogenetic study on Asian isolates (Cui et al., 2004). The archetypal isolates used were four Af1, six Af2-a, 10 EU-a1, four B1-c, 12 CY-a, three MY-a, eight MY-b and 11 SC-f isolates (Loeber & Dörries, 1988; Agostini et al., 1997, 1998a, 1998c; Kato et al., 2000; Saruwatari et al., 2002; Sugimoto et al., 2002a; Suzuki et al., 2002; Zheng et al., 2003, 2004c; Takasaka et al., 2005). Each PML-type VP1 amino acid sequence was then compared with its genotypic prototype to find out whether there were any amino acid changes.


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Table 6. Amino acid residue variations in VP1 sequences detected in the brains of PML patients

All isolates had unique rearranged CRs. The BC, DE and HI loops are structural elements of the JCPyV VP1 defined by amino acid sequence similarity to SV-40 VP1 (Chang et al., 1996). See text for explanation of the consensus sequence.

 
In total, VP1 amino acid substitutions were detected in 13 (81 %) of the 16 PML-type JCPyV isolates for which complete VP1 sequences were reported (Table 6). These substitutions were all located in the outer loops (BC and HI) of the VP1 protein, five in the BC loop and eight in the HI loop. One residue (269) represented hot spots where substitutions occurred most frequently. All consensus sequences representing various genotypes turned out to have the same amino acids at the positions (residues 55, 60, 66, 265, 267 and 269) where substitutions were detected in PML-type isolates (Table 6). In addition, most of the detected amino acid substitutions, excluding the substitution of asparagine with threonine in the SA21-01 VP1, caused changes in the amino acid properties defined based on a Venn diagram grouping of amino acids (Betts & Russell, 2003).

As described above, the VP1 sequences of archetypal isolates belonging to each genotype were identical (or essentially identical). This finding suggests that VP1 loop mutations occur rarely in archetypal JCPyV circulating in the human population.

Lack of VP1 loop mutations in JCPyV isolates from the urine of immunosuppressed patients
JCPyV DNAs recovered from the urine of immunosuppressed patients were then examined to determine whether they carried VP1 loop mutations. Complete JCPyV DNA clones were established in the present and a previous study (Yogo et al., 1991b) from urine samples of 13 Japanese and two Chinese renal-transplant patients. These clones were classified as CY-a (n=2), CY-b (n=6), MY-b (n=5), B1-c (n=1) and SC-f (n=1) according to phylogenetic analysis based on their DNA sequences (data not shown). Complete DNA sequences or partial sequences encompassing the VP1 gene were determined in this and previous studies (Zheng et al., 2004c) and the VP1 amino sequences were deduced from these DNA sequences. These VP1 amino acid sequences were then compared with their genotypic prototypes, generated as consensus sequences of representative archetypal isolates belonging to the same genotypes (data not shown). No VP1 amino acid substitution was detected in the 15 isolates derived from the urine of immunosuppressed patients, suggesting that the immunological state is not directly associated with the induction of VP1 loop mutations.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many complete JCPyV DNA clones obtained from brain tissue from three PML cases were sequenced. Multiple coding sequences were identified, distinguished in each case by the presence or absence of one or two nucleotide substitutions and a short duplication (only in case 3). It is unlikely that variations were introduced during molecular cloning because of the high fidelity of DNA synthesis in bacterial cells. To clarify the relationships among the detected coding sequences, two analyses were performed (see above). From these results, it is concluded that 1-1, 2-1 and 3-1 were the parental sequences in cases 1, 2 and 3, respectively, from which the other coding sequences (e.g. variant coding sequences) were generated. A comparison between the parental and variant coding sequences in each PML case enabled us to detect VP1 loop mutations. Furthermore, 16 reported VP1 sequences from PML-type isolates were compared with their genotypic prototypes, generated as consensus sequences of representative archetypal isolates belonging to the same genotypes. It was found that 13 VP1 proteins underwent amino acid changes in the surface loops. From these findings, it can be concluded that PML-type JCPyV frequently undergoes amino acid substitutions in the VP1 loops.

The frequency of JCPyV DNA clones with VP1 loop mutations varied significantly among the three cases. Thus, 25, 14 and 86 % of the analysed clones in cases 1, 2 and 3 showed the VP1 loop mutations. In cases 1 and 2, the patients died within 6 months of the onset of symptoms, whereas the patient in case 3 survived for about 2 years. The observations noted above suggest that the ratio of VP1 loop mutations increases with time as virus propagation continues in the central nervous system.

Although amino acid changes in the VP1 loop occurred frequently in JCPyV isolates derived from brain tissue and CSF of PML patients, they occurred rarely in JCPyV isolates derived from the urine of both healthy individuals and immunosuppressed patients. These findings suggest that the VP1 loop mutations are somehow associated with the development of PML.

By analogy with the crystallized VP1 structure of a related polyomavirus, i.e. SV-40 (Liddington et al., 1991), the VP1 loops of JCPyV are considered to be involved in functions such as interactions with cell receptors and antigenic responses. Therefore, there are two possible explanations for the frequent occurrence of VP1 loop mutants in PML-type JCPyV isolates. First, VP1 loop mutants have a higher affinity for receptors on the cell surface, thereby being able to grow more efficiently. Second, VP1 loop mutants are escape mutants that are not neutralized by the antibodies against JCPyV with non-mutated VP1 loops.

Gee et al. (2004) reported that arginine-56 and arginine-75 on the BC loop and arginine-273 on the HI loop are the potential sialic acid-binding sites for JCPyV infection. None of these amino acids was found to be altered in the PML-type JCPyV shown in this study (Tables 5 and 6). This would suggest that these PML-type JCPyV underwent no change in their ability to bind to cellular receptors, if the indirect effect of the VP1 loop mutations detected in this study could be excluded.

In summary, it was found that VP1 loop mutations occur frequently in JCPyV isolates from the brain and CSF of PML patients and that these mutations occur rarely in isolates from the urine of patients, regardless of their immunological state. These polymorphisms should serve as a new marker for the identification of JCPyV isolates associated with PML. The biological significance of these mutations, however, remains unclear.


   ACKNOWLEDGEMENTS
 
This study was supported in part by grants from the Ministry of Health, Labour and Welfare, Japan.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 28 December 2004; accepted 24 March 2005.



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