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Short Communication |
1 Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, RE 213C, 330 Brookline Avenue, Boston, MA 02215, USA
2 Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, RE 213C, 330 Brookline Avenue, Boston, MA 02215, USA
Correspondence
Igor J. Koralnik
ikoralni{at}bidmc.harvard.edu
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ABSTRACT |
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). This disease occurs in immunosuppressed individuals, for example in people with AIDS or leukaemia, or in organ-transplant recipients. JCV causes a lytic infection of oligodendrocytes and a restrictive infection of astrocytes. Until recently, this virus was thought to have a host-cell range limited to human glial cells, as a productive infection of neurons had never been observed. However, we have described a lytic infection of cerebellar granule cell neurons by JCV in a human immunodeficiency virus (HIV)-infected patient with PML (Du Pasquier et al., 2003; Tyler, 2003). This neuronal infection resulted in focal loss of granule cell neurons leading to cerebellar atrophy. This was associated with a clinically apparent cerebellar syndrome, which was distinct from PML, as classic demyelinating lesions were found only in the hemispheric white matter of the cerebrum, and not of the cerebellum, of this patient.
To estimate the JCV load in cerebellum and hemispheric white matter, a quantitative PCR was performed on DNA extracted from fresh-frozen samples as described previously (Du Pasquier et al., 2006); this indicated that the JCV load in the cerebellum and hemispheric white matter was 3.45x102 and 1.88x107 copies (µg DNA)–1, respectively.
We then attempted unsuccessfully to rescue JCV by co-cultivation of autopsy brain and cerebellum samples with human SVG astroglial cell and human astrocyte progenitors as described previously (Major et al., 1985). Therefore, we sought instead to clone full-length JCV sequences from cerebellum and hemispheric white matter. To achieve this goal, long PCR was performed on cellular DNA extracted from these two locations [as described by Koralnik et al. (1999)] using primer pair Eco7 (5'-AGAATTCCACTACCCAATCTAAATGAGGAT-3', nt 1721–1750) and Eco12 [5'-TGGAATTCTGGCCACACTGTAACAAG-3', nt 1729–1704, reverse complement (RC); Agostini & Stoner, 1995]. A full-length JCV clone was obtained from the hemispheric white matter (JCVHWM). Due to the low copy number of JCV DNA in the cerebellum, long PCR amplification failed to amplify a full-length product from this location. We therefore designed primers according to the sequence of JCVHWM to sequence the JCV strain present in granule cell neurons (JCVGCN1) in small overlapping fragments.
We analysed the regulatory region (RR) sequences (from nt 5118 to 276; see Fig. 1a) of JCVHWM and JCVGCN1. PCR amplification of the JCV RR was performed using primer pair JCS4913 (5'-TGTTTCCCCATGCAGATCTATCAA-3', nt 4913–4936) and JCR755 (5'-GCCCCCGGAGCTCCAGTT-3', nt 755–738 RC). As shown in Fig. 1(a), the predominant JCV RRs found in hemispheric white matter and granule cell neurons comprised a tandem-repeat pattern with two incomplete 98 bp elements, the first of which had an intact 23 bp insert and the second a truncated 23 bp and an intact 66 bp insert. In addition, JCVHWM had a deletion in the late promoter. These data confirmed and expanded our previous analyses performed with PCR primers encompassing a smaller area of the RR (Pfister et al., 2001).
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; Loeber & Dorries, 1988). In addition, there was a new point mutation in both JCVHWM and JCVGCN1, resulting in an amino acid change from Gly (Mad 1) to Ala (JCVHWM and JCVGCN1) at position 62 of the VP2 protein (see Table 1 for details).
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). Such a deletion in the T gene has not been reported previously. To rule out the possibility that this deletion was caused by an artefact of the long PCR, amplification of the DNA extracted from the brain hemispheric white matter was performed using PCR primers that amplified a 106 bp fragment encompassing this area of the T gene. Amplified products were distinctly smaller than the Mad 1 control DNA and sequencing of four clones confirmed that they all had this deletion. However, this in-frame deletion in the variable domain of the T protein did not seem to affect the phenotype of the JCVHWM isolate, as the clinical, radiological and histological presentation of lesions in the hemispheric white matter was classic for PML.
Interestingly, JCVGCN1 had a unique deletion of 10 bp starting at nt 2503 (compared with the JCV Mad 1 sequence), resulting in an open reading frame shift and a complete change of the deduced last 10 aa of the C terminus of the VP1 protein, as well as the addition of 3 aa, from RYGQLQTKML to SCRQKCCNQKPLL (Fig. 1c). To rule out the possibility that this deletion was caused by a PCR or sequencing artefact, amplification of the DNA extracted from the cerebellum was performed using the PCR primer pair CJS2465 (5'-CAGGAGACCCAGATATGATGAGATA-3', nt 2465–2489) and CJR2578 (5'-TGGTTATACTTTATTAAAATGTACTGCATATT-3', nt 2578–2547 RC; nucleotide numbering based on the sequence of JCVHWM), which amplify a 104 bp fragment encompassing the deleted area of the VP1 gene, compared with a 114 bp fragment for the non-deleted VP1 gene. Sequencing of eight clones confirmed that they all contained this 10 bp deletion. Using these PCR primers, JCVGCN1 was the only strain found in this patient's cerebrospinal fluid (CSF) sample (Fig. 2a). This was verified by sequence analysis of 3/24 cloned fragments of identical size. In contrast, this strain was only detectable 107 days later as a minor species in his peripheral blood mononuclear cells (PBMCs) (Fig. 2a, PBMC #2, lower band, indicated by an arrow). In this sample, only 1/52 clones had the VP1 deletion, verified by sequencing.
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. CSF samples from seven PML patients showed only the intact VP1 gene. However, in one patient (Fig. 2b, lane 8, faint lower band, indicated by an arrow), the VP1 deletion coexisted with an intact VP1 gene (Fig. 2b, lane 8, upper band) in the same sample. DNA sequencing determined that the lower band was identical to the JCVGCN1 VP1. This patient had classic PML lesions in the white matter of the cerebellum and a cerebellar syndrome. Therefore, it was not possible to determine whether the presence of a VP1 deletion mutant in his CSF was clinically relevant in this case. To determine whether VP1 deletion mutants could also be found in the blood of other individuals, we screened stored PBMCs or plasma DNA of PML patients available in our laboratory. Of 25 JCV PCR-positive samples, eight (32 %) showed an intact VP1 gene only, whereas 17 (68 %) had two bands on the gel, a major upper band for the intact VP1 and a fainter lower band corresponding to the deleted form of VP1. Sequence analysis was performed on amplified PCR products of one of these patients and showed that 1/8 clones had a GCN1 deletion identical to that described in our index case. The deleted VP1 form was not found in isolation in the PBMCs of any PML patient. There was no association between the presence of the GCN1 mutation in blood and a cerebellar syndrome.
While JCV can enter a variety of cell types, the restricted host-cell range of this virus is attributable mostly to the cellular requirements for transcription of the viral genome (Raj & Khalili, 1995). The JCV RR, which is required for viral expression and DNA replication, contains numerous binding sites for cellular proteins. Some of these proteins, such as NF1 class D, are predominantly expressed in glial cells (Sumner et al., 1996). If the RR of JCVGCN1 is responsible for its ability to grow in granule cell neurons, one would have expected to find a uniquely different RR sequence in these cells. However, in the present case, the RR of JCVGCN1 had a classic tandem-repeat pattern, as seen in PML isolates such as JCVHWM isolated in the classic PML lesions of the same patient. These results suggested that the specific tropism of JCVGCN1 for neuronal cells may not be attributed to its RR.
JCV entry in cells occurs via a clathrin-dependent endocytosis and requires interaction with 2,6-linked sialic acids and the serotonin 5HT2a receptor (Elphick et al., 2004; Komagome et al., 2002; Pho et al., 2000), which are present on granule cell neurons (Pasqualetti et al., 1998). Based on the crystal structure of the related polyomavirus Simian virus 40 and on studies of mouse polyomavirus (Stehle et al., 1994, 1996), the capsid of JCV is formed from 72 pentamers of VP1 protein, which are associated on their inner surface with either the VP2 or the VP3 protein. Although the exact site of interaction of JCV VP1 protein with its receptors remains unknown, it is unlikely that the GCN1 mutation, located in the C terminus, leads to a direct alteration of this site, as this portion of the VP1 protein is not exposed on the outer surface of the capsid.
In contrast, the C-terminal arms of the VP1 protein form the principal interpentamer contacts by extending away from the subunit of origin and reaching out to a subunit of an adjacent VP1 pentamer. Therefore, each pentamer extends five C-terminal arms to surrounding pentamers and receives five invading arms in return (Stehle et al., 1996). This lattice effectively acts as the ‘grout’ that keeps the pentamers together. The deletion of the VP1 gene appears to change the deduced tertiary structure of the VP1 C terminus dramatically, as resolved by EXPASY (Expert Protein Analysis System), resulting in the loss of a 
-pleated sheet in this area (data not shown). Therefore, we hypothesize that this mutation results in an alteration of the properties of the viral capsid. However, classic JC virions assembled in crystal structure were seen by electron microscopy in the granule cell neurons of this patient (Du Pasquier et al., 2003). This finding suggests that the VP1 deletion may not prevent formation of the capsid, but rather may alter post-viral entry events such as uncoating, transport and replication of JCV DNA, which in turn could enable a productive infection in granule cell neurons. Similarly, a change in phenotype and host-cell range was demonstrated in a mouse polyomavirus isolate that had a single amino acid change in the VP1 protein (Bauer et al., 1995; Freund et al., 1991).
JCVGCN1 was found in 25 % of CSF samples and 68 % of PBMC samples from our PML patients. These results suggest that this deletion may originate outside of the central nervous system, perhaps at the level of the bone marrow. Nevertheless, these data indicate that VP1 deletion mutants frequently co-exist as a minor species with intact JCV strains in the blood of patients with PML.
Finally, our findings open up a new avenue of investigation, which may shed some light on the pathogenesis of medulloblastomas. Indeed, JCV induces an abortive infection of granule cell neurons in neonatal hamsters, causing medulloblastomas (Kim et al., 2003; Ressetar et al., 1990). These tumours arise precisely from the granule cell layer of the cerebellum (Kim et al., 2003) and account for 20 % of brain tumours in children. Therefore, researchers have tried to determine whether polyomaviruses could have an aetiological role in medulloblastomas. In one study, investigators reported that 22/23 medulloblastomas (96 %) contained JCV DNA sequences, and JCV T antigen was detected by immunohistochemistry in 4/16 (25 %) samples tested (Krynska et al., 1999). However, these results were not confirmed by others (Kim et al., 2002; Weggen et al., 2000). Therefore, the availability of a granule cell neuron-tropic strain of JCV would be extremely valuable to clarify the potential role of this virus in human cerebellar tumours.
Since the publication of our index case, we and others have reported a productive infection of granule cell neurons by JCV in other patients (Gahaffar et al., 2005; Koralnik et al., 2005). We are now investigating whether this mutation confers a different phenotype on JCV in vitro.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 February 2006;
accepted 19 May 2006.
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