1 Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, RE 213B, 330 Brookline Avenue, Boston, MA 02215, USA
2 Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham, UK
3 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, RE 213B, 330 Brookline Avenue, Boston, MA 02215, USA
4 Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, RE 213B, 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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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences determined in this work are AY628224AY628238.
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INTRODUCTION |
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Since PML was identified as a major opportunistic infection at the beginning of the AIDS epidemic, JCV pathogenicity has been widely studied and full-length sequences of more than 250 strains of this virus have been analysed. Conversely, BKV has only been recognized as an important human pathogen more recently and until now the full-length sequences of only three strains have been reported. BKV strains have been classified into four different genotypes, which correspond to distinct serotypic profiles on the basis of nucleotide changes within a small fragment of the VP1 gene.
Little is know about polyomavirus sequence variation within individuals. There have been a few reports showing the co-existence of multiple JCV-coding region sequences in a single patient (Agostini et al., 1996; Ferrante et al., 2001
; Martin & Foster, 1984
). BKV sequence variations were observed on sequential kidney biopsy specimens from patients with polyomavirus nephropathy (Randhawa et al., 2002
). However, it has been suggested that polyomaviruses have co-evolved with their hosts (Shadan & Villarreal, 1993
; Soeda et al., 1980
) and, consistent with this, the major genotypes of JCV appear to have diverged in parallel with ancient human migrations (Agostini et al., 1997
; Guo et al., 1998
). Under the assumption of hostvirus co-evolution, the rate of evolution of polyomaviruses has been estimated to be in the range of 4x107 to 4x108 synonymous nucleotide substitutions per site per year (Hatwell & Sharp, 2000
; Yasunaga & Miyata, 1982
). These rates are so slow as to suggest that almost no genetic polymorphisms would be expected to be found among the viral sequences within a given individual, unless this person had been infected by more than one strain. Thus, a more specific analysis of intra-host genetic diversity of BKV is needed.
We have recently reported the case of a renal transplant recipient infected with a novel BK-related virus that had a tropism for capillary endothelial cells (Petrogiannis-Haliotis et al., 2001). This infection resulted in a vasculopathy that led to a capillary leak syndrome, myocardial infarction and death. To determine whether this BKV isolate was different from the known BKV strains and to explore BKV variability in vivo, we have sequenced nine full-length clones of this virus amplified from the heart and striated muscle DNA of this patient. Since the three published full-length BKV sequences have been derived from isolates that had been cultured in vitro, which may promote artificial mutations and lead to the selection of laboratory-adapted strains, we characterized the sequences of six novel full-length BKV clones amplified from the urine of one human immunodeficiency virus type 2 (HIV-2)-positive and one healthy individual and used them as additional controls. Our results indicated that a surprising amount of BKV sequence variation exists within a given individual. This diversity sheds new light on our understanding of BKV evolution.
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METHODS |
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Qualitative BKV PCR.
The presence of BKV DNA in the clinical samples was determined by PCR. A total of 200 ng DNA extracted from tissue or urine samples was used in a 50 µl reaction consisting of Platinum PCR Supermix (Invitrogen) and 10 pmol of each of the oligonucleotide primers BK2116F and BK2415R (Table 1), which amplified a 300 bp fragment of the BKV VP1 gene, or JCV-specific primers VP11 and VP12 (Koralnik et al., 1999
), or SV40-specific primers SP15 5'-TTAGCAGCTGAAAAACAGTTTACAGAT-3' (nt 17121738) and SP13 5'-TTAACAGTAACAGCTTCCCACATCA-3' (nt 18301854), which amplified a 181 bp fragment of the JCV and a 143 bp fragment of the SV40 VP1 genes, respectively. The amplification was carried out in a GeneAmp PCR System 9700 (PE Applied Biosystems) with a first denaturing step of 2 min at 94 °C, followed by 40 cycles consisting of 30 s denaturation at 94 °C, 30 s annealing at 55 °C and 1 min elongation at 72 °C, with a final elongation period of 7 min at 72 °C. Amplified products were analysed by electrophoresis on a 1 % agarose gel.
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Determination of the error rate of the Taq Plus Long PCR system.
To determine the error rate of the long PCR technique used for the BKVCAP, BKVHI and BKVHC strains, the Taq Plus Long PCR system was used to amplify the BKV Dunlop reference strain (BKVDUN) obtained from the American Type Culture Collection (ATCC). The pBR322-BKVDUN plasmid (containing the full-length BKVDUN DNA inserted at the BamHI site) was digested with BamHI to separate the BKV genome from the plasmid and with AvalI and PvuI to degrade the vector for 2 h at 37 °C in a water bath. After inactivation of the restriction enzymes at 70 °C for 30 min, 25 ng BKVDUN genome DNA and 1 µg human genome DNA were added to each PCR. The long PCR conditions were as described above. The 5 kb PCR-amplified products from 10 individual PCRs were purified from 0·5 % agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and cloned into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen). A 1360 bp (nt 21543513) fragment of one clone from each PCR was sequenced on both strands with sequencing primers BK2723F and BK3184R.
Laser capture microdissection (LCM).
This was performed on a fresh-frozen autopsy sample of the heart from the patient with BKV vasculopathy. Frozen tissues were cryo-sectioned on a Laser Pressure Catapulting 1·35 µm thin polyethylene film (PALM Microlaser Technologies), fixed in 70 % ethanol for 5 min and stained with 0·5 % toluidine blue in water.
Enlarged capillary endothelial cells containing nuclear inclusions or myocytes that appeared normal were captured using the Microbeam Laser apparatus (PALM Microlaser Technologies). One to six cells were captured in each microdissection. DNA from approximately 100 cells was extracted using the Gentra Systems Puregene DNA Isolation kit and resuspended in 40 µl Gentra DNA hydration solution. PCR amplification was performed using 10 pmol of the BK2217F and BK2320R primers (Table 1), which amplified a 104 bp fragment of the BKV VP1 gene, or the VP11 and VP12 primers, which amplified a 181 bp fragment of the JCV VP1 gene (Koralnik et al., 1999
), in a 50 µl reaction volume containing the Platinum PCR Supermix and 10 µl DNA solution. Amplified products were subjected to direct sequencing.
Evolutionary analysis of BKV sequences.
Previously published BKV sequences were obtained from GenBank (Table 2). DNA sequences were aligned and compared using CLUSTAL W (Thompson et al., 1994
). Maximum-likelihood phylogenetic analyses were performed using DNAML from the PHYLIP package (Felsenstein, 1993
). The input order of sequences was shuffled in 10 replicate analyses and the transition-to-transversion ratio was optimized at 1·9. The extent of synonymous and non-synonymous nucleotide substitution within coding sequences was estimated by the method of Li (1993)
and compared with previous estimates for JCV (Hatwell & Sharp, 2000
).
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RESULTS |
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Detection of BKV DNA in capillary endothelial cells after LCM
To confirm that these polyomavirions were BKV, capillary endothelial cells harbouring enlarged nuclei were sampled by LCM and their DNA was extracted and subjected to PCR amplification. A BKV, but not JCV, VP1 gene fragment was successfully amplified from capillary endothelial cells, but not from myocytes collected by LCM (data not shown), which confirmed that BKV was indeed located exclusively in the capillary endothelial cells of this patient (Petrogiannis-Haliotis et al., 2001).
Only three BKV strains, DUN (Gardner et al., 1971; Seif et al., 1979
), MM (Takemoto et al., 1974
; Yang & Wu, 1979
) and AS (Coleman et al., 1980
; Tavis et al., 1989
), have been entirely sequenced. These and most partially sequenced BKV strains published to date have been derived from isolates that have been cultured in vitro (Table 2
), which may induce mutations in their DNA, especially at the level of their non-coding regulatory region (Johnsen et al., 1995
). Since our goal was to compare the entire genome of this BKV variant with other strains of BKV ex vivo, we screened urine samples from 45 individuals (15 HIV-positive and 30 HIV-negative) for the presence of BKV DNA. Samples from 3/15 (20 %) HIV-positive (one HIV-2 and two HIV-1) and 2/30 HIV-negative (6·6 %) individuals had positive results. This was comparable to the percentage of BKV-positive urine samples reported in HIV-positive (Sundsfjord et al., 1994
) and HIV-negative individuals (Tsai et al., 1997
). DNA extracted from fresh-frozen autopsy samples of the heart and striated muscle of the BKV vasculopathy patient, as well as DNA extracted from the urine of one HIV-2-positive patient and one HIV-negative healthy control subject who tested positive by qualitative BKV PCR, were subjected to long PCR amplification.
Long PCR amplification of full-length BKV genomes
BKVDUN genomic DNA consists of a single copy of double-stranded circular DNA of 5153 bp (Seif et al., 1979). We therefore adapted a long PCR technique initially developed for JCV (Agostini & Stoner, 1995
) and SV40 (Lednicky et al., 1997
) to amplify full-length BKV genomes in a single reaction. Amplification of BKV DNA extracted from the heart and muscle of the patient with BKV vasculopathy and from the urine samples of one HIV-2-positive patient and one healthy control subject each yielded a 5 kb fragment. To study the potential genetic diversity of these viral isolates, amplified fragments were cloned and sequenced. Full-length sequences were obtained from five BKV clones from the muscle and four BKV clones from the heart of the patient with BKV vasculopathy (BKVCAP) and from three clones from the urine of the HIV-2-positive patient (BKVHI) and the healthy control subject (BKVHC), respectively (Table 2
).
Variation within the regulatory region
The regulatory regions of BKVCAP, BKVHI and BKVHC consistently had an O1142-P168-Q139-R163-S163 pattern identical to the archetype BKVWW regulatory region (Markowitz & Dynan, 1988; Moens et al., 1995
; Sugimoto et al., 1990
) in all clones, except for minor deletions from nt 182 to 190 in one BKVHI clone (HI-u8) and from nt 224 to 272 in one BKVHC clone (HC-u2), which did not affect the T antigen binding sites (Moens et al., 1995
). Single isolated nucleotide substitutions were also found in CAP-m13 (T
C at nt 45) and CAP-m5 (A
G at nt 278), the latter being also present in HI-u6 and HI-u8 clones. This indicates that, unlike other BKV strains with expanded tropism (Stoner et al., 2002
), infection of capillary endothelial cells by BKVCAP was not caused by changes in its regulatory region. In addition, six isolated substitutions were found in all HC clones compared with BKVWW including T
G at nt 41, G
A at nt 86, A
T at nt 160, T
C at nt 173, A
G at nt 253 and A
G at nt 330.
Variation within the coding region
The coding region sequences of the BKVCAP, BKVHI and BKVHC strains had unique nucleotide differences in each clone. There was an unusual pattern of sequence variation among the nine CAP clones (Fig. 1). At 61 of 63 variable sites, one clone differed from the other eight; at the other two sites, the difference was shared by just two clones. The lack of variation shared among multiple clones has implications concerning the structure of the viral population within a host (discussed below). It also implies that the consensus sequence is the best estimate of the sequence of the common ancestor of the various clones. Interestingly, the five muscle clones exhibited greater divergence from this consensus (mean 10·4 differences) than the four heart clones (mean 3·3). The differences from the consensus can be used to examine the pattern of mutation within the BKV population. These mutations were highly non-random (Table 3
). The vast majority (59/63=94 %) of changes occurred at A or T sites. In addition, most of the changes (55/63=87 %), including most of the changes at A or T sites (52/59=88 %), were transitions. Of note, it was not possible to distinguish, for example, whether an A
G change on the strand sequenced represented an A
G mutation on that strand or a T
C mutation on the complementary strand. Thus, it was possible to distinguish six categories of mutation: among the CAP clones, 52 of the total of 63 changes (83 %) belonged to just one category, transitions from A or T.
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Phylogenetic analysis and genotypic classification of BKV sequences
The BKV strains from these three patients were compared with the three previously published full-length sequences in a phylogenetic analysis (Fig. 2a). Even though the extent of diversity among different CAP clones overlapped that between CAP and HI (Table 4
), the multiple clones from each patient formed monophyletic clades, suggesting that each patient was infected from a single source.
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Full-length VP1 sequences were available from an additional six viruses. These included one sequence (GenBank accession no. Z19534) described as being the original BKV isolate (Jin et al., 1993a). Preliminary comparisons indicated that this sequence was recombinant: across the first 320 codons this sequence differed from strain MM at only 2 nt and from strain SB at 43 nt, whereas in the other 43 codons it differed from MM at 11 nt but was identical to the SB sequence. Therefore, we excluded this sequence from the phylogenetic analysis of VP1 sequences (Fig. 2b
). The other five additional sequences included one, SB, previously designated as genotype II (Gibson & Gardner, 1983
); the other four appeared to be genotype I and consisted of WW (Chauhan et al., 1984
), DIK (Goudsmit et al., 1981
), JL (Pauw & Choufoer, 1978
) and MT (Sugimoto et al., 1989
).
The VP1 phylogeny indicated that three of these genotype I sequences fell within the radiation of the strains CAP, HI and HC; surprisingly, one strain (JL) fell within the radiation of HC clones. JL differed from the HC-u5 and HC-u9 sequences at only one site, while HC-u2 differed from HC-u5, HC-u9 and JL at a single other site. This degree of identity among sequences from two individuals in different countries is surprising given the amount of diversity seen among the CAP clones (Tables 2 and 4).
The other interesting feature of the VP1 phylogeny was that the genotype II (SB) and III (AS) strains exhibited less divergence than different genotype I strains. The various BKV strains included in the VP1 phylogeny were obtained from individuals in Africa, Asia, Europe and North America (Table 2). SB and AS were both derived from individuals in the UK, but otherwise among the genotype I strains there seemed to be no correlation between geographical origin and phylogenetic relationships.
Recently, it has been suggested that two subgroups of genotype I (Ia and Ib) can be defined on the basis of differences at three nucleotide positions within a larger region surrounding the typing region (Stoner et al., 2002). However, all three sites are third positions of codons and in each case the differences proposed to be diagnostic of subgroups Ia and Ib were synonymous changes. Thus, these three differences cannot lead to any serological distinction. Furthermore, because synonymous substitutions generally occur more frequently than non-synonymous substitutions, these sites may not be reliable phylogenetic markers. Consideration of these sites within the VP1 sequences of strains in Fig. 2(b)
confirmed this. The DUN and WW strains represent the proposed subgroups Ia and Ib: DUN had T-G-T at the three sites, compared with A-A-C in strain WW. Strain MT had A-G-T, different from both of the other two; since MT is phylogenetically distinct from DUN and WW (Fig. 2b
), it might seem reasonable to classify this strain as a third subgroup. However, while eight of the nine CAP clones had A-A-C (as for subgroup Ib), the other (CAP-m9) had A-A-T; clearly this clone should not be classified as a fourth subgroup. The CAP-m9 clone fell within the radiation of CAP sequences, but at these three sites differed from other CAP sequences to the same degree that it differed from MT or even from genotypes II and III (both A-C-T). Thus, variations at these three sites do not seem to form a sound basis for classification into subgroups.
Analysis of VP1 proteins
BKVCAP tropism for vascular endothelial cells may have been caused by changes in the amino acid sequence composition of the major capsid protein, VP1. We therefore aligned the deduced VP1 sequences of all BKVCAP clones and compared them with BKVDUN, as well as the novel BKVHI and BKVHC clones (Table 5a). BKVCAP clones had three to seven VP1 amino acid changes compared with BKVDUN. Some of these amino acid changes were unique to a given clone, but none of them was present in more than one of the BKVCAP clones and not in the BKVHI and BKVHC clones.
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The only crystal structure of the VP1 capsid protein of polyomaviruses that has been characterized is that of SV40 (Liddington et al., 1991). We therefore examined the structure of SV40 VP1 pentamers at positions that correspond to the amino acid changes seen in the VP1 protein of BKVCAP clones compared with BKVHI and BKVHC. None of these changes were at positions likely to be exposed on the surface of the virions and, therefore, no conformational differences could be expected to result in a novel interaction with potential cellular receptors. In addition, the C terminus of the capsid protein VP2, which contains its binding site with the VP1 pentamers, showed no significant differences among the clones of these three viruses.
Analysis of T antigen sequences
The large T protein is the main regulatory protein of BKV. It has a crucial role in the life cycle of this virus since its presence is required for the initiation of BKV DNA replication and for activation of the switch from early to late gene transcription. It also contains binding sites for nuclear proteins, as well as a host-range domain. Therefore, changes in the T antigen structure may be responsible for the vascular tropism of the BKVCAP strain.
Although three to seven amino acid changes compared with BKVDUN could be seen in the T antigen of BKVCAP clones, none of them was present in all BKVCAP clones, nor in the BKVHI or BKVHC clones (Table 5b). Analysis of the T antigen binding site showed no mutations at positions 4298 and 4299, previously described in the virulent BKVCin strain in an AIDS patient with end-stage renal disease (Smith et al., 1998
). In addition, no significant amino acid changes were detected in any of the BKVCAP clones in the DNA-binding domain, zinc-finger domain, p53-binding domains, host range domains or phosphorylation sites. Therefore, changes of the amino acid sequence of the T antigen of BKVCAP clones could not readily explain the difference in cell tropism of this novel BKV strain. In contrast, three unique amino acid changes were found in the BKVHC clones only and one amino acid change was found in BKVHI clones only.
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DISCUSSION |
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To circumvent the need for virus culture altogether, we chose to amplify the complete genome of BKV isolates in a single PCR. Moreover, we sequenced three to nine clones from each patient's isolates, to gain insights regarding their genetic diversity. The availability of 15 novel full-length molecular clones of BKV thus radically expands our knowledge of the genetic diversity of this virus. In addition, comparison of multiples clones derived simultaneously from the same patient sheds a new light on BKV evolution. Indeed, there are few data available on the intra-patient genetic diversity of the coding region of BKV, since previously published studies used direct sequencing of PCR products, which aims to reveal only the most prominent species present in a sample and renders the detection of polymorphisms difficult (Jin et al., 1993b; Randhawa et al., 2002
).
The extent of sequence variation among clones from individual patients in our study was surprising. The most divergent sequences from patient CAP differed at 0·55 % of sites. BKV infections often occur at a very early age (Tavis et al., 1990). This patient with BKV vasculopathy was 52 years old when the samples were taken. If the initial BKV infection arose from a single strain, two viruses from this BKV population could have been diverging for about 50 years. A divergence of 0·55 % then implies a rate of nucleotide substitution of around 5x105 substitutions per site per year [i.e. 0·55 %/(2x50) to account for divergence along two lineages from the common ancestor]. Even taking the lower mean divergence seen in the two other patients (i.e. around 0·15 %) who could each have been infected for a little over 40 years yields a rate of evolution around 2x105 substitutions per site per year. These rate estimates are surprisingly high for a DNA virus and about two orders of magnitude greater than the rate of 4x107 per site per year estimated for synonymous substitutions among different JCV genotypes (Hatwell & Sharp, 2000
).
Several factors could contribute to these results. First, the possibility that the genetic diversity observed among the BKV clones was artificially inflated by errors of the Taq polymerase during the PCR and by sequencing artefacts must be considered. However, the fidelity of the Taq Plus DNA polymerase we used for the long PCR amplification is extremely high, with an error rate of only 1·3x106±2·2 (Cline et al., 1996). Therefore, on average, the PCR enzyme should give rise to only 1 nt change per 150 full-length BKV clones under ideal conditions. However, this error rate may only pertain to experiments conducted under certain optimal conditions. In our hands, sequencing of long PCR-amplified products of the BKVDUN reference strain from 10 individual reactions revealed a somewhat higher error rate (7·35x105). Nevertheless, the rate of mutations observed in the same region among the nine BKVCAP clones was more than 20 times higher than this (1·63x103) and thus this high intra-strain genetic diversity cannot be accounted for by errors of the Taq polymerase. Furthermore, sequencing artefacts could be discounted because the analyses were all performed on both strands of each clone. Surprisingly, we also found a single point mutation compared with the published BKVDUN sequence (position 3472, silent mutation A
T) in all 10 clones. We believe that this mutation, which is present in all BKVDUN clones from 10 separate PCRs, was not caused by an error of the PCR enzyme, but existed already in the plasmid obtained from the ATCC. Indeed, this A
T change has already been reported in BKV strains MM and AS, and was seen in this study in all CAP, HI and HC clones.
Secondly, although there are no obvious aspects of BKV and JCV biology that would be expected to lead to large differences in their evolutionary rates, the rate of evolution of BKV may be higher than that of JCV because of a higher mutation rate and/or a higher replication rate. Consistent with this, the diversity among BKV strains is higher than that among JCV strains; for BKV, the differences among genotypes are extensive enough to give rise to distinct serotypes, whereas that is not the case for JCV. The extent of divergence between BKV genotypes I and III is about twice that seen between the most divergent JCV genotypes, which is only a little higher than that seen within BKV genotype I (Table 6). In contrast to JCV, there is as yet no clear association between genetic diversity and geographical origin or ethnic background for BKV strains, perhaps because the broader host cell range of BKV renders it more readily transmissible, and so the historical epidemiology of the two viruses may have been different. Therefore, the greater diversity in BKV than in JCV may merely reflect a greater length of time since the most recent common ancestor for BKV strains, rather than a faster evolutionary rate.
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Our analyses also revealed that the previously published VP1 gene sequence described as being from the original BKV isolate (Jin et al., 1993a) is a likely recombinant of genotype I and genotype II sequences. Dual infection with more than one subtype has been described (Jin et al., 1995
), which may permit recombination to occur. A recombinant JCV sequence has been identified (Hatwell & Sharp, 2000
). However, for the BKV sequence, as with the JCV sequence, it is not yet clear whether the sequence reflects a recombinant virus or recombination in the laboratory.
Extensive analyses of potential molecular determinants of the vascular tropism of BKVCAP remained inconclusive. Our results indicate that the regulatory region is not responsible for the new phenotype of this isolate. Indeed, the regulatory region of all nine BKVCAP clones sequenced had an O-P-Q-R-S pattern identical to the archetype BKVWW regulatory region (Markowitz & Dynan, 1988; Moens et al., 1995
; Sugimoto et al., 1990
) found in primary urine isolates, including the novel BKVHI and BKVHC strains described in this study. This contrasts with JCV, where central nervous system strains usually have a regulatory region with a typical tandem repeat pattern and variable deletions compared with the archetype regulatory region found in the kidney (Major et al., 1992
).
We also explored whether amino acid changes in the capsid proteins of the BKVCAP clones could be responsible for conformational differences that may lead to their preferential entry in capillary endothelial cells. Such dramatic extension of host cell range has been demonstrated in mouse polyomavirus, where a single amino acid change (GE) at position 92 in the VP1 protein induces an increase in tumour incidence in a broader spectrum of tissues (Freund et al., 1991
). However, we could not identify amino acid changes present in the VP1 or VP2 proteins of most BKVCAP clones that were not previously identified in non-vasculotropic BKV strains (Table 5
). In addition, conformational analysis of the VP1 protein of BKVCAP clones failed to show any changes at the surface of the virions that could result in a novel interaction with potential cellular receptors. To our surprise, we found that each BKVCAP clone had several unique amino acid differences. Since BKVCAP was not detected in renal tubular cells but exclusively in the capillary endothelial cells of the kidney, muscle and heart of this patient, it is unclear which, if any, of these changes is responsible for the initial vascular tropism of this particular isolate. It is indeed possible that some of these changes are the consequence, instead of the cause, of the expanded host cell range of this virus.
In addition to cell entry, a productive infection by polyomaviruses requires a successful interaction between the cellular machinery and both the virus regulatory region and the T antigen, which is necessary to initiate the expression of capsid proteins and viral DNA replication. Again, no unique amino acid changes could be identified in the T antigen of all BKVCAP clones that were not already described in non-vasculotropic isolates (Table 5b) and a similar diversity was found in their T antigen as in their VP1 protein (Table 5a
).
Therefore, the event that led to successful entry and replication of BKV in capillary endothelial cells remains unclear. It may be that a unique feature of the patient himself, such as mutation in a cellular protein present in capillary endothelial cells, was in fact responsible for this peculiar clinical presentation. Furthermore, the mechanisms by which BKV caused an increased vascular permeability in this patient are not resolved. Direct action of the virus on the cell membrane or possibly an immune-mediated mechanism may play a role in this syndrome. Studies of the phenotype of BKVCAP clones in vitro are currently in progress in our laboratory to address these issues.
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ACKNOWLEDGEMENTS |
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Received 18 December 2003;
accepted 11 May 2004.