Download Human polyomavirus JC control region variants in persistently

Journal
of General Virology (1998), 79, 789–799. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Human polyomavirus JC control region variants in persistently
infected CNS and kidney tissue
Cornelia Elsner and Kristina Do$ rries
Institut fu$ r Virologie und Immunbiologie der Universita$ t Wu$ rzburg, Versbacher Str. 7, D-97078 Wu$ rzburg, Germany
The question of a possible role for JC virus (JCV)
genomic rearrangements in the pathogenesis of
progressive multifocal leukoencephalopathy (PML)
was addressed by analysis of the genomic complexity and the transcriptional control region (TCR)
of the JCV DNA population in persistently infected
CNS and kidney tissue. After cloning of full-length
viral DNA, no extensive changes were detected in
the coding regions of the JCV genome by restriction
analysis suggesting an intact JCV DNA population.
For further analysis of the distribution of JCV
subtypes, the non-coding region was amplified by
PCR. Molecular analysis revealed homogeneous JCV
TCR populations in almost 50 % of the individuals.
Heterogeneity was found in two CNS samples with
three and five different JCV subtypes, respectively,
Introduction
Infection with the human polyomavirus JC virus (JCV) is
usually acquired in the first decade of life with seroconversion
rates of more than 90 % in the adult (Arthur & Shah, 1989 ;
Kitamura et al., 1990 ; Major et al., 1992). Primary contact leads
to lifelong asymptomatic persistence in the kidney (Chesters et
al., 1983 ; Do$ rries & Elsner, 1991 ; Flaegstad et al., 1991) and the
CNS (Chesters et al., 1983 ; Elsner & Do$ rries, 1992). Additionally, JCV DNA is found in tonsillar cells, leukocytes and
the bone marrow (Do$ rries et al., 1994 ; Monaco et al., 1996 ;
Myers et al., 1989). Activation of persistent virus infection
occurs frequently without causing disease (Coleman et al.,
1980). Only in cases of severe impairment of immunocompetence (Schmidbauer et al., 1990 ; Walker & Padgett,
1983) may cytolytic JCV infection of oligodendrocytes cause
the fatal CNS disease progressive multifocal leukoencephalopathy (PML). Although the number of cases is steadily
increasing due to the AIDS epidemic, the pathomechanisms
leading to PML are not yet understood. As well as the role of
Author for correspondence : Kristina Do$ rries.
Fax ­49 931 201 3934. e-mail Doerries!mail.uni-wuerzburg.de
and in four kidney specimens with two TCR subtypes.
Altogether, seven TCR subtypes were identified.
One in each group represented single promoter
element TCRs without duplication of sequences. The
TCR of the major variant JCV-W1 was comparable in
sequence and structure to that of the PML prototype
JCV Mad-1 DNA. The identification of dominant
PML-derived JCV TCR subtypes in most persistently
infected individuals suggests that rearrangements
of the JCV TCR can be associated with the persistent
state of infection. However, it appears unlikely that
PML-associated JCV subtypes are generated anew
in each individual host in the course of persistence.
The findings rather suggest that a limited number of
stable JCV subtypes circulate in different geographical regions of the world.
severe impairment of immunological surveillance, genomic
heterogeneity of JCV DNA was assumed to be associated with
development of the disease.
Characterization of JCV genomes in PML tissue revealed
that coding DNA segments are conserved with changes at the
nucleotide level not necessarily leading to changes in the
amino acid composition of proteins. Some of the changes in
the carboxy-terminal segments of VP1, T antigen and the
intergenic region are conserved in clustered geographical
groups. Therefore, they are used as a basis for the so-called VT genotyping scheme (Agostini et al., 1997 ; Ault & Stoner,
1992 ; Frisque et al., 1984 ; Loeber & Do$ rries, 1988). The noncoding DNA segment containing the origin of DNA
replication and the transcriptional control region (TCR) is
conserved with respect to the nucleotide sequence, but
heterogeneous in its structure (Agostini et al., 1997 ; Grinnell et
al., 1983 ; Martin et al., 1985 ; Matsuda et al., 1987). Promoter
elements in PML are classified into TCR type I and TCR type
II DNA. A prerequisite for TCR type I JCV genomes is the
fusion of the TATA box and following sequences leading to
repetition of elements that include the TATA box. TCR type
II JCV DNA has one TATA box proximal to the early mRNA
initiation site that is not included in the repeated elements.
0001-5074 # 1998 SGM
HIJ
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C. Elsner and K. Do$ rries
Table 1. Patients and organ material analysed for the presence of JCV DNA in
persistently infected organs
PCR products
Case
CNS
B2
B14
B17
B20
B23
Kidney
K1
K3
K4
K6
K7
K8
K9
K10
Age/sex*
T
antigen
Control
region
34}m
Not known
74}m
84}m
69}m
­
­
­
­
­
­
­
­
­
­
Multiple sclerosis
Liver carcinoma
Rectum carcinoma
Heart failure
Heart failure
59}m
11}m
50}m
58}m
77}f
64}m
77}f
32}m
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
HSV encephalitis
Leukaemia
Creutzfeldt-Jacob disease
Aneurysm
Liver cirrhosis
Leukaemia
Cardio-pulmonal failure
Renal transplantation,
aneurysm
Disease
* Age is given in years ; m, male ; f, female.
Both subtypes have conserved factor binding sites in common
that are framed by highly variable sequences preferentially
used as sites of recombination (Agostini et al., 1997 ; Ault &
Stoner, 1993 ; Loeber & Do$ rries, 1988). TCRs with unique
DNA sequences of single promoter elements were detected in
kidney and urine (Flaegstad et al., 1991 ; Loeber & Do$ rries,
1988 ; Tominaga et al., 1992 ; Yogo et al., 1990, 1991 b). In
addition to genotype and TCR variants, defective polyomavirus genomes were observed in semipermissive cell cultures
and tissue. Assuming that such molecules might be generated
in the course of persistent JCV infection, a possible role of a
defective DNA population with extensive deletions or substitutions within coding sequences has been repeatedly
discussed (Grinnell et al., 1982 ; McCance, 1983 ; Yoshiike et al.,
1982).
Comparison of JCV DNA populations with a high rate of
virus replication in PML brain, and those exhibiting low rates
in persistently infected kidney tissue demonstrated JCV DNA
molecules of different TCR types but with highly related
coding sequences in the respective tissue of one patient (Ault
& Stoner, 1994 ; Do$ rries, 1984 ; Loeber & Do$ rries, 1988).
Promoter elements of TCR types from cytolytically infected
brain tissue included complex rearrangements. In contrast,
virus from histopathologically healthy kidney tissue exhibited
control regions without extensive sequence duplications. From
these findings, the assumption was drawn that the parental
rather attenuated JCV archetype may circulate in the human
population, invading the CNS during persistence. It appeared
HJA
to be likely that the PML-type of JCV variants might
subsequently be generated anew in each host from the
archetype TCR during asymptomatic activation processes (Iida
et al., 1993 ; Kato et al., 1994 ; Yogo et al., 1990). The detection
of minor heterogeneity in JCV populations from PML brain
(Yogo et al., 1994) supported this hypothesis. However, the
time-scale of rearrangements in the course of infection and the
extent of genomic changes in the CNS prior to disease
remained uncertain.
In an attempt to elucidate the extent of the genetic
heterogeneity in persistently infected organs, we determined
the genomic complexity of JCV genomes in CNS and kidney
tissue without histopathological changes typical for polyomavirus-induced disease. Analysis of JCV rearrangements in the
TCR revealed the presence of one dominant JCV TCR subtype
among minor TCR variants in both organs. The TCR was
found to be identical in sequence to that of PML-derived JCV
TCR types, thus demonstrating that PML-type viruses can
similarly invade the CNS and the kidney prior to disease. The
characterization of a PML-type TCR as the dominant virus
type in peripheral tissue of immunocompetent individuals
suggests that rearranged TCR types are circulating in the
human population and are not necessarily generated in each
host.
Methods
+ Patient material. Autopsy material was analysed from CNS and
kidney of 13 patients from Western Europe. Brain samples were examined
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JC virus variants in persistently infected tissue
from patients with multiple sclerosis, heart failure and carcinomas of the
rectum and liver. Kidney material was from eight patients with herpes
simplex virus (HSV) encephalitis, Creutzfeldt-Jacob disease, liver cirrhosis,
leukaemia, aneurysm and cardio-pulmonal failure. Cellular DNA was
prepared by standard procedures (Do$ rries et al., 1994 ; Elsner & Do$ rries,
1992).
+ Cellular gene libraries from human brain and kidney tissue.
JCV genomes were cloned from tissue with the restriction enzyme BamHI
in the phage lambda replacement vector NM1151 (Elsner & Do$ rries,
1992). Cellular DNA was cleaved to completion and cloning was
performed essentially as described by Sambrook et al. (1989). Between
1¬10& and 6¬10& clones}µg cellular DNA were achieved by in vitro
packaging ; 1–3¬10' clones per specimen were analysed by plaque
hybridization with radioactive genomic JCV DNA probes (Elsner &
Do$ rries, 1992). Characterization of cloned JCV genomes was performed
by restriction endonuclease cleavage and followed by Southern blot
analysis.
+ Amplification of JCV-specific DNA by PCR. PCR was performed
with primer pairs either covering a DNA fragment of the early coding
region (TEP1}PEP2) or the non-coding region flanked by conserved
coding sequences (JCV71}72). Cellular DNA (1 µg) was amplified in
2±5 mM MgCl in a reaction volume of 50 µl with 1 U of Taq polymerase.
#
Cycling was as described previously (Do$ rries et al., 1994 ; Elsner &
Do$ rries, 1992). As a precaution against PCR contamination, all reactions
were performed in parallel with a complete set of controls. Negative
controls included testing of the buffer components in duplicate prior to
and after preparation of experimental reactions in laboratories used
exclusively for PCR. Characterization of amplification products was
performed in another laboratory by electrophoresis and radioactive
hybridization of Southern blots with a JCV-specific internal oligonucleotide or a specific ssRNA probe (Do$ rries et al., 1994 ; Elsner &
Do$ rries, 1992).
+ Cloning and typing of JCV control region elements. For
cloning, JCV-specific products were amplified by PCR with the primer
pair JCV71}72 containing an artificial BamHI and EcoRI restriction site,
respectively (Elsner & Do$ rries, 1992), and inserted into the bacterial
vector pBluescript SK(­). Recombinants were selected by colony
hybridization with a radioactive ssRNA probe (Elsner & Do$ rries, 1992).
JCV prototypes were cloned by the same strategy (Frisque et al., 1984 ;
Loeber & Do$ rries, 1988). The characterization of JCV inserts was
performed with the restriction enzymes BamHI}EcoRI and StyI followed
by electrophoresis on agarose gels.
+ Analysis of cloned JCV DNA segments by nucleotide
sequencing. DNA sequencing of the complete JCV control region
between the start codon of the putative agnogene and that of the T
antigen gene was performed from one-tube plasmid mini-preparations
(DelSal et al., 1988). Isolated DNA was denatured with 0±2 M NaOH
(10 min, 65 °C), neutralized by 0±4 vol. 5 M ammonium acetate (pH 5±5)
and ethanol-precipitated. For sequencing virus-specific internal oligonucleotides JCV 69, 87, 60, 53 and pBluescript primers 16 and 31 were
used (Do$ rries et al., 1994). Sequencing was performed with the enzyme
Sequenase and $&S-dATP (USB, Amersham) essentially as recommended
by the supplier.
Results
Complexity of JCV DNA in persistently infected brain
and kidney tissue
For characterization of the JCV DNA population in
persistently infected tissue, autopsy specimens were selected
from brain parenchyma and from the renal medulla of patients
with CNS diseases other than PML or with systemic disorders
(Table 1). In none of the cases, neurological symptoms or
histopathological patterns indicative of PML were observed.
Cellular DNA was subjected to amplification of JCV DNA.
One primer pair was specific for the T antigen coding region
and resulted in a fragment of 199 bp in length. A second
primer pair spanning the control region amplified a fragment of
about 600 bp in length (Elsner & Do$ rries, 1992). The specificity
of electrophoretically separated products was confirmed by
Southern blotting and JCV-specific radioactive hybridization.
PCR analysis revealed the presence of JCV-specific DNA
sequences in at least one of five brain specimens and one of
four kidney specimens from each case (Table 1).
In order to analyse the complexity of JCV DNA molecules
in persistently infected organs, the complete genome was
examined by cloning of JCV genomes from CNS tissue in two
cases (B20, B23) and from kidney tissue in three cases (K1, K3,
K4). The single BamHI restriction site in the early coding
region of the JCV genome was used for insertion in phage
lambda NM1151. Characterization of cloned JCV molecules
was performed by restriction cleavage with the enzyme BamHI
and combinations of BamHI with EcoRI, PstI, BglII, XbaI, NsiI,
HpaII, HincII or HindIII followed by Southern blot analysis.
The analyses revealed a JCV insert of 5 kb in length in all
clones from the brain and kidney. Comparison of the DNA
fragment pattern with that of the JCV prototypes showed the
characteristic JCV pattern with all enzymes used (data not
shown). This finding demonstrated that cloned viral DNA
molecules in persistently infected tissue were intact without
major rearrangements of coding sequences.
Comparison of JCV TCR subtype populations in brain
and kidney tissue by restriction mapping
Analysis of full-length JCV DNA with the enzyme
combination BamHI}PvuII focussed on the TCR. Palindromes
of the restriction enzyme PvuII are located close to and within
the non-coding region of the JCV genome resulting mainly in
three DNA fragments spanning conserved coding DNA
segments and a variable fragment C including the viral control
region. In one case, two out of 16 brain-derived clones
exhibited heterogeneous mobility of fragment C. One was not
related to the prototype segment C, whereas the second was
comparable in length to that of JCV GS}B (991 bp) (Fig. 1 a,
lanes 4 and 10). In the remaining clones, fragment C was
comparable to that of JCV Mad-1 (1093 bp). In contrast, clones
from the second brain specimen and all kidney-derived clones
from three cases had an identical fragment pattern comparable
in length to that of JCV Mad-1 (Fig. 1 b).
To determine the TCR heterogeneity further, JCV DNA
was amplified by PCR on brain tissue from three and on kidney
tissue from five additional cases (Table 2). Products were
directly cloned to avoid loss of less common JCV subtypes
involved. Twenty to 40 JCV-specific clones per case were
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HJB
C. Elsner and K. Do$ rries
(a) CNS
( b) Kidney
Fig. 1. Analysis of JCV genomes from
persistently infected organs ; (a) CNS and
(b) kidney. Restriction cleavage was
performed with BamHI/PvuII followed by
separation on a 1 % agarose gel and
Southern blot hybridization with a
radioactively labelled JCV-specific RNA
probe. JCV genomic DNA was cloned
from CNS and kidney tissues. Prototype
DNA GS/B, Mad-1 and archetype GS/K
DNA are shown on the left. Numbers at
the top of each lane represent individual
clones. BamHI/PvuII fragments A, B, D
represent coding DNA segments ;
fragment C includes the non-coding
region.
isolated, thus giving a representative view on the JCV
population present. A total of 91 clones was established from
CNS tissue, and 227 clones were achieved from kidney tissue.
Restriction cleavage of virus-specific inserts with the cloning
enzymes BamHI}EcoRI revealed the presence of heterogeneous
JCV TCR fragments in 67 % of the CNS samples and in 80 % of
the kidney samples.
A detailed analysis of the JCV control region was
performed with the restriction enzyme StyI. The enzyme
cleaved the insert into 5–6 fragments (Fig. 2 a, b). The
fragments A and B spanned sequences upstream of the origin
of DNA replication and downstream of late coding sequences.
Promoter elements were divided into 2–4 fragments as
HJC
demonstrated by comparison with those of prototype TCR
type I (JCV Mad-1), type II (JCV GS}B) and archetype DNA
(JCV GS}K). In CNS and kidney tissue, seven and two different
TCR StyI patterns were identified, respectively (Figs 2 and 3 a,
b, Table 2). Pattern JCV-W1 was comparable to that of
prototype JCV Mad-1. JCV-W6 was similar to that of
prototype JCV GS}B, and JCV-W7 was comparable to that of
archetype JCV GS}K. Four additional patterns, JCV-W2 to
JCV-W5, were detected. The JCV-W2 type exhibited DNA
fragments related to those of JCV Mad-1, although the entire
insert length was about 100 bp longer ; this was due to a
duplicated band. Fragment pattern JCV-W4 was also related to
the Mad-1 subtype. The missing 100 bp band was reflected in
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JC virus variants in persistently infected tissue
Table 2. JCV TCR variants in persistently infected CNS and kidney tissue
Tissue
Case
No. of
clones
analysed*
CNS
B20
16a
B23
6a
B2
17b
B14
14b
B17
38b
5
K1
91
16a
K3
7a
K4
6a
K6
43b
K7
38b
K8
42b
K9
39b
K10
36b
Totals
Kidney
Totals
8
227
JCV TCR type I†
JCV TCR type II†
W1
W2
W4‡
W6
W5
W3
W7‡
14}16
(87±5 %)
6}6
(100 %)
17}17
(100 %)
–
1}16
(6±3 %)
–
1}16
(6±3 %)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3}38
(7±9 %)
40}91
16}16
(100 %)
7}7
(100 %)
6}6
(100 %)
40}43
(93 %)
37}38
(97 %)
41}42
(97±6 %)
39}39
(100 %)
27}36
(75 %)
213}227
–
–
1}91
–
1}91
–
14}14
(100 %)
12}38
(31±6 %)
26}91
–
1}38
(2±6 %)
1}91
–
1}38
(2±6 %)
1}91
–
21}38
(55±3 %)
21}91
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3}43
(7 %)
1}38
(3 %)
1}42
(2±4 %)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
9}36
(25 %)
14}227
–
–
–
–
0}227
0}227
0}227
0}227
–
–
–
–
0}227
* a, isolated by direct cloning ; b, isolated after PCR amplification.
† Type determined by restriction analysis and sequencing.
‡ Related sequence with single elements.
the reduced length of the entire insert. JCV-W5 had a pattern
with a smaller fragment C that showed similarities to that of
the archetype DNA. JCV-W3 clones exhibited a pattern that
was not comparable to any of the other JCV TCR types. Thus,
in addition to a new as yet unknown TCR pattern, control
regions similar to those of PML-derived JCV genomes at both
sites of infection were found.
Nucleotide sequence of variant control regions from
persistently infected tissue
The detection of JCV TCR segments of the PML-type in
persistently infected tissue was rather unexpected. Therefore,
the JCV control region elements were further characterized by
DNA sequencing (Fig. 4). To improve the comparison of
divergent DNA sequences, the JCV control region was divided
into the origin of DNA replication (ori) and seven DNA
segments (A–E, F1, F2) with reference to Ault & Stoner (1993).
On the early side of all TCR types, ori sequences were highly
conserved. The length of TCR type I control regions varied
between 726 and 522 bp (Fig. 2). The DNA segment A, a
TATA box element, was fused to nucleotides of segment C
and segment E. In TCR type II DNA, the control region varied
in length between 581 and 662 bp. Segment A was fused to
segment B and the following segment C. In TCR type I,
segment C was conserved, whereas segment C of TCR type II
was variable in length with deletions of 8–21 bp at the 5« end.
Segment B separated segments A and C leading to repeated
elements without TATA box sequences. These conserved
sequence elements were framed by variable sequences leading
to differentially constructed repeated elements. Downstream
sequences included a 16–18 bp segment E in both TCR types.
In TCR type II, these were followed by the 3«-truncated forms
of the 66 bp archetype segment D. The sequence of segments
F1, F2 and Agno downstream of the initiation codon of the
putative agnogene was conserved in six out of seven subtypes
identified. Rearranged segments E through to Agno were
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HJD
C. Elsner and K. Do$ rries
(a) CNS
(a)
(b)
(b) Kidney
Fig. 2. Restriction endonuclease mapping of the JCV subtype control
region. PCR amplification of the control region and flanking early and late
coding sequences was carried out with degenerative primers containing
restriction sites for BamHI and EcoRI, respectively ; the amplification
products were cloned in the bacterial vector pBluescript SK(­). (a)
Positions of StyI restriction sites in the control region are indicated in the
prototype JCV Mad-1, GS/B and archetype GS/K DNA segments. A–E
represent StyI restriction fragments ; ORI, origin of DNA replication ; n,
nucleotide position ; a, numbering according to Loeber & Do$ rries (1988).
Early and late genes show the orientation of the TCR. (b) StyI fragment
composition of JCV subtype control regions ; *, values normalized after
nucleotide sequence analysis.
identified only once (Fig. 5, W6). This TCR subtype was
previously observed in PML tissue (Loeber & Do$ rries, 1988).
Single point mutations were repeatedly detected. However,
comparison of different clones of the same subtype revealed
random distribution of the changes. This suggested that single
point mutations were probably introduced at least in part by
the PCR amplification step. The genotype defining single base
change on the late side of ori at the archetype TCR position
133 was represented by the nucleotide adenosine in all cases.
This is a common finding in American and European isolates
(Yogo et al., 1990).
JCV TCR subtype lineages in persistently infected
individuals
The JCV control regions identified so far were divided into
three types. Type I and type II both contain repeated conserved
elements that might even include the TATA box or extend to
late coding sequences. In contrast, the so-called archetype TCR
is formed by unique sequences and a complete segment D that
is located downstream of the conserved region C. As shown in
Fig. 5, the major JCV subtype W1 carried a type I TCR. The
HJE
Fig. 3. Characterization of JCV TCR subtypes from CNS (a) and kidney (b)
tissue after PCR amplification and subcloning. JCV control regions were
amplified by PCR with a degenerative primer pair containing BamHI and
EcoRI cloning sites and cloned in the bacterial vector pBluescript SK(­).
BamHI/EcoRI/StyI restriction fragments were separated and compared with
the electrophoretic mobility of the control region fragments of JCV Mad-1,
GS/B and GS/K ; the JCV Mad-1 fragment length is indicated in bp. B2,
B14 and B17, brain tissue of individual patients ; K7, K6, K8 and K9,
kidney tissue ; numbers represent individual clones of one case.
DNA nucleotide sequence was almost identical to that of the
JCV prototype Mad-1. Additionally, two rearranged TCR
type I regions were identified differing only in the number of
the first conserved element. In JCV-W2, this element was
present in triplicate. JCV-W4 represented a single TCR element
identical in sequence to that of the first conserved type I
element. The downstream segments E, F1 and F2 were all
similarly arranged. Four TCR type II variants were identified
by the presence of segment B following the TATA box. JCVW6 was identical in sequence and structure to that of JCV
GS}B (Loeber & Do$ rries, 1988). In the early segment, JCV-W5
was highly related to JCV-W6, whereas the late part, including
the segments F1 through to Agno, was comparable to that of
the archetype. JCV-W5 and JCV-W6 both carried truncated
forms of the archetype related segment D suggesting a close
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JC virus variants in persistently infected tissue
Fig. 4. Alignment of the nucleotide sequence of JCV subtypes isolated from persistently infected individuals. The nucleotide
sequences of JCV TCR subtypes W1, W2, W4, W3, W5, W7 from German individuals are aligned from the core origin of DNA
replication to the start codon of the agnogene. Nucleotide numbering is according to Loeber & Do$ rries (1988). The DNA
strand has the polarity of the late mRNA. The start codon for the agnogene is indicated by an arrow. The sequence is divided in
seven boxed segments (A, B, C, D, E, F1, F2) ; shading is identical to that in Fig. 5. ‘ A ’ represents the TATA box and T antigen
binding sites are indicated. Deletions are marked by a dash.
Fig. 5. Structure of the promoter
elements of JCV TCR type I (JCV W1, W2,
W4) and type II (JCV W6, W5, W3, W7)
isolated from persistently infected
individuals. The DNA sequence of the
A–F2 segments is identical to that in Fig.
4. The initiation codon for the putative
agnogene is indicated by an arrow. Agno
represents the putative agnogene coding
DNA segment repeated in the JCV-W6
and the GS/B prototype TCR. Segment
length is given in bp ; AT represents the
TATA box ; ori, origin of DNA replication.
*, Nucleotides are numbered according to
Loeber & Do$ rries (1988). v, Deletion of
23 bp ; –, conserved sequences.
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HJF
C. Elsner and K. Do$ rries
relationship between these TCR variants. In JCV-W3, segment
D is eliminated and a deletion at the 3«-end of segment C and
at the 5«-end of the second B element could be observed. JCVW7 represented the archetype, which is thought to be the
ancestral type of all differentially arranged TCRs. The TCR
classification demonstrates that both lineages of rearranged
TCR types and the archetype TCR were present in persistently
infected individuals in Western Europe.
Distribution of JCV TCR subtypes in infected organs
Comparison of the TCR types in brain tissue revealed the
presence of major and minor subtypes (Table 2). The rate of
minor JCV types varied between 2±6 and 30±8 %. Major types
were found at a rate between 53 and 100 %. The PMLassociated subtypes JCV-W1 and JCV-W6 were the dominant
types in brain tissue with rates between 87±5 and 100 %.
Interestingly, the archetype JCV-W7 was present at a rate of
53 % in one brain specimen. This was higher than the closely
related duplicated TCR JCV-W6 (30±8 %) in the same specimen.
Although this finding suggested extensive rearrangements in
this patient, variant molecules were observed only in two out
of five cases. Thus, rearranged JCV TCRs are probably not
regularly present in persistently infected brain tissue. In the
kidney, JCV TCR populations were similarly homogeneous in
about 50 % of all cases (seven out of 13 cases). The dominant
TCR was the PML-derived prototype Mad-1. Only one minor
variant was detected at rates between 2±4 and 25 % in the four
heterogeneous DNA populations. This subtype contained a
single element TCR closely related to the Mad-1 subtype. The
same subtype was additionally detected in brain tissue ;
therefore the TCR type is not tissue-specific.
TCR JCV-W1 was the major type identified in 12 of the 13
patients irrespective of the tissue analysed. This revealed that
PML-type variants persisted in the CNS and the kidney of
individuals without JCV-associated disease. From the distribution of TCR subtypes in kidney and brain tissue,
rearrangements in the CNS of individual patients cannot be
excluded. However, it appears to be unlikely that changes in
the TCR are restricted to the CNS. The identification of the
JCV Mad-1 (JCV-W1) prototype as the only variant in the
majority of cases rather suggested the presence of conserved
stable JCV TCR subtypes circulating in the human population.
Discussion
The extensive divergence of the JCV control region in PML
patients gave rise to the hypothesis that the generation of
heterogeneous TCRs might be involved in virulence during
the pathogenic process. It was believed that JCV genomes may
change from an attenuated persisting subtype with single
promoter elements to a virulent virus type with rearranged
duplicated promoter elements growing efficiently in the CNS.
This hypothesis implicated that the state of disease is preceded
by progressive selection of JCV subtypes leading to a
heterogeneous JCV population in persistently infected CNS
HJG
tissue. Besides a hypervariability of the JCV TCR in the noncoding region of the genome, DNA rearrangements of coding
sequences were described in brain tissue of a PML patient and
as a result of JCV growth in tissue culture (Elsner & Do$ rries,
1992 ; Martin et al., 1982 ; Matsuda et al., 1987 ; Rentier Delrue
et al., 1981). Although detected in vivo only in rare cases and
in the late stages of disease, it was conceivable that JCV
genomes with major changes in the coding region of the
genome might become prevalent in the attenuated persistent
state of infection. To further evaluate the role of JCV variants
in the pathogenesis of PML, we examined the genomic
structure of JCV DNA in tissue of patients without histopathological changes indicative for JCV-associated disease.
Persistent infection was confirmed by PCR amplification of
JCV DNA. However, amplification of JCV DNA fragments
alone does not give evidence of the complexity of viral
genomes, and full-length genomic JCV PCR is not sensitive
enough for JCV detection in persistence (own observations).
Therefore, JCV DNA from tissue was cloned in a phage lambda
gene library and then analysed. Characterization of JCVspecific inserts revealed exclusively the presence of full-length
JCV genomes. Extensive restriction enzyme mapping gave no
indication of major changes within coding DNA segments.
This confirmed the high conservation rate of the genomic
structure (Grinnell et al., 1982 ; Martin et al., 1982 ; Rentier
Delrue et al., 1981) and suggested that rearranged coding
DNA segments are not regularly generated in the persistent
state of infection. Therefore, it appears rather unlikely that
defective JCV genomes play a major role in polyomavirus
persistence.
In contrast to the conserved coding region, restriction
analysis of the non-coding region revealed heterogeneity of
DNA segments carrying the TCR. Most of the genomes
revealed fragment lengths similar to that of the rearranged
PML prototype Mad-1 DNA. In view of reports on JCV
archetype TCRs with single elements in healthy individuals
(Markowitz et al., 1991 ; Yogo et al., 1990, 1991 b), this finding
suggested a more extensive involvement of PML-associated
promoter elements in persistent infection than thus far assumed
(Sundsfjord et al., 1994). To evaluate this finding further, the
TCR populations were analysed by PCR of the non-coding
region and following molecular characterization. Loss of minor
JCV variants was avoided by cloning of the products prior to
further analyses. Using this strategy, the clone number per
individual sample probably reflected the concentration of JCV
DNA, which appeared to be higher in the renal medulla than in
the CNS (Kitamura et al., 1994 a, 1994 b ; Stoner et al., 1996 ;
Tominaga et al., 1992).
Detailed sequence analysis of the JCV control region
revealed the presence of seven different TCRs in persistently
infected tissue samples from 13 patients. The most prominent
types at both sites of infection were identical to those of the
prototypes JCV Mad-1 and GS}B isolated from an American
and a German PML brain. The archetype structure described
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JC virus variants in persistently infected tissue
earlier as the dominant TCR type in renal infection was
detected as a minor population in brain tissue. This confirmed
the results from the cloning experiments above, but is in
contrast with the assumed predominance of JCV archetype
TCRs in persistently infected individuals (Markowitz et al.,
1991 ; Sundsfjord et al., 1994 ; Yogo et al., 1991 b, 1990).
In the attempt to find an explanation for this discrepancy,
the possible generation of artificial amplification products by
PCR-dependent recombination was taken into consideration.
Although recombination rates of almost 5 % were suggested,
depending on the PCR target DNA used (Meyerhans et al.,
1990), in our experiments the major TCR subtypes were not
only detected by PCR amplification but also in the cloned fulllength genomes. Therefore, PCR recombination and PCR
contamination appeared to be rather unlikely. Besides the
extensive control of the PCR experiments, the strongest
argument against contamination is the detection of the same
rate of JCV Mad-1-like TCRs in the classical cloning approach.
Additionally, analysis of the V-T intergenic genotype linked to
the described TCRs has been performed. In contrast to the
Mad-1 genome isolated from a PML patient in the USA, the
JCV-W1 genomes from persistently infected tissue are linked
to coding regions of V-T genotype 2. This confirms that the
JCV-W1 molecules are unique isolates. Interestingly, V-T
genotype 1 sequences were described in urine of individuals
from Northern Europe (Yogo et al., 1991 a). In contrast, in the
mixed American population almost all genotypes were found,
with a predominance of genotypes 1 and 2 in white Caucasians
(Agostini et al., 1997 ; Ault & Stoner, 1992). Therefore, the
detection of genotype 2 in our group might be representative
of the geographical distribution of JCV genotypes in Western
Europe.
The often propagated hypothesis that each individual
carries a unique JCV TCR subtype generated from the
circulating archetype (Yogo et al., 1990) is not only contradicted by the data presented here, but also by reports from
the USA demonstrating a predominance of the Mad-1 type in
persistently infected patients (Myers et al., 1989 ; White et al.,
1992). This points to a higher prevalence of the Mad-1 TCR in
selected groups in the USA, whereas in others the archetype
was found to be the dominant TCR. In Japan, almost exclusive
archetype-related TCRs with minor changes were described in
persistently infected tissue (Markowitz et al., 1991 ; Yogo et al.,
1991 b). These findings support the assumption that a limited
number of TCR types dominate the virus population in
different geographical regions of the world. The overgrowth
of distinct TCRs might be explained by differential stability of
TCR structures. Conserved stable TCR segments can be
observed in type I and type II TCRs. In case of the Mad-1
isolate, even the complete control region exhibits high stability
after a prolonged tissue culture history. This is demonstrated
by conservation of the DNA sequence and structure of the
direct JCV Mad-1 isolate (Br-1) and the cultured virus (Mad-1)
from the same PML brain (Grinnell et al., 1983 ; Martin et al.,
1982 ; Rentier Delrue et al., 1981). In contrast to this stable
organization, highly heterogeneous TCR populations and
defective genomes were found after tissue culture passages of
other TCRs (Howley et al., 1976 ; Martin et al., 1979 ; Osborn
et al., 1974). Therefore, it is conceivable that TCR structures
may have evolved which are more or less susceptible to
rearrangement events. Whether this is linked to the biological
function of the respective promoter element is not yet
determined.
Predominantly homogeneous TCRs or subtype populations
with limited heterogeneity were detected at both sites of
persistent infection. The major TCR subtype identified in
healthy CNS was similar to that in lytically infected PML
tissue. Therefore, a neurotropic selection of highly active JCV
TCRs as a pathogenic event is rather unlikely. This does not
exclude occasional rearrangements of TCRs in the course of
persistence. The conditions leading to the development of
variant TCR types are not yet known. In case of the archetype
TCR, sequence rearrangements with only small variations from
the archetype were observed in immunosuppressed renal
transplant patients (Kitamura et al., 1994 b ; Yogo et al., 1990).
This suggested that immunosuppression may support TCR
rearrangement. However, in the cases described here a
correlation of immune impairment by malignancies and the
presence of variant JCV DNA populations could not be found.
In a search for other possible factors responsible for TCR
rearrangements it became obvious that most studies concentrated on brain and kidney tissue, organs not exhibiting
cellular replication activity. In this context, the characterization
of lymphoid cells as a site of persistent JCV infection was
noticeable (Do$ rries et al., 1994 ; Major et al., 1992). This cell
compartment includes actively dividing cell populations and it
is conceivable that JCV TCR variants might be generated in
the replicating cells.
The occurrence of a major PML-derived JCV TCR
population in persistently infected tissue of individuals without
PML suggests that highly active JCV TCRs circulate in
Western Europe. They might already infect the individual host
on primary contact and distribute to the target organs of
infection. Therefore, it is not likely that active virus growth in
the oligodendrocytes depends on the selection of glia cellspecific promoter elements. Activation of infection and
induction of disease may rather be controlled by conditions
closely related to the immunological competence of the viral
host.
This study was supported by the Deutsche Forschungsgemeinschaft,
Sonderforschungsbereich SFB 165}B3 and by the Wilhelm Sander
Stiftung, Grant 91.061.
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Received 9 July 1997 ; Accepted 10 November 1997
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HJJ