Journal of General Virology (1995), 76, 1571-1581. Printedin Great Britain
1571
Subpopulations of non-coding control region variants within a cell
culture-passaged stock of BK virus: sequence comparisons and
biological characteristics
John Inge Johnsen, 1 0 l e Morten Seternes, 1 Terje Johansen, 2 Ugo Moens, 1 Rauno M/intyj/irvi 3 and
Terje T r a a v i k 1.
Departments of 1 Virology and 2 Biochemistry, Institute of Medical Biology, University of Tromso, N-9037, Norway
and 3Department of Clinical Microbiology, University of Kuopio, Kuopio, Finland
In the circular DNA genome of the human polyomavirus
BK an approximately 400 bp non-coding control region
(NCCR) separates the early and late genes. The NCCR
contains the origin of replication as well as the
promoter/enhancer with a mosaic of cis-acting elements
involved in the regulation of both early and late
transcription. The NCCR has been shown to be very
heterogeneous between different BK virus (BKV)
strains. This may affect the host cell permissivity and
oncogenic potential of a given BKV strain. Our previous
studies with BKT-1B, a continuous cell line established
from a BKV (Gardner) -induced hamster fibrosarcoma,
revealed that the BKV DNA is integrated in the host
genome in multiple copies. The sequence of the
integrated BKV NCCR was substantially different from
(and even contained sequences not found in) that of the
BKV (Gardner) strain supposedly used to establish the
BKT-1B cell line. PCR amplification, cloning and
subsequent sequencing revealed that the original BKV
(Gardner) stock contained at least seven different
subpopulations of viral genomes. None of them had a
control region 'anatomy' which was identical to either
the BKV (Gardner) strain, the variant found integrated
in BKT-1B cells or any previously published NCCR. In
order to study the biological significance of these new
BKV NCCR variants we developed a simple cassette
model allowing the NCCRs of the new variants to be
cloned in an identical genomic background of BKV
protein-coding sequences and performed transfection
studies with the recombinant genomes in non-permissive
rodent cells and in semi-permissive monkey cells. The
results demonstrated that the NCCR variants conferred
striking differences, in both transforming capacity and
host cell permissivity, to the recombinant BKV genomes.
Sequence comparisons suggested genetic explanations
for these differences, as well as evolutionary relationships between BKV NCCRs.
Introduction
During the last few years a number of different BKV
strains have been isolated. DNA sequencing and restriction enzyme analyses have, with few exceptions,
revealed a strong sequence conservation in the proteincoding regions of the genome (Seif et al., 1979; Yang &
Wu, 1979a, b; Tavis et al., 1989; Sugimoto et al., 1990;
Jin et al., 1993), whereas the NCCRs exhibit considerable
variation between different BKV isolates. It should be
added though, that relatively few BKV strains have been
sequenced completely and variation in coding sequences,
with biological consequences, has been demonstrated for
mouse polyomavirus (Dubensky et al., 1991 ; Freund et
al., 1991). Biochemical and genetic studies have identified
an assortment of individual genetic elements that may
contribute to the biological activities of the BKV NCCR
(Watanabe & Yoshiike, 1986; Deyerle et al., 1987; Cassil
& Subramani, 1988; Deyerle & Subramani, 1988; Grinell
et al., 1988; Markowitz & Dynan, 1988; Cassil et al.,
The approximately 5.2 kbp double-stranded, circular
genome of the ubiquitous (Brown et al., 1975) human
polyomavirus BK (BKV) is organized and expressed as
in other polyomaviruses (Fig. 1), with the coding regions
for early (T, t antigens) and late (agno, capsid proteins
VP1 3) genes separated by a non-coding control region
(NCCR) of approximately 400 bp. The NCCR contains
the origin of replication as well as a promoter/enhancer
with a mosaic of cis-acting elements involved in the
regulation of both early and late transcription (reviewed
in Yoshiike & Takemoto, 1986).
* Author for correspondence. Present address: Department of
Virology, Institute of Medical Biology,MH-Bygget,Breivika, University of Tromso, N-9037 Tromso, Norway. Fax +47 776 45350.
e-mail [email protected]
0001-2939 © 1995 SGM
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1572
J. L Johnsen and others
VP2/3 m
Large T mRNA
Fig. 1. Physical and functional map of the BKV (Gardner) genome.
Thick lines represent the coding sequences for BKV proteins. The
putative agnoprotein has been omitted. The broken lines represent the
spliced-out regions. The numbering is according to Yoshiike &
Takemoto (1986), but includes the 43 bp region which is not present in
the Dunlop strain (Yang & Wu, 1979b). The restriction sites for the
endonucleases EcoRI (E), HindlII (H) and SacI (S) are indicated.
1989; Chakraborty & Das, 1989). It was proposed that
the NCCR hypervariability was due to recombinational
events during replication, resulting in different duplications and deletions of the same basic sequences
(reviewed in Yoshiike & Takemoto, 1986). Most BKV
NCCRs that have been PCR amplified and sequenced
directly from human urine, without cell culture passages,
contain no duplications and/or deletions of common
sequence motifs (Rubinstein et al., 1987; Sundsfjord et
al., 1990, 1994; Fla~gstad et al., 1991 ; Markowitz et al.,
1991 ; Negrini et al., 1991). BKV strains with such linear
NCCR anatomy became considered to be BKV archetypes, the prototype of which is B K ¥ (WW) (Rubinstein
et al., 1987). The promoter/enhancer of such strains was
arbitrarily divided into three sequence blocks called,
starting from the early side, P(68 bp)-Q(39 bp)-R(63 bp)
(Markowitz & Dynan, 1988). This nomenclature was
later extended by including the origin/early leader, O
(142 bp), and the late leader, S(63 bp), sequences (J. I.
Johnsen et al., unpublished results; Sundsfjord et al.,
1994; Ferguson & Subramani, 1994). The nomenclature
and anatomy of BKV NCCRs are illustrated in Fig. 3.
All BKV NCCRs analysed so far may be envisaged as
having evolved from that of BKV (WW) by repeated
duplications and deletions of the basic O - P - Q - R - S
blocks, although the exact mechanism is unknown
(Markowitz et al., 1990). Mixed infection studies with
NCCR variants of simian virus 40 (SV40) have shown
that illegitimate recombination between heterologous
viral genomes can give rise to new variants (Clarke &
Herr, 1987). It may be speculated that the NCCR
heterogeneity of different BKV strains is biologically
reflected in the ability of polyomaviruses to adapt to new
cellular environments, because duplications, deletions
and new junctions of the P, Q and R sequence blocks
create new, both proven and putative, binding sites for
transcription factors. It is conceivable that such variation
may influence the relative efficiency of viral transcription
and replication in different host cells and hence their
permissivity and oncogenic potential in cell cultures as
well as in the human organism.
BKV was first isolated from the urine of a kidney
transplant recipient (Gardner et al., 1971). This BKV
strain remained a laboratory reference strain for biological experiments. Part of its genomic DNA was
sequenced several years and many cell culture passages
later (Dhar et al., 1978; Yang & Wu, 1979a). As
indicated in Fig. 3 it contained an NCCR with the
anatomy O142-P6s-Pso-P6s-Q39-S63. In this communication we demonstrate the NCCR heterogeneity in a cell
culture passaged stock of supposed BKV (Gardner),
document seven new NCCR variants and propose a
model for their evolution. Having developed a cassette
cloning strategy, we made recombinant viral genomes
with the new NCCR variants cloned into an identical
genomic background and demonstrated distinct
differences in host cell permissivity and transforming
potential governed by the individual NCCR variants.
Methods
Origin o f the B K V stock. The BKV-transformed hamster cell line
BKT-1B has been used for studies on transcriptional control and
expression (Moens et al., 1990). This cell line was established, in the
laboratory of one of us (R. M.), by subcutaneous inoculation of a Vero
cell-passaged BKV (Gardner) stock received from Dr Sylvia Gardner,
and originating from the first BKV isolate (Gardner et al., 1971). Local
fibrosarcomas developed and the turnout tissue was used to establish
the BKT-1B cell line, which has a malignantly transformed phenotype
(Sten et al., 1976), expresses large T antigen constitutively and contains
multiple copies of integrated BKV DNA sequences in the genome
(Moens et al., I990). Comparison of the BKV NCCR found integrated
in the BKT-1B cellular genome with that of BKV (Gardner) used to
establish the cell line, revealed major differences (see Fig. 3 b). Most
notable was the presence of R-block sequences, which are totally absent
in the published BKV (Gardner) sequence (Dhar et al., 1978;Yang &
Wu, 1979a).
With this background, it was decided to analyse the BKV NCCRs in
the Veto cell passage used to establish the BKT-1B cell line. As it
turned out, the specific BKV (Gardner) preparation used to inoculate
the hamsters was no longer available. A harvested, infectious Vero
culture medium at the same passage level was available and was made
the basis for the studies described. This BKV (Gardner) preparation
had been stored at - 7 0 °C since the hamster inoculations were
performed in 1973.
P C R amplification and D N A sequencing o f B K V NCCRs. Prior to
PCR amplification 5 ~tl of the virus preparation was diluted to 100 ~tl
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B K virus non-coding control region
with distilled H20 and boiled for 10 min to disrupt cells and virus
particles. Of each sample, 2 lal was amplified with 5 pmol of each
primer BKTT-1 (5' AAGGTCCATGAGCTCCATGGATTCTTCC
3') and BKTT-2 (5' CTAGGTCCCCCAAAAGTGCTAGAGCAGC
3'). These primers are complementary to the early T / t antigen and the
late VP2-coding sequences, respectively (Fl~egstad et al., 1991). PCR
was performed in a 50 gl reaction volume containing 1.25 U of Taq
polymerase (Perkin Elmer Cetus) in the recommended buffer with
2.5 mM-MgC12. Amplification of viral DNA was achieved by 40 cycles of
1 min at 94 °C, 2 min at 55 °C and 3 min at 72 °C following an initial
5 min denaturation step. All reactions were performed in parallel with
negative and positive controls, in a laboratory exclusively used for
PCR. All reagents were pretested for the presence of polyomavirusspecific DNA and the general precautions outlined by Kwok (1990)
were followed.
For each sample 10gl of the amplified DNA product was
electrophoresed on 2 % agarose gels and visualized by UV fluorescence
after staining with 1 lag/ml ethidium bromide. Different DNA
fragments were purified from agarose and digested with Sad, snbcloned
into M 13 vectors (Pharmacia) and sequenced by the dideoxynucleotide
chain-termination method using Sequenase (USB).
Plasmid constructions. The cassette model for examination of
different BKV NCCR variants in a common background of BKV
protein-coding sequences was constructed from a p G E M 3 - Z f ( - )
derived plasmid, pBKV(TU), containing the BKV(TU) genome cloned
into the unique EeoRI site. The BKV genome contains three SacI
restriction sites. Two of the sites are located on either side of the NCCR
(Fig. 1). Partial digestion of pBKV(TU) with SacI generates a 7860 bp
DNA fragment containing only the protein-coding sequences of BKV,
allowing ligation of SacI-digested NCCR PCR products. Recombinant
genomes were made with the following NCCR variants, CRV 102 105,
PQ, BKT-1B (Moens et al., 1990), TU (Sundsfjord et al., 1990) and
WWT (Fl~egstad et al., 1991). The resulting plasmids were called
pBKV102, pBKVI03, pBKV104, pBKV105, pBKVPQ, pBKV-1B,
pBKV(TU) and pBKVWWT.
Prior to DNA transfections the recombinant BKV-containing
plasmids were digested with EcoRl (to free the BKV DNA), purified by
gel electrophoresis and GeneClean (BIO101), and recircularized.
Cell culture conditions, transfection assays and quantification o f BKVcontaining cells. Vero (ATCC CCL81) cells were cultured in Eagle's
MEM supplemented with 1% HEPES, 2 mM-glutamine, 200 U
penicillin, 100 mg streptomycin/ml and 5% fetal calf serum (FCS),
whereas NIH 3T3 (ATCC CCL1658) cells were cultured in Dulbecco's
MEM containing the same supplements as above.
Vero cells were transfected at 60 % confluence by the DEAE-dextran
method using 50 ng of recircularized BKV DNA, whereas NIH 3T3
cells were transfected by a modified calcium-DNA coprecipitation
method using 400 ng DNA (Luthman & Magnusson, 1983). Transfections were performed triplicate in three different series of experiments to ensure reproducibility.
Forty-eight hours after transfections the Vero cells were overlaid
with 15 % carboxymethyl cellulose in order to prevent secondary
spread of virus from the initially transfected cells. An indirect
immunoperoxidase staining method was applied to quantify the
number of Vero cells expressing BKV structural proteins (Fla~gstad &
Traavik, 1987), using rabbit antisera against purified BKV virions as
primary antibodies (Christie et al., 1987).
NIH 3T3 cells were seeded as a suspension in 0.35% SeaPlaque
agarose onto 35 mm wells (1000 cells/well) containing 0.7 % solidified
agarose for examination of transformational potential. Colonies were
counted after 3 weeks of growth. NIH 3T3 cells stably transformed
with v-src served as a positive control. Mock- and pUC18-transfected
cells were used as negative controls in both Vero and NIH 3T3
transfections.
1573
Results
The B K V stock preparation is heterogeneous and
contains at least seven new N C C R variants
PCR amplification of NCCRs in the viral stock preparation generated several DNA products ranging from
approximately 760-520 bp (Fig. 2). Isolation, cloning
and subsequent sequencing of the individual DNA
products revealed that the BKV (Gardner) stock contained at least seven different subpopulations of BKV
NCCRs. The O-P Q-R-S anatomy and the sequences
from these new variants, as well as from BK(WW), BKV
(TU), BKV (Gardner) and BKT-1B are given in Fig. 3.
None of the isolated NCCRs were identical to that of
BKV (Gardner), or to the integrated BKT-1B NCCR or
any other published variant. All these new NCCR
variants have intact O m block sequences, with one
exception (see below). One of the NCCRs has intact
P68-Q39-S~3 blocks, but totally lacks R-block sequences.
This variant was accordingly named PQ and has an
identical NCCR anatomy to the described laboratory
constructed p8303/p8326 clones (Hara et al., 1985),
d1504 (Watanabe & Yoshiike, 1985) and the pBK68
clone (Deyerle & Subramani, 1988). All the others have
duplications and/or deletions of the sequence blocks,
and were named CRV (control region variant) 101 to
6
5
4
3
2
1
Fig. 2. Analysis of PCR amplified BKV NCCRs from BKT-1B cells
and infectious Vero culture medium. The sizes of the ~bX174 HincII
ladder fragments are indicated in bp. Lane 1, ~bX174 HincII ladder;
lane 2, negative control; lane 3, positive control; 50 fg pBK 33.1
containing BKV(Proto-2); lane 4, infectious Vero culture medium
isolated before hamster inoculation; lane 5, infectious Vero culture
medium isolated two passages after inoculation in hamster; lane 6,
BKT-1B cells.
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1574
J. I. J o h n s e n a n d o t h e r s
T/t antigen
O(142/
G C~\AAAAT~GCAAAAGAATAGGGA~T~`C(?CCAAATAG'~TFFG(~`A~AGAA`4.AAGCC'~CCACACCC~TACTACTTGAGAGAAAGGGTGGAGG~AGAGGCGGC
P(68)
(71"CGGCC['CTFATATA'I'L,~,TAA A A AAAAAGGCICACAGGGAGGAGC'rGCTFACCCATGGATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACT]
Q(39)
~
GGGCAGCCA
R(63)
GCCAGTGGCAGT•AATAGTGAAACCCCGCC•CC'FAAAATFCTCAAATAAACACAAGAGGAAGTGGAAACrGGCCAAAGGAGTGGAAAGCAGCCA•GACAGACATG
ITII G
S(63)
CGAGCCTAGGA
ATCTTGGCCI
TGTCCCCAGTTAAACTGGACA
AAGGCC
[
Agno
P(68)
Q(39)
R(63)
S(63)
I';'";';';:;~";'~///////////////A w w
1
I
7 26
I
1
Gardner
lI . . . . . . . . . . . .
r-"---2--"-'--i iI
I
I
I
110
I
43 47
BKT-1B
..... L. . . . . . .
64, ' r - ~ ; f - - - r ~
I
I
~PQ
I
I
I
25
----'-'-
I-
19
....
25
I
I
I
---4l
CRV101
14
i
25
]
55
CRV102
I
25
I
I
CRV103
19
I
]
I
i
25
I
60
AA .....
30 36
CRV104
I
I
I
l
I
I
I 14
--,~-
25
l
--t" . . . . . . . . .
.... * .............
1
25
55
I. . . . .
I
[CRV106
I
I
I
I
I
!
" ......
16 . . . . . . .
CRV105
!
I
I
I
I
14__.- .......
i .i ....
!
36
. . . .
"~". . . .
|
12
52
ITU
106. C R V 1 0 3 a n d C R V 1 0 6 t o t a l l y l a c k R - b l o c k
sequences. T h e l a t t e r is similar b u t n o t identical to the
p u b l i s h e d B K V ( G a r d n e r ) N C C R ( D h a r et al., 1978;
Y a n g & W u , 1979a), in h a v i n g t r i p l i c a t e d P - b l o c k
sequences. T h e m i d d l e P - b l o c k o f B K V ( G a r d n e r ) h a s a n
18 b p deletion, r e m o v i n g nucleotides 8 to 25, whereas the
m i d d l e P - b l o c k o f C R V 1 0 6 lacks the first 13 nucleotides
(Fig. 3b). T h e last f o u r C R V s (101, 102, 104 a n d 105)
Fig. 3. Nucleotide sequence of BKV WWcon. (a) The consensus sequence is
based upon previously published WW NCCR sequences (Rubinstein et al.,
1987; Sundsfjord et al., 1990; Fbegstad et al., 1991, Markowitz et al., 1991;
Negrini et al., 1991). The promoter/enhancer region have been arbitrarily
divided into sequence blocks called P, Q and R (Markowitz & Dynan, 1988).
Also included are the origin of replication (O) and the late leader designated
S. The number of nucleotides in each sequence block and the orientation with
respect to early and late expression are indicated. (b) Comparison of the
different BKV NCCR variants used. The structure of the archetype WW is
presented using the P, Q, R and S nomenclature. The numbers in parenthesis
indicate the length of each sequence block in base pairs (Markowitz &
Dynan, 1988; Ferguson & Subramani, 1994; Sundsljord et al., 1994). The
NCCRs of the other variants are shown as thick lines. Parallel lines indicate
a repeated sequence and horizontal dashed lines indicate deletions relative to
the archetype WW sequence. The vertical dashed lines represent the
boundaries of each sequence block. The numbers indicate the beginning or
end of a block. The insertion of two A nucleotides between the partial
repeated Q-block and P-block sequences in the CRV105 variant is shown.
c o n t a i n R - b l o c k sequences. A l l are unique, b u t C R V 1 0 5
is evidently related to the p r e v i o u s l y p u b l i s h e d N C C R
v a r i a n t BKV9, which was c l o n e d f r o m a p r e p a r a t i o n o f
B K V ( G a r d n e r ) after p a s s a g e in P H F G cells ( C h u k e et
al., 1986). C R V 1 0 5 is u n i q u e in two respects: it has
d u p l i c a t e d S - b l o c k sequences a n d a 1 b p d e l e t i o n in the
O - b l o c k . T h e i n t e g r a t e d B K T - 1 B N C C R is h e a v i l y
r e a r r a n g e d a n d o n l y d i s t a n t l y r e l a t e d to a n y p r e v i o u s l y
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B K virus non-coding control region
1575
T a b l e 1. Growth efficiency, transforming potential and number o f proven transcription
f a c t o r binding sites in B K V N C C R variants
BKV NCCR variants
Characteristics
WWT
TU
BKT-1B
PQ
101
102
103
104
105
106
0
9
100
9
4
4
0
4
NO
ND
14
3
46
4
5
1
2
17
NO
ND
6
1
0
1
1
1
6
1
0
1
1
1
7
1
0
1
1
1
4
1
0
1
1
1
8
0
0
1
1
1
4
0
0
1
1
1
6
1
0
1
1
1
6
0
0
1
1
1
5
0
0
1
1
1
6
1
1
1
1
1
Growth efficiency*
Transforming potential*
Transcription factort
NF-I
Spl
AP-1
ER
PR
GR
* Results are expressed as percentages (see Methods).
i" Results indicated the number of binding sites found. Key: ER, oestrogen receptor; PR, progesterone
receptor; GR, glucocorticoid receptor.
ND, Not done.
T a b l e 2. Putative transcription f a c t o r binding sites located at sequence block junctions in the B K V N C C R variants
used in these studies*
Strain
Junction
Factor
Consensus motif1"
BKV motif
Reference
WV¢
P68-Q39
NF-I
TGGMNNNNGCCAA
TGGAATGCAGCCAA
Markowitz & Dynan (1988);
Gronostajski (1987)
Gardner
Q39-R63
R63S6a
P1 7-P26-68
none
GH1
NF-I
TCTGTCTG
TGGMNNNNGCCAA
TCTGTCTG
TGGAATGCAGCCAA
P-P
Q39S6a
AP-1
Spl
TGANTMA
CCCGCC
TGACTCA
CCCGCC
hsp70
CCCGCC
CCCGCC
Khalili et al. (1988)
Markowitz & Dynan (1988);
Gronostajski (1987)
Faisst & Meyer (1992)
Jones & Tjian (1985); Greene
et al. (1987)
Wu et al. (1987)
BKT-1B
R1_loR4~47
R43 47-P6~68
Q1 25-Ra~ 63
none
none
Hi-conserved
AAACACA
AAACACA
Wells (1986)
CRV101
Qa_25-P~6_n8
QI_2sR14_6a
none
TFIID/TBF
TATAAA
TATAAA
Breathnach & Chambon (1981);
Wu et al. (1987)
CRV102
Q1 25-Rs6-G3
none
CRV103
Q1 25-P19-n8
Q39-$63
Q1 25 R14 ~3
none
none
TFIID/TBF
TATAAA
TATAAA
Breathnach & Chambon (1981);
Wu et at. (1987)
QI 25-R5~ 68
S 1 36-$6a
P3~36-$63
P68-Px4_68
none
OctB2
GH1
XRE
R1 12 11716-68
Q1 3~-R52 63
c-Myc
Spl
CTTGCAT
TCTGTCTG
CACGCW
TCTCTTA
CCCGCC
CTTGCAT
TCTGTCTG
CACGCA
TCTCTTA
CCCGCC
Rosales et al. (1987)
Khalili et al. (1988)
Shirayoshi et al. (1988)
Ariga et al. (1989)
Jones & Tjian (1985); Greene
et al. (1987)
Wu et al. (1987)
Kim et al. (1987)
Martin et al. (1985)
CRVI04
CRV105
CRV106
TU
CCCGCC
CCCGCC
JCV repeat sequence G G G N G G R R
hsp70
LSF(SV40)
CCCGCC
CCCGCC
GGGCGGGG
* Sequence analysis was performed using the GCG Sequence Analysis Software Package 7.3 (Ghosh, 1990). Analysis allowing no mismatches was
used to identify the transcription factor binding sites created at junctions between the sequence blocks.
t Key: M = A or C; N = A or C or G or T; R = A or G; W = A or T.
p u b l i s h e d v a r i a n t , h o w e v e r it is r e l a t e d t o C R V 1 0 1 a n d
CRV104 in having the
f i g u r a t i o n (see Fig. 3 b).
distal
QI_~5-Rla 6a-S6a
con-
A closer look at the sequences of the different blocks
in the NCCR variants revealed only three mutations
when compared with the consensus sequence based on
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1576
J. L Johnsen and others
the published WW sequences (Rubinstein et al., 1987;
Sundsfjord et al., 1990; Fl~egstad et al., 1991 ; Markowitz
et al., 1991 ; Negrini et al., 1991). CRV105 has a deletion
in the O-block that removes nucleotide 127. CRV106 has
a point mutation (AG3-~G) in its first P-block, whereas
BKT-1B has a transition (A4G-+G) in the R~a~47segment.
No mutations were found in the Q- or S-block for any of
the variants described here.
twodupiicatiotls
P68 + one deletion
~
+
Two duplicationsof P68 [o/f
one deletion ~
~ G a r d n e ~
T
The new N C C R variants confer divergent host cell
permissivity and transforming potential to B K V
In order to investigate the biological properties of the
new BKV NCCRs and to compare them with some
previously sequenced NCCRs, we developed a cassette
model for cloning any NCCR variant into a common
genomic background of BKV protein-coding sequences.
The resultant recombinant viral genomes were transfected into both semi-permissive Vero cells and nonpermissive NIH 3T3 cells for the examination of viral
permissivity and transforming capacity, respectively. As
shown in Table 1, considerable differences were detected
between individual NCCR variants in both their ability
to propagate in Vero cells and to transform NIH 3T3 to
anchorage-independent growth in soft agarose.
The recombinant BKV with the TU NCCR, which
was used as a reference strain, multiplied most efficiently
in Vero cells, giving a final result of 2.2 x 104 BKVproducing cells/tag transfected DNA at day 21 after
transfection. CRV103 also grew reasonably well, with an
efficiency which was approximately 46 % of that of BKV
(TU). The only difference between this variant and PQ,
which had no activity in Vero cells, is the insertion of a
Q1 25-P19-~8 sequence, which hence may contain cisacting elements with positive functions.
The efficiency of the other NCCRs tested was low
compared to that of BKV (TU). WWT and PQ showed
no viral multiplication at all. CRV104 demonstrated
5%, BKT-1B 4% and CRV105 only 1.4% efficiency
compared with BKV (TU). It is interesting to note that
CRV104 multiplied to some extent. This NCCR variant
may be viewed as a simple deletion mutant compared to
BKV (WWT) (Fig. 3 b), which showed no activity at all
(Table 1). This strongly indicates that the deletion, which
removes distal Q and proximal R sequences, may contain
cis-acting elements with negative function in Vero cells
(see Discussion).
In order to reveal differences in transforming capacities
conferred by NCCR variants, the recombinant BKV
genomes were transfected into N1H 3T3 cells, which
were then tested for the ability to form colonies in soft
agarose. In these experiments a clone of the same NIH
3T3 stock, stably transformed with the powerful transforming oncogene v-src (Johansen et al., 1994) was used
Duplicationand
deletionP/Q
~u~__lCRV103]
of
WW-like a r c h e t y ~
Deletion of
f
} CRV102 ]
Duplication and deletion
~
Duplieation and
deletion
I Rvi0, I
Fig. 4. Hypothetical model for the generation of the new N C C R
variants from the archetype (WW) assuming a minimum of duplication
and deletion events.
as a positive control. The transformation efficiency (i.e.
number of transformed colonies/lag transfected DNA)
of the BKV NCCRs is shown in Table 1 as percentage
values relative to the results obtained with v-src. The
recombinant genome with the NCCR variant CRV105
transformed NIH 3T3 cells with an efficiency of 17 %
compared to v-src. All the other NCCR variants tested
were also transforming to some degree, with efficiencies
ranging from 1% (CRV104) to 9% (TU). The BKT-1B
variant, which was found integrated in the hamster
fibrosarcoma cell line, had an intermediate transformation efficiency (4 %) in this assay.
The evolution and sequence characteristics o f the new
B K V N C C R variants
Sequence alignments of the new NCCR variants with
each other, and with two key NCCR variants, BKV
(Gardner) and the archetype BKV (WW) (Rubinstein et
al., 1987; Sundst~ord et al., 1990, 1994; Fl~egstad et al.,
1991 ; Markowitz et al., 1991 ; Negrini et at., 1991), give
some striking indications about origins and evolutionary
relationships (Fig. 3 b). Comparisons of the new sequence
block junctions that have been created, and conserved,
strengthen these indications further (Table 2). Taken
together, the results led us to the model outlined in Fig.
4. The model assumes that an archetypal, BKV (WW)like, strain has been present in the viral stock at some
earlier stage of the passage history (see Discussion). For
the sake of completeness, the variant PQ has been
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B K virus non-coding control region
1577
Table 3. E x p e r i m e n t a l l y p r o v e n and p u t a t i v e binding sites f o r transcription f a c t o r s in the P, Q, and R blocks in the
NCCR
o f the B K V
Transcription factor
WW genome*
Consensus motif
BKV motif
Binding site
JCV repeat sequence
NF-I
GGGNGGRR
GGGAGGAG
T G G M N N N N N G C C A A TGGATGCAGCCAA
P5-12
Pz4-37
c-mos
H-2RIIBP/T3R-~
Histone-H4
PEA3
IE1.2
AP-2
Lymphokine
NFrB
NF-GMA
Spl
CAAACCA
GAGGTC
YCCTGA
AGGAAG
GGAAAG
GGSCWSSC
RTGRAAYCYC
GDRRADYCCC
GRGRTTKCAY
CCCGCC
P35-~1
P43-~s
P~v-sz
P4,-s4
P~-59
Qt-8
Q27-36
Q27-36
Q36-~7
Q34-39
hsp70
LSF (SV40)
Hi-conserved
EF-1A
CCCGCC
CCCGCC
AAACACA
RNMGGAWGT
GAGGAA
SMGGAWGY
SAGGAAGY
AGGAAG
RRARNNGAAACT
GTGGAAA
CAAACCA
GAGGTC
TCCTGA
AGGAAG
GGAAAG
GGGCAGCC
GTGAAACCCC
GTGAAACCCC
GGGGTTTCAC
CCCGCC
NF-I
CCCGCC
CCCGCC
AAACACA
AGAGGAAT
GAGGAA
GAGGAAGT
GAGGAAGT
AGGAAG
GAAAGTGGAAACT
GTGGAAA
GTGGAAA
T G G M N N N N N G C C A A TGGATGCAGCCAA
Q~4-39
Q3~-39
R17_23
Rz4_32
R25_30
R25_32
Rz~_32
R26_3a
R2s_39
R3o_36
R50_56
R3~_45
NFBK,5
SV40 enhancer-core
IE1.2
A G T G G A A A G C A G C C AGTGGAAAGCAGCC
TGGAAAG
TGGAAAG
GGAAAG
GGAAAG
R4~_6~
R~,1_~7
R5~_57
PU. 1
Ets-1
TCF-2
PEA3
ICFbf
Insulin
Reference
Martin et al. (1985)
Markowitz& Dynan (1988);
Gronostajski (1987)
van der Hoorn (1987)
Marks et al. (1992)
Clerc et al. (1983)
Martin et al. (1988)
Ghazal et al. (1987)
Mitchell et al. (1987)
Stanley et al. (1985)
Leonardo& Baltimore (1989)
Shannonet al. (1988)
Jones & Tjian (1985); Greene et al.
(1987)
Wu et al. (1987)
Kim et al. (1987)
Wells (1986)
Bruder & Hearing (1989)
Klemsz et al. (1990)
Faisst & Meyer (1992)
Faisst & Meyer (1992)
Martin et al. (1988)
Shirayoshiet al. (1988)
Ohlsson& Edlund (1986)
Markowitz& Dynan (1988);
Gronostajski (1987)
Markowitz& Dynan (1988)
Weiher et al. (1983)
GhazaI et al. (I987)
* Computer analysis using the GCG Sequence Analysis Software Package version 7.3 with the TF Sites file from the Transcription Factor
Database (Ghosh, 1990) allowing no mismatches was performed to identify the putative binding sites. The positions of the binding motifs in each
block are indicated by numbers.
t Key:D=AorGorT;K=GorT;M=AorC;N=AorCorGorT;R=AorG;S=CorG;W=AorT;Y=CorT.
included in the model. However, a recombinant BKV
with this N C C R does not grow in Vero cells. PQ may
therefore represent an end stage rather than an intermediate. The variants CRV103 and CRV106 can,
however, easily be conceived as being derived from BKV
(WW) by two independent events. The variants CRV102
and CRV104 may have evolved directly from BKV
(WW) by two independent deletion events. CRV 101 may
have evolved from CRV104, and CRV105 from CRV102,
by consecutive duplication and deletion events. This
conclusion is strengthened by the presence of the conserved Qr 25/R13 63 (CRV104 and 101) and Q1 2~/R55-6a
(CRV102 and 105) junctions. Such junctions are rare
among the previously sequenced N C C R variants, but the
former is present in the integrated BKT-1B and the latter
in BKV9 (Chuke et al., 1986).
The biological importance of BKV N C C R variation
probably ties with the resultant divergence in number,
types and interdistance of cis-acting binding motifs for
trans-acting factors (reviewed in Markowitz et al., 1990).
Previous studies (Nowock et al., 1985; Grinnell et al.,
1988; Markowitz & Dynan, 1988; Chakraborty & Das,
1991 ; Moens et al., 1994) have proven functional binding
of the transcription factors Spl (Qa~-39), AP-I (P-P
junction), NF-1 (P24-38, Ps-s/2r 3s in the P-block with
internal 18 bp deletion, at the P Q junction, R32 46,
R52_63, $31_~ and 862_~gnogene) , the oestrogen receptor, the
progesterone receptor and the glucocorticoid receptor
(distal part of S-block). We deduced the number of
binding sites for these proven transcription factors in the
N C C R variants discussed here. The results are presented
in Table 1. All strains contain several NF-1 (four to
eight) binding motifs, while most of them have a single
Spl site. CRV101, 102, 104 and 105 lack an Spl site
because they lack the distal sequences of the Q-block.
The AP-1 site which is located at the P - P junction is only
found in Gardner (two sites) and in CRV106 (one site).
In addition to proven binding motifs, we analysed the
N C C R for the presence of fully consensus c/s-acting
elements for known transcription factors. These results
are summarized in Table 3. Some binding motifs are
present at the junctions of the different sequence blocks
(AP-1 at the P - P junction, Spl at the P - Q junction). This
prompted us to examine aberrant sequence junctions for
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1578
J. I. Johnsen and others
the presence of putative binding motifs. The results are
summarized in Table 3. Some new binding motifs were
created, but their biological significance remains to be
investigated.
Discussion
The first isolation of BKV was made after inoculation of
Veto cells with urine from a renal allograft recipient on
immunosuppressive therapy (Gardner et al., 1971). In
1978, following many cell culture passages, an NCCR
from a stock of this isolate was sequenced (Dhar et al.,
1978; Yang & Wu, 1979a). BKV with such NCCR
sequence was thereafter termed prototype BKV
(reviewed by Howley, 1980). Our experiments were based
on a cell culture passaged 'prototype' BKV (Gardner)
preparation. This preparation was used to study the
oncogenic properties of BKV in hamsters (N/ise et al.,
1975). The BKT-1B cell line established from fibrosarcomas evoked by this supposed BKV (Gardner) was
later (Moens et al., 1990) shown to contain monoclonal,
integrated BKV DNA with an NCCR very different
from the published BKV (Gardner) sequence (Dhar et
al., 1978; Yang & Wu, 1979 a). The presence of R-block
sequences, totally absent in BKV (Gardner), made it
clear that the integrated BKV DNA could not have
evolved from the Gardner strain.
Our NCCR analysis of the stock virus preparation
strongly indicates that neither BKV (Gardner) nor the
integrated BKV BKT-1B NCCRs were present. We
found the BKV stock preparation to be heterogeneous
and to contain at least seven new CRV variants (PQ,
CRV101-106). Previously, other NCCR variants have
been isolated from 'Gardner' stocks: BKV9 (Chuke et
al., 1986), P2 (Berg et aL, 1988), Proto-2 (Rubinstein et
al., 1987) and pm522, -526 and -527 (Watanabe et al.,
1979; Watanabe & Yoshiike, 1986). Unlike Gardner,
which does not contain R-block sequences, BKT-1B,
CRV101, CRV102, CRV104, CRV105, BKV9 and P2
do. Mixed infection studies with NCCR variants of
another polyomavirus (SV40) have shown that illegitimate recombination between heterologous virus
genomes can give rise to new variants (Clarke & Herr,
1987). Fig. 4 outlines a hypothetical model for the
generation of these new NCCR. The model is based on
the assumption that an archetypal BKV (WW)-like
strain, although never detected by us, must have been
present in earlier passages of the stock or in the urine of
the patient from which BKV (Gardner) was originally
isolated (Gardner et al., 1971). This is not unlikely since
BKV (WW)-like strains have been isolated from human
urine on many occasions (Rubinstein et al., 1987;
Sundsfjord et al., 1990, 1994; Fl~egstad et al., 1991;
Markowitz et al., 1991 ; Negrini et al., 1991). All strains
can be derived from BKV (WW) through deletion and
duplication events. Deletion of the complete R-block
created the PQ variant. This strain forms the rudiments
for BKV (Gardner), CRV103 and CRV106. Two
duplications, with partial deletions in the middle Pblock, gave rise to BKV (Gardner) and CRV106. Partial
duplication of the P- and Q-block in the PQ variant
created CRV103. The observation by Watanabe &
Yoshiike (1986) that their d1504 strain, which has a PQ
NCCR anatomy, gave rearranged control regions after
passage in human embryonic kidney cells support this
assumption. Variants CRV101, 102, 104 and 106 were
generated from the WW archetype. Deletion of Q - R
sequences created CRV102 and CRV104. Further duplications and deletions led to the genesis of CRV105 and
CRV101, respectively. This conclusion is strengthened
by the conserved QI_~5-R14 63 (CRV104 and CRV101)
and Ql_2~-R55_63(CRV102 and CRV105)junctions. The
former junction is also found in the previously described
strains E, KS85, KS122 (Negrini et al., 1990), IR
(Pagnani et al., 1986), and BKT-1B (Moens et al., 1990),
while the latter junction is also present in BKV9 (Chuke
et al, 1986) and P2 (Berg et al., 1988).
Employment of the described cassette cloning technique will make it feasible to compare the biological
characteristics contributed to BKV by any NCCR
variant, and in any transfectable cell type. As illustrated
by the sequence analyses of the new NCCR variants and
our experiments with recombinant viruses in Vero and
NIH 3T3 cells, even rather simple duplication/deletion
events may have dramatic biological consequences.
Sequence comparisons between NCCR variants that
must be evolutionarily very closely related, but showed
very divergent biological characteristics, gave us the
ability to identify sequences and cis-acting sequence
elements that may positively and negatively contribute to
divergent Vero cell permissivity. This opportunity was
offered by comparisons between two sets of NCCR
variants: CRV102 and CRV104 are simple deletion
mutants of BKV (WWT), whereas CRV103 is an
insertion mutant of the PQ NCCR variant. In both cases
the mutations provide gain of activity compared to the
'parental' NCCRs, which are both totally inactive in
Vero cells. Consequently, we may directly deduce that
deletion of the sequence Q26_z9-Rl_13 (CRV104) seems to
remove cis-acting elements with a negative effect on the
archetypal BKV (WWT). On the other hand, by
comparing the NCCR variants WWT and PQ, it becomes
evident that deletion of the whole R-block does not
result in any gain of activity. However, deletion of the
whole R-block combined with a partial duplication of
P/Q sequences (Qx_~5-P19_6s)gives rise to CRV103 with a
considerable activity in Vero cells. Based on computerized searches we have made efforts to identify
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B K virus non-coding control region
responsible cis-acting elements/trans-acting factors. A
listing of the putative consensus elements removed or
added by these deletion and duplication processes are
given in Table 2. However, this does not allow us to draw
any definite conclusions at present. It should also be
noted that many trans-acting factor binding motifs allow
at least one mismatch, making the situation even more
complex. Only further, focused experimental studies may
identify the exact cis-acting sequence motifs responsible
for the observed positive and negative effects on Vero cell
permissivity.
All the NCCR variants tested, irrespective of their
permissivity in Vero cells, had a significant capacity to
transform NIH 3T3 cells. The outstanding performer in
this context was CRV105, which may be viewed as an
insertion mutant of CRV102 (see Fig. 4). The insertion
(S1 36-P60-68-Ql-~s-R~ G3) seems to diminish the
efficiency in Vero cells, but distinctly enhances the
transforming potential, which was three- to fivefold
higher than for any of the other NCCR variants. It has
been reported that decreasing the number of P-blocks
enhances transforming ability (Watanabe & Yoshiike,
1985). This indicates that the duplicated S-block
sequences may be responsible for the higher transforming
capacity of the CRV105 variant. The only other variant
containing repeated S-block sequences is BKV9 (Chuke
et al., 1986). This strain has an six- to sevenfold higher
transformation efficiency than BKV (Gardner) in Rat-2
cells (Tavis et al., 1990).
In conclusion, seven new BKV NCCR variants have
been isolated from a BKV (Gardner) stock, none of them
being Gardner. Comparative studies of these variants
within the same protein-coding background revealed
biological differences which are probably the result of the
creation or removal of binding motifs for known transacting factors. Only further, focused experimental studies
may identify which of these cis-acting sequence motifs
are functional.
The technical assistance of Bjarne Johansen is gratefully
acknowledged. This work was supported by funds from the Norwegian
Cancer Society, The Norwegian Research Council and Erna and Olav
Aakres Foundation for Fighting Cancer. One of us (J.I.J.) has a
fellowship from the Norwegian Cancer society.
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