Download Analysis of splice sites in the early region of bovine polyomavirus

Journal of General Virology(1992), 73, 2879-2886. Printedin Great Britain
2879
Analysis of splice sites in the early region of bovine polyomavirus: evidence
for a unique pattern of large T mRNA splicing
Rob Schuurman,* Marcel Jacobs, Ans van Strien, Jan van der Noordaa and Cees Sol
Department of Virology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 A Z Amsterdam,
The Netherlands
The genetic organization of the early region of bovine
polyomavirus (BPyV) was studied by analysis of the
splice sites used in early m R N A maturation, using
reverse transcription-polymerase chain reaction and
DNA sequencing techniques. When compared to other
polyomaviruses, the BPyV early region appears to have
an uncommon organization. In the major early mRNA
molecule two small intron sequences of 71 and 77
nucleotides, separated from one another by an 80
nucleotide exon sequence, were identified. Through
splicing out both introns, a m R N A molecule is
generated that contains an open reading frame with the
capacity to encode 619 amino acids. Comparisons with
the simian virus 40 large T antigen suggested that this
mRNA molecule encodes the BPyV large T antigen.
Remarkably, no m R N A product encoding a protein
with a size comparable to that of the small t antigens of
other polyomaviruses was detected. Another transcript
was observed from which only the 77 nucleotide intron
sequence had been removed, thereby creating a m R N A
molecule with the capacity to encode only 45 amino
acids. Whether this mRNA product represents a
mature transcript which is translated in BPyV-infected
cells or is an intermediate in the formation of the large
T m R N A molecule is not known. Analysis of BPyVspecific early m R N A products isolated from BPyVtransformed murine cells revealed only the amplification product representing the putative large T antigen
transcript.
Introduction
pre-mRNA molecules. As a result, it was proposed that
the BPyV early region would be organized as outlined in
Fig. 1 (b) (Schuurman et al., 1990). In the current report,
splice junctions present in the BPyV early m R N A
molecules were mapped using reverse transcriptionpolymerase chain reaction (RT-PCR) and subsequent
sequencing of the amplification products. Furthermore,
the splicing pattern observed in BPyV-transformed
murine cells was compared to that observed in permissively infected monkey kidney ceils. The results of these
experiments indicate that the organization of the early
region of BPyV is different to that anticipated (Schuurman et al., 1990). BPyV large T antigen seems to be
encoded by an m R N A molecule assembled from three
exons. No evidence was obtained for the presence of an
m R N A molecule encoding a protein of a size approximating that of the small t antigens encoded by other
polyomaviruses.
Many cellular and viral mRNAs, including those which
encode the early antigens of polyomaviruses, are formed
by alternative splicing (Leffet al., 1986). In this process,
several mature m R N A molecules are generated from one
precursor transcript by the use of different combinations
of splice donor and acceptor sites. For instance, the
m R N A molecules encoding the large T and small t
antigens of simian virus 40 (SV40) are formed by the
selective use of two splice donor sites in combination
with a single splice acceptor site (Fig. I a; Noble et al.,
1986).
In the nucleotide sequence of the early region of
bovine polyomavirus (BPyV; Schuurman et al., 1990;
EMBL/GenBank/DDBJ accession number D00755),
several stretches of nucleotides have been identified that
exhibit close similarity to the splice site consensus
sequences defined by Mount (1982). After translation of
each of the potential spliced mRNAs into protein, the
sizes of the encoded proteins and their similarity to those
of other polyomaviruses were compared. Based on these
results, it was determined whether a splice site could be a
potential candidate for use in the splicing of BPyV early
Methods
Cell and virus culture. Monkey kidney cell cultures derived from
Macacafascicularis kidneyswereculturedin MEM supplementedwith
0001-1018 © 1992SGM
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2880
R. S c h u u r m a n a n d others
Earle's salts and 2% Ultroser-G (Gibco) in lieu of foetal calf serum
(FCS).
Cell cultures of embryonic mouse cells (ME cells) and embryonic
mouse cells transformed by the early region of bovine polyomavirus
(ME-RBPy clones l, 2 and 3; Schuurman et al., 1992) were cultured in
Iscove's modified Dulbecco's medium supplemented with 8% FCS.
The FCS used for cell culture had previously been screened for the
presence of BPyV using a BPyV-specific PCR assay (Schuurman et al.,
1991).
Total cellular RNA isolation. Isolation of RNA from cells cultured in
vitro was performed essentially as described by Chirgwin et al. (1979).
Six days after infection, when cytopathology was observed throughout
the cell culture, total cellular RNA was isolated from monkey kidney
cells infected with BPyV (0-1 TCIDso/cell). Total cellular RNA was
also isolated from subconfluent cultures of either BPyV-transformed
murine embryo cells (Schuurman et al., 1992) or from untransformed
(ME) cells.
Amplification primers. The location and sequence of the primers used
in RT and amplification reactions are presented in Fig. 1(d and e).
Each primer contained at least 20 nucleotides complementary to the
BPyV genome. The 5' ends of all three primers contained a naturally
occurring or an engineered restriction enzyme recognition sequence to
enable cloning of the amplification products into a plasmid vector. The
primers were synthesized using an Applied Biosystems synthesizer
(model 381A).
R T and amplificationprocedure. RT reactions were performed in 20 gl
volumes using 100 ng of either primer r120 or r55. The reaction mixture
consisted of 75 mM-KCI, 50 mM-Tris-HCl pH 8-3, 3 mM-MgC1z, 10 mMDTT, 1 mM each of four dNTPs, 20 units (U) of RNasin (Boehringer
Mannheim), 5 U avian myeloblastosis virus reverse transcriptase
(Boehringer Mannheim) and 1 to 3 ~tg of total cellular RNA. RT
reactions were incubated at 42 °C for 60 min. Subsequently, the
reaction vessels were incubated at 95 °C for 5 min to inactivate the
enzyme. Thereafter, 80 ~tl of 1 x PCR buffer containing 50 mM-TrisHCI pH 8-3, 25 mM-KCI, 2 mM-MgCI2, 100 p~g/ml BSA (Boehringer
Mannheim), 100 ng of the 5' primer (1120) and 1 U of AmpliTaq DNA
polymerase (Cetus) was added to the reaction mixtures. Amplification
reactions were performed using a thermal cycler (Perkin-Elmer Cetus)
according to the following procedure. After 5 min of denaturation at
95 °C, 35 amplification cycles were performed (1 min at 95 °C, 1 min at
55 °C, 2 min at 72 °C), followed by a final incubation at 72 °C for 8 min.
Subsequently, 10% of the amplified material was analysed on a 2%
agarose gel containing ethidium bromide, using the Tris-acetateEDTA buffer system (Maniatis et al., 1982). A 123 bp ladder (Gibco)
was used as a size marker.
Cloning and sequencing of amplification products. Amplification
products were excised from the agarose gels and subsequently purified
using guanidinium isothiocyanate and silica particles, as described by
Boom et al. (1990). After digestion with BamHI and EcoRI, the
fragments were cloned into pGEM-7 (Promega) and subsequently
sequenced using Sequenase 2.0 (United States Biochemicals) according to the protocol described by Schuurman & Keulen (1990).
Results
Analysis o f B P y V early m R N A splice sites by R T - P C R
B a s e d o n t h e n u c l e o t i d e s e q u e n c e o f B P y V , it h a s b e e n
suggested that the genes encoding the putative large T
and small t antigens contain an intron sequence
(a)
LargeT
1
/
=:z4918
Small T
1
~
11
AA AA
4571
A
4636 4571
AAAA
(b)
Large T 1 . - ~
4593
"~
111
AAAA
4346
Small T 1 ~
11
4574c==z4503
AAAA
(c)
Large T
111
AAAA
4574 4503
4423 4346
1
A
4423 4346
AAAA
(d) ~4>
<~
<2=
Primerlocations
..... )1_29_. . . . . . v.55 .. ~1_29. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(e)
Primer
Sequence
Location
r120
TTGAATTCTTCCCACCACTGTTCCCACTGTG
4296~4326
1120
GCGGATCCATGGAATTAACATCTGAGGAAT
4697 4676
r55
GCGTTTAAGCTTATATATCCAATAATTAAG
4385 441 I
Fig. 1. Schematic representation of the genetic organization of the
early regions of SV40 and BPyV. Numbering of SV40 is according to
Buchman et al. (1981); numbering of BPyV is according to Schuurman
et al. (1990). Thick lines represent protein-coding sequences, thin lines
non-coding and intron sequences. Roman numerals above the
sequences represent the reading frames used in mRNA translation.
Open boxes below the antigens represent the HPDKGG motif
described in the text. AAAA, polyadenylation signal sequence. (a)
Genetic organization of the early region of SV40. (b) Proposed genetic
organization of the early region of BPyV as described by Schuurman et
al. (1990). (c) Genetic organization of the early region of BPyV as
determined experimentally (this study). (d) Schematic location of the
primers used for reverse transcription (r120 and r55) and subsequent
amplification (r120, r55 and 1120) of BPyV early mRNA sequences. (e)
Actual sequences of the primers used in RT-PCR experiments and
their location on the BPyV genome. Letters in italics represent
endonuclease recognition sequences.
( S c h u u r m a n et al., 1990). B o t h m a t u r e m R N A s w e r e
e x p e c t e d to b e f o r m e d t h r o u g h t h e use o f d i f f e r e n t splice
d o n o r a n d a c c e p t o r sites (Fig. 1 b). T o d e t e r m i n e t h e
splice j u n c t i o n s p r e s e n t in t h e e a r l y t r a n s c r i p t s o f B P y V ,
t h e m R N A s e q u e n c e s e n c o d e d by t h e B P y V g e n o m e
b e t w e e n n u c l e o t i d e s 4697 a n d 4297 w e r e a m p l i f i e d u s i n g
R T - P C R . P r i m e r r120 w a s u s e d to r e v e r s e t r a n s c r i b e
early mRNA
molecules isolated from either BPyVi n f e c t e d m o n k e y k i d n e y cells o r f r o m m u r i n e cells
t r a n s f o r m e d b y t h e e a r l y r e g i o n o f t h e virus. T h e
r e s u l t i n g c D N A m o l e c u l e s w e r e a m p l i f i e d for 35 cycles
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BPyV early mRNA splicing
using primers r120 and 1120 (Fig. 1d). According to the
model outlined in Fig. 1 (b), this R T - P C R was expected
to generate two amplification products of 162 and 338
nucleotides, originating from putative mRNA molecules
encoding the large T and small t antigen respectively
(Schuurman et al., 1990).
However, as shown in Fig. 2, the 162 bp product could
not be detected after RT and subsequent amplification of
mRNA sequences isolated from BPyV-infected monkey
kidney cells. Instead, an unexpected product of approximately 261 nucleotides was amplified. Additionally, a
minor amplification product was detected which appeared to be approximately 338 nucleotides in size and
therefore was expected to originate from the putative
small t m R N A molecule. The amplification product of
approximately 409 nucleotides, observed in reactions
with and without reverse transcriptase, most probably
originated from DNA molecules or from unspliced BPyV
R N A molecules present in the sample. In addition, a
very faint band was observed in reactions with reverse
transcriptase which migrated slightly faster than the 409
bp product. We do not expect this amplification product
to represent a spliced BPyV early mRNA product
because no combination of potential splice donor and
acceptor sites was theoretically capable of generating an
amplification product of this size. Further evaluation of
the nature of this minor amplification product was
unsuccessful; several attempts to clone the fragment
have failed.
Upon performing the R T - P C R using R N A isolated
from three independent lines of BPyV-transformed
murine cells, we observed an amplification product of
approximately 261 nucleotides in each of the reactions
with reverse transcriptase (Fig. 3). No other amplification products were observed with any of these samples
except the 409 bp full-length product which was
observed in the reactions without reverse transcriptase.
The 261 bp RT-PCR product represents BPyV large T
mRNA
Upon D N A sequence analysis of the 261 bp R T - P C R
products amplified from either permissive BPyV infected cells or from BPyV-transformed cells, it appeared
that two intron sequences had been spliced out (Fig. 4a
and 1c). An intron of 71 nucleotides located between
nucleotides 4574 and 4503 (intron I) was spliced out
together with a second intron of 77 nucleotides, located
80 nucleotides further downstream between nucleotides
4423 and 4346 (intron II). The presence of both splice
junctions in one and the same recombinant clone
strongly suggests that both introns were excised from the
same pre-mRNA molecule. The splice donor and
acceptor sites surrounding intron I closely match the
1
2
2881
M
Fig. 2. Results of RT PCR experiment performed with 1 to 3lxg of total
cellular RNA isolated from BPyV-infected monkey kidney cells.
cDNA synthesis was performed using primer r120. Subsequent
amplification of cDNA molecules was performed for 35 cycles using
primers r120 and 1120. Lane 1, reaction without reverse transcriptase;
lane 2, reaction with reverse transcriptase; lane M, 123 bp ladder.
ME
1
2
M
MERBPy-I
MERBPy-2
1
1
2
2
MERBPy-3
1
2
M
bp
--409
--261
Fig. 3. Results of RT-PCR experiment performed with 1 to 3 ~tg of
total cellular RNA isolated from three expanded colonies of BPyVtransformed embryonic murine cells (ME-RBPy cells) and from ME
cells, cDNA synthesis was performed using primer r120. Subsequent
amplification of cDNA molecules was performed for 35 cycles using
primers r120 and 1120. Lanes 1 and 2, reactions with and without
reverse transcriptase, respectively; lanes M, 123 bp ladder.
consensus sequences of splice donor and acceptor sites as
defined by Mount (1982), and correlated exactly with
those expected to be used in the formation of the m R N A
molecule encoding the putative BPyV small t antigen
(Schuurman et al., 1990). To excise the 77 nucleotide
intron II sequence from the early primary transcript, a
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R. Schuurman and others
2882
(a)
4I
4697
ATG GAA TTA ACA IC? GAG GAA TAT GAG GAG CTT AGG GGG CTC ITA GGA ACC CCT GAT ATT
M E L I S E E Y E E L R G L L G ~ P D I
4637
GGC AAT GCA GAT ACT TTG AAA AAG GCA ?TC CTG AAG GCA TGC AAG GTG CAT CAT CCA GAI
G
~
A
D
T
L
K
K
A
F
L
K
A
C
K
V
H
H
P
D
4577
AAA ~ A GGG AAT GAA GAA GCA ATG AAA AGA CTT CTG TAT TIG TAT AAT AAA GCA AAA ATT
K
G
G
N
E
E
A
M
K
R
L
L
Y
L
Y
N
K
A
K
I
4446
GCT GCA AGT GCC ACT ACT AGC CAG GTT CCA GAA TAT GGC ACC TCA CAG ?GG GAA CAG TGG
A A S A T T S Q V P E Y G T S Q W E Q W
4309
TGG GAA GAA TTC
W E E F ~
BPyV large T
SV40 large T
1
1
ME--LTSEEYEELRGLLGTPDI°-GNADILKKAFLKACKVHHPDKGGNEEAMKRLLYLYN
BPyV large T
SV40 large T
57
61
KI
411
a
KA AASAT . . . . . . . . . .
TSQVPEYGTSOWEQWt4EEFNQGFDEQDLHCDEELEPSDNEE
KMEDGVKYAHQPDFGGFWOATEIPTYGTDEWEQW14N
. . . . AFNEENLFCSEEMPSSDDEA
gPyV large T
$V40 large T
107
117
b
ENPAGSQAPGSOATPPKKPRT---SPDFPEVLKEYVSNALFTMRTYNCFIIFTTAEKGKE
T . . . . . . ADSQHSTPPKKKRKVEDPKDFPSELLSFLSHAVFSNRTLACFAIYTTKE~L
.
*****
.
***
.
. . • ***
** . ** **
8FyV Large T
SV40 Large T
171
MDKVLNREESLQLMDLLGLERSAWGNIPLMRKAYLKKCKEFHPDKGGDEEKMKKMNTLYK
*
*
**
*
***
**
** ** ** ****** ** **
**
c
(b)
4697 ATG GAA TTA ACA TCT GAG GAA IAT GAG GAG CTT AGG GGG CTC TTA GGA ACC CCT GAT ATT
M
4637
E
L
T
S
E
E
Y
E
E
L
R
G
L
L
G
T
P
D
I
GGC AAT GCA GAT ACT TTG AAA AAG GCA TIC CTG AAG GCA TGC AAG GTG CAT CAT CCA GAT
G N A
D
I
L
K
K
A
F
L
K
A
C K
V
H
H P
D
4577 AAA GGT AAA TAT ATT TAG TAT TGA TCT GTA CAT GCA AAT CTT GTT TAC AGG CAG TAA ATC
K
4517
4457
G
K
Y
I
164
EAYNVCC--TFELISQNIQGGLPSSFFNPVQEEE-KSVNWKLISEFACSIKCTOPLLLMA
LMYSALTRDPFSVIEESLPGGLKEHDFNPEEAEETKQVSWKLVTEYAMETKCDDVLLLLG
*
*
*
***
***
** * * ***
* *
** * ***
BPyV Large T
SV40 Large T
221
BPyV Large T
SV40 Large T
281
291
LYLEFTTAPEACKVCDNPRRLEHRRHHTKDHTLNALLFQDSKTQKTICNQACDTVLAKRR
MYLEFQYSFEMCLKCIKKEQPSHYKYHEKHYA-NAAIFADSKNQKTICQQAVDTVLAKKR
****
* *
*
.
* *
**
. *** ***** ** ****** .
BPyV Large T
SV40 Large T
341
d
LDMKTLTRNELLVQRWQGLFQEMEDLFGARGEEHLAHRMAAVMWLNALHPNMPDVIFNYI
VDSLQLTREQMLTNRFNDLLDRMDIMFGSTGSADIEE~AGVAWLHCLLPKMDSVVYDFL
231
350
U
TGA TTT TAT TTT TAG GAG GGA ATG AAG AAG CAA TGA AAA GAC TTC TGT AIT TGT ATA ATA
LYPCIQAAYKCTFIALYMYNGDSVLYIITVGKHRVNAMENLCSKKCTVSFLQAKGVLKPQ
LYKKIMEKYSVTFISRHNSYNHNILFFLTPflRHRVSAINNYAQKLCTFSFLICKGVNKEY
**
*
*
***
*
*
***
*
*
* ** ***
*** *
*
***
*
*
*
*
**
*
**
*
**
*
*
*
*
BPyV l a r g e T
SV40 l a r g e T
401
410
KMVVENKPKQRYLLLKGPVNCGKTTVAAGLIGLCGGAYLNINCPPERLAFELGMAIDQFT
KCMVYNIPKKRYWLFKGPIDSGKTTLA/L~LLELCGGKALNVNLPLDRLNFELGVAIDQFL
BPyV Large T
SV40 l a r g e T
461
470
VVFEDVKGKKSSKSSLQTGIGFENLDNLRDHLPGAVPVNLERKHQNKVTQIFPPGIVTCN
VVFEDVKGTGGESRDLPSGQGINNLDNLRDYLDGSVKVNLEKKHLNKRTOIFTPGIVTMN
gPyV Large T
SV4D l a r g e T
521
EYDIPLTVKIRMYQKVELLHNYNLYKSLKNTEEVGKKRYLQSGIIWLLLLIYFRSVDDFT
EFSVPKTLQARFVKQIDFRAKDYLKHCLERSEFLLEKRIIQSGIALLLMLIWYRPVAEFA
*
* *
*
*
*
*
**
**** ** ** * *
*
BPyV l a r g e T
SV40 l a r g e T
581
590
QSIQSRIVEWKERLOKEFSLSVYQKMKFNVAMGIGVLO~LRNSDDDDEDSQENADKNEDG
620
650
...........................................................
GEKNMEDSGHETGIDSQSQGSFQAPQSSQSVHDMNQPYHICRGFTCFKKPPTPPPEPET
AAG CAA AAA TTG CTG CAA GTG CCA CTA CTA GCC A~ITTc CAG AAT ATG GCA CCT CAC AGT
4330 GGG AAC AGT GGT GGG AAG AAT TC
Fig. 4. Nucleotide sequence and amino acid sequence of the R T - P C R
product representing the splice I/II molecule (a) and the splice II
molecule (b). The sequences are shown in the sense of the early m R N A
molecule. Numbering is according to Schuurman et aL (1990). The
location of the splice junctions is indicated by vertical arrows. I, Intron
I; II, intron If. In (a), continuation of the O R F 3' to the indicated
sequence of splice I/lI is indicated by a horizontal arrow.
splice donor site at nucleotide 4423 (5' CAGIGUAUGG
3') was used that had not been included in the initial
model for BPyV early mRNA splicing outlined in Fig.
1 (b). The 261 bp fragment will be referred to as the splice
I/II product.
The mRNA molecule giving rise to the splice I/1I
product contains an open reading frame (ORF) encoding
619 amino acids (nucleotides 4697 to 2693; Fig. lc),
assuming no other splice reactions occur. This mRNA
most probably encodes the BPyV large T antigen because
alignment of the sequence of the encoded protein with
that of the SV40 large T antigen revealed a significant
degree of identity (42~) between them (Fig. 5).
Excluding the C-terminal part of the SV40 large T
antigen, regions of amino acid conservation were
observed in most parts of the molecules. As indicated in
Fig. 5, several domains of the SV40 large T antigen to
which a functional role has been assigned are well
conserved in the BPyV large T antigen.
Analysis of amplification products expected to represent
small t mRNA molecules
As shown in Fig. 2, a minor amplification product of
approximately 338 nucleotides was observed when RNA
isolated from permissively infected ceils was used as
template in the RT-PCR. In contrast, this minor product
BPyV l a r g e T
SV40 l a r g e T
530
EKLQECVVKWKERIEIE. . . . . . . . . . . . . . .
*
*
****
*
VGDM-WLLTMKENIEQGKNILEK. . . .
*
**
*
*
Fig. 5. Comparison between BPyV large T antigen and SV40 large T
antigen using the Clustal program (Higgins & Sharp, 1988). Vertical
arrows refer to splice junctions present in the m R N A s encoding BPyV
and SV40 large T antigens. Identical amino acid residues in BPyV and
SV40 are indicated by an asterisk. Functional domains identified in
SV40 large T antigen are underlined and denoted a to d. a, Putative
PP2A interaction site; b, pRB-binding domain; c, nuclear localization
signal; d, zinc finger motif.
was not observed in experiments in which RNA isolated
from BPyV-transformed cells was used (Fig. 3). The size
of this amplification product indicates that it might
represent the small t mRNA molecule (depicted in Fig.
1 b) from which the 71 nucleotide intron I sequence had
been excised. However, nucleotide sequence analysis of
several recombinant plasmids containing this amplification product demonstrated that in all clones a 332 bp
insert instead of the expected 338 bp product was
present, and that the 77 nucleotide intron II sequence
had been removed (Fig. 4b and 1 c) instead of intron I. In
the RNA molecule observed, which will be referred to as
splice II, the size and location of the excised intron
correlates exactly with the 77 nucleotide intron II
removed from the mRNA molecule encoding the
putative BPyV large T antigen, as is schematically shown
in Fig. 1 (c). Translation of the nucleotide sequence
encoded by this splice II molecule resulted in a protein
with a maximum size of 45 amino acids (nucleotides 4697
to 4562; Fig. 4b and 1 c). In analogy to the situation in
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BPyV early mRNA splicing
1
2
M
3
4
Fig. 6. Results of an RT-PCR experiment performed with 1 to 3 ~tg of
total cellular RNA isolated from BPy¥-infected monkey kidney cells.
cDNA synthesis was performed using either primer r55 (lanes 1 and 2)
or primer r120 (lanes 3 and 4). Subsequent amplification of the cDNA
molecules was performed for 35 cycles in combination with primer 1120
for all reactions. Lane M, 123 bp ladder; lanes 2 and 4, reaction without
reverse transcriptase; lanes I and 3, reaction with reverse transcriptase.
SV40 small t m R N A splicing, the intron sequence
removed from the splice II molecule was located
downstream of the translation termination codon (nucleotide 4561).
As described, in none of the clones sequenced was an
insert present that represented the proposed small t
mRNA from which the 71 nucleotide intron I sequence
had been removed. Since the ORF created by removing
this intron sequence from the primary transcript potentially encodes a 124 amino acid protein, which could
possibly represent BPyV small t antigen, additional
experiments were performed to investigate the presence
of this mRNA molecule in BPyV-infected cells. For this
purpose, RT reactions were performed using primer r55,
which is complementary to the central part of the intron
II sequence. Since the nucleotide sequence recognized by
this primer had been removed from m R N A molecules
from which the intron II sequence had been excised,
splice II and splice I/II mRNA molecules could not be
reverse transcribed using r55.
Upon performing an RT reaction with primer r55 to
synthesize BPyV cDNA from R N A sequences isolated
from permissively infected monkey kidney ceils, followed by 35 cycles of PCR amplification using primers
r55 and 1120, we observed a single amplification product
of 320 nucleotides, originating from either unspliced
m R N A or from D N A (Fig. 6). No amplification product
of 249 bp, expected to originate from a small t antigenencoding transcript, was observed. In parallel control
amplification reactions using primers r120 and 1120,
minor reaction products of about 332 to 338 nucleotides
were again observed. These experiments indicate that
the concentration of splice I RNA, if it exists, is probably
low when compared to that of splice II and splice I/II
transcripts.
4697
2883
-
4574
-
4502
-
4423
-
4345
-
4563
-
4423
-
4345
-
~ l c o I/
(laroe
T
2693 --
~,Jo--
Fig. 7. Physical map of the BPyV genome. The genetic organization of
the B PyV genome was originally proposed by Schuurman et al. (1990);
in this map, the early region of the genome has been modified
according to the present results. Nucleotide numbering is according to
Schuurman et al. (1990). Putative first and last protein-coding
nuc|eotides are indicated.
Organization of the BPyV genome
The results of the experiments described in this paper
constitute evidence that the organization of the gene
encoding the large T antigen of BPyV is quite different
from that of the SV40 gene and from the model proposed
by Schuurman et al. (1990). Therefore, the physical map
of the BPyV genome was modified, as is shown in Fig. 7.
Whether the splice II m R N A molecule is translated in
BPyV-infected cells is not known. For this reason, the
splice II transcript has not been designated as encoding a
specific antigen.
Discussion
The genetic organization of the BPyV large T antigen
appears to differ from the general organization of genes
encoding polyomaviral large T antigens. In contrast to
the characteristic presence of only one intron of 250 to
400 nucleotides, two small introns (introns I and II)
separated by an 80 nucleotide exon sequence have been
identified in the gene encoding BPyV large T antigen.
The most upstream splice donor site used in BPyV early
m R N A splicing is located at position 4574 and, in
combination with the acceptor site at position 4503, is
involved in the removal of the 71 nucleotide intron I
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R. Schuurman and others
sequence. For mRNA maturation, another, not previously predicted (Schuurman et al., 1990), splice donor
site located at position 4424 was involved. This donor
site is used in combination with the acceptor site at
position 4346, thereby removing the 77 nucleotide intron
II sequence from the primary transcript. The splice
donor site at position 4424 (5' CAGIGUAuGg 3')
matches the consensus sequence for splice donor sites (5'
MAG[GUAAGU 3') defined by Mount (1982) at six of
eight positions (indicated by capital letters), and is
identical at the most critical nucleotides, those flanking
the exon-intron junction.
Upon alignment of the sequences of the large T
antigens of BPyV and SV40, as shown in Fig. 5, highly
conserved sequence motifs could be identified in several
regions of the proteins, excluding the C-terminal part of
the SV40 large T antigen. About 64 amino acid residues
encoded by the SV40 C-terminal region are most
probably not encoded by the BPyV genome. In SV40,
this region is thought to contain a host range control
element and a so-called adenovirus helper function, and
to be involved in capsid assembly (Pipas, 1985; Tornow
et al., 1985; Khalili et al., 1988; Cole et al., 1979). The
single transcription termination and polyadenylation
signal (AAUAAA) present on the BPyV early coding
strand is located only 48 nucleotides downstream of the
translation termination codon proposed to be used in
large T antigen production. We do not expect splicing in
this region, for which no apparent or degenerate splice
sites could be identified, to add amino acid residues to
the large T antigen of BPyV. The 619 amino acidencoding ORF of the splice I/II mRNA was expected to
encode a protein of approximately 70K. This has
recently been proven by Western blotting (Schuurman et
al., 1992).
One of the two introns excised for the formation of the
putative large T antigen mRNA molecule, the 71
nucleotide intron I, had previously been postulated to be
involved in the formation of the putative small t antigen
mRNA molecule (Schuurman et al., 1990 and Fig. 1b).
Splicing intron I but not intron II from the early premRNA molecule would generate a RNA molecule with
an ORF with the potential capacity to encode 124 amino
acids. However, no experimental proof for the presence
of this small t mRNA was obtained from the experiments
presented. Whenever the mRNA molecule encoding the
putative small t antigen was present in the BPyVinfected cells used for mRNA isolation, its concentration
was probably very low compared to that of the splice I/II
and splice II molecules.
Besides the splice I/II mRNA product, a splice II
mRNA product, from which only the 77 nucleotide
intron II sequence had been removed, was detected in
permissively infected cells. It is not known whether the
splice II mRNA molecule, which contains an ORF
encoding 45 amino acid residues, is actually translated in
BPyV-infected cells. However, all experiments performed to identify the splice sites in the early mRNA
molecules of BPyV were performed on total cellular
RNA. Therefore, the possibility that the splice II mRNA
molecule is a relatively abundant nuclear intermediate in
the formation of the mature large T mRNA molecule, for
which both intron sequences have to be removed, cannot
be excluded.
Apart from the splice I/II molecule, no additional
early transcripts were detected in the three BPyVtransformed murine cell lines, suggesting that transformation of these cells can be achieved by BPyV large T
antigen only. However, the possibility that other viral
early antigens encoded by mRNA molecules of low
abundance are synthesized in these cells cannot be
excluded. In all experiments performed, the strength of
the signal of the observed amplification product of large
T mRNA molecules isolated from BPyV-transformed
murine cells was at least equal to that observed with
similar quantities of RNA isolated from permissively
infected cells. Therefore, the relative concentration of
the splice II molecules and other potential early viral
transcripts was most probably lower in BPyV-transformed cells than in permissively infected cells. A
mRNA molecule that would encode a small t-like protein
could also have been missed using primers r120 and 1120,
in cases in which a splice acceptor site located
downstream of the 3' primer (rl20) was used. However,
no amino acid sequence homology with SV40 small t
antigen could be identified in any of the sequences
encoded by the reading frames downstream of the 3'
primer.
Some clearly defined functional domains of the SV40
large T antigen seem to be well conserved in the BPyV
large T antigen, domains a to d in Fig. 5. Domain b has
been shown to be involved in the interaction between
SV40 large T antigen and at least two cellular proteins:
the product of the retinoblastoma tumour suppressor
gene (pRB; DeCaprio et al., 1988) and p107 (Dyson et
al., 1989; Ewen et al., 1989). Although no experimental
evidence has yet been obtained, the presence of a pRBbinding domain in the BPyV large T antigen strongly
suggests that the latter is capable of complexing with
pRB. Such an interaction might be an important step in
cell transformation (Chen & Paucha, 1990). In this
respect, it is worth mentioning that primary rodent cells
can be transformed by the early gene products of BPyV
(Schuurman et al., 1992).
Apart from a putative pRB-binding domain, a wellconserved nuclear localization signal can be identified in
the BPyV large T antigen between residues 120 and 126
(Kalderon et al., 1984); and an amino acid sequence
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B P y V early m R N A
containing some characteristics of a zinc finger motif is
located between 292 and 307 (Loeber et al., 1989).
Furthermore, a high degree of amino acid conservation
in BPyV large T antigen was observed in the sequences
flanking both sides of the splice junction between exons 1
and 2, as well as in the sequences immediately
downstream of the junction between exons 2 and 3 (Fig.
5). In this respect, it is worth mentioning that splicing out
intron I from the BPyV early p r e - m R N A sequence
creates an amino acid motif ( H P D K G G ) which is
completely conserved in the large T, small t and middle T
antigen sequences of all polyomaviruses sequenced. The
H P D K G G motif is located in the BPyV large T antigen
between residues 38 and 43.
Two cellular proteins with Mrs of 61K and 37K have
been shown to associate with the small t antigens of SV40
(Yang et al., 1979) and human polyomavirus B K
(Rundell et al., 1981), and with the small t and middle T
antigens of polyomavirus (Grussenmayer et al., 1985;
Pallas et al., 1988). As has been demonstrated recently,
these two proteins represent the regulatory (A) and
catalytic (C) subunit of the protein phosphatase 2A
(PP2A; Pallas et al., 1990; Walter et al., 1990). The
interaction of SV40 small t antigen with a complex of
PP2A subunits A and C inhibits the catalytic activity of
the C subunit (Yang et al., 1991). Inhibition of PP2A
activity is also achieved upon binding of the 55K B
subunit to the AC complex of PP2A, indicating that the
interaction of either SV40 small t antigen or the B
subunit of PP2A with the AC complex might have
analogous effects on enzyme activity (Yang et al., 1991).
From comparisons between mouse polyomavirus
small t antigen and the sequence of PP2A subunit B, no
regions of significant amino acid sequence homology
were noted (Pallas et al., 1992). However, we detected a
stretch of four amino acids ( D K G G ) in the sequence of
the B subunits of PP2A proteins originating from several
m a m m a l i a n species which is the same as the four Cterminal residues of the aforementioned polyomavirus
H P D K G G motif. Whether the presence of this sequence
motif explains in part the common effect of small t
antigen and PP2A subunit B on PP2A activity is not
known. When the BPyV splice II molecule is translated,
an H P D K G K motif is created. The C-terminal residue
of the D K G G motiL a neutral glycine residue, is
replaced by a polar lysine (K). Such a change could have
dramatic influence on the function of the H P D K G G
module. Although the H P D K G G motif is also present in
the amino acid sequences of the large T antigens of all
other polyomaviruses, no interactions of these antigens
with the PP2A subunits have been reported. It might be
possible that the motif is hidden within the threedimensional structure of large T antigens, thereby
making it inaccessible to interaction with the PP2A
splicing
2885
subunits. An interaction of the PP2A subunits with the
mouse polyomavirus middle T antigen has been observed
previously (Pallas et al., 1990; Walter et al., 1990),
indicating that in this case PP2A interacts with a viral
early antigen which has been shown to play a crucial role
in mouse polyomavirus-mediated cell transformation. It
is important to note that the H P D K G G sequence
present in BPyV large T antigen is created by joining the
first and the second exon. This phenomenon, together
with the apparently very high degree of intraspecies
conservation of the motif in the early antigens, suggests
an essential role for this sequence in the virus life cycle,
and perhaps also for cell transformation. It could be
speculated that BPyV large T antigen, like mouse
polyomavirus middle T antigen, might be capable of
interacting with PP2A, thereby taking over the function
otherwise performed by small t antigen.
The authors would like to thank Wim van Est for excellent artwork.
This work was supported by the Netherlands Technology Foundation
(STW).
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