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Journal of Virology, September 1999, p. 7761-7768, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein
Binds to Single-Stranded DNA near the 5' End or within the Genome of
Feline Parvovirus and Can Modify Virus Replication
Dai
Wang and
Colin R.
Parrish*
James A. Baker Institute, College of
Veterinary Medicine, Cornell University, Ithaca, New York 14853
Received 16 March 1999/Accepted 28 May 1999
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ABSTRACT |
Phage display of cDNA clones prepared from feline cells was used to
identify host cell proteins that bound to DNA-containing feline
panleukopenia virus (FPV) capsids but not to empty capsids. One gene
found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar
in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to
oligonucleotides representing a sequence near the 5' end of the genome
which is exposed on the outside of the full capsid but did not bind
most other terminal sequences. Adding purified DBP40 to an in vitro
fill-in reaction using viral DNA as a template inhibited the production
of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region
between nt 295 and 330 of the viral genome which has been associated
with transcriptional attenuation of the parvovirus minute virus of
mice, which is mediated by a stem-loop structure of the DNA and
cellular proteins. Overexpression of the protein in feline cells from a
plasmid vector made them largely resistant to FPV infection.
Mutagenesis of the protein binding site within the 5' end viral genome
did not affect replication of the virus.
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INTRODUCTION |
Animal viruses have many specific
interactions with their host cells which allow them to infect the
cells, replicate their genomes, express their genes, and assemble new
infectious particles. Autonomous parvoviruses have genomes of about
~5,000 nucleotides (nt) of single-stranded (ssDNA) DNA which encode
two genes and as few as four proteins, and they depend on the host cell
for most of their replicative and metabolic additional functions
(15, 16), while the adeno-associated viruses (AAV) of the
genus Dependovirus also require functions supplied by helper
viruses, including adenoviruses or herpesviruses (5).
Successful parvovirus replication depends on the mitotic and
transformed state of the cell, as well as on its specific
differentiated phenotype and host of origin (13, 14).
Several different cellular proteins which interact with the genome of
the autonomous parvoviruses or with nonstructural protein 1 (NS1) have
been defined; they include the parvovirus initiation factors, which
bind within the 3'-terminal palindrome of minute virus of mice (MVM)
(12); HMG and SGT, which interact with NS1 (18,
19); and others that are not yet identified associated with
various regions of the viral genome (49, 50). Members of the
14-3-3 family of proteins interact specifically with NS2 (8).
In the case of AAV, the associations of cellular proteins or proteins
from the helper adenovirus or herpesviruses may occur through binding
the larger nonstructural proteins Rep68 and Rep78 or directly with the
viral DNA. Cellular proteins involved in DNA replication and gene
expression that bind the Rep proteins include the TATA binding protein
and the transcription coactivator PC4 (26, 55). Another
protein involved in DNA replication and controlling the fill-in of the
incoming viral DNA strand is a ~53,000-Da protein termed the D-box
binding protein which binds a sequence between nt 125 and 145 in the
AAV genome, in a reaction that is regulated by tyrosine phosphorylation
of the protein (41). A heterogeneous nuclear
ribonucleoprotein (hnRNP)-related protein has been identified as
binding to the NS1 protein of MVM (25). The hnRNP family
includes many different members that bind DNA or RNA, and they may
control gene expression, DNA metabolism, pre-mRNA processing, or
nucleocytoplasmic transport (20, 24). More than 20 different
hnRNPs have been identified in vertebrate cells; they are designated A
through U, according to their positions in two-dimensional protein gel
electrophoresis (20).
Few cellular proteins or other ligands that bind the parvovirus capsids
in either their assembled or unassembled forms have been defined.
Canine parvovirus (CPV) binds to a number of cellular proteins in
virus-overlay protein blots (2, 3). Several parvoviruses,
including the feline panleukopenia virus (FPV) and CPV, bind
efficiently to carbohydrates including sialic acids on a number of
glycoproteins (2, 51). AAV type 2 binds the heparan sulfate
proteoglycan, and it also uses the 
V
5
integrin and/or the human fibroblast growth factor receptor 1 as
coreceptors for infection (40, 47, 48).
CPV and FPV are closely related parvoviruses that differ in host range,
antigenic structure, and a number of other biological properties
(37, 52). Both viruses efficiently infect cat cells, but
only CPV infects dog cells. The viral determinants of host range
include a small number of sequence differences in the coat protein
gene, which result in differences in the structure of the surface of
the capsid. Infection by FPV in the nonpermissive dog cells is blocked
at an early stage of the replication cycle, between virus uptake from
the cell surface and the amplification of the viral DNA within the
nucleus (11, 27).
We used cDNA expression library screening to identify an hnRNP-related
protein that binds viral DNA sequences near the 5' end of the viral
genome outside the capsid. Although that particular sequence near the
5' end of the genome did not appear essential for virus replication,
the protein also bound sequences in other regions of the genome, and
overexpression of the protein in cells blocked successful FPV replication.
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MATERIALS AND METHODS |
cDNA library preparation and screening.
mRNA was isolated
from the feline CRFK cell line, and poly(dT) priming was used to
prepare cDNA. The cDNA was ligated to adapter sequences so that it
could be cloned in a directional manner into the T7Select system
(Novagen, Madison, Wis.). The products of cDNA sequences fused in frame
with the 3' end of the T7 phage coat protein gene are therefore
displayed as fusion proteins on the surface of the phage capsid. The
phage were grown and titrated in the capsid protein-complementing host
BLT5615, and the phage library containing 107 independent
clones was amplified once. Full (DNA-containing) FPV capsids purified
by repeated sucrose gradient banding at 5 µg/ml were bound to
polystyrene 96-well plates, which were then incubated with 0.5% bovine
serum albumin (BSA) in phosphate-buffered saline (PBS). A sample of
109 phage were incubated with the virus in PBS with 2 mM
CaCl2, 1 mM MgSO4, and 0.1 to 0.5% Triton
X-100. After washing in the same buffer, the bound phage were grown in
BLT5615 cells and then repanned sequentially four times on full FPV
capsids. Inserts in individual phage were sequenced by using a T7Select
capsid protein gene primer and automated DNA sequencing, and the
sequences were used to search the GenBank database by using the BLAST
2.0 search algorithm (1).
Recombinant phage prepared from bacterial lysates were incubated with
full or empty FPV capsids in 96-well trays; after washing, the bound
phage were detected with an anti-T7 capsid antibody. In some cases the
capsids were pretreated for 30 min with micrococcal nuclease (100 U/ml)
or DNase I (100 U/ml) prior to use in enzyme-linked immunosorbent assay (ELISA).
Protein expression.
Several phage that bound the FPV capsids
encoded the same DNA binding protein, and one clone (referred to here
as DBP40) appeared to be missing only six amino acids from its amino
terminus by comparison with closely related proteins in the database.
That gene was recovered and expressed in Escherichia coli
from the pET-28b (+) expression vector (Novagen) with both the
polyhistidine tag and T7 epitope tag fused to its amino terminus. The
expressed protein was purified by binding to a nickel column, eluted
with 2 mM Tris-HCl (pH 7.9)-0.5 mM NaCl-1 M imidazole
(53), and then dialyzed against 10 mM Tris-HCl (pH 7.9)-100
mM NaCl.
ssDNA binding and in vitro fill-in assays.
For
electrophoretic mobility shift assays (EMSA), synthetic
oligonucleotides representing sequences near the 3' and 5' ends of the
viral genome (Table 1) were 5'-end
labeled with polynucleotide kinase and [
-32P]ATP. The
labeled oligonucleotides were incubated with 20 ng of the purified
DBP40 in 20 mM HEPES-NaOH (pH 7.9)-100 mM NaCl-5 mM
MgCl2-1 mM dithiothreitol-10% glycerol-1 µg of
poly(dI-dC)-1 µg of BSA and then electrophoresed in 6%
nondenaturing polyacrylamide gels, which were dried and exposed to
X-ray film (45). Competition EMSAs were performed as
described above, but with 10- or 100-fold excess of unlabeled
oligonucleotides added.
To map other DBP40 binding sites in the genome, immune coprecipitation
of FPV DNA fragments with the protein was performed.
An FPV infectious
clone in plasmid pGEM3Z (Promega, Madison, Wis.)
was digested with
DdeI and
HinfI, and the 3' ends were
32P labeled with the Klenow fragment of
E. coli
DNA polymerase I
(Gibco/BRL, Gaithersburg, Md.) and
[
-
32P]dATP. After boiling for 10 min, the DNA was
transferred to a
dry ice-ethanol bath and incubated for 30 min with 0.1 µg of
E. coli-expressed DBP40 at 30°C for 1 h; then
the protein and any
associated DNA were immunoprecipitated with
antibody against the
T7 epitope (Novagen). After incubation with 10 µg of proteinase
K for 30 min, the DNA was electrophoresed in 2%
alkaline agarose
gels, which were then dried and exposed to X-ray
films.
We examined the effect of DBP40 on the availability of FPV DNA as a
template in a DNA fill-in assay. Viral ssDNA was prepared
from full
capsids by boiling for 10 min, then extracted with phenol
and
chloroform, and ethanol precipitated. The ssDNA was incubated
with or
without DBP40 for 30 min at 30°C in EMSA buffer with BSA
as the
carrier nonspecific protein. Two units of the Klenow fragment
(Gibco/BRL), 50 µM each dATP, dGTP, dCTP, and dTTP, and 5 µCi
of
[
-
32P]dCTP (4 Ci/mmol) were added, and the reaction
mixture was incubated
for a further 20 min at 37°C; 20 mM EDTA and
1% sodium dodecyl
sulfate were added to stop the reaction. After
phenol-chloroform
extraction, the DNA was ethanol precipitated and then
digested
with
SnaI or
BseRI, which cut nt 289 and
469 from the genomic
3' end, respectively. The DNA was electrophoresed
in neutral polyacrylamide
gels, which were then dried and exposed to
X-ray film or to phosphorimager
plates (Fuji Medical Systems, Stamford,
Conn.).
DNase I footprint assays were performed according to standard
procedures (
7). ssDNA fragments of the negative- and
positive-strand
orientations generated by asymmetric PCR using primers
flanking
the region from nt 268 to 507 were gel purified. After 5'-end
labeling with [
-
32P]dATP, 20,000 cpm of each probe was
preincubated for 30 min with
20, 50, or 100 ng of DBP40; then 0.22 U of
DNase I (Promega) was
added. After incubation at room temperature for 1 min, 200 mM
NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate, and 100 µg
of yeast
tRNA per ml were added; then the mixture phenol-chloroform
extracted
and the DNA was ethanol precipitated. Samples were loaded on
sequencing
gels along with G+A ladders prepared according to the
Maxam-Gilbert
sequencing protocol (
36).
Effect of protein expression on viral replication.
The DBP40
gene fused to the T7 epitope tag was cloned into the mammalian
expression vector pcDNA3.1 (
) (Invitrogen, Carlsbad, Calif.), and
that plasmid was used to transfect CRFK cells by electroporation. The
cells were stained for the T7 epitope with a mouse monoclonal antibody
and also with rabbit antibody against the capsid protein and then with
specific anti-mouse or anti-rabbit immunoglobulin G (IgG) labeled with
fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate.
CRFK cells transfected with the DBP40 expression plasmid were incubated
for 2 days and then inoculated with FPV. The cells
were suspended by
trypsinization 24 h later, fixed with 2.5% paraformaldehyde,
and
then incubated with 0.1% Triton X-100 and 0.5% BSA in PBS.
The cells
were incubated with rabbit serum against the FPV capsid
protein and a
mouse monoclonal antibody against T7 epitope for
1 h at 37°C and
then washed twice with PBS. The cells were subsequently
stained with
FITC-conjugated goat anti-rabbit IgG and phycoerythrin-conjugated
goat
anti-mouse IgG and analyzed by flow
cytometry.
For the cell cycle analysis, DBP40-transfected cells were fixed and
permeabilized in methanol and acetone (1:1) and then stained
with a
monoclonal antibody against the T7 epitope for 1 h at 37°C
followed by FITC-conjugated goat anti-mouse IgG. After secondary
antibody staining, the cells were treated with 1 mg of RNase per
ml at
37°C for 30 min, and then the DNA was stained with 0.01
mg of
propidium iodide per ml-0.02% Triton X-100. The DNA content
of the
cells was examined with a FACScan flow cytometer (Becton
Dickinson, San
Jose, Calif.), and cell cycle parameters were obtained
by using the
ModFit LT program (Verity Software House Inc., Topsham,
Maine).
Mutagenesis of the DBP40 5'-end binding site.
The infectious
plasmid clone of FPV b strain was mutagenized to alter the predicted
DBP40 binding site. The GeneEditor site-directed mutagenesis system
(Promega) was used to replace the TCTATAAGGTGAACT sequence
on the inboard side of the 5'-terminal palindrome with TCTATTCAGTGAACT, so that the sequence within both arms of
the palindrome would be altered after virus replication (Table 1; Fig.
3D). The mutated sequence was recloned into the FPV infectious clone,
and the region replaced was sequenced. The mutant plasmid was
transfected into CRFK cells, and infectious virus yields were determined by 50% tissue culture infective dose in CRFK cells (42).
Nucleotide sequence accession number.
The DBP40 sequence has
been deposited in GenBank (accession no. AF153444).
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RESULTS |
hnRNP-related DNA binding protein interacts with FPV full viral
particles.
After five rounds of panning of the recombinant library
on full FPV capsids, 24 phage DNAs were prepared, sequenced, and
compared with sequences in GenBank. Fifteen clones encoded proteins
with high homology to A/B-type hnRNP proteins, and the genes were fused in frame with the T7 capsid gene, although with five different fusion
sites. The phage bound to the viral DNA and not the capsid, as shown by
the finding that they bound strongly to full capsid preparations but
weakly to empty capsids, and the binding was reduced >90% by
pretreatment of the capsids with micrococcal nuclease or DNase I (Fig.
1). The same phage bound in a similar
manner to CPV full capsids (data not shown).
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FIG. 1.
ELISA optical density at 450 nm (OD450) of
T7 phage 40 screened from the library by panning on full FPV capsids.
The phage supernatant was incubated in wells coated with full FPV
capsids, empty FPV capsids, or capsids pretreated with DNase I,
nuclease A, or micrococcal nuclease. The bound phage were detected with
a horseradish peroxidase-conjugated antibody against the T7 capsid
protein.
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The longest cDNA clone isolated was completely sequenced that clone
showed high homology to members of the hnRNP A/B subfamily
proteins; it
was most similar to the CArG binding factor A (CBF-A)
and the chicken
apolipoprotein D-box binding factor (ssDBF) (Fig.
2) (
30,
46).
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FIG. 2.
Alignments of the translated product of the largest
clone of the DBP40 gene with mouse CBF-A and chicken apoVLDL II
promoter single-stranded D-box binding factor (ssDBF), the most similar
sequences in the GenBank database. The shaded sequences indicate the
residues which differ between DBP40 and either of the other two
proteins.
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Recombinant DBP40 binds specifically to the 5' end of the virus
genome.
The full capsid of FPV is very stable, and the viral DNA
is packaged within the capsid with about 24 nt of the 5' end of the genome outside of the capsid, most likely with NS1 bound to the 5' end
when it is first encapsidated (17). EMSA with purified recombinant DBP40 showed that it bound specifically to the
single-stranded 5'-end sequence of the negative strand and to its
complementary sequence, but no binding was seen to oligonucleotides
representing 3'-end sequences or the genomic region corresponding to
the D-box sequence of AAV (which is distinct from apoVLDL gene promoter D box) (Table 1) (39, 41). Several members of hnRNP A/B
proteins have been identified as binding specifically to the telomeric DNA sequence TTAGGG and the complementary motif
CCCTAA (21, 45). Comparing the sequence of
the 5'-end upper probe with other identified specific binding sequences
of hnRNP proteins showed a common motif of TAAGG, which is
similar to the protected TTAGG sequence seen in the CArG box, TTTGG in
the apoVLDL single-stranded D box, and TTAGG in the rat heptocyte
telomeric sequence (21, 30, 46). Binding to the 5'-terminal
probe was also competed by CArG box and apoVLDL gene promoter
D-box oligonucleotides, by (TAAGGG)4 tetramers,
and to a much lesser degree by the mutated (TAAGAG)4
tetramer oligonucleotide (Fig.
3). DBP40 appeared to have a
similar affinity for the CArG box oligonucleotide compared with the
5'-end sequence, but competition was considerably lower with the
TAAGAG tetramer, indicating that DBP40 has specificity for
the TAAGG motif (Fig. 3).
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FIG. 3.
EMSA to test the binding of DBP40 to
32P-labeled oligonucleotides (Table 1). (A) Positions of
the oligonucleotide probes in the FPV genome. (B) EMSA of
32P-labeled probes with 50 ng of DBP40. The labeled probes
were incubated with the protein for 30 min and then electrophoresed in
a 6% native acrylamide gel. (C) Competition EMSA between
32P-labeled probe 1 and 10- or 100-fold excess of various
oligonucleotides. (D) Position of the mutation in the inboard end of
the FPV 5'-terminal palindrome. After replication of the DNA in cells,
synthesis of the outboard arm of the palindrome from the inboard
sequence in the plasmid results in the mutation in both arms.
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A number of heat-denatured restriction fragments from the FPV
infectious plasmid were immunoprecipitated when denatured DNA
was
incubated with purified DBP40. The regions of the genome precipitated
most efficiently under these conditions included the 5' end of
the
genome, nt 795 to 2015 and 2189 to 3249 (Fig.
4). The latter
fragment contained three
TAAGG sequence motifs, two of which were
within the splice donor
sequences of the viral gene transcripts.
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FIG. 4.
Protein binding to various sequences from the FPV genome
identified by immunoprecipitation of the protein-DNA complex. (A) The
DdeI and HinfI restriction maps of the FPV genome
in plasmid pGEM3Z. (B) The complete FPV genome in pGEM3Z was digested
with DdeI or HinfI and the fragments were
32P labeled. After denaturation, the DNA was incubated with
100 ng of purified DBP40 and then immunoprecipitated with an antibody
against the T7 epitope fused to the N terminus of the protein. After
incubation with proteinase K, the DNA was electrophoresed in 2%
alkaline agarose gels, which were dried and exposed to X-ray film.
Differing buffer conditions resulted in slightly slower migration of
the digested DNA. Total DNA, original digestion; IP, the
immunoprecipitated DNA.
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DBP40 inhibited virus genome fill-in in vitro.
DBP40 added to
an in vitro fill-in reaction of FPV DNA inhibited the production of
second-strand DNA by about 40% (Fig.
5A). When the DNA was digested with
SnaI (cleaving nt 289 from the 3' end), incorporation into
that fragment was seen to be reduced by ~50%, while formation of
double-stranded DNA (dsDNA) past the BseRI site (nt 469)
fragment production was reduced by >90% (Fig. 5B), indicating that
extension by polymerase was greatly attenuated between nt 289 and 469 in the presence of the added protein (Fig. 5).
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FIG. 5.
Effect of DBP40 on the in vitro DNA filled-in of FPV
ssDNA by the Klenow fragment of DNA polymerase I. Viral DNA recovered
from purified virions was incubated with the polymerase in the presence
of deoxynucleoside triphosphates, [32P]dCTP, and either 0 or 50 ng of DBP40. (A) The product generated was electrophoresed in a
1% agarose gel, with the number of disintegrations per minute (in
phosphorimager units) in the total DNA product shown. (B) dsDNA
produced was digested with SnaI (nt 289) or BseRI
(nt 469) and electrophoresed in a 5% nondenaturing acrylamide gel,
which was exposed to X-ray film. Incorporation into the lower band is
shown below each lane; size markers are indicated in base pairs. (C)
Positions of SnaI and BseRI sites relative to the
3'-end palindrome of the FPV ssDNA genome.
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To define the nucleotides that interact with DBP40 in this region, we
next performed the DNase I protection studies. Since
some of the hnRNP
proteins, such as CBF-A (
30) and qTBP42 (
45),
bind specifically to both 5'-TTAGG-3' and its complementary sequence
5'-CCTAA-3', and CBF-A protected both strands in the methylation
interference studies, we generated both negative (viral ssDNA,
complementary to mRNA) and positive ssDNA representing the sequences
between nt 268 and 507 by asymmetric PCR and examined them for
protection from DNase I cleavage by DBP40. The sequence on the
negative
strand between nt 296 and 330 was protected. Protection
of positive
strand in the region between nt 296 and 330 was hard
to assess, as that
region contained only a few sites susceptible
to DNase I under the
conditions of the assay (Fig.
6A). To
further
investigate the presence and specificity of binding,
oligonucleotides
representing each strand in the region of DNA
protection were
synthesized and labeled, and both bound DBP40 protein
(Fig.
6C).
Together, the footprinting and EMSA data suggested that
DBP40
interacts with both positive and negative strands between nt 296
and 330. The positive-strand region contained a TATGG motif which
was
located within the sequence AGTTATGGAG and that differed in
only one nucleotide from the AAV D-box sequence AGTGATGGAG
(Fig.
6B).
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FIG. 6.
Protein binding and protection of sequences within the
FPV genome. (A) DNase I protection of the negative and positive strands
of the region from nt 290 to 370 of the FPV genome by increasing
amounts of DBP40, in the region where the block to DNA polymerization
appeared to occur. Numbers represent nucleotides in the complete FPV
genome. (B) Sequence of the FPV genome between nt 250 and 340, showing
the approximate region protected by the bound DBP40. (C) EMSA showing
binding of 20 ng of DBP40 to 32P-labeled oligonucleotides
containing the sequences from the negative (ve) and positive (+ve)
strands of the viral DNA in the region protected and competition with
10- and 100-fold excess of the same unlabeled oligonucleotide.
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The sequence of the DBP40 binding region had a high likelihood of
making a stem-loop structure in its single-stranded form,
and the DNase
I-protected sequences were within the predicted
loop structures (Fig.
7B). That region has been associated with
attenuation of transcription of the MVM genome (
31,
32), and
so the protected sequence of DBP40 was compared with the homologous
sequences from MVM and LuIII virus and shown to differ in much
of the
sequence in that region (Fig.
7A).
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FIG. 7.
(A) Alignment of FPV, MVM, and LuIII virus sequences in
two regions of the genome identified as containing binding sequences
for FPV in these studies. The sequence in FPV that appears to be the
consensus binding sequence of the NS1 is underlined. The negative DNA
strand sequences are shown in each case. Numbering is from the complete
FPV genome sequence, starting at the left-hand end. (B) Predicted
folding of the negative strand of the FPV DNA in the region of the
genome between nt 260 and 360, as predicted by the mfold program
(44). Arrows indicate the DNase I-sensitive sites. Numbering
is from the complete FPV genome sequence, starting at the left-hand
end.
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DBP40 overexpression blocked virus replication in cells.
The
T7-tagged DBP40 recombinant protein was primarily found in the nucleus
when expressed in CRFK cells, although it was also found in the
cytoplasm of some cells (Fig. 8A). When
transfected cells were inoculated with FPV and examined for the
coincidence of recombinant DBP40 expression and CPV infection, few
cells expressing DBP40 were infected (Fig. 8A). Flow cytometry analysis
showed that the nontransfected cells were infected with an efficiency of 12.5%, while only 1% of the DBP40-expressing cells became
infected, indicating a >90% reduction in efficiency of infection by
FPV after DBP40 expression (Fig. 8C). The experiments were repeated four times, with transfection efficiencies ranging from 5.75 to 8.46%
and infection efficiencies ranging from 12.1% to 28.54%; all showed
greater than 85% reduction of susceptibility in transfected cells. No
difference in the cell cycle distribution was seen between the
transfected and nontransfected cells (results not shown).
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FIG. 8.
Infection of feline cells expressing DBP#40. The cells
were stained for the T7 epitope fused to the N-terminus of the DBP#40
and for FPV capsid protein antigens. (A) Two different fields showing
CRFK cells transfected with a plasmid expressing DBP#40, and then
inoculated with FPV. The cells were then stained for T7-epitope on the
DBP#40 and for the FPV capsid protein. Rhodamine (red) stained cells
are FPV infected cells, and fluorescein (green) stained cells show T7
epitope fused to DBP#40. Yellow stain indicates overlapping cells,
but none of the transfected cells were found to be infected by the
fluorescent microscopy. (B) Flow cytometric analysis of the cells
without DBP#40 transfection. The T7 epitope tagged with DBP#40 is
shown in the vertical direction, and FPV antigen is shown in the
horizontal direction, and DBP#40 expressing FPV-infected cells would
be in the upper right field. The percentage of cells in each quadrant
is shown. (C) Flow cytometric analysis of the cells with DBP#40
transfection, infected and analyzed as in (B).
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Mutation of the DBP40 binding site near the 5' end of FPV did not
affect replication.
The sequence of the inboard 5'-terminal
palindrome of the FPV infectious clone was replaced with the equivalent
sequence from MVM, resulting in a substitution of the complementary
outboard sequence in the palindrome after viral DNA replication (Fig.
3D). The mutant virus was readily recovered and replicated to the same titers in CRFK cells as wild-type FPV (results not shown).
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DISCUSSION |
We show that an hnRNP A/B protein binds to several positions in
the genome of FPV and CPV, that the protein can attenuate DNA
polymerase fill-in of the viral DNA in vitro, and that overexpression of the protein in cells significantly reduces the infection or replication of the virus. The initial binding site identified on full
capsids could be removed by treatment with nucleases, indicating that
it was likely to be part of the 5' end of the genome extended to the
outside the viral capsid (17). We were not able to identify
any function for that binding, and a three-base alteration of that
binding sequence in an infectious FPV clone did not reduce the
infectivity of the virus or the viral yields obtained in tissue
culture. However, the binding site was very close to the 5' nick site
of the genome predicted by comparison with the well-defined sequences
within the MVM 5'-terminal palindrome (Fig. 7A), suggesting that there
may still be some association between binding of the protein and
replication at the 5' end (9, 16, 18, 43, 50).
Expression of DBP40 in cells from a plasmid vector resulted in the
cells becoming largely resistant to infection or replication (Fig. 8).
This effect was due to some direct or indirect effect of DBP40 on the
virus but was not due to arrest of the cell cycle, as the
DBP40-expressing cells showed the same proportion of cells in S phase
as the wild-type cells (results not shown). It is possible that DBP40
binds directly to the viral genomic DNA during replication, and in
vitro the protein was seen to bind to at least three regions within the
genome, either by coimmunoprecipitation of DBP40 DNA fragments along
with the protein (Fig. 4) or by blocking the fill-in of the viral
genomic DNA by the Klenow fragment of DNA polymerase I (Fig. 5). The
block to fill-in appeared to occur at least in part between the
SnaI site at nt 289 and the BseRI site at nt 469. Surveying the positive and negative strand sequences within that region
for protein binding using DNase I footprinting showed protection of a
20- to 30-base region around nt 300 and 315 on the negative strand
(Fig. 6). That region had been previously shown in studies of MVM to be
responsible for attenuation of transcription of mRNA synthesis from the
P4 promoter in a reaction that involved the binding of unidentified
cellular proteins (4, 32). That region of the MVM genome was
predicted to form an extended stem-loop structure in the ssDNA, and
this was also seen for FPV (Fig. 7) (4). Some features of
that predicted folded DNA structure were confirmed by the DNase I
digestion profile, as most of the predicted stem structure between nt
295 and 330 was not digested by the DNase I under the conditions used
(which were optimized for digestion of the ssDNA), and cleavage
occurred mostly at predicted mismatches around nt 315, 316, and 300 (Fig. 7B).
DBP40 protein belongs to the hnRNP A/B subfamily, which have two RNA or
DNA binding domains in the central part of the protein and an auxiliary
glycine-rich region with one RGG box in the C terminus (10,
20). These two nucleic acid binding domains are very conserved,
while the N- and C-terminal domains are more diverged. A variety of
hnRNP A/B proteins that associate with pre-mRNA, forming the hnRNP
complex have been found, and many family members also bind to ssDNA or
dsDNA in sequence-specific manner (20, 21, 24, 46). Several
of the proteins, including the CBF-A and the VLDL D-box binding factor,
bind to DNA and regulate gene expression (30, 46), while
others bind to and regulate the synthesis of telomeric regions of the
genome (21, 33, 45). There are also similarities between
some hnRNP proteins and the adenovirus 72-kDa DNA binding protein,
which has an essential role in replication of AAV (29, 35,
54). The most closely related proteins were the CBF-A and the
chicken VLDL D-box binding factor (Fig. 2), and the binding motif of
DBP40 that we identified was TAAGG, similar to that of the CArG box
(TTAGG) and the VLDL D box (TATGG), which both bound well in EMSA,
although DBP40 clearly bound less efficiently to the sequence TAAGA
(Fig. 3C).
Although no essential role(s) for this protein in enhancing or
regulating viral replication has been defined, some possibilities are
suggested by the data obtained on viral DNA binding and on the effect
on replication and from the properties of similar proteins in this
family. The protein may bind the viral genome and be involved in
regulation of transcription or replication. The effect on replication or fill-in of the viral DNA may be similar to that seen for a cellular
protein which binds the AAV genome just inside the 3' hairpin and
blocks viral DNA fill-in during infection (22, 23, 41),
where binding is regulated by tyrosine phosphorylation of the protein
(34, 39). Some hnRNP proteins are also phosphorylated on
tyrosine by tyrosine kinases, or on serine or threonine by protein
kinase C or casein kinase, and those modifications can regulate the
binding or other functions of the proteins (6, 28, 38). We
have not determined whether this has any effect of DBP40. The
association of the protein with the 5' end of the encapsidated genome
may indicate a role in DNA packaging or in the location or transport of
the full capsid within the cell. DBP40 may also play a role in the
replication or resolution of the genomic 5' end. Similar to the case
for MVM, it is likely that FPV NS1 binds to the ACCA sites and
introduces a nick at specific site in the genome adjacent to the DBP40
binding site, but we have not yet tested whether it can bind that
sequence in the double-stranded form or whether there is any direct
effect on that process.
| |
ACKNOWLEDGMENTS |
We thank Wendy Weichert for expert technical assistance.
This work was supported by NIH grant AI28385 to C.R.P.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: James A. Baker
Institute, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. Phone: (607) 256-5649. Fax: (607) 256-5608. E-mail: crp3{at}cornell.edu.
| |
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Abstract
Introduction
