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Journal of Virology, September 2001, p. 8082-8089, Vol. 75, No. 17
National Veterinary Research Institute,
Pulawy,1 and Department of Internal
Diseases, Faculty of Veterinary Medicine, Agricultural University,
Warsaw,2 Poland; Department of Applied
Biochemistry and Biology, Faculty of Agronomy, B-5030 Gembloux,
Belgium3; and Department of Veterinary
Microbiology and Pathology, Washington State University, Pullman,
Washington 99164-70404
Received 17 January 2001/Accepted 1 June 2001
The cytoplasmic tail of bovine leukemia virus (BLV) transmembrane
protein gp30 has multiple amino acid motifs that mimic those present in
signaling proteins associated with B-cell and T-cell receptors. The
proline-rich motif of gp30, PX2PX4-5P, is analogous to the recognition site of Src homology 3 (SH3) domains of
signaling molecules. Using site-directed mutagenesis of an infectious
molecular clone of BLV, point mutations were introduced which changed
three of the prolines of the motif to alanines. The influence of these
mutations on the pathogenicity of BLV was studied in sheep which
received either (i) plasmid DNA with provirus containing
proline-to-alanine mutations (pppBLV), (ii) plasmid DNA with wild-type
provirus (wtBLV), or (iii) transfection reagent alone. Although all of
the BLV-injected animals seroconverted at approximately the same time,
viral loads at later time points were high in five of five of the wtBLV
group and two of five of the pppBLV group but low in three of five of
the pppBLV group, as determined by semiquantitative PCR. Viral
expression was lower in the pppBLV-transfected sheep, as measured by
p24 antigen enzyme-linked immunosorbent assay in cultured cells, and
serologic titers were lower. Thirty-one months after transfection, four
of four wtBLV-transfected sheep had died of leukemia and lymphoma, and
all five of the pppBLV-transfected sheep were clinically healthy and
had normal peripheral blood lymphocyte counts. These data indicate that
the proline-rich motif of gp30 is not required for viral infectivity
but is important for high viral load in vivo, suggesting that
SH3-mediated gp30 interactions are critical for viral pathogenesis
following infection. Absence of interactions with the proline-rich
motif may prevent or delay tumorigenesis in sheep.
Infection of cattle with bovine leukemia virus
(BLV), a member of the BLV-human T-cell leukemia virus (HTLV) group of
retroviruses, results in persistent lifelong infection, the mechanism
of which is still poorly understood. The main target of BLV infection
is the B lymphocyte expressing surface immunoglobulin M (IgM)
(7). The clinical manifestations of this persistent
infection are polyclonal expansion of B cells in many of the infected
animals and lymphosarcoma in a small percentage of infected animals
(4, 17). Available data suggest two possible mechanisms
for this expansion: (i) the activity of the BLV transactivating protein
Tax, and (ii) interactions of other BLV proteins with cellular
proteins. It is indeed well documented that although BLV does not
possess a typical oncogene in its genome, BLV Tax can behave as such
(30, 31). Transforming properties of Tax are better
documented in HTLV biology where it has been shown to transactivate a
variety of genes or long terminal repeat (LTR) sequences,
transcriptional enhancers, oncogenes, and interleukins
(3). Another possible mechanism is the direct interaction
of viral proteins other than Tax with lymphocyte signaling pathways,
resulting in an increased rate of proliferation and/or reduced
B-cell apoptosis. Both phenomena are documented, although detailed
interactions and proteins involved at each step are still not known
(8, 9, 13).
BLV gp30, the transmembrane component of envelope glycoprotein, can
participate in signaling interactions (1, 2, 6, 29). BLV
gp30 has a long cytoplasmic tail with several motifs, including an
immunoreceptor tyrosine-based activation motif (ITAM), an
immunoreceptor tyrosine-based inhibition motif (ITIM), and an upstream
proline-rich motif (Src homology [SH] 3 recognition site motif)
(5, 25). SH2 and SH3 motifs are found in a diverse collection of cellular proteins and are involved in downstream signaling events of receptors for growth factors, cytokines, hormones, antigens, and extracellular matrices in the control of cell growth, differentiation, migration, and death (20). ITAMs and
ITIMs are recognition sites for the SH2 motif and are shared among a number of signaling proteins associated with the B-cell and T-cell antigen receptors (25) and in several viruses that infect
B cells (5). Proline-rich sequences, especially with the
sequence PX2PX4-5P, where X represents any
amino acid, are recognition sites for the SH3 motif (24).
The proline-rich motif in proximity to an ITAM is found not only in BLV
gp30 but also in proteins of other viruses that infect B cells,
including the herpesviruses Epstein-Barr virus LMP2A and herpesvirus
papio LMP2A and the orbiviruses African horsesickness virus VP7 and
epizootic hemorrhagic disease virus VP7 (5). The presence
of this motif in unrelated viral groups led us to hypothesize that the
proline-rich motif is essential for viral survival and replication in B cells.
To test the significance of the proline-rich
PX2PX4-5P motif in BLV gp30, we changed three
of the prolines to alanines in an infectious molecular clone of BLV.
The influence of the proline-rich motif on viral load and pathogenicity
was studied in sheep.
Provirus mutants.
The source of BLV provirus was the
infectious molecular clone pBLV344H, as previously described
(32). In this clone, the proline-rich motif has the
sequence PHFPEISFPPK. In other isolates of BLV, there is an L instead
of F, or a T or A instead of the third P (18). Mutations
were performed using PCR and resulted in changing three prolines
(positions 471, 474, and 479 according to Rice et al.
[26]) to alanines. Briefly, two pairs of
oligonucleotides were used in mutagenesis: flanking primers OL and OR,
and internal primers OC1 and OC2 that contained altered bases encoding
the P In vitro activity.
To determine if the mutated virus was
functional in vitro, approximately 2 × 105 canine
osteosarcoma cells (D17) were transfected by calcium
phosphate-precipitated plasmid DNA (ProFection Mammalian Transfection
System; Promega). Cells were cotransfected with pBLVLTR-CAT and either
pBLV344H or pppBLV344H. The cells were then washed and cultivated in
minimal essential medium (Gibco) supplemented with 10%
heat-inactivated fetal calf serum. After 48 h the cells were
harvested and washed with phosphate-buffered saline (PBS) and one-half
of the cells were lysed by three cycles of freeze-thaw. After
centrifugation, chloramphenicol acetyltransferase (CAT) activity was
determined from the supernatants (as described in reference
32). The other half of the transfected cells were lysed by
one cycle of freeze-thaw and used for p24 antigen titration by an
enzyme-linked immunosorbent assay (ELISA) procedure as described
previously (21, 22). Briefly, 96-well microtiter plates
were precoated with the anti-p24 monoclonal antibody (MAb) 4'G9. The
antigen mixtures to be tested were then added to the wells. After
washing of the plates, the p24 antigen was revealed by colorimetric
assay using two antibodies (2'C1 and 4'F5) conjugated with peroxidase.
In vivo transfection of sheep.
Before starting the
experiments, the animals were adapted to the housing and feeding
conditions in the experimental herd in Pulawy, Poland. During this
period, sheep were treated with parasiticides, and absence of parasites
was confirmed.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.8082-8089.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of the Proline-Rich Motif of Bovine Leukemia
Virus Transmembrane Protein gp30 in Viral Load and Pathogenicity
in Sheep
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
A mutations. The first round of amplification consisted of two
separate reactions, using two primer sets. The first primer pair
consisted of upstream OL (5'-ATC AAC AAT GGA TGA CAA CAT-3') and downstream OC1 (5'-CGA AGG AGA TTT CAG
CGA AGT GGG CAG CCT GC-3'). The second
pair was upstream OC2 (5'-GCC CAC TTC GCT
GAA ATC TCC TTC GCC CCT AAA C-3') and downstream OR
(5'-GAG GGT GGA ATA AAA AGA AAG-3'). Underlined bases
designate those changed to cause the P A mutations. After the first
round of amplification using two sets of primers (OL plus OC1 and OR
plus OC2), the products of each reaction were mixed and used as
template in a second round of amplification with only the flanking
primers OL and OR. This resulted in a 2-kb amplicon that contained the
desired PA mutations. The new amino acid sequence, starting with
amino acid 471 (26) is
AHFAEISFAPK. We took advantage of
the presence of NcoI and XbaI restriction sites
in the amplicon, and after cutting of the NcoI-XbaI fragment from the whole amplicon we
cloned it into the original pBLV344H. The resultant construct is
designated pppBLV344H. Correctness of the construct was verified by
sequencing. For the transfection experiments, plasmids were purified
using the Qiagen Plasmid Mega kit.
Serological examination. Serologic response to BLV proteins was evaluated by agar gel immunodiffusion (AGID) assay, which detects both gp51 and p24 antibodies (Dr. Bommeli AG, Liebefeld Switzerland). Antibodies to p24 were determined by ELISA (ELISA BLV kit; Bioveta, Ivanovice na Hane, Czech Republic). Additionally, serial dilutions were prepared to measure the p24 antibody titer using the same ELISA kit. Sera from pppBLV-injected sheep with no detectable p24 antibodies were tested again to confirm results by using a second ELISA kit that also detects p24 antibodies (Institut Pourquier, Montpellier, France).
BLV p24 titration in PBMC cultures. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples using Histopaque (Sigma) density gradient centrifugation and cultured for 48 h at a concentration of 3 × 106/ml of Eagle medium supplemented with 10% heat-inactivated calf serum, L-glutamine, gentamicin, and amphotericin B. Cell-free supernatants were prepared by centrifugation for 10 min at 900 × g. The p24 major Gag antigen was then titrated from the culture supernatants by ELISA (21, 22).
PCR analysis of proviral sequences in blood samples.
DNA for
PCR was isolated from Histopaque-purified sheep PBMCs using the Genomic
DNA Prep Plus kit (DNA-Gdansk II s.c., Gda
sk, Poland). The
composition of the reaction mixture at a volume of 50 µl was
PrimeZyme polymerase buffer (Biometra Ltd, Goettingen, Germany), 1.5 mM
Mg2+, 0.5 µM concentrations of each primer, 1 mM
deoxynucleoside triphosphates, and 2 U of PrimeZyme polymerase
(Biometra). DNA (0.5 µg) was added to the reaction mixture and the
total volume was covered with 2 drops of mineral oil. The amplification
process was performed in a programmed thermal cycler (UnoII; Biometra
Ltd). To examine proviral load, semiquantitative PCR was performed on
the PBMCs. The number of PCR cycles was restricted to 25 in order to
eliminate the "plateau effect" and to allow comparison between
amplification of abundant and scarce BLV sequences. The reaction was
started with denaturation at 94°C for 4 min followed by 25 cycles of
40-s denaturation at 94°C, 40-s primer hybridization at 65°C, and
1-min elongation at 72°C. The amplification was finished with a 5-min elongation. Two oligonucleotides were used: MCF-1
(5'-GCGAGAAACCATTCATTCTG-3') and MCR-2
(5'-CAAGAAGAGGCTTGTGATGG-3'). Amplification products of the
BLV DNA were detected electrophoretically in a 2% agarose gel. The
specificity of the PCR was confirmed by Southern blot hybridization of
amplified DNA using a molecular probe (SacI insert of
previously cloned provirus DNA) (23) followed by
autoradiography. DNA samples from an FLK cell line infected
persistently with BLV and from a persistently lymphocytotic,
ELISA-positive cow were the positive controls. To more sensitively test
for the presence of proviral DNA, a nested PCR was used. Initial
template DNA (0.5 µg), approximately 5,000 cell-equivalents, was
initially amplified with upstream primer 5'-ATCAACAATGGATGACAACAT
and downstream primer 5'-GAGGGTGGAATAAAAAGAAAG.
Denaturation was at 94°C for 4 min, followed by 30 cycles of
1-min denaturation at 94°C, 30-s primer hybridization at 57°C, and
1-min elongation at 72°C and concluding with a 7-min elongation.
One-tenth of the initial amplification was used as a template for the
second PCR, using primers MCF-1 and MCR-2 as described above. In the
second reaction, 30 cycles were performed as above, except with a 40-s
denaturation at 94°C and 40-s primer hybridization at 65°C.
Amplification products were detected electrophoretically in a 2%
agarose gel. As a control of sensitivity, known dilutions of plasmid
DNA (pBLV344H) were prepared in water.
Flow cytometry.
Cytometric analysis was performed using a
FACStrak flow cytometer (Becton Dickinson Immunocytometry Systems), and
the percentages of B-cell and T-cell subpopulations were recorded using
Simulset and PC Lysis programs. Leukocytes were gated with MAbs
directed against ovine antigens CD2 (MUC2A), CD4 (GC50A1), CD8
(CACT80C), B-B2 (BAQ44A), and WC1-N2 (BAQ4A) (VMRD Inc., Pullman,
Wash.). WC1-N2 is a determinant on WC1+ 
T cells, the
predominant population of peripheral blood T cells in sheep.
Debris was excluded from the analysis by the conventional scatter
gating method.
Statistical analysis. Analysis of variance was conducted by using the Statgraphics Plus statistical analysis package, version 2.0. The results are presented as the mean ± 1 standard deviation of the absolute number and percentage of each lymphocyte subpopulation. The statistical significance of the observed differences in the numbers and percentages of lymphocyte subsets between three experimental groups as well as between estimated time points within each group was evaluated by Student's t-test. A P value of <0.05 was considered statistically significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of mutated provirus.
We constructed a mutant of
bovine leukemia provirus by introducing three PA point mutations
within the proline-rich motif upstream of the gp30 ITAM. The location
of the mutations in the BLV gp30 and the mutated sequence are presented
in Fig. 1. Before in vivo injection, wtBLV was compared
with pppBLV in vitro by transient cotransfection of either wtBLV or
pppBLV, together with pLTRCAT in the canine D17 osteosarcoma cell line.
Expression of the transactivator Tax, as determined by activation of
BLV LTR-CAT, and expression of the capsid p24 protein, as determined by
ELISA, did not reveal significant differences (p > 0.2) between the proline-mutated and wild-type provirus (data not
shown).
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Initial infectivity.
Seroconversion as measured by AGID assay
and ELISA occurred in all of the animals transfected with wild-type or
proline-mutated provirus 4 to 7 weeks after plasmid injection (Table
1). Negative control animals injected
only with DOTAP remained seronegative. The five sheep transfected with
wild-type provirus (wtBLV) seroconverted at a mean of 5.6 ± 1.1 and 4.8 ± 0.8 weeks, as measured by AGID and ELISA, respectively,
while the five sheep transfected with the proline-mutated provirus
(pppBLV) seroconverted at a mean of 5.8 ± 1.3 and 6.0 ± 1.6 weeks, as similarly measured. The differences between the time of
seroconversion of sheep transfected with wtBLV versus pppBLV were not
significant, regardless of the type of assay used. Similarly, there
were no significant differences in the time to onset of p24 antigen
expression between wtBLV- and pppBLV-infected sheep (5.4 ± 3.3 and 7.7 ± 2.3 weeks, respectively) as measured in supernatant of
cultured PBMCs. In the wtBLV-transfected group of sheep, the shortest
period to onset of p24 antigen expression was 3 months, while in the
pppBLV-transfected group of animals it was 5 months.
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Long-term serologic response.
Despite an initial similarity in
infectivity, as determined by the time of seroconversion, the dynamics
of serum titers varied considerably after infection (Fig.
2, Table 1). Two of the five pppBLV-transfected sheep
(animals 5 and 7) were serologically positive by AGID and ELISA for 4 and 2 months, respectively, but then became seronegative again. The
other three pppBLV-transfected sheep (animals 8, 9, and 10) remained
seropositive for the duration of the study. Twelve months from the time
of transfection, the mean titer in the wtBLV-transfected group was four
times higher than the mean titer for the three seropositive animals in
the pppBLV-transfected group (273,060 versus 68,260). The difference between the group of animals (sheep 5 and 7) producing the low p24
antibody titers and the remaining pppBLV-infected sheep (numbers 8, 9, and 10) was also evident when the lymphocyte phenotype was compared
(see below).
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p24 expression in cultured PBMCs.
Comparison of BLV p24
expression in supernatant from cultured PBMCs from the wtBLV and pppBLV
groups revealed similar tendencies. There was a much higher mean p24
expression level from the wtBLV-transfected animals compared to the
three long-term-seropositive, pppBLV-transfected animals (Fig.
3). Significantly, the two sheep that became
seronegative also failed to express p24 protein after culture of PBMCs.
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Semiquantitative PCR to compare viral loads.
To examine
proviral load, semiquantitative PCR was performed on PBMCs. The number
of PCR cycles was restricted to 25, and dilutions of infected bovine
PBMCs were used as a positive control. A 337-bp fragment from the
env gene that is present in both the wild-type and
proline-mutated proviral BLV was amplified. To increase sensitivity of
detection, the PCR products were analyzed by Southern blotting using a
BLV-specific probe (23), followed by nonradioactive detection using the ECL system (Amersham Pharmacia Biotech). As expected, the 337-bp fragment was present in the PCR of all
wtBLV-transfected sheep throughout the whole experiment. At the same
time, however, the proviral sequence was weakly and inconsistently
detected in three of the five pppBLV-transfected sheep (numbers 5, 7, and 8) (Fig. 4). However, pppBLV-transfected sheep
numbers 9 and 10 had proviral loads equivalent to those of the
wtBLV-transfected animals. After initial infection, confirmed both by
the presence of specific antibodies and proviral sequences in PBMCs, we
were unable to detect BLV DNA in pppBLV-transfected sheep number 5 and
7 after 6 months posttransfection (Fig. 5). These
results correlate well with the serological results for these two sheep (Table 1). The sheep were serologically positive for 4 and 2 months,
respectively, but then became seronegative. The five negative control
(uninfected) sheep had no detectable BLV DNA, confirming that there was
no transmission among the groups of sheep.
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-actin gene as a control for
the integrity of the DNA samples. The primers do not amplify bovine
Leukocyte phenotype.
The wtBLV group had a statistically
significant increase in total numbers of lymphocytes and numbers of B
cells compared with both the pppBLV and uninfected control groups. No
significant differences were found between the pppBLV group and the
uninfected negative control group. In the wtBLV-transfected group, the
mean total lymphocyte count was (5.5 ± 0.3) × 109/liter, whereas in the pppBLV-transfected group, the
mean total lymphocyte count was (4.1 ± 0.3) × 109/liter [(4.4 ± 0.5) × 109/liter and
(3.8 ± 0.4) × 109/liter for pppBLVtr and
pppBLVlr, respectively], and the negative control group had a mean
total lymphocyte count of (4.1 ± 0.3) × 109/liter (Table 2). The mean B-cell count
was particularly increased in the wtBLV animals and was (2.4 ± 0.2) × 109/liter (46% of the total lymphocytes), while
the mean B-cell count of the pppBLV-transfected animals was (1.1 ± 0.2) × 109/liter (27% of the total lymphocytes)
[(1.0 ± 0.7) × 109/liter and (1.2 ± 0.6) × 109/liter for pppBLVtr and pppBLVlr, respectively], and
the mean B-cell count in the negative control group was (1.2 ± 0.2) × 109/liter (28% of the total lymphocytes).
Simultaneous lack of significant differences in absolute T-cell numbers
among groups and decreased T-cell percentage in the wtBLV group
confirmed that the elevation in total numbers of PBMCs in the wtBLV
group was due to increased numbers of B cells. No significant
differences were found in the absolute numbers of CD4+ T
cells, CD8+ T cells, or WC1-N2+ cells.
Therefore, the mean percentage of T cells, CD4+ T cells,
and CD8+ T cells decreased (34 versus 48%, 20 versus 26%,
and 11 versus 18%, respectively) in the wtBLV-transfected group
compared with the pppBLV-transfected group. In contrast to the
wtBLV-infected group of sheep, no differences between any lymphocyte
subpopulations in the overall pppBLV group and the negative control
group were evidenced (Table 2). Phenotyping of cells within the
pppBLV-infected group of sheep revealed differences between the
low-titer-producing group (sheep numbers 5 and 7) and high-titer group
(numbers 8, 9, and 10). In particular, significant differences were
observed between percentage of B-B2 and WC1-N2 antigen-bearing
cells. The percentage of B-B2 B cells was lower (mean, 22 versus 30%)
in the low-titer-producing group (sheep 5 and 7) than in the high-titer group (sheep numbers 8, 9, and 10). Simultaneously, the
low-titer-producing group of sheep showed an increase in percentage of
WC1-N2 lymphocytes compared with that of the high-titer group (15 versus 11%) (data not shown).
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Lack of reversions and/or transmission of pppBLV mutant.
The
sequence of the viral proline-rich motif was examined 7 months after
transfection to verify that in vivo reversions or sheep-to-sheep
transmission had not occurred (Fig. 6). DNA from PBMCs
was extracted, amplified by PCR, and sequenced. The sequence of gp30 in
the five pppBLV-transfected animals was consistent with the original
mutated and transfected sequence. At the mutated sites, peaks were
homogeneous, consistent with a pure population of mutated virus. To
verify that there were no reversions at later times after transfection,
the proviral gp30 sequence of sheep 9 and 10 was determined at 12 and
24 months posttransfection. The sequences were identical with that of
the original mutant pppBLV used for transfection.
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Progression to neoplasia. One wtBLV-transfected sheep (number 33) died of unrelated causes at 10 months posttransfection. This animal became listless and recumbent and was euthanatized, but a specific cause of death could not be determined. All four of the remaining wtBLV-transfected sheep died of leukemia and lymphoma within 31 months posttransfection. Sheep numbers 4 and 11 at the time of death showed marked lymphocytosis (~5 × 1011 to 7 × 1011/liter) as well as solid lymphomas in many organs, including lymph nodes, heart, kidney, and peritoneum. A third sheep (number 6) had a single spike of leukocytosis of up to 1012/liter that later subsided, and this sheep later developed solid lymphomas. The fourth sheep (number 32) also developed solid lymphomas and died. None of the pppBLV-transfected sheep or negative control sheep had developed leukemia or lymphoma 34 months after transfection.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the cytoplasmic tail of the BLV transmembrane protein, gp30, a proline-rich motif is located 12 amino acids upstream of the ITAM. Because of the observation that proline-rich motifs with nearly identical spacing of three prolines (PX(2)PX(4-5)P) are found in close proximity to ITAMs in five other viruses, including four that infect lymphocytes (5), we hypothesized that the proline-rich motif is necessary for a key step in the viral life cycle. To test this hypothesis in vivo, we mutated the first two conserved prolines and a third proline of an infectious molecular clone and transfected the plasmid into sheep.
Initial serologic responses of the pppBLV- and wtBLV-transfected animals were similar, suggesting that the mutation did not cause such dramatic changes in the virus that infectivity was significantly inhibited. Following initial infection, however, sheep transfected with pppBLV failed to maintain high levels of virus. In three of five sheep, semiquantitative PCR showed little or no proviral load in PBMCs, as compared with the wtBLV-transfected animals. In two pppBLV-transfected animals, proviral load in PBMCs was similar to that in the wtBLV-transfected animals. However, p24 expression in cultured PBMCs was less than in the wtBLV-transfected animals, and the serologic titers were lower as well in all of the pppBLV-transfected animals. Consistent with the reduction in proviral load and viral expression, the B-cell population in the pppBLV-transfected animals was similar to the uninfected control animals, unlike the expanded B-cell population of the wtBLV-transfected animals. One wtBLV-transfected animal died of unrelated causes, and all four of the remaining wtBLV-transfected animals developed leukemia and lymphoma. The pppBLV-transfected animals have not developed disease.
It is of interest that sheep transfected with pppBLV reacted differently. Two of the pppBLV-transfected animals were initially infected, seroconverted, and had PBMCs with detectable provirus by PCR. However, after 26 weeks these animals reverted to seronegativity, and no provirus could be detected in the PBMCs by semiquantitative PCR or by nested PCR. A third pppBLV-transfected animal remained seropositive and proviral load was low, as determined by semiquantitative PCR. In contrast, the fourth and fifth animals in this group had semiquantitative PCR levels similar to the wtBLV-transfected animals. Various mechanisms might explain these findings. Although the gp30 sequence in the pppBLV-transfected animals was identical to the original plasmid, indicating lack of reversion, it is possible that there was a compensatory mutation elsewhere in the viral genome that permitted increased viral growth. It is also possible that the host sheep differed genetically, thus facilitating viral growth in some of the animals. Alternatively, it may be that stochastic events in some mutant-transfected animals shortly after initial transfection lead to greater infection. Regardless of the mechanism, the pppBLV-transfected animals with higher proviral loads, as determined by semiquantitative PCR, did have lower serologic titers than the wtBLV-transfected animals. Moreover, p24 protein expression in the supernatant of cultured cells was lower and the animals were free of tumor development, compared with the wtBLV-transfected animals.
Previously, it has been shown that the ITAM of gp30 is essential for in vivo viral infectivity and maintenance of viral load (29). This study shows, in addition, that the PX2PX4-5P proline-rich motif is also important for maintenance of proviral load in vivo. It is intriguing that in viruses with both proline-rich motifs and ITAMs, the two motifs are in close proximity. It is not known what role the relative positions of these two motifs plays, but it is possible that BLV gp30 and the other viral proteins with both motifs act as a signaling scaffold by bringing signaling molecules with SH3 motifs together with molecules with SH2 motifs (12, 16).
Although this in vivo study demonstrates the importance of the proline-rich motif, the mechanisms of action of this motif have not been determined. There is considerable evidence that proline-rich motifs with PX2PX4-5P spacing are involved in signal transduction and are recognition sites of the SH3 motifs, which are common in a wide variety of intracellular signaling molecules, including the Src family of tyrosine kinases, Fyn, Lyn, and Hck, Vav, and others (11, 16, 24, 27). A variant of the proline-rich motif contains PY sequences and interacts with WW domains on signaling molecules (16). The PY motif is found in proximity to many of the viruses with ITAMs, but it is not present in BLV gp30 (5).
Functions of the proline-rich motif other than interactions with SH3 domains of signaling molecules also may be utilized in retroviruses. In human immunodeficiency virus type 1 (HIV-1) and other retroviruses, proline-rich motifs are essential in a variety of late processes in the viral life cycle, including efficient release of particles from the cell surface (14), viral maturation (10), and incorporation of the Pol proteins, reverse transcriptase (RT) and integrase, into the virion (10). In HIV-2, a proline-rich motif of the Vpx protein is necessary for nuclear localization of the preintegration complex (19). In other viruses, the P protein of Borna disease virus has two nuclear localization signals consisting of proline-rich motifs (28), and a proline-rich PPPY motif of vesicular stomatitis virus is necessary for a late step of virus release (15). Further studies will be needed to investigate if there are specific signaling defects in SH3-mediated signaling pathways or in other aspects of the viral life cycle in pppBLV-infected lymphocytes.
BLV is evolutionarily and biologically similar to HTLV-1 and -2. The value of the BLV model system is that viral mutations can be generated and tested in experimental animals. This approach can examine the in vivo significance of different putative viral signaling motifs and their interactions with host signal pathways. Identification of motifs that cause expansion of B-cell populations can lead to understanding of specific host signaling pathways that are altered and may be significant for cancer research and targeted drug development.
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ACKNOWLEDGMENTS |
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Funding was provided by the USDA Foreign Agricultural Service, Research and Scientific Exchanges Division; Maria Sklodowska-Curie Joint Fund II (PL-AES-284); and the Commissariat général aux relations internationales de la Communauté Wallonie-Bruxelles.
We thank Daniel Portetelle for providing the p24BLV MAbs. We also thank Malgorzata Zaborna and Sue Pritchard for excellent technical assistance and Arsène Burny and Diana Stone for critical reviews of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: National Veterinary Research Institute, Al. Partyzantow 57, 24-100 Pulawy, Poland. Phone: 48-81-8863051. Fax: 48-81-8862595. E-mail: reichert{at}piwet.pulawy.pl.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, September 2001, p. 8082-8089, Vol. 75, No. 17
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.8082-8089.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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