(Received for publication, December 24, 1996, and in revised form, May 20, 1997)
From the ¶ Department of Molecular Biology, Research
Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 and
the Laboratory for Neurovirology, Department of
Neurology, Anatomy and Neurobiology, and Microbiology and Molecular
Genetics, University of California, Irvine, California 92697-4290
Borna disease virus (BDV) is a newly classified
nonsegmented negative-strand RNA virus (order of Mononegavirales)
that persistently infects specific brain regions and circuits of
warm-blooded animals to cause behavioral disturbances. Viruses within
the order of Mononegavirales have phosphoproteins that typically serve
as transcription factors and are modulated in functional activity
through phosphorylation. To identify the kinases involved in BDV
phosphoprotein (BDV-P) phosphorylation, in vitro
phosphorylation assays were performed using recombinant
phosphoprotein produced in Escherichia coli as substrate
and cytoplasmic extracts from a rat glioma cell line (C6) or rat brain
extracts as sources of kinase activity. These experiments revealed that
BDV-P was phosphorylated predominantly by protein kinase C (PKC) and to
a lesser extent by casein kinase II. Partial purification of the PKC
from rat brain extract suggested that the BDV-P phosphorylating kinase
is PKC. A role for PKC phosphorylation in vivo was
confirmed by using the PKC-specific inhibitor GF109203X. Furthermore,
peptide mapping studies indicated that BDV-P is phosphorylated at the
same sites in vitro as it is in vivo.
Mutational analysis identified Ser26 and Ser28
as sites for PKC phosphorylation and Ser70 and
Ser86 as sites for casein kinase II phosphorylation. The
anatomic distribution of PKC
in the central nervous system may have
implications for BDV neurotropism and pathogenesis.
Borna disease virus (BDV)1 is the prototype of a new family, Bornaviridae, within the nonsegmented negative-strand RNA viruses (Mononegavirales) (1, 2), which is characterized by low productivity, neurotropism (3, 4), a nuclear localization for transcription and replication (5, 6), and posttranscriptional modification of subgenomic RNAs by splicing (7, 8). The potential host range for BDV is likely to include all warm-blooded animals. Accumulating evidence suggests that it may be a human pathogen (9-15).
Phosphoproteins of nonsegmented negative-strand RNA viruses are
typically integral components of the viral polymerase complex (16). The
activity of these proteins, and in some cases their three-dimensional
structure, is dependent upon phosphorylation (17-20). Casein kinase II
(CKII)-mediated phosphorylation of VSV-P leads to its multimerization
in vitro and promotes transcription (17, 20), possibly by
facilitating binding of VSV-P to the VSV polymerase (21). In human
respiratory syncytial virus (RSV), CKII-mediated phosphorylation of
phosphoprotein is also a prerequisite for transcriptional activity (18,
22). Although the human parainfluenza virus type 3 (HPIV3)
phosphoprotein is phosphorylated by protein kinase C (PKC) rather
than by CKII (19), the effect of HPIV3 phosphoprotein phosphorylation
is similar in significance to phosphorylation of VSV-P and RSV
phosphoprotein; inhibition of PKC
by pseudopeptides results in
abrogation of viral replication (19).
Because phosphorylation of the BDV phosphoprotein (BDV-P) is likely to
be an important step in the life cycle of BDV we have identified the
kinases involved in this process and mapped the BDV-P phosphorylation
sites. Our findings indicate that BDV-P is phosphorylated predominantly
by PKC and to a lesser extent by CKII.
BDV-infected C6 cells (23) (5 × 105) were washed twice with phosphate-buffered saline (PBS), incubated in modified RPMI medium 1640 (Irvine Scientific) without Pi (2 ml) in the presence or absence of GF109203X (1-5 µM/ml) (Biomol) for 6 h, and then labeled with inorganic 32P (100 µCi/ml) (NEN Life Science Products) for 3 h. Following two washes with PBS, cells were scraped into PBS, collected by centrifugation, and lysed in 100 µl of 10 mM Tris, pH 8.0, 2 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 µM okadaic acid (gift of A. Lepple, University of California, Irvine). BDV-P was immunoprecipitated overnight at 4 °C with rabbit antiserum to BDV-P diluted 1:400 in 10 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride. After incubation with Protein G-Sepharose (Sigma) for 1 h at 4 °C, bound protein was collected by centrifugation and then released by boiling in Laemmli buffer (24). Proteins were size-fractionated by 15% SDS-PAGE for analysis by autoradiography.
Plasmid ConstructionsBDV-P mutants were generated by PCR
using plasmid p23 (25) containing the complete phosphoprotein open
reading frame in pBluescript (Stratagene). The BDV-P mutant P11
(lacking 11 amino acids at the amino terminus) was generated using a
5-primer containing an NdeI site (p23-NdeI) and
a 3
-primer containing a T7 RNA polymerase binding site (T7 primer).
All other BDV-P mutants were generated by amplifying overlapping PCR
fragments using the T7 primer or p23-NdeI primer and
combinations of the primers described below. In a final PCR, the
complete BDV-P ORF mutant sequence was amplified with the
p23-NdeI primer and T7 primer and cloned into the
NdeI/HindIII site of pET15b (Invitrogen).
Introduction of the correct sequence for each mutant was confirmed by
dideoxy sequencing. Primers used to generate the PKC mutants were
P-26/28 (5
-AACGAGCGGGGGCACCAAGACC-3
), P-55 (5
-ATCGCAGACCCAGAC-3
),
P-130 (5
-CTCCGATGCCATCAGAATCC-3
), and P-144
(5
-GGATCGCGCCATGAAGAC-3
). Primers used to generate the CKII mutants
were P-11 (5
-AGAATCATATGCTGGAGGACGAAGAA-3
), P-58
(5
-ATCGCAGACCCAGAC-3
), P-70 (5
-CTAGCGAATGATGAG-3
), and P-86
(5
-AATGCCATGATCGAGG-3
).
Histidine-tagged BDV-P (25) and BDV-P mutants were purified from the soluble supernatant of transformed E. coli by nickel-agarose chromatography as described previously except that Nonidet P-40 was not used (26). The amino-terminal histidine tract was removed using avidin-coupled thrombin (Invitrogen). BDV-P was incubated for 1 h with 0.5 unit of thrombin in 20 mM Tris-HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 10 mM 2-mercaptoethanol, 20% glycerol, 2.5 mM CaCl2, 250 mM imidazole. The thrombin digestion reaction was stopped with 2 mM phenylmethylsulfonyl fluoride, and the avidin-coupled thrombin was removed with biotin-agarose according to the manufacturer's protocols (Invitrogen). Cleaved BDV-P was bound to Q-Sepharose (Pharmacia Biotech Inc.) in 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2 and then eluted with 550 mM NaCl. Pilot studies indicated that phosphorylation activity was equivalent with uncleaved and thrombin-cleaved BDV-P; thus, thrombin-cleaved protein was used only for peptide mapping experiments. Methods used for purification of phosphoproteins of VSV and HPIV3 have been described (19).
Purification of PKC and CKII from Extracts of C6 Cells and Rat BrainPKC was purified from cytoplasmic extracts of C6 (rat glial) cells as described (19). Briefly, 108 cells were washed in PBS, collected in 4 ml of Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and lysed by three freeze-thaw cycles. Cell extracts were centrifuged at either 10,000 × g for 10 min (S10 extract) or 100,000 × g for 60 min (S100 extract) at 4 °C. The S100 supernatant was dialyzed against 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5% (v/v) glycerol, 0.1 mM EDTA, 1 mM dithiothreitol (Buffer A) overnight at 4 °C and then subjected to DEAE-cellulose chromatography. The highest PKC activity was present in fractions eluted between 200 and 250 mM NaCl. These fractions were pooled and loaded onto a phosphocellulose column equilibrated with Buffer A containing 0.2 M NaCl. Whereas PKC activity was found in the flow-through fraction of this column, heparin-sensitive kinase (CKII) activity was eluted with increasing concentrations of NaCl starting at 300 mM. The flow-through fraction of the phosphocellulose column was again subjected to DEAE-cellulose chromatography, and PKC was eluted as already described. The pooled fractions were dialyzed against 10 mM K2PO4, pH 7.5, 5% glycerol, 1 mM dithiothreitol and applied to a hydroxylapatite column equilibrated with the same buffer. PKC activity was eluted between 250 and 350 mM potassium phosphate.
Methods used to purify PKC from rat brain were similar to those employed to purify PKC from C6 cells except that whole rat brain was lysed by Dounce homogenization in buffer A rather than by alternate freeze-thaw cycles.
SDS-PAGE Immunoblot AnalysesSDS-PAGE immunoblot analyses
were performed using 10-20 µg of cell lysates (S10 extract) or 500 ng of purified protein fraction from the final hydroxylapatite column
(rat brain material). Polyclonal antibodies against PKC, PKC
,
PKC
(Life Technologies, Inc.), and PKC
(Panvera) were used at
concentrations suggested by the manufacturer. Recombinant PKCs (PKC
,
PKC
, and PKC
) expressed in a baculovirus vector system were a
gift of W. Koelch, München.
For kinase experiments, 100-200 ng
of BDV-P and variable amounts of cellular kinases or recombinant PKCs
were incubated in 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.25% bovine serum albumin, 50 µM ATP, 5-10 mCi of [-32P]ATP (3000 Ci/mmol), and when indicated, 200 mM CaCl2,
phosphatidylserine (PS, 10 µg/ml), or diacylglycerol (DAG, 100 µg/ml) for 60 min at 30 °C in a total volume of 30 µl. After
addition of 1.5 µl of 10 mM ATP and 10 µl of 4 × Laemmli buffer, reactions were analyzed by SDS-PAGE and
autoradiography.
Peptide phosphorylation assays were performed as described (39).
Briefly, the kinase activities were assayed under standard reaction
conditions (30-µl volume) in the presence of PS (10 µg/ml), DAG
(100 µg/ml), EGTA (0.2 mM), and substrate peptides at 200 µM or substrate protein at 200 µg/ml. After incubation
for 30 min at 30 °C, a 3-µl aliquot was spotted onto P81 paper
(Whatman). Radioactivity bound to paper was counted after washing for 5 min with 75 mM H3PO4 five times.
Peptides AcMBP-(4-14) (Boehringer Mannheim),
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide, Sigma), -peptide [Ser-25]PKC-(19-31) (Life Technologies, Inc.) and
-peptide
[Ser-159]PKC-(149-164) (Peninsula Laboratories), and proteins
histone H1 (Boehringer Mannheim), and histone IIA (Boehringer Mannheim)
were used as substrates.
32P-Radiolabeled BDV-P was fractionated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membrane-bound BDV-P was digested in situ with trypsin, and peptides were recovered as described by Boyle et al. (27). Trypsin-digested peptides were fractionated on cellulose plates (J. T. Baker Inc.) by electrophoresis at pH 1.9 at 900 V for 25 min in a precooled peptide map apparatus and then subjected to chromatography in 1-butanol (37.5%, v/v), pyridine (25%, v/v), glacial acetic acid (7.5%, v/v), and deonized water (30%, v/v) for 6-8 h. Plates were dried and analyzed by autoradiography.
Recombinant BDV-P was purified from E. coli to use
as substrate to study phosphorylation of BDV-P in vitro.
Incubation of BDV-P with an S10 extract of C6 cells resulted in its
phosphorylation (Fig. 1A,
lane 1). Initially, two kinase inhibitors were used to gain
insight into the nature of the kinase that phosphorylates BDV-P.
Whereas staurosporin, a potent inhibitor of PKC and other kinases (28),
had a strong inhibitory effect on BDV-P phosphorylation (Fig.
1A, lanes 4 and 5), heparin which
efficiently inhibits CKII had only a minimal effect (Fig.
1A, lanes 2, 3, and 6). The
combination of staurosporin and heparin resulted in nearly complete
inhibition of BDV-P phosphorylation (Fig. 1A, lane
6). Incubation with an antibody to the conserved catalytic subunit
of PKC (PKC1.9) resulted in a concentration-dependent
inhibition of BDV-P phosphorylation (Fig. 1B, lanes 2 and 3). The extent of inhibition with saturating concentrations of
PKC1.9 (2 µg) was similar to that observed with
400 nM staurosporin (Fig. 1B, lane
4). Inhibition was also observed following addition of the
specific PKC inhibitor GF109203X to 6.0 µM final
concentration. CKII-mediated phosphorylation of VSV-P was not affected
by
PKC1.9 antibodies (data not shown). These results indicate that a
cellular kinase of the PKC family is the major kinase involved in the
incorporation of phosphate into the BDV-P.
PKC-dependent Phosphorylation of BDV-P Is Calcium-independent
The PKC enzymes include two subgroups, which
differ in their response to calcium (29). To determine whether
phosphorylation of BDV-P was mediated by a
calcium-dependent PKC (PKC, -
, and -
) or
calcium-independent PKC (PKC
, -
, -
, and -
) (29), phosphorylation experiments with C6 or rat brain S100 extracts were
performed in the presence of EGTA or supplemental calcium. Neither 0.5 mM EGTA nor calcium to 200 µM concentration
had any effect on kinase activity (data not shown).
The
BDV-P-phosphorylating PKC was partially purified from C6 cells (data
not shown) and rat brain by sequential chromatography through columns
composed of DEAE, phosphocellulose, DEAE, and hydroxylapatite. Although
elution profiles were similar with C6 and rat brain extracts, levels of
phosphorylation activity were higher in fractions representing rat
brain extracts. Fig. 2 displays results
with rat brain extracts. Phosphorylation of BDV-P by the kinase in the
elution fractions of the second DEAE column (Fig. 2A,
upper panel) correlated with the presence of PKC in each fraction as judged by Western immunoblot (WIB) using a PKC
subtype-specific antibody (Fig. 2A, lower panel).
In contrast, WIB analysis of the same fractions using PKC
subtype-specific antibodies revealed a different elution pattern, which
did not correlate with the BDV-P phosphorylation activity (data not
shown). In the final chromatography step, the kinase was eluted with
250 mM potassium phosphate (Fig. 2B, fraction 30 and beyond). The highest concentration of protein was eluted at 180 mM potassium phosphate (data not shown). BDV-P
phosphorylation activity by the kinase eluted in fraction 40 was
inhibited by staurosporin and
PKC1.9 antibodies but not by heparin
(data not shown). To identify the PKC present in fraction 40, PKC
subtype-specific antibodies (PKC
, PKC
, PKC
, PKC
) were used
for WIB analysis. Only PKC
was detected in fraction 40 (Fig.
2C, lane 2).
To assess whether the purified BDV-P-phosphorylating PKC might be
sensitive to the known PKC activators PS or DAG, the kinase activity
was measured in experiments where substrate was not a limiting factor
(Fig. 3A). Addition of 10 µg/ml PS (lane 1), 100 µg/ml DAG (lane 2),
and 10 µg/ml PS and 100 µg/ml DAG in the presence (lane
3) or absence (lane 4) of 200 µM calcium
stimulated phosphorylation of BDV-P 3-4-fold.
Comparison of Enzymatic Activity of the Recombinant PKC
The characteristics of the BDV-P protein kinase present
in extracts from C6 cells and rat brain suggested its identity as PKC. To further investigate the activity of individual PKC isotypes with respect to BDV-P, recombinant PKC
, PKC
, and PKC
were
examined for the capacity to phosphorylate BDV-P (Fig. 3) and a panel
of phosphate acceptors (Table I). When
employed at concentrations normalized for phosphorylation of PKC
-peptide, the efficiency of PKC
in BDV-P phosphorylation was
5-10-fold higher than PKC
and PKC
(Fig. 3B). Whereas
the efficiencies of BDV-PK and PKC
in PKC
-peptide
phosphorylation were similar (BDV-PK, 106%; PKC
, 118%),
phosphorylation of
-peptide by PKC
and PKC
was less efficient
(PKC
, 60%; PKC
, 80%). BDV-PK and PKC
were also similar in
inefficiency of AcMBP phosphorylation (BDV-P, 10%; PKC
, 13%). In
contrast, PKC
and PCK
were more efficient in AcMBP
phosphorylation (PKC
, 58%; PKC
, 45%). All kinases tested
(BDV-PK, PKC
, PKC
, and PKC
) were inefficient in
phosphorylation of histone H1 and histone IIA (4-8%), phosphate
acceptors that are phosphorylated by cAMP- or
cGMP-dependent protein kinase rather than PKC.
|
Phosphorylation of proteins
may differ in vitro or in vivo. Therefore, to
test whether BDV-P is also phosphorylated by PKC in vivo,
infected C6 cells were incubated with the specific PKC inhibitor
GF109203X and inorganic [32P]phosphate. BDV-P was
immunoprecipitated from these cells using a monospecific antibody to
BDV-P and subjected to SDS-PAGE and autoradiography. Increasing
concentrations of GF109203X resulted in decreased BDV-P phosphorylation
(Fig. 4, lanes 3-6)
indicating a role for PKC in phosphorylation of BDV-P.
Peptide mapping studies were pursued to determine whether BDV-P is
phosphorylated by PKC at the same sites in vivo and in vitro. For this purpose, BDV-P from infected C6 cells
phosphorylated in vivo and recombinant BDV-P phosphorylated
in vitro using a PKC-enriched fraction (C6 cells, second
DEAE column) were separated by SDS-PAGE, transferred to nitrocellulose
membranes, digested in situ with trypsin, and separated on
thin layer chromatography plates for analysis by autoradiography.
Peptide maps were identical (Fig. 5A) with three dominant
spots. Similar maps were observed when recombinant BDV-P was
phosphorylated with the crude S10 extract of C6 cells (data not
shown).
Previous work had shown that BDV-P is phosphorylated in vivo at serine residues (30). Thus, the five potential sites of BDV-P phosphorylation were investigated using BDV mutants in which serine residues were changed to alanine by PCR mutagenesis. Experiments with a C6 S100 extract depleted of CKII revealed that the double mutant BDV-P (26/28) was not efficiently phosphorylated (Fig. 5B). Peptide mapping of BDV-P (26/28) revealed that this site corresponds to the major peptide spot 1 (Fig. 5A). Due to the lack of a trypsin cleavage site between Ser26 and Ser28 it was not possible to differentiate between the two potential phosphorylation sites with peptide maps.
The Heparin-sensitive Kinase Is CKIIThe observations that
staurosporin did not completely block phosphorylation of BDV-P and that
the residual kinase activity was heparin-sensitive (Fig. 1) suggested
the possibility of minor phosphorylation by CKII. To investigate this
further, CKII was purified from the C6 S100 extract by phosphocellulose
chromatography. The majority of PKC-mediated kinase activity was found
in the void volume (Fig. 6A,
lane 1). This activity was inhibited by staurosporin (Fig.
6A, lane 3) but not by heparin (Fig.
6A, lane 2). In contrast, kinase activity in
fraction 50 was inhibited by heparin (Fig. 6B, lane
2) and not by staurosporin (Fig. 6B, lane
3). The kinase activity in fraction 50 was also inhibited by
antibodies to CKII (Fig. 6B, lane 4). To identify
the sites of CKII phosphorylation, BDV-P mutants were generated by PCR
that either lacked the first 11 amino acids (deleting two potential CKII sites) or substituted alanine for serine residues at positions 58, 70, and 86. Incubation of these BDV-P mutants and wild-type recombinant
BDV-P with a commercial CKII revealed that the serine residues at
positions 70 and 86 are the major sites of phosphorylation by CKII
(Fig. 6C). Neither mutation had an impact on the
PKC-dependent phosphorylation (data not shown).
Similarly, mutations at sites found to impair PKC-dependent phosphorylation of BDV-P (residues 26 and 28) had no effect on CKII-mediated phosphorylation (data not shown). Phosphorylation of wild-type BDV-P by a commercial CKII was similar in the presence or absence of S10 C6 cell extracts (data not shown).
The objective of this study was to characterize the cellular
kinases responsible for phosphorylation of BDV-P. In vitro
experiments with recombinant BDV-P and crude extracts from C6 cells
indicated that BDV-P was phosphorylated by both PKC and CKII; however,
phosphorylation appeared to be mediated primarily by PKC. BDV-P
phosphorylation was largely inhibited in vitro by antibodies
directed against the catalytic subunit of PKC. Exposure of infected
cells to increasing concentrations of GF109203X, a specific PKC
inhibitor (31), resulted in decreased phosphorylation of BDV-P.
Analysis of extracts from C6 cells and rat brain enriched for BDV-P
phosphorylation activity revealed that the BDV-PK is PKC. The
enzymatic activity of BDV-PK was correlated with the presence of PKC
rather than that of PKC
, PKC
, or PKC
. In addition, the
in vitro enzymatic activity profiles of purified BDV-PK and
recombinant PKC
were similar with respect to BDV-P and a panel of
phosphate acceptors. Peptide maps of BDV-P phosphorylated in
vitro and in vivo were identical and revealed one major
and two minor phosphopeptides. Experiments with BDV-P mutants lacking
potential PKC sites indicated that the major phosphopeptide represents
a PKC site.
The major phosphopeptide found in peptide maps from in vivo phosphorylated BDV-P contained the potential PKC sites, Ser26 and Ser28. We have only examined phosphorylation of BDV-P from BDV strain He/80. Because this viral strain lacks a suitable cleavage site between Ser26 and Ser28, it was not possible to directly determine which site was phosphorylated through peptide mapping. However, the observation that Ser26 is not present in BDV strain V (32) suggests that BDV-P is likely to be phosphorylated at Ser28 in vivo.
Site-directed mutagenesis of recombinant BDV-P established Ser70 and Ser86 as the principal sites for phosphorylation by CKII in vitro. A potential basis for the observation that CKII-dependent phosphorylation is less pronounced than phosphorylation mediated by PKC is that CKII sites are more sensitive to phosphatases. However, this explanation appears to be unlikely given that levels of BDV-P phosphorylation with recombinant CKII did not differ in the presence or absence of crude C6 cell extract. Alternatively, the difference in the efficiency of PKC and CKII phosphorylation in vitro may reflect accessibility of individual sites in the folded protein. Whether BDV-P is phosphorylated by CKII in vivo remains to be determined; however, the observation that kinase activity cannot be eliminated in vivo using PKC inhibitors is consistent with a role for CKII in BDV-P phosphorylation.
Phosphoproteins of other nonsegmented, negative-strand RNA viruses are
typically phosphorylated in vitro only by one kinase, CKII
(e.g. measles (33) and RSV (34)) or PKC (e.g.
HPIV3 (19)). An exception is VSV-P where initial phosphorylation by
CKII induces a conformational change of VSV-P (New Jersey) that opens
sites for secondary phosphorylation by an L-associated
kinase (35). BDV-P is phosphorylated in vitro by both PKC
and CKII. Although there are no data concerning the possibility that
phosphorylation by one kinase effects a conformational change in BDV-P,
the activities of these kinases appear to be independent because
mutants lacking PKC sites are phosphorylated by CKII, and conversely,
mutants lacking CKII sites are phosphorylated by PKC.
The observation that BDV-P is predominantly phosphorylated by PKC
does not imply that CKII phosphorylation is inconsequential. There is
precedent for functionally significant phosphorylation of individual
cellular transcription factors by more than one kinase. For example,
phosphorylation of cyclic AMP-responsive element binding proteins Jun
and Fos by different kinases has been shown to regulate their
activities (36). We can only speculate as to the role of
phosphorylation of BDV-P at multiple sites by different kinases. BDV is
the only nonsegmented negative-strand RNA virus known to have a nuclear
localization for transcription and replication. Perhaps phosphorylation
of BDV-P by one kinase impacts its translocation to the cell nucleus.
Consistent with such a hypothesis is the observation that the PKC
phosphorylation sites are located within the putative nuclear
localization signal of phosphoprotein. Phosphorylation by a second
kinase might then trigger assembly with other cellular or viral
proteins (for example, the BDV polymerase) to form an active
transcription factor complex. Finally, it is intriguing to speculate
that phosphorylation events may play a role in BDV tropism for limbic
circuitry. Indeed, the anatomic distributions of PKC
(37) and BDV
are similar in rat brain (3, 38), the best described model system for
Borna disease. As recombinant BDV systems are established these
hypotheses will be tested using BDV-P mutants lacking specific
phosphorylation sites.
We thank C. Glabe and R. A. Bradshaw for intellectual and material support.