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Journal of Virology, September 2001, p. 8854-8858, Vol. 75, No. 18
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.18.8854-8858.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Varicella-Zoster Virus ORF47 Protein Serine Kinase:
Characterization of a Cloned, Biologically Active Phosphotransferase
and Two Viral Substrates, ORF62 and ORF63
T. K.
Kenyon,1
J.
Lynch,2
J.
Hay,2
W.
Ruyechan,2 and
C.
Grose1,*
Departments of Microbiology and Pediatrics,
University of Iowa, Iowa City, Iowa,1 and
Department of Microbiology, State University of New York,
Buffalo, New York2
Received 24 April 2001/Accepted 11 June 2001
 |
ABSTRACT |
Varicella-zoster virus (VZV) codes for a protein serine kinase
called ORF47; the herpes simplex virus (HSV) homolog is UL13. No
recombinant alphaherpesvirus serine kinase has been biologically active
in vitro. We discovered that preservation of the intrinsic kinase
activity of recombinant VZV ORF47 required unusually stringent in vitro
conditions, including physiological concentrations of polyamines. In
this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62
protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV
ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein
from the kinase assay supernatant.
| |
TEXT |
Varicella-zoster virus (VZV)
contains two genes, the ORF47 gene and the ORF66 gene,
with classical serine/threonine kinase motifs (26). Two
VZV immediate-early proteins, ORF62 and ORF63, are functional homologs
of herpes simplex virus (HSV) regulatory proteins ICP4 and ICP22,
respectively. ORF62 is the major VZV transcriptional regulatory
phosphoprotein during primary infection (22). VZV ORF62 is
also present in significant and readily detectable quantities in the
viral tegument (12). ORF63 is expressed as a major
transcript and protein during VZV latency (2, 3, 9, 10,
13), frequently in conjunction with the transcripts for ORF4,
ORF21, ORF29, and ORF62 (2, 10). The ORF63 protein has
been shown to be phosphorylated by casein kinase II (CKII) in
vitro (30).
Previously, all in vitro data concerning ORF47 have been obtained with
infected cell lysate immunoprecipitations or recombinant viruses.
With ORF47 and ORF62 proteins purified from VZV-infected MeWo cells,
ORF47 phosphorylated ORF62 (18). Under these conditions, the possibility existed that a cellular protein kinase bound to ORF47
or ORF62. In recombinant VZV where stop codons truncated ORF47, ORF47 was not necessary for virus replication in cultured cells and not required for in vivo phosphorylation of VZV ORF63 (1). However, ORF47 is required for virus replication in
fetal skin and thymus implants in the SCID-hu mouse model and for
efficient replication in human T lymphocytes (15, 27).
Previous attempts to express an active recombinant alphaherpesvirus
kinase have been unsuccessful (17). Herein, we define in
vitro conditions for the VZV ORF47 protein kinase and document that two
VZV regulatory proteins are authentic substrates.
ORF47.12 required polyamines in the protein kinase reaction.
ORF47 has often been compared to CKII, so conditions that increase CKII
phosphorylation may stimulate ORF47 kinase activity (4, 7,
18). To investigate the effect of basic molecules on ORF47.12
kinase activity, various polyamines were added at a final concentration
of 1 mM to ORF47 in vitro kinase reactions (8). To aid
recovery, the 3B3 epitope of VZV gE was inserted into ORF47
(GenBank accession number AAK19253) by PCR mutagenesis, and the new
construct was called ORF47.12 (6, 25). The sequence of the
ORF47.12 gene was confirmed at the University of Iowa DNA facility.
ORF47.12 was subcloned into pCAGGS (19).
HeLa cells (5 × 105) (ATCC CCL2) seeded
into 35-mm tissue dishes were transfected with 2 µg of plasmid DNA
with Lipofectin (Gibco BRL) and variations (21). Sixteen
hours later, after 15 min of pretreatment with 1 ng of okadaic acid
(Sigma)/ml, the cells were lysed in radioimmunoprecipitation
assay buffer with 50 mM NaF, 1 mM
Na3VO4, 1 mM PMSF, 1 mM
benzamidine, 1 mM leupeptin, and 0.025 trypsin inhibition unit
each of aprotinin and I-S soybean trypsin inhibitor (Sigma) and
immunoprecipitated with monoclonal antibody (MAb) 3B3 (25,
32). Immunoprecipitates were washed twice with lysis buffer and
twice with kinase buffer (17). The reaction mixture
contained 25 µl of kinase buffer, 1 µl of a 30 mM stock of the
indicated polyamine, and, where indicated, 1 µl of a 125-mg/ml
poly-DL-lysine stock (Sigma). Radiophosphate (0.5 µCi of
[
-32P]ATP; Amersham) initiated the
kinase reaction (1 h, 30°C). The reaction was halted by adding
ice-cold lysis buffer. After being washed, the samples were boiled for
5 min in reducing sample buffer, electrophoresed on sodium dodecyl
sulfate-10% polyacrylamide gel electrophoresis gels
(17), and analyzed with an HP InstantImager. Data are
presented as counts per minute per square millimeter minus
counts per minute per square millimeter of background. The stabilizing
conditions described herein were the result of numerous experiments
defining exact conditions under which ORF47.12 was an active kinase.
All polyamines stimulated ORF47.12 autophosphorylation above background
levels (Fig.
1, lanes 1 to 4). Trivalent
spermidine
stimulated ORF47.12 kinase autophosphorylation by 18-fold,
while
the tetravalent spermine increased autophosphorylation by
15-fold.
Stimulation of ORF47.12 autophosphorylation was not merely due
to the presence of a cation, as Mg
2+ does not
stimulate ORF47 kinase and kinase assays using
Ca
2+ did not stimulate the kinase (data not shown
and reference
17).
We interpret this data to mean that the
polyamines stabilized
either the tertiary structure of ORF47.12 or the
formation of
dimer complexes of ORF47.12, thus facilitating auto- or
allophosphorylation.
Polyamines are basic cations synthesized in cells
from ornithine.
In a cell, positively charged polyamines stabilize
protein-protein
interactions and protein complex formation
(
8).
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FIG. 1.
Polyamines in the ORF47.12 protein kinase assay. Vector
pCAGGs-transfected (diagonally striped columns and lower gel slice) or
pCAGGs-ORF47.12-transfected (black columns and upper gel slice) lysates
were immunoprecipitated with MAb 3B3 and reacted in ORF47 kinase
buffer, which is supplemented with manganese (17). The
indicated polyamine (lane 1, no polyamines; lane 2, putrescine; lane 3, spermidine; lane 4, spermine) was added to a final concentration of 1 mM in each kinase reaction mixture. The gel slice shows the area at 54 kDa, the molecular mass of ORF47 (17), and the graph above
depicts the amount of radioactivity as quantified with the HP
InstantImager and presented as counts per minute per square millimeter
minus background (bk). In the results shown in the last three lanes of
the gel, 1 µl of a 125-mg/ml poly-DL-lysine stock was
added to the polyamine-stimulated kinase reactions (lane 5, putrescine
and lysine; lane 6, spermidine and lysine; lane 7, spermine and
lysine). The first three cells of the table below the gel slice show
the fold increase of ORF47.12-transfected radioactivity incorporation
in counts per minute per square millimeter minus background above that
of vector-transfected lysate (ORF47.12/V). The last three cells show
percent decrease due to adding
poly-DL-lysine to the polyamine-stimulated kinase reaction
mixture, for example, 100%  (putrescine + lysine/putrescine only).
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Poly-
DL-lysine also stimulates CKII kinase activity
(
7). When poly-
DL-lysine alone was added to
the ORF47.12 in vitro kinase
reactions, phosphorylation levels remained
undetectable (data
not shown). When poly-
DL-lysine was
added to ORF47.12 in vitro
kinase reactions with polyamines,
autophosphorylation decreased
by an average of 48% (Fig.
1, lanes 5 to
7), and the most polyamine-stimulated
samples were reduced the most by
poly-
DL-lysine. Poly-
DL-lysine
may be competing
for the same epitope to which the polyamines
bind on ORF47.12, yet
the poly-
DL-lysine did not stimulate the
kinase autophosphorylation. Because poly-
DL-lysine is much
larger
than the polyamine compounds, this reduction in ORF47.12
stimulation
may be due to steric hindrance. ORF47.12 is a monomer;
therefore,
ORF47.12 may not have the conformational flexibility of
CKII,
which is a tetramer stimulated by poly-
DL-lysine.
PCR mutagenesis of the invariant lysine reduced ORF47.12 protein
kinase autophosphorylation.
Kinases specify an invariant
lysine upstream of the kinase activation domain that is required for
kinase activity (26). To definitively attribute the
phosphotransferase activity to ORF47, the invariant lysine (codon
169) near the kinase catalytic domain was replaced with an aspartic
acid by PCR mutagenesis, and the mutant was designated ORF47.12d
(31). VZV gE was selected as a positive control in these
experiments because VZV gE coprecipitates with and is heavily
phosphorylated by CKII (20). Ten percent of the total
lysate was immunoblotted with primary MAb 3B3 with SuperSignal
West Pico Chemiluminescent Substrate (Pierce) (data not shown and
reference 25). Equivalent amounts of transfected VZV
gE, ORF47.12, and ORF47.12d proteins were produced.
As assessed by the in vitro kinase assay, the lysine substitution in
ORF47.12d reduced autophosphorylation by 63% compared
to
autophosphorylation of wild-type ORF47.12 (Fig.
2, lanes 2
and 3). The observed protein
kinase activity cannot be attributed
to contaminating CKII because (i)
kinase activity was not inhibited
by heparin (references
14
and
17 and data not shown), (ii)
CKII requires magnesium
for activity (
7) and the ORF47 kinase
buffer supplied only
manganese, and (iii) mutation of the invariant
lysine in ORF47
drastically reduced the biological activity of
the cloned and expressed
kinase.
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FIG. 2.
Substitution of the invariant lysine (K169) in the
ORF47.12 protein kinase. For both the gels and the graph, HeLa cells
were transfected with pCAGGS-vector (lane 1), pCAGGS-ORF47.12 (lane
2), pCAGGS-ORF47.12d (lane 3), or pTM1-VZV gE (lane 4). Ninety percent
of each lysate was immunoprecipitated with MAb 3B3 and reacted with
[-32P] ATP in an in vitro kinase assay in the presence
of 1 mM spermine in a suitable kinase, ORF47, or CKII buffer, which was
supplemented with magnesium and separated by sodium dodecyl
sulfate-10% polyacrylamide gel electrophoresis (20). The
remaining 10% of the whole-cell lysate was immunoblotted with MAb 3B3
(data not shown). The gel slice for lanes 1 to 3 shows that the
molecular mass of ORF47.12 and ORF47.12d is 54 kDa, and that for lane 4 shows that the molecular mass of VZV gE is 98 kDa. Radioactivity was
quantified with an HP InstantImager. Results are shown as counts per
minute per square millimeter minus background (bk) in the same units.
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The ORF47.12 protein kinase phosphorylated exogenous, recombinant
baculovirus-expressed VZV ORF62.
Previously, wild-type ORF47
immunoprecipitated from VZV-infected cells was found to phosphorylate
coimmunoprecipitated VZV ORF62 (18). Expression and
purification of VZV ORF62 in Sf21 insect cells by a recombinant
baculovirus vector have been described (28). Using the
stringent reaction conditions, we added 2 µg of ORF62 to the ORF47.12
in vitro kinase reactions (Fig. 3) and 1 µl of a 1-µg/µl heparin stock (Sigma) where indicated to
inhibit any contaminating CKII. A 30-µl aliquot of the
kinase reaction supernatant was removed before washing and precipitated
in 80% acetone (20°C).
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FIG. 3.
Phosphorylation of the VZV ORF62 protein by ORF47.12 and
CKII. HeLa cells were transfected with pCAGGS-vector (lane 1),
pCAGGS-ORF47.12 (lane 2), or pTM1-VZV gE (lanes 3 and 4). MAb 3B3
immunoprecipitates were reacted with exogenous baculovirus-produced
ORF62. The samples were incubated in suitable kinase buffer (ORF47
buffer or CKII buffer) with spermine and 1 mM heparin where indicated
(+hep). The gel slice shows radiolabeled ORF62. The graph above depicts
radioactivity in the gel slice as quantified by the HP InstantImager.
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|
The ORF47.12 kinase heavily phosphorylated the full-length ORF62.
Phosphorylated ORF62 was recovered from the supernatant
at levels of
radioactivity 10-fold above background. In control
samples, CKII
phosphorylated ORF62 at levels threefold higher
than those in the
ORF47.12 samples. Heparin reduced CKII phosphorylation
of ORF62
drastically, as expected. With another set of in vitro
kinase assays,
we utilized truncation mutants of ORF62 that included
only the first 42 amino acids and therefore the potential phosphorylation
site at S16.
These ORF62 constructs were derived from the first
half of the acidic
transcription activation domain (
23). In
these
experiments, ORF47.12 failed to phosphorylate the mutants
or
glutathione
S-transferase (GST) alone (data not
shown). Thus,
this site either was not utilized or was inaccessible to
the ORF47
kinase under these
conditions.
ORF47.12 protein kinase phosphorylated VZV ORF63.
ORF63 was
excised from the plasmid pCMV63 (a gift of Paul Kinchington, University
of Pittsburgh), inserted into pGEX-4T-1 (Pharmacia), expressed in
Escherichia coli, and purified by glutathione-Sepharose 4B
affinity chromatography (Pharmacia). A 2-µg aliquot of GST-ORF63 was added to ORF47.12 in vitro kinase assays.
ORF47.12 phosphorylated GST-ORF63 and, even after extensive washing,
precipitated GST-ORF63 from the kinase reaction supernatant
and
retained it in the pellet (Fig.
4).
Indeed, 71% of the total
radiolabeled ORF63 was found in the reaction
pellet (lane 2).
CKII also phosphorylated GST-ORF63 (lane 6), though
phosphorylation
observed with CKII was substantially less than that
observed with
ORF47.12. The addition of 1 or 2 mM heparin to the CKII
in vitro
kinase reactions reduced CKII phosphorylation of GST-ORF63 by
73 and 72%, respectively (lanes 7 and 8).
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FIG. 4.
Phosphorylation of the VZV ORF63 protein by ORF47.12 and
CKII. HeLa cells were transfected with pCAGGS-vector (lanes 1 and 5),
pCAGGS-ORF47.12 (lanes 2 to 4), or pTM1-gE (as a source of CKII) (lanes
6 to 8). MAb 3B3 immunoprecipitates were reacted with 2 µg of
GST-ORF63 (all lanes) and 1 mM (lanes 3 and 7) or 2 mM (lanes 4 and 8)
heparin. The upper gel slice shows ORF63 in the reaction mixture
supernatant, while the lower gel slice shows ORF63 retained in the
kinase reaction pellet. (Lanes 1 through 4) The horizontally striped
columns on the graph show ORF47.12-phosphorylated GST-ORF63 in the
kinase reaction mixture supernatant. The solid black columns
show the amount of ORF47.12-phosphorylated GST-ORF63 precipitated by
the kinase even after extensive washing. Lane 1 (negative control) was
reacted under ORF47 kinase conditions. (Lanes 5 through 8) The gray
areas of the columns show CKII-phosphorylated GST-ORF63 in the kinase
reaction mixture supernatant. The pixilated columns show
the amount of CKII-phosphorylated GST-VZV ORF63 in the kinase reaction
pellet. Lane 5 (negative control) was reacted under CKII
kinase conditions. The table shows the amounts of incorporated
radioactivity as quantified with the HP InstantImager.
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When 1 or 2 mM heparin was added to ORF47.12 in vitro kinase assays to
inhibit any contaminating CKII, ORF47.12 phosphorylation
of GST-ORF63
increased (Fig.
4, lanes 3 and 4). We hypothesize
that the ORF47.12 was
allowed greater access to the exogenous
GST-ORF63, and GST-ORF63
phosphorylation due to ORF47.12 increased
by 2.5-fold. Also, in the
presence of 1 mM heparin, the coprecipitation
of GST-ORF63 by ORF47.12
increased from 71 to 75%. When heparin
was increased to 2 mM,
phosphorylation of the exogenous GST-ORF63
was very nearly the same as
for the 1 mM sample, though more of
the phosphorylated GST-ORF63 was
retained in the kinase reaction
pellet (Fig.
4).
Using this in vitro system that preserved the biological activity of
cloned ORF47.12, we determined with certainty that the
VZV ORF62
protein (homolog of HSV ICP4) and the ORF63 protein
(homolog of HSV
ICP22) were authentic viral substrates of the
ORF47.12 protein kinase.
Phosphorylation-dephosphorylation events
have long been
considered to be important modifications in transcriptional
regulatory
proteins (
5). In addition, the observation that
ORF47
bound so tightly to ORF63 that ORF47 precipitated ORF63
from the kinase
reaction supernatant was an unusual finding. Although
kinases may often
be identified by precipitation of substrate
with concurrent
coprecipitation of kinase, only rarely can phosphorylated
substrates be
identified by precipitation of the relevant kinase
(
24).
Typically, the substrate is released immediately after
phosphorylation.
Thus, the latter result suggested that ORF47
and its substrate ORF63
existed as a complex. Subsequent experiments
with addition of exogenous
heparin demonstrated that ORF47 and
the cellular kinase CKII
competed for ORF63 binding, a possible
regulatory
interaction.
The fact that ORF62, ORF63, and ORF47 are present in the viral tegument
is of great interest because HSV phosphorylation analyses
have
demonstrated that protein phosphorylation and release from
the tegument
occur concurrently (
11,
12,
16,
29). Presumably,
therefore, at least one functional site of the ORF47 kinase is
within
the tegument. Finally, these studies add more information
about the
likely phylogeny of the herpesvirus protein serine/threonine
kinases
and their relatedness to mammalian protein kinases (
5).
| |
ACKNOWLEDGMENTS |
We thank Jolinda Traugh (UC-Riverside) for sharing additional
information about her published articles.
This research was supported by NIH grants AI36884, AI22795, and AI18449.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University
Hospital, 2501 JCP, 200 Hawkins Dr., Iowa City, IA 52242. Phone: (319)
356-2270. Fax: (319) 356-4855. E-mail:
charles-grose{at}uiowa.edu.
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K. M. Duus,
J. Peng,
D. H. Price, and C. Grose.
1999.
Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependant kinase.
J. Virol.
73:1320-1330[Abstract/Free Full Text].
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Journal of Virology, September 2001, p. 8854-8858, Vol. 75, No. 18
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.18.8854-8858.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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