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J Biol Chem, Vol. 274, Issue 50, 35999-36008, December 10, 1999
From the In vitro mRNA synthesis of Sendai
virus is almost entirely dependent on the addition of cellular proteins
(host factors). Previous studies indicated that the host factor
activity from bovine brain was resolved into at least two complementary
fractions, one of which may be tubulin. In this study, the host factor
activity that stimulates the transcription in the presence of tubulin
was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent
Mr of 46,000 (p46). From the amino acid
sequence, biochemical, and immunological analyses, p46 was identified
as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native
PGK from rabbit and yeast, and a recombinant human PGK substituted for
p46. Although, as previously suggested, tubulin was involved in the
transcription initiation complex formation by being integrated into the
complex, p46 and its complementary factor had little effect on the
complex formation. On the other hand, when p46 and the complementary
factor were added to the RNA chain elongation reaction from the
isolated initiation complex formed with tubulin, mRNA synthesis was
dramatically stimulated. The enzymatic activity per se of
PGK did not seem to be required for its activity. West-Western blot
analysis showed that PGK could directly interact with tubulin. These
data suggest that PGK stimulates Sendai virus mRNA synthesis at the
elongation step, probably through its interaction with tubulin in the
initiation complex.
Sendai virus (SeV),1 a
member of the Paramyxovirus family in the order Mononegavirales,
contains a monopartite negative strand RNA genome, which consists of
six genes encoding the viral proteins, nucleocapsid protein (NP),
phosphoprotein (P), matrix protein (M), fusion glycoprotein (F),
hemagglutin-neuraminidase glycoprotein (HN), and large protein (L)
(reviewed in Refs. 1-3). The viral envelope encloses a
ribonucleoprotein complex (RNP), which mainly contains the RNA genome
of 15.3 kilobases and three viral proteins: NP, P, and L. The RNA
genome encapsidated with the NP proteins serves as the template to
synthesize a positive strand leader RNA and mRNAs as well as a
full-length positive strand anti-genomic RNA, which serves as the
template for the negative strand RNA genome (1). The genetic
information of the RNA genome is expressed through at least six
monocistronic mRNA species. The P gene mRNA products which
encode the C and V protein families, in addition to the P protein, use
multiple translation initiation sites as well as an open reading frame
newly generated by RNA editing (1). According to a current model (1),
the viral RNA synthesis starts with a viral RNA-dependent
RNA polymerase entering at the 3' end of the genome RNA, where the
viral RNA polymerase initiates the sequential synthesis of a leader RNA
and mRNAs or synthesizes a full-length anti-genome RNA. However,
little is known about the mechanism of mode switching of the RNA
polymerase from transcription to replication, although it has been
shown that three viral proteins, the NP, P, and L proteins are required
for transcription and replication of the genome RNA (4-6) and that the
L protein interacts with the P protein to form the polymerase complex
for replication and transcription (4, 7). It has also been proposed
that, during replication, P protein acts as a chaperon for the NP to
encapsidate newly synthesized genome or anti-genome RNAs (8).
Several studies using in vitro transcription systems of
paramyxoviruses, e.g. human parainfluenza virus type 3 (HPIV3) (9, 10), mumps virus (11), measles virus (12), respiratory
syncytial virus (13), rinderpest virus (14), and canine distemper virus (15), suggest the involvement of cellular proteins in the viral RNA
synthesis in addition to viral proteins (reviewed in Refs. 16 and 17).
We have established an accurate and efficient in vitro
mRNA synthesizing system using purified SeV particles or viral RNP
complexes, in which viral mRNA synthesis is almost completely
dependent on the presence of host cell proteins (host factors) (18). We
have demonstrated that cellular tubulin, which is essential for
in vitro transcription of SeV, is integrated into the
transcription initiation complex and activates viral mRNA synthesis
(18, 19). The involvement of tubulin in SeV RNA synthesis was also
supported by the inhibition of in vitro transcription of SeV
by a monoclonal anti-tubulin antibody (20). On the other hand, De
et al. (21, 22) showed that the polymeric form of actin
binds viral RNP of human parainfluenza virus type 3 (HPIV3) and
activates in vitro transcription. They also found that the
HPIV3 RNPs are specifically localized on the actin microfilaments in
the virus-infected cells (23). The participation of tubulin or actin in
transcription has also been observed in measles virus (12) and
respiratory syncytial virus (24, 25), respectively. Furthermore,
cellular protein kinases such as casein kinase II and protein kinase C
isoform In this study, we purified the host factor activity complementary to
tubulin, and showed it has at least two components, and we identified
one of them as phosphoglycerate kinase (PGK), a glycolytic enzyme. This
is the first example showing the involvement of a glycolytic enzyme in
the transcription of paramyxoviruses. We have shown that PGK stimulates
viral mRNA synthesis at the elongation step, probably through its
interaction with tubulin integrated into the initiation complex.
Tubulin stimulated both mRNA synthesis and leader RNA synthesis of
SeV, while PGK failed to stimulate leader RNA synthesis, suggesting
that mRNA and leader RNA synthesis may be regulated by different
sets of host proteins.
Materials
ATP, GTP, UTP, and CTP were purchased from Yamasa Shoyu, Japan.
Hypatyte C was obtained from Clarkson Chemical Co.
[ Viruses
Sendai virus strain Z, propagated in the allantoic cavity of
10-day-old chicken eggs, was purified as described by Shibuta et
al. (31). Vesicular stomatitis virus strain New Jersey was kindly
supplied by Dr. H. Shibuta (Institute of Medical Science, University of
Tokyo, Tokyo, Japan).
Preparation of Bovine Brain Extract (S100 Fraction)
The 100,000 × g supernatant fraction (S100)
from bovine brain was prepared as described by Takagi et al.
(19).
Purification of Cellular Tubulin
Microtubular proteins were prepared form bovine brain through
three assembly-disassembly cycles as described by Shelanski et
al. (32). Purified tubulin free from microtubule-associated protein was obtained as a heterodimer by phosphocellulose column chromatography of microtubular proteins as reported by Weingarten et al. (33).
In Vitro Transcription of SeV and Vesicular Stomatitis Virus
(VSV)
In vitro mRNA synthesis of Sendai virus or
vesicular stomatitis virus was carried out under standard conditions as
described by Mizumoto et al. (18), with 50 µM
[ Preparation of Sendai Virus Ribonucleoprotein (RNP)
Purified Sendai virus particles (300 µg) were solubilized by
incubation in 500 µl of a solution containing 40 mM
HEPES-KOH (pH 7.9 at 20 °C), 30 mM NaCl, 30 mM KCl, 6 mM MgCl2, 2 mM DTT, and 0.1% Nonidet P-40 for 30 min at 30 °C. The
reaction mixture was centrifuged in a 2-ml tube in a Beckman TLS55
rotor containing 750 µl cushion A (30% (v/v) glycerol, 20 mM Tris-HCl (pH 7.9), 70 mM KCl, 10 mM MgCl2, 2 mM DTT, and 0.05%
Nonidet P-40) layered on a 750-µl cushion B (50% (v/v) glycerol, 20 mM Tris-HCl (pH 7.9), 70 mM KCl, 10 mM MgCl2, and 2 mM DTT), at
100,000 × g for 120 min at 4 °C. The RNP pellets
were resuspended in 60% sucrose/TNE (10 mM Tris-HCl (pH
7.4), 100 mM NaCl, and 1 mM EDTA), and then subjected to in vitro transcription. The amounts of the
pelleted viral RNP used for transcription were determined by adjusting NP protein contents in each RNP to that of 6 µg (total protein) of
virus particles using SDS-polyacrylamide gel electrophoresis.
Isolation of Transcription Initiation Complexes
The transcription initiation complex was isolated as described
by Takagi et al. (19). Purified Sendai virus particles (30 µg) were incubated for 30 min at 30 °C with the host factor in a
125-µl reaction mixture containing 40 mM HEPES-KOH (pH
7.9 at 20 °C), 30 mM NaCl, 50 mM KCl, 6 mM MgCl2, 2 mM DTT, and 0.1% Nonidet P-40, and the reaction mixture was centrifuged in a 0.8-ml ultraclear tube (Beckman), which contained two cushions, A and B (200 µl each), at 100,000 × g for 120 min at 4 °C in a
Hitachi P55ST-2 rotor with adapters. The viral RNP pellets were
resuspended in cushion B and were subjected to the elongation reaction
without additional host factors.
Purification of Host Factors
All operations were performed at 4 °C.
Step 1: Hydroxylapatite Column Chromatography--
Bovine brain
extract (S100) (36 mg, 3.6 ml) was loaded onto a hydroxylapatite
(Hypatite C) column (inner diameter, 1.6 × 16 cm)
pre-equilibrated with 1 mM potassium phosphate buffer (1 mM KH2PO4-K2HPO4 (pH 6.9 at 20 °C), 0.1 mM EDTA, 5 mM
2-mercaptoethanol, 20% glycerol) as described by Mizumoto et
al. (18), and proteins were step-eluted with 75, 150, 300, and 600 mM potassium phosphate buffer at a flow rate of 26 ml/h.
Proteins in each fraction were concentrated with ammonium sulfate (80%
saturation) and then used for in vitro transcription assays.
The 75, 150, 300, and 600 mM potassium phosphate eluates
were referred to as HA75, HA150, HA300, and HA600, respectively. For
further purification of host factors in HA75, hydroxylapatite column
chromatography was done in four batches without ammonium sulfate
precipitation. The HA75 fractions from four batches were combined and
then dialyzed against TEMG (20 mM Tris-KCl (pH7.9 at
20 °C), 0.5 mM EDTA, 5 mM 2-mercaptoethanol, 20% glycerol) containing 20 mM KCl. Approximately 172 ml
(0.5 mg/ml) of the HA75 was obtained from 14.4 ml of S100 fraction.
Step 2: Blue-Sepharose Column Chromatography--
A portion (43 mg, 86 ml) of the HA75 fraction was loaded onto a Blue-Sepharose column
(inner diameter, 1.6 × 18 cm) pre-equilibrated with TEMG/20
mM KCl, and proteins were step-eluted with TEMG containing 20, 150, and 300 mM KCl at a flow rate of 18 ml/h. The 20, 150, and 300 mM KCl eluates were referred to as BS20,
BS150, and BS300, respectively. This column gave two active,
complementary fractions, BS20 and BS150. The BS150 fractions from two
batches were combined and dialyzed against TEMG/150 mM KCl.
The dialysate of BS150 (50 ml, 0.26 mg/ml) was subjected to
Heparin-Sepharose column chromatography. The BS20 fraction was kept at
Step 3: Heparin-Sepharose Column Chromatography--
A batch
(6.5 mg, 25 ml) of the BS150 fraction was loaded onto a
Heparin-Sepharose column (inner diameter, 0.8 × 9.6 cm)
pre-equilibrated with TEMG/150 mM KCl. After washing the
column with 10 ml of the same buffer, proteins were eluted with a 50-ml
linear gradient of 150-350 mM KCl in TEMG at a flow rate
of 6 ml/h and fractions of 0.54 ml were collected. The active fractions
(220 mM KCl eluate, referred to HS220) containing a single
polypeptide with Mr of 46,000 (p46) (0.035 mg/ml, 19.5 ml from two batches) were pooled and stored at
Amino Acid Sequence Analysis
Highly purified p46 (90 µg, ~2 nmol) was digested with
TPCK-treated trypsin (2 µg) at 1:45 ratio (enzyme/substrate, w/w) in 180 µl of a trypsin-digestion buffer (50 mM Tris-HCl (pH
8.0), 10 mM CaCl2) at 30 °C overnight. The
tryptic peptides were fractionated by reverse-phase high performance
liquid chromatography. The individual peptides were sequenced by
automated Edman degradation using an Applied Biosystems model 477A
sequencer. The obtained amino acid sequences were compared with
sequences in the protein data base (PIR) by using the BLAST program.
Assay for Phosphoglycerate Kinase Activity
PGK activity was assayed in a coupled reaction with GAPDH
reaction as described by Lee (34). The assay was performed at room
temperature (~25 °C) in a total volume of 0.5 ml containing 0.1 M Tris-HCl (pH 7.9), 10 mM MgCl2,
0.15 mM NADH, 2 mM ATP, 6 mM
3-phosphoglycerate, 0.1 mg/ml BSA, 50 µg of GAPDH, and 5-30 ng of
PGK fraction. The consumption of NADH is monitored at 340 nm. One unit
of PGK activity was defined as the amount of enzyme that catalyzes the
formation of 1 µmol of 1,3-bisphosphoglycerate (= oxidation of 1 µmol of NADH) per minute, using a molar extinction coefficient for
NADH of 6.3 × 103 M Preparation of Antibody against Rabbit Muscle PGK
Rabbit muscle PGK (300 µg, obtained from Sigma) was mixed with
an equal volume of Freund's complete adjuvant and injected subcutaneously into a New Zealand White rabbit (2 kg) four times at
about 3-week intervals. After 11 weeks from the first immunization, polyclonal rabbit muscle PGK antibodies were affinity-purified using a
rabbit muscle PGK-immobilized affinity column that had been prepared by
coupling of PGK to 2-fluoro-1-methylpyridinium toluene-4-sulfonate-activated Cellulofine according to the
manufacture's protocol.
Bacterial Expression of Human PGK-1
The coding sequence of human PGK-1 (GenBank accession nos.
L00159 and L00160) was amplified from human leukocyte RNA (35) by
RT-PCR using a sense primer, 5'-CCA GGA TCC ATG
TCG CTT TCT AAC AAG C-3' (BamHI site underlined) and an
antisense primer, 5'-GGT AGA TCT AAT ATT GCT
GAG AGC ATC CAC-3' (BglII site underlined). The amplified
PCR fragments of 1269 base pairs were digested with BamHI
and BglII, inserted into BamHI and
BglII sites of the pQE-16 plasmid (Qiagen), and sequenced to
confirm their sequences and orientations. The resultant plasmid,
pQE-hPGK, was transformed into Escherichia coli strain
XL1-Blue. E. coli XL1-Blue harboring the plasmid pQE-hPGK
were grown in LB medium containing ampicillin (100 µg/ml) at 37 °C
until the A600 reached 0.6. Expression of the
recombinant human PGK with the hexahistidine tag at the carboxyl
terminus was induced by adding
isopropyl-1-thio- Immunoblotting
SDS-polyacrylamide gel electrophoresis was performed essentially
as described by Laemmli (36). For Western blotting, proteins were
resolved by electrophoresis in a 10% polyacrylamide gel containing SDS, and electroblotted onto a PVDF membrane. The membrane was probed
with monoclonal Protein Concentration
Protein concentrations were determined by the methods of
Bradford (37) using BSA as the standard.
Purification of the Host Factors--
Cellular proteins (host
factors) have been considered to have essential roles in the in
vitro mRNA synthesis of SeV. We have previously shown that the
host factor activity in the bovine brain extract is separated into two
complementary fractions by hydroxylapatite column chromatography (18).
These two fractions synergistically stimulated the in vitro
mRNA synthesis of SeV, and one could be replaced by highly purified
tubulin (18). In order to identify and characterize the host factor(s)
that acts complementally to tubulin, we attempted to purify the active
components from the bovine brain extract. In this study,
hydroxylapatite column chromatography was performed as the first step
essentially as described by Mizumoto et al. (18) with some
modifications. Bovine brain extract (S-100 fraction) was loaded onto a
hydroxylapatite (Hypatite C) column, and proteins were step-eluted with
75, 150, 300, and 600 mM potassium phosphate buffer, as
illustrated in Fig. 1A. In
these conditions, the host factor activity was separated as observed
before (18) into two complementary fractions, the 75 and 300 mM potassium phosphate eluates (referred to HA75 and HA300,
respectively). Western blot analysis with monoclonal anti-tubulin
antibody revealed that tubulin was mainly detected in HA300, but almost
not in HA75 (data not shown). Although the addition of HA75 alone (Fig.
1B, lane 2) or HA300 alone
(lane 8) to the in vitro transcription mixture containing SeV particles (lane 1) gave
weak transcription-stimulatory activities, the combinatorial addition
of HA75 and HA300 synergistically stimulated the reaction to give 18 S
size transcript (lane 9). To purify the host
factor(s) contained in HA75, the fraction was loaded onto a
Blue-Sepharose column, and proteins were step-eluted with 20, 150, and
300 mM KCl (Fig. 1A). The 20, 150, and 300 mM KCl eluates are referred to as BS20, BS150, and BS300,
respectively. When BS fractions were added to the transcription
reaction mixture without HA300, each BS fraction alone (Fig.
1B, lanes 3-5) or in combination
(lanes 6 and 7) supported little, if
any, stimulatory activity. However, the simultaneous addition of BS20
and BS150 (lane 13) or all Blue-Sepharose column
fractions (lane 14) in the presence of a
saturating amount of HA300 restored the transcription-stimulatory activity to levels comparable to that obtained with the combination of
HA75 and HA300 (lane 9), while each
Blue-Sepharose column fraction alone failed to stimulate transcription
(lanes 10-12), even in the presence of HA300.
These results indicate that the host factor activity in HA75 can be
further separated into the BS20 and BS150 fractions.
One of the two complementary fractions from Blue-Sepharose column,
BS150, was further purified through a Heparin-Sepharose column. As
shown in Fig. 2A, proteins
were eluted with a linear gradient of 150-350 mM KCl, and
the transcription-stimulatory activity of each fraction was assayed in
the presence of HA300 and BS20. The activity was eluted as a single
peak at 220 mM KCl. As seen in Fig. 2B, the
active fractions (fractions 110-126) contained a single polypeptide
with an apparent Mr of 46,000 (p46), which was
coeluted with the transcription-stimulatory activity (lanes 5-9). To investigate the complementarity of the host
factors for the transcription stimulation in a more purified system,
p46 and BS20 were subjected to an in vitro transcription
system reconstituted with highly purified tubulin instead of HA300
using viral particles or viral RNP (Fig.
3). In the case of transcription with
virions (Fig. 3B), the addition of BS20 alone
(lane 2), p46 alone (lane 3), or their combination (lane 4)
caused a weak stimulation activity. In the presence of a saturating
amount of highly purified tubulin, which by itself had some stimulatory
activity (lane 5), neither BS20 (lane
6) nor p46 (lane 7) altered the
reaction. However, the combination of three factors, BS20, p46, and
tubulin, resulted in significant stimulation of mRNA synthesis
(lane 8). In order to characterize the host
factors using a more purified viral transcription machinery, the viral
RNP complex was isolated from detergent-disrupted viral particles as
described under "Experimental Procedures" (Fig. 3A,
lane 2) in which most of the envelope
glycoproteins were removed, and used for transcription in place of
viral particles (Fig. 3C). Transcription was similarly and
effectively stimulated depending on the addition of three factors:
tubulin, p46, and BS20 (lane 8). These data
suggest that the simultaneous presence of p46, tubulin, and unknown
factor(s) in BS20 is required for the maximal transcription of the SeV
genome, and that these factors act directly on the viral RNP to
stimulate transcription.
Identification of p46 as PGK--
To identify p46, we performed
amino acid sequence analysis of tryptic peptides of p46 and then
searched for similar sequences in the protein data base (PIR) using the
BLAST program. Surprisingly, amino acid sequences of three peptides
were highly homologous to those of a eukaryotic glycolytic enzyme, PGK
(ATP:3-phospho-D-glycerate 1-phosphotransferase, EC
2.7.2.3) (Fig. 4A). These
sequences almost completely matched the sequences of mammalian PGK,
such as mouse PGK-1 (38) and human PGK-1 (39). The molecular weights of
various PGKs were reported to be 46,000 ± 2,000 by
SDS-polyacrylamide gel electrophoresis (40), which also corresponds to
the Mr of p46 (also see Fig.
5A).
PGK catalyzes the reversible interconversion of ADP + 1,3-bisphosphoglycerate to ATP + 3-phosphoglycerate. To assess p46 as bovine PGK, we examined the enzymatic activity of PGK at various steps
during purification of p46. As shown in Table
I, a majority of PGK activity was
copurified with transcription-stimulatory activity of p46. The specific
activity as the enzyme of the final column fraction, HS220 (481 units/mg protein) was comparable to that of rabbit muscle PGK (431 units/mg protein) or of yeast (S. cerevisiae) PGK (646 units/mg protein). An effective purification of PGK (p46) was achieved
by three steps of column chromatography with an increase of 125-fold in
the specific activity and with 59% recovery. Furthermore, PGK activity
was coeluted with the transcription-stimulatory activity from a
Heparin-Sepharose column (Fig. 4B). Since p46 was not
separated from the other complementary factor(s) until Blue-Sepharose
column chromatography, the specific activities of transcription in the
presence of BS20 and tubulin were measured for the last two steps of
purification. The final step of purification led to a 15-fold increase
in the specific activity of transcription-stimulatory activity, which
was nearly comparable to a 13-fold increase in PGK activity. As shown
in Fig. 4C, affinity-purified polyclonal anti-rabbit muscle
PGK antibodies, which reacted with rabbit muscle PGK and yeast one
(lane 9), recognized p46 in BS150
(lane 1) as well as in HS fractions
(lanes 3-7).
To investigate whether the transcription-stimulatory activity of p46
can be replaced by PGK from other sources, highly purified PGKs from
rabbit muscle or Saccharomyces cerevisiae instead of p46
were added to the transcription reaction in the presence of complementary factors, tubulin and BS20 (Fig. 5). Each preparation from
rabbit muscle (lane 2) or yeast (lane
3) contained a single polypeptide with approximate
Mr values of 43,000 or 48,000, respectively (Fig. 5A). As shown in Fig. 5B, PGK either from
rabbit muscle or from yeast was also capable of stimulating SeV
transcription in the presence of tubulin and BS20 in a
dose-dependent manner as was p46, with a similar specific
activity. By contrast, the addition of the rabbit muscle type GAPDH
with tubulin and BS20 had no effect on the SeV transcription. To
further provide evidence in support of a direct involvement of PGK in
the SeV transcription, we tested whether a recombinant PGK can
substitute for p46. We constructed human PGK-1 expression vector to
produce His-tagged recombinant protein and purified the protein to
almost homogeneity, which had an apparent Mr of
46,000 (Fig. 5A, lane 4) corresponding to the calculated Mr (46,112) of the tagged
protein. The purified recombinant PGK exhibited the PGK activity (363 units/mg protein). As seen in Fig. 5B, the purified
recombinant human PGK also stimulated the SeV transcription reaction in
the presence of tubulin and BS20 with a specific activity similar to
those obtained with native PGKs. From these biochemical and
immunological data, we conclude that p46 is the bovine PGK.
Functional Properties of the Host Factors--
It is interesting
to see whether the enzymatic activity of PGK is directly involved in
the transcription-stimulatory activity or not. We tested the effects of
3-phosphoglycerate, a substrate, and
DL-glycerol-3-phosphate, a competitive inhibitor against
3-phosphoglycerate, on the transcription-stimulatory activity of p46 in
the range of 1-3 mM (Table
II), since the Km
value for 3-phosphoglycerate (34, 40) and the Ki
value for DL-glycerol-3-phosphate (41) were reported to be
1 and 0.7 mM, respectively. Increasing the concentrations
of both reagents had little effect on the transcription-stimulatory activity of p46. Thus, the enzymatic activity per se of PGK
does not seem to be required for its activity.
To elucidate the functions of the host factors in SeV mRNA
synthesis, their effects on transcription initiation and elongation reactions were examined. First, viral particles were incubated with the
host factors in various combinations without four NTPs, followed by
ultracentrifugation to obtain viral RNPs (initiation complexes) as the
pellets. Four NTPs were then added to the isolated viral RNPs to
determine their mRNA synthesizing activity (Fig. 6A). We have previously shown
that transcription initiation complex formed with bovine brain extract
contains tubulin, an essential factor required for mRNA synthesis
(19). When virus particles were preincubated with highly purified
tubulin instead of bovine brain extract, the isolated viral RNP could
also synthesize viral mRNAs (lane 5). These
data further confirmed the notion that tubulin plays an important role
in the initiation complex formation (19). In contrast, when virus
particles were preincubated with BS20 (lane 2), p46
(lane 3), or their combination (lane
4), neither RNP pellets obtained by centrifugation supported
mRNA synthesis. BS20 and p46 had little effect on the initiation
complex formation with tubulin (lanes 6-10). We
analyzed these RNP pellets by electrophoresis in a SDS-polyacrylamide
gel followed by silver staining (Fig. 6B) and Western
blotting using anti-tubulin antibodies as the probe (Fig.
6C). All the active initiation complexes formed in the
presence of tubulin contained nearly identical amounts of tubulin (Fig.
6B, lanes 7-10), which was confirmed
by Western blotting (Fig. 6C, lanes
7-10), indicating that neither BS20 and p46 affected
integration of tubulin into the initiation complexes. Thus, it seems
that p46 and the unknown factor(s) in the BS20 fraction participate in
transcription after the initiation complex formation.
We analyzed the effect of host factors on the mRNA chain elongation
reaction from the isolated initiation complexes formed in the presence
of tubulin (Fig. 7). The initiation
complexes were prepared by incubating virus particles with or without
highly purified tubulin, followed by ultracentrifugation, and then
subjected to transcription reactions in the presence or absence of the
host factors. As mentioned above, the initiation complex formed with tubulin showed a transcription activity, although at a low level, without addition of the other host factors (Fig. 7, lane
6), and further addition of tubulin to the complex had no
effect on transcription activity (lane 10),
indicating that a saturating amount of tubulin was integrated into the
initiation complex. Either BS20 alone (lane 7) or
p46 alone (lane 8) also failed to stimulate the
transcription from the isolated initiation complex. However, when p46
(PGK) and BS20 were simultaneously added to the RNA chain elongation reaction from the isolated initiation complex, mRNA synthesis was
dramatically stimulated (lane 9). On the other
hand, addition of p46 (PGK) and BS20 to RNA chain elongation reaction
from the RNP, which had not been preincubated with tubulin, showed only a little mRNA synthesizing activity (lane 4).
From these data, it seems that p46 and BS20 act cooperatively on the
tubulin-containing active initiation complex at the level of RNA chain
elongation.
To investigate whether these factors can interact with tubulin,
West-Western blot analysis was performed using highly purified tubulin
as the probe (Fig. 8). Fig. 8A
shows the patterns of protein transfer of BS20, p46, tubulin, and virus
particles from the gel to a PVDF membrane monitored by Amido Black
staining. For West-Western blotting, proteins were similarly blotted on
a PVDF membrane, then the membranes were pretreated with
(lanes 5-8) or without (lanes
1-4) highly purified tubulin. After extensive washing of the membrane, tubulin remaining on the membrane was detected with an
anti- Host Factors Act Specifically on mRNA Synthesis of Sendai
Virus--
Finally, to confirm a specific roles of host factors in SeV
mRNA synthesis, we examined the effects of host factors on the in vitro plus leader RNA synthesis of SeV (Fig.
9) and in vitro mRNA
synthesis of VSV, which belongs to the Rhabdovirus family in the order
Mononegavirales (Fig. 10). The
plus-sense leader RNA was transcribed from the 3'-end of a SeV genome.
We have developed an in vitro plus leader RNA synthesis
system using virus particles as described elsewhere.2 In
this system, artificially prematurely terminated plus leader RNA was
detected to confirm that transcription took place from the genome
leader sequence. Using this system, we have shown that plus leader RNA
synthesis also requires host cell proteins. We tested whether the host
factors for mRNA synthesis isolated in this study can also
stimulate the leader RNA synthesis or not. As shown in Fig. 9, tubulin
stimulated not only mRNA synthesis of SeV but also plus leader RNA
synthesis (lane 5). In contrast, BS20 potently
repressed tubulin-mediated leader RNA synthesis (lane
6), while p46 (PGK) failed to stimulate tubulin-mediated leader RNA synthesis with (lane 8) or without
(lane 7) BS20. In addition, no significant effect
of these factors on the mRNA synthesis of VSV was observed (Fig.
10). These results suggest that p46 (PGK) and factor(s) present in BS20
may be specifically involved in the mRNA synthesis of SeV.
In our in vitro mRNA synthesizing system using
purified SeV particles, viral mRNA synthesis was almost completely
dependent on the presence of host cell proteins (host factors), one of
which was suggested to be tubulin, a cytoskeletal protein (18). In this
study, we attempted to purify the host factor(s) which acts complementarily to tubulin to stimulate viral mRNA synthesis and found the activity can be separated into two complementary fractions (BS20 and BS150) by Blue-Sepharose column chromatography (Fig. 1).
These results suggest that the host factor is composed of at least
three factors including tubulin. One of them was further purified to a
single polypeptide with an apparent Mr of 46,000 (p46) by Heparin-Sepharose column chromatography of BS150 (Fig. 2), and
was identified as phosphoglycerate kinase (PGK), a glycolytic enzyme by
biochemical and immunological analyses (Fig. 4 and 5). The fact that
the bacterially expressed PGK could substitute for p46 also supported
the direct involvement of PGK in SeV transcription. To our knowledge,
this is the first instance of functional identification of PGK as a
host factor for mononegavirus transcription.
Since the molecules of PGK (0.1 µg or 2.2 pmol per assay), required
for SeV transcription in the presence of sufficient amounts of tubulin
and BS20, is lower than that of tubulin (3 µg or 30 pmol as
heterodimers per assay) (Fig. 3), and is roughly the same order as that
of L protein present in an assay mixture, it seems that PGK acts
catalytically rather than stoichiometrically. PGK catalyzes the
transfer of a phosphoryl group from the acyl phosphate of
1,3-bisphosphoglycerate to ADP, thereby forming ATP and
3-phosphoglycerate. However, the enzymatic activity of PGK does not
seem to be involved in the transcription-stimulatory activity of PGK,
because the substrates, 1,3-bisphosphoglycerate and 3-phosphoglycerate
were not present in the transcription reaction mixture. Additionally, the addition of 3-phosphoglycerate, a substrate, or
DL-glycerol-3-phosphate, a substrate analogue inhibitor,
did not affect transcription (Table II). Transcription of SeV was
stimulated by both mammalian PGKs and yeast PGK, although the identity
of overall primary amino acid sequences between mammalian PGK and yeast
PGK is about 64% (38, 42).
Moyer et al. (20) reported that SeV transcription is
inhibited by a monoclonal antibody against We have previously shown that a transcription initiation complex formed
with bovine brain extract contains tubulin molecules (about 250 copies/genome), and that the amounts of tubulin integrated into the
complexes correlate with their transcriptional activity (19). In the
present work, we showed that highly purified tubulin is also integrated
into the viral RNP complex at the transcription initiation step and
activates transcription from the complex (Fig. 6), confirming that
tubulin is directly involved in transcription initiation complex
formation. By contrast, p46 (PGK) and BS20, when added together,
dramatically stimulated chain elongation from the initiation complex
formed with purified tubulin, without affecting the initiation complex
formation (Fig. 7). Moreover, direct interactions between p46 (PGK) and
tubulin was shown by West-Western blot analysis. These observations
suggest that p46 (PGK) stimulates SeV mRNA synthesis at the
elongation step, probably through the interaction of p46 (PGK) with
tubulin that has been integrated into the initiation complex.
Alternative possibility is that p46 (PGK) and BS20 may function in the
reutilization of the viral RNA polymerase.
De et al. (21) purified a host factor for in
vitro transcription of human parainfluenza virus type 3 (HPIV3), a
paramyxovirus closely related to SeV, from the cytoplasmic extract of
uninfected cells, and identified it as actin, a cytoskeletal protein.
Furthermore, they found that both polymeric and monomeric forms of
actin are integrated into the viral RNP complex, but only the polymeric form can activate HPIV3 transcription, and HPIV3 transcription is not
stimulated by tubulin (22). In our SeV mRNA synthesizing system,
highly purified actin also stimulated mRNA synthesis, but specific
activity of tubulin dimer was 7-fold higher than that of the actin
monomer (data not shown). On the other hand, they reported that GAPDH,
a glycolytic enzyme, interacts with the 3'-terminal leader sequence of
the genome as well as the plus leader RNA (43). It is interesting to
note that GAPDH catalyzes formation of 1,3-bisphosphoglycerate, which
is in turn the substrate for its downstream enzyme, PGK, in the
sequential glycolytic reaction. It was reported that GAPDH is able to
interact with not only PGK (44) but also tubulin (45). However,
significant effect on the transcription with SeV particles was not
observed by the addition of rabbit muscle type GAPDH in the various
combinations with the host factors (tubulin, PGK, and BS20) for SeV
transcription (Fig. 5, data not shown). We could not detect GAPDH in
SeV particles either by Western blotting with anti-GAPDH antibody or by
the assay for its enzymatic activity (data not shown).
Most of the glycolytic enzymes have been shown to interact with
filamentous actin (46-49) as well as microtubules (50-52), suggesting that it is a common feature of these enzymes to bind to cytoskeletal proteins. We also demonstrated that PGK interacts with tubulin (Fig.
8). However, physiological significance of binding of the glycolytic
enzymes to cytoskeletal proteins are still unknown. Many glycolytic
enzymes were suggested to interact with the carboxyl-terminal tails of
the tubulin subunits (53), which is important sites for regulation of
the assembly of microtubules through the binding of
microtubule-associated proteins (reviewed in Ref. 54). Previous studies
suggested that the carboxyl-terminal tails of the tubulin subunits,
which are highly negatively charged and exposed on the surface of the
tubules, may be involved in SeV transcription (16, 18). It is important
to investigate the significance of the acidic tails of tubulin in the
transcription-stimulatory activity and the recruitment of other host
factors such as PGK. Tubulin may have multiple roles in SeV mRNA
synthesis, e.g. tubulin 1) dissociates M protein, which acts
as the negative regulator for RNA synthesis from the viral RNP; 2)
stabilizes the RNP structure to maintain the template as a
transcriptionally active state; and 3) recruits other host factors to
the viral RNP. It remains to be seen at which precise steps of viral
transcription PGK and BS20 act, and to which viral or cellular proteins
they interact.
Our studies indicate that tubulin functions as a common host factor for
both mRNA synthesis and plus leader RNA synthesis of SeV, while PGK
acts specifically for mRNA synthesis (Fig. 9). Moyer et
al. (20) also reported that purified tubulin stimulates the
synthesis of leader-like RNA from the detergent-disrupted SeV
particles. Interestingly, crude BS20 fraction, which exhibits the
mRNA synthesis-stimulatory activity, was found to strongly repress
leader RNA synthesis. Preliminary purification of BS20 revealed that
both activities are separated into different fractions (data not
shown). It is interesting to note that the negative factor which
inhibits leader RNA synthesis, was found to be distinct from an RNase
or a proteinase, and that it may act specifically to inhibit plus
leader RNA synthesis, because this factor did not inhibit in
vitro mRNA synthesis of SeV or of VSV. In addition, we
previously reported an inhibitory factor that potently inhibits SeV
mRNA synthesis from rat liver extract (55). Thus, mRNA
synthesis as well as plus leader RNA synthesis may be regulated by both positive and negative factors.
The specific action of the host factors, tubulin, PGK, and BS20, in SeV
mRNA synthesis also comes from the following observations. These
factors did not show any effect on the in vitro mRNA
synthesis with VSV particles (Fig. 10). Although either purified VSV
particles or the viral RNP from virions are capable of transcribing
mRNA in vitro, soluble cell extracts increase the level
of mRNA synthesis (56). We also observed that in vitro
transcription with VSV particles was stimulated 2-3-fold either by the
addition of bovine brain extract or HeLa cell extract (data not shown),
suggesting that at least tubulin, PGK, and BS20 are not responsible for
the stimulation VSV transcription by crude cell extracts in our system. Hill et al. (56, 57) reported that in vitro
transcription and replication of VSV was stimulated by
microtubule-associated proteins derived from HeLa cells or bovine
brain, but not by tubulin. By contrast, Moyer et al. (20)
reported that in vitro transcription of VSV was stimulated
by purified tubulin and was inhibited by a monoclonal antibody against
Identification of a host factor as PGK will be helpful for
understanding not only the regulatory mechanisms of SeV RNA synthesis but also the precise replication site of SeV in the cell. Since PGK is
abundant and ubiquitous in cytoplasm of eukaryotic cells, its usage as
a transcription factor seems to be feasible for the cytoplasmic
replication of SeV. It is important to note that PGK was also
identified as a subunit of primer recognition proteins, which are
cofactors of DNA polymerase *
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
81-3-5791-6245; Fax: 81-3-3444-6198; E-mail:
mizumotok@pharm.kitasato-u.ac.jp.
2
M. Iwama and K. Mizumoto, manuscript in preparation.
The abbreviations used are:
SeV, Sendai virus;
PGK, phosphoglycerate kinase;
RNP, ribonucleoprotein;
VSV, vesicular
stomatitis virus;
HPIV, human parainfluenza virus;
DTT, dithiothreitol;
BSA, bovine serum albumin;
PVDF, polyvinylidene difluoride;
HRP, horseradish peroxidase;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
TPCK, N-tosyl-L-phenylalanyl
chloromethyl ketone.
Involvement of a Cellular Glycolytic Enzyme, Phosphoglycerate
Kinase, in Sendai Virus Transcription*
§,,,,, and¶
Department of Biochemistry, School of
Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku,
Tokyo 108-8641, Japan and the § Research Center for
Biologicals, Kitasato Institute, Shirokane, Minato-ku,
Tokyo 108-8642, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
have been shown to phosphorylate the P protein (26-30).
However, the precise mode of action of these host factors in activating
and regulating the RNA polymerase of paramyxoviruses remains to be
studied. In addition, there seem to be other factors required for the
transcription as well as replication of paramyxovirus genome that have
not been identified yet (18, 19). Therefore, the purification and the
functional analysis of each host factor may, of course, provide an
important step toward understanding the molecular mechanism of
transcription and replication of the paramyxovirus genome.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP (400 Ci/mmol), [-32P]GTP
(400 Ci/mmol), Blue-Sepharose CL-6B, Heparin-Sepharose CL-6B, mouse
monoclonal anti-chicken brain 
-tubulin antibody (IgG1, clone DM1B),
horseradish peroxidase (HRP)-linked protein A, and the ECL Western
blotting system were from Amersham Pharmacia Biotech. N-Tosyl-L-phenylalanyl chloromethyl ketone
(TPCK)-treated bovine pancreatic trypsin was from Worthington. Yeast
(Saccharomyces cerevisiae) phosphoglycerate kinases (PGK)
and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were
obtained from Roche Molecular Biochemicals. Rabbit muscle PGK,
3-phosphoglycerate, and DL-glycerol-3-phosphate were from
Sigma. Nickel-nitrilotriacetic acid-agarose and 4 CN membrane
peroxidase substrate system were obtained from Qiagen and Kirkegaard & Perry Laboratories, respectively. HRP-conjugated goat anti-mouse IgG
polyclonal antibody was from Promega. Immobilon P polyvinylidene
difluoride (PVDF) membrane was from Millipore. Freund's complete
adjuvant and Freund's incomplete adjuvant came from Difco.
2-Fluoro-1-methylpyridinium toluene-4-sulfonate-activated Cellulofine
was the product of Seikagaku Corp.
-32P]UTP (3,000-5,000 cpm/pmol) and 6 µg of
purified Sendai virus particles, or with 50 µM
[-32P]UTP (500-1,000 cpm/pmol) and 1 µg of purified
vesicular stomatitis virus particles. After incubation for 120 min at
30 °C, the reaction mixtures were treated with proteinase K. The
transcripts were extracted with phenol-chloroform and precipitated with
ethanol, and then electrophoresed in an 1.2% agarose gel after
denaturation with glyoxal. In vitro plus leader RNA
synthesis of Sendai virus was carried out with 10 µM
[-32P]GTP (40,000-50,000 cpm/pmol) and 6 µg of
virus particles under the same conditions for mRNA synthesis except
that UTP was omitted, as described
elsewhere.2 After incubation for
120 min at 30 °C, the RNA products were denatured with formamide,
and analyzed by electrophoresis in a 20% polyacrylamide gel containing
8 M urea.
80 °C.
80 °C.
1
cm1.
-D-galactopyranoside to a final
concentration of 1 mM and cells grown for another 2 h
before harvesting. The His-tagged human PGK was purified using a
nickel- nitrilotriacetic acid-agarose column according to the manufacturer's protocol (Qiagen).
-tubulin antibody (1:500 dilution) and HRP-conjugated goat anti-mouse IgG polyclonal antibody for the detection of tubulin, or with affinity-purified polyclonal PGK antibody
(1:100 dilution) and HRP-linked protein A for the detection of PGK. The
immunocomplex was detected by ECL detection system. To detect the
protein band(s) that interacts with tubulin by West-Western blotting,
the blotted membrane was first blocked with 3% BSA in a binding buffer
(20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol)
at 4 °C overnight, and then incubated with or without highly
purified tubulin (50 µg/ml) in a binding buffer containing 0.1% BSA
at room temperature for 3 h. After extensive washing of the
membrane, tubulin on the membrane was detected by using
anti--tubulin monoclonal antibody and 4-chloro-1-naphthol detection system.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of host factor activity for SeV
mRNA synthesis in the HA75 by Blue-Sepharose column
chromatography. A, purification scheme for the host
factors, required for the mRNA synthesis of SeV, from bovine brain
extract (S-100). The host factors were purified by successive column
chromatography using Hypatite C and Blue-Sepharose, as described under
"Experimental Procedures". The 75, 150, 300, and 600 mM
potassium phosphate eluates from Hypatite C column are referred to as
HA75, HA150, HA300, and HA600, respectively. The 20, 150, and 300 mM KCl eluates from Blue-Sepharose column are referred to
as BS20, BS150, and BS300, respectively. B, Hypatite C
column 75 mM potassium phosphate eluate (HA75, 43 mg),
containing an activity that stimulates SeV mRNA synthesis in the
presence of HA300, was loaded onto a Blue-Sepharose column (inner
diameter, 1.6 × 18 cm) pre-equilibrated with TEMG (20 mM Tris-KCl (pH 7.9 at 20 °C), 0.5 mM EDTA,
5 mM 2-mercaptoethanol, 20% glycerol) containing 20 mM KCl, and proteins were step-eluted with TEMG containing
20, 150, and 300 mM KCl. Proteins in each column fraction
were concentrated and subjected to in vitro mRNA
synthesis reactions with SeV particles (6 µg) in the absence
(lanes 1-7) or presence (lanes
8-14) of HA300 (10 µg) as indicated on the top of the
figure. 32P-Labeled transcripts were resolved by
electrophoresis in an 1.2% agarose gel and detected by
autoradiography. Lane 1 indicates transcription
products with virus particles alone (SeV alone). Lanes
2 and 9 showed products from reactions with
sample before column chromatography (HA75, 10 µg) without or with
HA300, respectively. Proteins used in these assays were 8.0, 1.5, and
0.5 µg for the 20 (BS20), 150 (BS150), and 300 (BS300) mM
KCl eluates, respectively.
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Fig. 2.
Isolation of a host factor, p46, by
Heparin-Sepharose column chromatography. A, the 150 mM KCl eluate (BS150, 6.5 mg) from Blue-Sepharose column
was loaded onto a Heparin-Sepharose column (inner diameter, 0.8 × 9.6 cm) pre-equilibrated with TEMG/150 mM KCl. After
washing the column with 10 ml of the same buffer, proteins were eluted
with 50 ml of linear gradient of 150-350 mM KCl in TEMG.
The aliquots (0.3 µl) of each column fraction were subjected to
in vitro mRNA synthesis with Sendai virus particles (6 µg) in the presence of HA300 (10 µg) and BS20 (8.0 µg). The
transcription-stimulatory activity is expressed as the amounts of
[32P]UMP incorporated into the 18 S mRNA band ( 
).
The activity was eluted as a single peak at 220 mM KCl.
B, proteins in each column fraction were analyzed by
electrophoresis in a 10% SDS-polyacrylamide gel followed by Coomassie
Brilliant Blue staining. Lanes 1-3 indicate 30 µg of bovine brain extracts S-100 (BE), 20 µg of
Hypatite C column 75 mM potassium phosphate eluate
(HA75), and 3.0 µg of Blue-Sepharose column 150 mM KCl eluate (BS150), respectively.
Lanes 4-10 indicate 6-µl aliquots of column
fractions. The positions of marker proteins are shown on the
left.
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Fig. 3.
The factor requirement of SeV
transcription. A, Sendai virus particles (virion;
lane 1, 6 µg) and ribonucleoprotein complexes
(RNP, lane 2) that had been purified from
detergent-disrupted virions were electrophoresed in 10%
SDS-polyacrylamide gel followed by staining with Coomassie Brilliant
Blue. Positions of viral proteins (NP, P, M, F1, HN, and L)
and marker proteins are shown on the left and
right, respectively. B and C, in
vitro transcription of Sendai virus was carried out with virions
(B) or RNPs (C), in the absence (lanes
1-4) or presence (lanes 5-8) of
tubulin (3.0 µg), 0.1 µg of p46 (lanes 3,
4, 7, and 8) and 8.0 µg of BS20
(lanes 2, 4, 6, and
8) as indicated above each lane.
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Fig. 4.
Identification of p46 as phosphoglycerate
kinase. A, the tryptic peptides of p46 were
fractionated by reverse-phase high performance liquid chromatography.
Three (peptides 1-3) of the obtained peptides were subjected to the
amino acid sequence analysis, followed by the homology search from the
protein data base using the BLAST program. Partial amino acid sequences
of the three peptides were shown with sequences of phosphoglycerate
kinases from mouse, human, and yeast (S. cerevisiae).
Numbers indicate the amino acid positions of each PGK.
Identical residues are marked by vertical lines.
An amino acid that could not be identified is indicated by a
question mark. B, the aliquots (0.3 µl) of the Heparin-Sepharose column fractions (Fig. 2A)
was assayed for PGK activity ( 
) by measuring the rate of the
formation of 1,3-bisphosphoglycerate (nmol/min) and in vitro
mRNA synthesis () as described under "Experimental
Procedures." C, Western blot analysis for PGK. BS150
(lane 1, 3.0 µg) and Heparin-Sepharose column
fractions (lanes 2-8, each 6.0 µl) were
immunoblotted with an affinity-purified anti-rabbit muscle PGK
polyclonal antibody. In lane 9, a mixture of PGKs
(each 0.1 µg) from rabbit muscle and yeast was used as the positive
control.
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Fig. 5.
SeV mRNA synthesis-stimulatory activity
of p46 and PGK from various sources. A, p46
(lane 1, 0.5 µg), rabbit muscle PGK
(lane 2, 0.5 µg), yeast PGK (lane
3, 0.5 µg), and recombinant human PGK (lane
4, 0.4 µg) were resolved by 10% SDS-polyacrylamide gel
electrophoresis and stained by Coomassie Brilliant Blue. The positions
of marker proteins are shown on the left. B,
various amounts (10, 30, and 100 ng) of p46 ( ), rabbit muscle PGK
(
), yeast PGK (), or recombinant human PGK (
) were added to
SeV transcription reactions supplemented with tubulin (3.0 µg) and
BS20 (8.0 µg). Glyceraldehyde-3-phosphate dehydrogenase (
) from
rabbit muscle was used as the negative control. The
transcription-stimulatory activity is expressed as the amounts of
[32P]UMP incorporated into the 18 S mRNA band.
Purification of p46 (PGK) as a host factor for mRNA synthesis of
Sendai virus from bovine brain extract (S-100)
Effects of a substrate and an inhibitor of PGK on mRNA
synthesis of Sendai virus
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Fig. 6.
Isolation of an active transcription
initiation complex of SeV. A, detergent-disrupted SeV
particles (30 µg) were preincubated with 15 µg of tubulin
(lanes 5-8), 0.5 µg of p46 (lanes
3, 4, 7, and 8), and 40 µg of BS20 (lanes 2, 4,
6, and 8) in various combinations, as shown above
each lane. After preincubation, the viral RNPs were isolated as pellets
by ultracentrifugation, and then subjected to RNA chain elongation by
adding nucleoside triphosphates, as described under "Experimental
Procedures." Lane 1 indicates transcription
products from the reaction with a pellet obtained after incubation of
virus particles alone. B, the viral RNP pellets
(lanes 3-10) corresponding to lanes
1-8 in panel A were analyzed by
electrophoresis in a 10% SDS-polyacrylamide gel. Lanes
1 and 2 indicate 0.05 µg of tubulin and 0.4 µg of virus particles (SeV), respectively. After electrophoresis,
proteins were detected by silver staining. Closed
arrowhead indicates the migration position of tubulin
incorporated into the complexes. Positions of viral proteins (NP, P, M,
and L) and marker proteins are shown on the right and
left, respectively. C, samples shown in
panel B were immunoblotted with an anti-chicken
brain -tubulin monoclonal antibody. The migration position of
-tubulin is shown by a closed arrowhead.
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Fig. 7.
Effect of p46 and BS20 on the chain
elongation step. The initiation complex were prepared by
incubating SeV particles (300 µg) without (lanes
1-5) or with (lanes 6-10) tubulin
(150 µg), followed by ultracentrifugation as described under
"Experimental Procedures." Aliquots of the RNP pellets
(corresponding to 6 µg of staining with SeV particles) were subjected
to the chain elongation reaction in the presence of indicated factors.
Factors used were tubulin (3.0 µg), p46 (0.1 µg), and BS20 (8.0 µg). After elongation reaction, RNAs were analyzed by agarose gel
electrophoresis and autoradiographed.
-tubulin monoclonal antibody. In the case of the pretreatment of the membrane without tubulin, no positive band was observed. By
contrast, pretreatment of the membrane with tubulin allowed visualization of tubulin-binding proteins, p46 (lane
6) and two other proteins with apparent
Mr values of 52,000 (p52) and 27,000 (p27) in
the BS20 fraction (lane 5). These data suggest
that p46 (PGK) and factor(s) in BS20 stimulate SeV mRNA synthesis
at the elongation step, possibly through their interaction with tubulin that has been integrated into the active initiation complex. However, it is not clear whether tubulin-binding proteins in BS20 are directly involved in transcription-stimulating activity.
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Fig. 8.
Detection of tubulin-binding proteins by
West-Western analysis. A, BS20 (lane
1, 20 µg), p46 (lane 2, 0.5 µg),
SeV particles (lane 3, 6 µg), and tubulin
(lane 4, 0.5 µg) were resolved by
electrophoresis in a 10% SDS-polyacrylamide gel. After
electrophoresis, proteins on the gel were transferred to an Immobilon P
membrane, followed by staining with Amido Black. Positions of viral
proteins (NP, P, F1, and HN) and marker proteins are shown
on the right. B, after the pretreatment of the
membrane without (lanes 1-4) or with
(lanes 5-8) tubulin (50 µg/ml), tubulin
remaining on the membrane was detected by using an anti-chicken brain
-tubulin monoclonal antibody. Closed and open
arrowheads indicate the positions of tubulin-binding
proteins (p52 and p27) in BS20 (lane 5) and p46
(lane 6), respectively.
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Fig. 9.
Effects of tubulin, p46, and BS20 on leader
RNA synthesis of SeV. In vitro plus leader RNA
synthesis of SeV was carried out with 6 µg of virus particles under
the same conditions for mRNA synthesis except that one of the
nucleotide substrates, UTP, was omitted and in the presence of
indicated factors. Factors used were tubulin (3.0 µg), p46 (0.1 µg), and BS20 (8.0 µg). After transcription reaction,
32P-labeled short transcripts were resolved by
electrophoresis in a 20% polyacrylamide gel containing 8 M
urea and detected by autoradiography. Lane 1 indicates transcription products with virus particles alone.
Closed arrowhead indicates the band of the major
prematurely terminated leader RNA species (about 30 nucleotides). The
positions of the xylene cyanol (XC) and bromphenol blue
(BPB) dyes are shown on the left.
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Fig. 10.
Effects of tubulin, p46, and BS20 on
mRNA synthesis of VSV. In vitro mRNA synthesis
of VSV was carried out under the same conditions for SeV using 1 µg
of VSV particles instead of SeV particles in the presence of various
factors as indicated. The following amounts of factors were added to
the reaction: tubulin (3.0 µg), p46 (0.1 µg), and BS20 (8.0 µg).
After reaction, 32P-labeled transcripts were resolved by
electrophoresis in an 1.2% agarose gel and detected by
autoradiography. Lane 1 indicates transcription
products with virus particles alone (VSV alone).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin in an in
vitro transcription system with SeV-infected cell lysate. They
also mentioned that when the cell extract from uninfected BHK cells was
added to detergent-disrupted SeV particles, several classes of viral
mRNA were synthesized. By contrast, although addition of purified
tubulin alone to SeV particles led to the synthesis of leader-like RNA
and mRNA for the 5'-proximal NP gene, downstream mRNAs were not
transcribed. Similar results were reported for measles virus (12, 16).
Thus it seems that an additional factor(s) may be required for complete
transcription of these viral genomes. These observations are in
agreement with our results presented before and in this work. At
present, however, it is not clear whether the complementary factors PGK
(p46) and BS20 preferentially stimulate transcription of downstream
mRNAs rather than that of mRNA for the 5'-proximal NP gene.
-tubulin. At present, we do not know the reason for this discrepancy
in the requirement of the host factors for in vitro mRNA
synthesis of VSV. Recently, a reconstituted VSV transcription system
was created using genome RNA encapsidated with nucleocapsid protein (N)
and recombinant L and P proteins (58). In those studies, it was
revealed that phosphorylation of P protein with cellular casein kinase
II (59) and the association of L protein with protein synthesis
elongation factor EF-1 (60) are essential for mRNA synthesis of
VSV, and these host factors are packaged within the viral particles.
Therefore, to elucidate precise functions of the host factors for
transcription of SeV, it is important to develop a transcription system
reconstituted with highly purified viral proteins and host factors.
and may have a role in lagging strand
DNA replication in nuclei (61, 62). These observations including the
involvement of PGK in cellular DNA replication and in viral
transcription have suggested that PGK may contribute to regulation of
multiple cellular as well as viral processes in addition to glycolysis.
In this regard, a detailed study along the precise function of PGK and
other host factors for SeV multiplication should lead to better
understanding of host-virus interaction processes. Bacterially
expressed recombinant PGK will make it possible to map the domain(s) of
PGK required for the SeV transcription through site-directed mutational
analysis. Experiments are in progress to address the significance of
these interactions by delineating the role of PGK and BS20 in SeV
transcription using a reconstituted RNA synthesizing system.
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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