|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 47, 45306-45314, November 22, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, July 9, 2002, and in revised form, September 5, 2002
Efficient transcription and replication of the
influenza virus genome are dependent upon host-derived factors. Using
an in vitro RNA synthesis system, we have purified and
identified Hsp90 as one of the host factors that stimulate viral RNA
polymerase activity. Hsp90 interacted with the PB2 subunit of the viral
RNA polymerase through the amino-terminal chaperone domain and the middle region containing a highly acidic domain. The acidic middle region was also responsible for its stimulatory activity. We found that
a portion of Hsp90 is re-localized to the cell nucleus after viral
infection. A PB2 fragment containing a Hsp90 binding domain inhibited
viral gene expression in a dominant-negative manner. These
results suggest that Hsp90 is a host factor for the influenza virus RNA polymerase.
Influenza A virus belongs to the Orthomyxoviridae family, and its
genome consists of eight segmented, single-stranded RNA of negative
polarity (1). The transcription promoter and the replication signal of
the viral genome exist at the 3' and 5' termini of each of the eight
segments. Components associated with ribonucleoprotein complexes
(vRNP)1 purified from virions
are the minimum factors required for primary transcription. The genome
RNA forms vRNP with the viral RNA polymerases consisting of three
subunits, PB2, PB1, and PA (2), and nucleocapsid protein (NP).
Transcription of the influenza virus genome is initiated with
host-derived oligo RNA containing a cap structure. PB2 contains cap
recognition domains at its carboxyl-terminal region. The capped RNA
bound to PB2 is cleaved by the PB1 subunit 10-15 bases downstream from
the 5' end (2-4), and the capped RNA fragment serves as a primer for
viral mRNA synthesis catalyzed by PB1 (5). Elongation of the RNA
chain proceeds until the polymerase reaches a polyadenylation signal
consisting of 5-7 uracil (U) residues located near the 5' terminal
region of the vRNA (6). The viral RNA polymerase polyadenylates the
nascent RNA chain possibly by a slippage mechanism at the U-stretch
(7). Replication of the vRNA is thought to take place by a
primer-independent, two-step reaction, namely the complementary RNAs
(cRNA) are first synthesized from vRNA templates, and then the progeny
vRNAs are amplified from cRNA templates. Genetic analyses suggest that
PA participates in the replication process (8). However, vRNP complexes
isolated from virions are incapable of catalyzing replication reactions.
A variety of host proteins have been identified as factors involved in
the regulation of the RNA synthesis of viral genomes of
Paramyxoviridae, the genome of which contains non-segmented and
single-stranded RNA of negative polarity. Tubulin, an acidic cytoplasmic structural protein, is one of the host factors for RNA
synthesis of the measles virus, VSV, and Sendai virus genomes (9, 10).
RNA synthesis of these viral genomes is catalyzed by viral RNA
polymerases consisting of L and P subunits. Tubulin interacts with L
protein, a catalytic subunit of the viral RNA polymerase, and is
present in isolated transcription initiation complexes (11). Because
replication and regulated transcription of the influenza virus genome
do not occur only by influenza viral components associated with
virions, it has long been thought that some factor(s) present in
infected cells is required to carry out these processes. In fact,
several host cellular proteins were identified as factors that interact
with influenza virus NP and nonstructural protein 1 (12).
We have identified host factors that are involved in the regulation of
influenza virus RNA synthesis; these factors were derived from
uninfected HeLa cell nuclear extracts by biochemical complementation using an in vitro RNA synthesis assay with vRNP complexes
isolated from virions (13, 14). In this system, we have used a
53-base-long exogenously added model viral genome (53-merVwt) that
contains 15 nucleotides of the 3' terminal sequence and 22 nucleotides of the 5' terminal sequence of A/Puerto Rico/8/34 segment 8 vRNA. The
stimulatory activity in the extract was examined and fractionated by
measuring RNA synthesized from this model viral genome template. With
this system, we identified host factors designated as RAF (RNA polymerase activating
factor)-1 and RAF-2 that stimulate viral RNA synthesis
(14). Subsequently, we revealed that RAF-2 consists of two polypeptides
designated as RAF-2p48 and RAF-2p36; moreover, we found that RAF-2p48
is identical with UAP56/BAT1, a factor that is generally thought to be
related to RNA splicing (15). RAF-2p48 interacts with NP and is
involved in NP-RNA complex formation, thereby stimulating viral RNA
synthesis by viral RNA polymerase.
Here, we purified RAF-1 and identified it as Hsp90, a known heat shock
protein that functions as a molecular chaperone, together with some
conjugating factors (16). Recently, it was reported that Hsp90
functions as a capacitor of phenotypic variation in both insects as
well as plants (17, 18). In this report, we demonstrated that Hsp90
interacts with the PB2 subunit through its amino-terminal chaperone
domain and its middle region containing a highly acidic domain. The
RAF-1 stimulatory activity of Hsp90 depended on this acidic middle
region. Furthermore, we found that a portion of Hsp90 is re-localized
to the nucleus after viral infection. In this context, we have
addressed the function of heat shock proteins in terms of host factors
involved in the replication of viruses.
In Vitro Viral RNA Synthesis--
In this report, all viral
resources were derived from influenza A/Puerto Rico/8/34 virus. vRNP
was purified from virions as previously described (19) and was used as
the enzyme source in the in vitro RNA synthesis assays. The
53-base-long model vRNA designated as 53-merVwt
(5'-AGUAGAAACAAGGGUGUUUUUUCAUAUCAUUUAAACUUCACCCUGCUUUUGCU-3') was synthesized by transcription with MEGAscript T7 Kits (Ambion) and
synthetic DNA templates, as previously described (13). In vitro RNA synthesis was carried out at 30 °C for 60 min in 25 µl of a reaction mixture containing 50 mM HEPES-NaOH (pH
7.9); 3 mM MgCl2; 50 mM KCl; 1.5 mM dithiothreitol (DTT); 500 µM each of ATP,
GTP, and CTP; 25 µM UTP; 5 µCi of
[ Preparation of Vectors and Recombinant Proteins--
The
nucleotide sequences of the plasmids used in this study were confirmed
by DNA sequencing. The plasmids containing full-length Hsp90
The plasmids thus constructed were used for the transformation of the
Escherichia coli BL21 strain for the preparation of recombinant proteins. Expression and purification of histidine- or
GST-tagged proteins were carried out according to the instructions from
the manufacturers (Novagen and Amersham Biosciences, respectively). Recombinant proteins were further purified by Mono Q column
chromatography as native RAF-1 and dialyzed against Buffer H (50 mM HEPES-NaOH (pH 7.9), 10% glycerol, 1 mM
DTT) containing 50 mM KCl. Recombinant Hsp70 protein was
prepared from purified GST-Hsp70 by digestion with PreScission protease
(Amersham Biosciences) and dialyzed as described above. Concentrations
of these recombinant proteins were determined by electrophoretic
separation on 10% SDS-polyacrylamide gels and Coomassie Brilliant Blue staining.
The labeled PB2 was prepared by in vitro translation of PB2
mRNA with rabbit reticulocyte lysates (TNT quick
coupled transcription/translation systems, Promega), and
[35S]methionine (1,000 Ci/mmol at 10 mCi/ml), according
to the instructions from the manufacturer. PB2 mRNA was synthesized
in a transcription and translation reaction mixture that contained T7
RNA polymerase, and plasmid pET3a-PB2 containing PB2 cDNA, which
was prepared from the A/Puerto Rico/8/34 PB2 gene. The PB2 fragments,
N515 and N383, were prepared with mRNAs synthesized from pET3a-PB2 digested with AvaI and PvuII, respectively.
GST Pull-down Assay--
Ten picomoles of GST or GST-tagged
recombinant protein was fixed on 10 µl (bed volume) of
glutathione-Sepharose beads (Amersham Biosciences). The binding
reaction was carried out at 37 °C for 60 min in a final volume of
100 µl, which contained 50 mM HEPES-NaOH (pH 7.9), 3 mM MgCl2, 50 mM KCl, 1.5 mM DTT, and the affinity beads in the presence or absence
of 5 µl of vRNP (~250 ng of the NP equivalent). After adsorption,
the beads were washed three times with a NETN buffer containing 20 mM Tris-HCl (pH 7.9), 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40. Proteins bound to the affinity beads were eluted by boiling them in an SDS-PAGE loading buffer, and then they were subjected to 7.5% SDS-PAGE. To identify each viral RNA polymerase subunit, rabbit anti-PB1, anti-PB2, and
anti-PA antisera (gifts from Dr. T. Toyoda) were used for the
immunoblotting analyses.
To calculate the amount of 35S-labeled PB2 bound to the
affinity beads, the proteins were separated by 10% SDS-PAGE. The gels were subjected to autoradiography, and the 35S-labeled PB2
was quantified using an image analyzer (BAS2000, Fuji Film).
Identification of RAF-1 as Hsp90
Next, we analyzed the amino acid sequences of five peptides that were
derived from the 90-kDa RAF-1 polypeptide by cleavage with a lysyl
endopeptidase. The determined sequences were exactly identical with
portions of human Hsp90
A limited elongation assay was carried out, in which UTP was omitted
from the reaction mixture of the in vitro RNA synthesis system, and only initiation and subsequent elongation reactions occurred, producing short oligoribonucleotides. The results of this
assay suggested that the RAF fraction recovered in fractions eluted
from the Mono Q column with 0.3 M KCl (Fig. 1A)
stimulated the initiation of viral RNA synthesis (14). In the limited
elongation assay with the 53-merVwt, the viral RNA polymerase proceeded
up to the first A residue on the template and generated 13-mer RNA (Fig. 2A, upper
panel) (14), because the sequence of the 53-merVwt contained
the 5' and 3' terminal sequences of segment 8 (13). To further confirm
the effects of RAF-1/Hsp90 on RNA synthesis in the UTP-limited system
and thereby to gain information about the function of RAF-1/Hsp90, we
designed a novel 35-mer RNA template (35-merV-a) (Fig. 2A,
lower panel). This RNA template consisted of 12 bases of the viral minimum promoter and an A-free 23-base-long tail,
such that a 35-mer run-off RNA product was to be synthesized both in
the presence and absence of UTP. Run-off RNA synthesis assays with the
35-merV-a were then carried out (Fig. 2B). As expected, the
35-mer RNA product was detected in the standard reaction mixture
containing 4 NTPs (lane 1), suggesting that this model viral RNA was functional. RAF-1/Hsp90 stimulated synthesis of the
35-mer RNA product (lane 2). In the limited
elongation condition lacking UTP, the 35-mer RNA was synthesized, along
with short RNAs of 12-19 bases derived from endogenous viral RNA
segments (lane 3). This finding suggests that the
RNA synthesis from exogenously added model RNA templates was catalyzed
by not only an RNA polymerase that proceeded toward and fell off of the
end of the endogenous RNA template, but also by an RNA polymerase that
paused and fell off at the first A residue. RAF-1/Hsp90 also stimulated
the limited elongation reaction and synthesis of the 35-mer RNA product
(lane 4). These results suggest that RAF-1/Hsp90
exerted its activity at the steps prior to the early elongation stage
(see "Discussion").
Interaction of Hsp90 with PB2, a Viral RNA Polymerase
Subunit--
Because RAF-1/Hsp90 was identified as a stimulatory
factor in the in vitro viral RNA synthesis system with vRNP
as viral factors, a target(s) of Hsp90 must have been the viral RNA
polymerase, NP, and/or the viral genome. When partially purified
RAF-1/Hsp90 was mixed with vRNP and incubated under the RNA synthesis
condition without ApG dinucleotide primer and NTPs, a portion of Hsp90
co-sedimented with vRNP in a glycerol density gradient centrifugation
assay (Fig. 3A,
arrowheads in lanes 6 and
7). To determine the viral factor that interacts with Hsp90,
we carried out GST pull-down assays with purified vRNP and a
recombinant Hsp90
Although RAF-1/Hsp90 was purified by monitoring the stimulatory
activity for the viral RNA polymerase, other heat shock proteins may
show similar activity. Thus, we tried to examine the effect of Hsp70
(heat shock 70-kDa protein 1A), one of the major Hsp70 proteins, in our
system. In the GST pull-down assays, Hsp70 was unable to bind to PB2 as
strongly as did Hsp90 (Fig.
4A). Slight GST-Hsp70 and PB2
interaction was detected only when a low salt buffer (containing 50 mM NaCl) was used for washing (lane
6). In contrast, this slight interaction was completely
abolished when a high salt buffer (containing 1.0 M NaCl)
was used for washing, a condition under which Hsp90 was bound to PB2
(lane 4). The other subunits were not bound to
Hsp70 under either the low or the high salt buffer conditions. We then
carried out in vitro RNA synthesis with recombinant Hsp70
(Fig. 4B, lanes 6-9), which could not
stimulate viral RNA synthesis as effectively as could Hsp90 Analysis of the Domains Involved in Hsp90
The stimulatory activities of these mutants were examined in an
in vitro RNA synthesis system using equal moles of Hsp90
Next, we tried to roughly estimate a Hsp90 binding site in PB2. GST
pull-down assays were carried out with PB2 proteins and either an
Hsp90 Re-localization of Hsp90 in Infected Cells and Involvement of Hsp90
in Viral Multiplication--
There appears to be a contradiction
regarding the observation that the majority of Hsp90 molecules is
localized in the cytoplasm of uninfected cells (Fig.
7B) (24), whereas viral RNA
synthesis takes place in the nuclei of infected cells (Fig.
7C). We hypothesized that the intracellular localization of
Hsp90 could be changed during viral infection. This was indeed the case
(Fig. 7D). In infected cells, Hsp90 was localized in both
the nucleus and the cytoplasm, although the total amounts of Hsp90
protein determined by Western blot analysis with rabbit anti-Hsp90
antiserum remained unchanged (data not shown). At present, the exact
mechanism of translocation of Hsp90 from the cytoplasm to the nucleus
upon infection has not been determined (see "Discussion").
Next, we tried to examine whether inhibition of the Hsp90-PB2
interaction has an effect on viral gene expression or not. It has been
shown with GST pull-down assays that the PB2 region spanning the area
between amino acid positions 383 and 515 was involved, in part, in the
interaction with Hsp90
Nuclear localization of a fraction of Hsp90 was also detected (Fig. 7,
J and N) in cells co-transfected with
pCAGGS-PB2-myc, encoding a PB2-containing carboxyl-terminal myc tag
(Fig. 7F), and pCAGGS-FLAG-Hsp90
HeLa cells that had been transfected with myc-258-401 or NP-myc
expression vector (Fig. 7, panels G,
K, and O and panels H, L, and P, respectively) were infected at 12 h after transfection with influenza virus A/Puerto Rico/8/34 at a
multiplicity of infection of 3. At 9 h after infection, cells were
fixed and subjected to indirect immunofluorescence assays. myc-258-401
was localized around the outside periphery of the nucleus (Fig.
7G), as shown in panel E. In
myc-258-401-negative cells, the cell membrane was reactive to
anti-influenza virus A/Puerto Rico/8/34 HA antiserum (Fig.
7K). In sharp contrast, myc-258-401-positive cells were markedly reduced in their expression of HA (Fig. 7K,
arrowheads). Expression of NP-myc (Fig. 7H) did
not have such an effect (Fig. 7, L and P,
arrowheads). This was also the case in cells expressing GFP
(data not shown). These observations suggest that the inhibitory effect
of myc-258-401 may be interpreted as related to the competitive binding of myc-258-401 to Hsp90, resulting in a decrease or loss of
the viral RNA synthesis stimulatory activity of Hsp90. Alternatively, the decrease in the Hsp90-PB2 interaction may cause destabilization of
nascent PB2. Basic subunits of the viral RNA polymerase would tend to aggregate and to be inactivated without an appropriate chaperone-like molecule (12).
We have demonstrated here the possibility that influenza virus
requires the host cellular stress protein, Hsp90, as a stimulatory host
factor for its RNA synthesis. Hsp90 is a typical molecular chaperone
highly conserved among higher eukaryotes, and mutations in this protein
cause serious disorganization of cellular metabolism (26). The ubiquity
of this protein would be advantageous to viral infections among
animals, such as the influenza virus. In this report, we demonstrated
that Hsp70 could not function as a stimulatory host factor, despite its
functional similarity as a molecular chaperone (Fig. 4B).
Hsp70 is highly conserved among species and also functions as a
molecular chaperone, as does Hsp90. Therefore, it is speculated that
the protein chaperone activity of Hsp90 may not be responsible for the
RAF-1 stimulatory activity. In fact, we found that the stimulatory
activity of Hsp90 resides in its middle region but not in its
amino-terminal chaperone domain (Fig. 5C), although both
regions would be required for stable interaction with PB2 (Fig.
5B). Furthermore, Hsp70 is known as a general molecular
chaperone, whereas interactors with Hsp90 are more specific to that
protein (27). Thus, the interaction between Hsp90 and PB2 might be
specific (Figs. 3 and 4), even if Hsp90 functions as a chaperone for PB2.
In this report, we used a newly developed 35-mer model viral RNA,
35-merV-a, to examine the mechanism of stimulation of viral RNA
synthesis. The efficiency of RNA synthesis with the 35-merV-a seemed to
be greater than that with the 53-merVwt template (Fig. 2B).
This observation could be a result of the assumption that the recycling
of the RNA polymerase on the run-off template was more effective than
that on the 53-merVwt template. In the limited elongation assay
condition lacking UTP, RAF-1/Hsp90 was capable of stimulating RNA
synthesis not only from 35-merV-a, but also from endogenous vRNA (Fig.
2B), suggesting that RAF-1/Hsp90 plays a role during the
steps leading to the early elongation stage. RAF-1/Hsp90 may facilitate
dissociation of the RNA polymerase on vRNP; RNA polymerase is located
at the promoter region but does not yet initiate RNA synthesis, or RNA
polymerase pauses on the template during elongation. However, this
hypothesis could not be the case, because the RAF fraction stimulated
RNA synthesis from the 53-base-long RNA template by vRNA-free purified
RNA polymerases (14). Instead, it is thought that RAF-1/Hsp90
facilitates the association of RNA-free RNA polymerases to template RNA
and/or stabilizes the RNA polymerase during its translocation between templates. This assumption is supported by our preliminary result that
pre-incubation of vRNP resulted in inactivation of the RNA polymerase
activity, whereas this inactivation was suppressed to some extent in
the presence of RAF-1/Hsp90 (data not shown).
The acidic middle domain of Hsp90 is involved in RAF-1 activity. We
have proposed the term "acidic molecular chaperone" to describe
proteins that contain highly acidic regions and function as chaperones
for basic proteins, thanks to their acidic properties, which allow them
to imitate the nature of a nucleic acid (12). Transcription and
replication from basic protein-nucleic acid complexes, such as
ribonucleoprotein and chromatin templates, require the dissociation and
re-association of nucleic acid-binding proteins from and to nucleic
acids. The acidic molecular chaperone prevents aggregation and
inactivation of basic proteins, and facilitates association and
dissociation of basic proteins to/from nucleic acids. A variety of host
factors involved in viral genome functions could be categorized as
acidic molecular chaperones. Acidic cytoskeletal proteins such as
tubulin (9, 10) and actin (28, 29) are stimulatory host factors for
viral RNA synthesis of vesicular stomatitis virus, Sendai virus, and
measles virus (tubulin) and for respiratory syncytial virus, human
parainfluenza III viruses (actin) (30). Interestingly, the P subunit of
the Paramyxoviridae family RNA polymerase contains the conserved
functional acidic domain at its amino-terminal region (31-34). The
amino-terminal region of VSV P protein, referred to as domain I, has
been shown to facilitate viral transcription in conjunction with
carboxyl-terminal regions designated as domains II + III in
trans (35). The acidic region of VSV P protein is functionally
exchangeable with a full-length of An alternative aspect of the Hsp90 function in influenza virus RNA
synthesis is that Hsp90 may be involved in the modulation of the
activity and structure of viral RNA polymerase. It is reported that
Hsp90 is involved in hepadnavirus reverse transcription (40). Hepatitis
B virus genome replication includes a reverse transcription step of the
pre-genomic RNA that is catalyzed by viral reverse transcriptase-Hsp90
complexes (41). The influenza virus RNA polymerase complex, comprising
PB2, PB1, and PA, is stably formed by strong binding between PB2 and
PB1 and between PB1 and PA (25). However, it has been reported using
cells expressing these subunits that a combination of PB2 and PB1 can
catalyze transcription, whereas the combination of PB1 and PA supports
replication (42, 43). Dissociation of the complex into each subunit has
not been observed under physiological conditions. It remains of
interest that the PB1 and PA subunits were found to be easily released from the polymerase complex that was bound to Hsp90 in GST pull-down assays (Figs. 3B and 4A). It seems likely that
binding of Hsp90 to PB2 in the polymerase complex loosens PB2-PB1
interactions. It would be worthwhile to examine whether or not Hsp90 is
one of the triggers involved in a possible conversion of the RNA
polymerase complexes as regards their structure and function.
Immunoprecipitation assays have revealed the interaction between Hsp90
and the RNA polymerase in
vivo.2 However, no Hsp90
molecule was detected in 1 µg of NP-equivalent purified vRNP or in
virions when tested by Western blot analysis with anti-Hsp90 polyclonal
antiserum and an enhanced chemiluminescence system (data not shown).
Therefore, we suggest here that there may be a mechanism(s) for the
dissociation of PB2-Hsp90 interactions when nascent vRNPs are packaged
in progeny virions and/or there may be a mechanism for the elimination
of the Hsp90-vRNP complex from the packaging process.
It was unexpected that Hsp90/RAF-1 could be purified from
uninfected HeLa cell nuclear extracts, because a major fraction of
Hsp90 is localized in the cytoplasm, and only a low level of Hsp90 is
present in nuclei (Fig. 7B) (24). We might have purified Hsp90 present in the nuclei, giving this minor fraction of Hsp90, and/or Hsp90 might have contaminated in the nuclear fractions during
the course of cell fractionation. In infected cells (Fig. 7D), Hsp90 is re-localized to nuclei, where the
transcription and replication of influenza virus take place. At
present, it remains unclear whether the re-localization of Hsp90 is
caused by cellular stress during infection or by co-migration with
viral nuclear protein(s). Recently, it was reported that, in vaccinia virus-infected cells, Hsp90 was transiently associated with virosomes that were localized in cytoplasm (44). Along these lines, it is
possible that the nuclear re-localization of Hsp90 in influenza virus-infected cells takes place as a co-migration with viral RNA
polymerase, possibly with the PB2 subunit. The possibility that newly
synthesized PB2 is a carrier candidate (Fig. 7N) is being
examined further by our group.
Expression of the PB2 deletion mutant, myc-258-401, exerted a
significant effect on the localization of co-expressed FLAG-Hsp90 We thank Drs. I. Yahara (Tokyo Metropolitan
Institute of Medical Science, Tokyo, Japan) and T. Toyoda (Kurume
University, Kurume, Japan), for the generous gifts of rabbit
anti-mouse Hsp90 (to I. Y.) and anti-PA, -PB1, and PB2 polyclonal
antisera (to T. T.). A portion of this study was carried out at
the Tokyo Institute of Technology.
*
This work was supported in part by a grant-in-aid from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan and by a grant for the Bioarchitect Research Program from RIKEN
(Institute of Physical and Chemical Research, Japan) (to K. N.),
and by a research fellowship from the Japan Society for the Promotion
of Science for Young Scientists (to F. M.).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. Current address: Dept.
of Infection Biology, Inst. of Basic Medical Sciences, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan. Tel.:
81-298-53-3233; Fax: 81-298-53-3134; E-mail:
knagata@md.tsukuba.ac.jp.
Published, JBC Papers in Press, September 10, 2002, DOI 10.1074/jbc.M206822200
2
F. Momose, T. Naito, and K. Nagata,
unpublished observation.
The abbreviations used are:
vRNP, virion
ribonucleoprotein complex;
NP, nucleocapsid protein;
GST, glutathione
S-transferase;
aa, amino acid(s);
VSV, vesicular stomatitis
virus;
DTT, dithiothreitol;
HA, hemagglutinin;
RAF, RNA polymerase
activating factor.
Identification of Hsp90 as a Stimulatory Host Factor Involved in
Influenza Virus RNA Synthesis*
§,
,,, and Kitasato Institute for Life Sciences,
Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, the
¶ Department of Infection Biology, Institute of Basic Medical
Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, the
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan, and the
§ Tokyo Institute of Technology, Nagatsuta, Yokohama
226-8501, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP (400 Ci/mmol); 10 units of RNase inhibitor;
25 µg/ml of actinomycin D; 250 µM ApG; 5 ng of a
53-merVwt; and vRNP (10 ng of NP equivalents) in the presence or
absence of the host factor fractions. A limited elongation assay was
carried out in the same reaction mixture, with the exception that UTP
and [-32P]UTP were omitted, and 500 µM
each of ATP and CTP were included, as well as 25 µM GTP,
and 5 µCi of [-32P]GTP (400 Ci/mmol). Run-off RNA
synthesis was carried out with a 35-mer adenine (A) residue-less
template (35-merV-a;
5'-GUUCUUCUUCUUCUUUCUUCUGGCCUGCUUUUGCU-3'), which
contained the 12-base-long conserved promoter sequence for the viral
RNA polymerase at the 3' terminal region and the subsequent 23-base-long sequence lacking the A-residues. The other materials for
the run-off RNA synthesis were the same as those used for the standard
or limited elongation assays.
and 
cDNAs were the kind gift of Dr. I. Yahara (Tokyo Metropolitan
Institute of Medical Science, Tokyo, Japan). The Hsp90 cDNA
portion was amplified using LA-Taq polymerase (TaKaRa), with
the plasmid as the template and specific primers,
5'-CCTGAGGAAACCCAGACCCA-3' and
5'-GCGTCTACTTCTTCCATGCGTGAT-3', corresponding to the Hsp90 amino-terminal and carboxyl-terminal regions, respectively. For the
preparation of the carboxyl-terminal hexahistidine-tagged Hsp90
expression vector, the amplified cDNA fragment was phosphorylated with polynucleotide kinase (Toyobo) and ligated into pET-14b (Novagen) that had been digested with NcoI and filled with Klenow
fragment (TaKaRa). For the preparation of the carboxyl-terminal
GST-tagged Hsp90 expression vector, the Hsp90 cDNA fragment
was ligated into a pET-GST vector, a pET-14b derivative containing GST
cDNA in place of the DNA sequence for the hexahistidine tag.
Hsp90 deletion mutant and Hsp90 expression vectors were
constructed via the same method as that used for constructing a
wild-type Hsp90 expression vector, with a primer combination
corresponding to the amino-terminal and carboxyl-terminal regions of
each mutant. The GST-tagged human Hsp70 (heat shock 70-kDa protein 1A,
GenBankTM accession number 5579469) expression vector,
pGEX6P-Hsp70, was constructed with a pGEX-6P-1 vector and an Hsp70
cDNA fragment. The Hsp70 cDNA was amplified from a HeLa cell
cDNA library using specific primers,
5'-GCGGATCCCATATGGCCAAAGCCGCGGCGA-3' and
5'-CCGGATCCTAATCCACCTCCTCAATGGTA-3', corresponding to the Hsp70
amino-terminal and carboxyl-terminal regions, respectively. The
amplified fragments and the pGEX-6P-1 vector were digested with
BamHI and were ligated.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ---
Using an
in vitro influenza virus RNA synthesis system with an
exogenously added 53-base-long model virus genome (53-merVwt), we found
stimulatory host factors for the viral RNA polymerase, designated as
RAF-1 and RAF-2, in nuclear extracts prepared from uninfected HeLa
cells (14). Previously, we demonstrated by biochemical fractionation
that RAF-2 consists of 48- and 36-kDa polypeptides; we found that the
former peptide is identical with BAT1/UAP56, a putative splicing factor
(15). Here, we purified RAF-1 as a host factor that stimulated viral
RNA synthesis; purification proceeded to apparent homogeneity through
sequential column chromatographies (14) (Fig.
1A). The native molecular mass
of RAF-1 was estimated as 350 kDa on gel filtration chromatography (14)
(data not shown), and the major polypeptide in the active fraction
showed a molecular mass of 90 kDa, as estimated by SDS-PAGE (Fig.
1B). This observation suggests that the purified RAF-1
exists as an oligomer, presumably a tetramer, of the 90-kDa
polypeptide.
View larger version (51K):
[in a new window]
Fig. 1.
Identification of the host factor that
stimulated influenza virus RNA synthesis activity. A, a
purification scheme of RAF-1. For the details regarding the column
chromatography and biochemical complementation assay, see Refs. 14 and
15. B, SDS-PAGE analysis of RAF-1 fractions of each
purification step. The loaded amounts were 10-fold those used in the
in vitro RNA synthesis assay; these amounts were adjusted to
the equal level of stimulatory activity attained in the in
vitro RNA synthesis assay. Lane 1,
uninfected HeLa cell nuclear extracts (5 µl); lane
2, 0.05 M KCl flow-through fraction from a
phosphocellulose column (7 µl); lane 3, 0.4 M KCl eluate from a Mono Q column (5 µl); lane
4, 0.3 M
(NH4)2SO4 eluate from a
phenyl-Superose column (2 µl); lane 5, 350-kDa
fraction of gel filtration chromatography (purified RAF-1 fraction) (1 µl); lane M, molecular size markers (Bio-Rad).
The gel was stained with Coomassie Brilliant Blue. C, the
stimulatory activity of recombinant Hsp90 . In vitro viral
RNA synthesis was carried out in the absence (lane
1) or the presence of purified RAF-1 fraction
(lanes 2-5), recombinant Hsp90
(lanes 6-10), or bovine serum albumin
(BSA, lanes 11-14). The amounts of 90 kDa polypeptide in purified RAF-1, recombinant Hsp90, and the bovine
serum albumin per assay were 4 ng (lanes 2,
6, and 11), 8 ng (lanes 3,
7, and 12), 16 ng (lanes 4,
8, and 13), 32 ng (lanes 5,
9, and 14), and 64 ng (lane
10). RNA products from endogenous viral RNA present in vRNP
are shown at the top of 10% polyacrylamide gel in the presence of 6 M urea, and the RNA products from the 53-base-long model
viral genome (53-merVwt) are indicated by arrowhead.
and Hsp90 (Table
I), indicating that RAF-1 is a mixture of
Hsp90 and Hsp90. Hsp90 is a typical molecular chaperone highly
conserved among species, and mainly exists in the cytoplasm as a
homodimer or as an oligomer consisting of or homodimers
(20-22). To perform a functional analysis of RAF-1/Hsp90 as a
stimulatory host factor for influenza virus RNA synthesis, we prepared
recombinant human Hsp90 and Hsp90, each containing a histidine
tag at the carboxyl terminus. We then examined their stimulatory
activity as regards the in vitro viral RNA synthesis. The
recombinant Hsp90 (Fig. 1C, lanes 6-10) demonstrated approximately the same level of
stimulatory activity as that of Hsp90 (data not shown), and
recombinant Hsp90 demonstrated slightly higher stimulatory activity
than did purified RAF-1/Hsp90 (lanes 2-5). We
assume that a portion of purified RAF-1/Hsp90 might have been
inactivated during the process of column chromatography. These results
confirmed that Hsp90 and Hsp90 are the active components in
RAF-1. Hsp90 and - have quite similar domain structure
organization, and the similarity of amino acid sequences between
Hsp90 and - is ~93%.
Amino acid sequences of the oligopeptides derived from the 90-kDa
polypeptide in a purified RAF-1 fraction
View larger version (21K):
[in a new window]
Fig. 2.
Limited elongation assay with a 35-mer model
viral RNA. A, illustration of in vitro viral
RNA synthesis without UTP (limited elongation) from the segment 8 RNA
and 53-merVwt template (upper) and 35-merV-a template
(lower), used as templates. In the absence of UTP, RNA
synthesis was paused at the first adenine residue on the template,
whereas RNA polymerase dropped off from the 5' end of the 35-merV-a
template. For additional details about this system, see the text.
B, RAF-1/Hsp90 stimulated viral RNA synthesis with a
35-merV-a template. RNA synthesis was carried out with 1.7 ng of
35-merV-a in the presence (lanes 1 and
2) or absence (lanes 3 and
4) of UTP and in the presence (lanes 2 and 4; 200 ng of Hsp90) or absence (lanes
1 and 3) of RAF-1/Hsp90. The short RNA derived
from the endogenous viral genome and the model RNA template are
indicated by arrowheads and numerals (12-mer,
segments 1, 3, and 7; 13-mer, segments 5 and 8; 14-mer, segment 6;
18-mer, segment 4; and 19-mer from segment 2 of A/Puerto Rico/8/34
strain). The RNA products that appeared between 19-mer and 35-mer
(lanes 2 and 4) may have been immature
and/or inaccurately initiated transcripts from the 35-merV-a
template.
that contains the glutathione
S-transferase at its carboxyl terminus (Hsp90-GST). After
binding and washing steps (see "Discussion"), one of the viral RNA
polymerase subunits, PB2, was observed bound to Hsp90-GST (Fig.
3B). A trace level of PB1 subunit was detected, whereas the
PA subunit remained for the most part unbound. The PB2-Hsp90 interaction was also detected in the presence of ribonuclease A (data
not shown), suggesting that these proteins do not interact via RNA.
Hsp90 also bound to PB2 as Hsp90, indicating that PB2 would
interact with a homologous region between Hsp90 and (Fig. 3C).
View larger version (53K):
[in a new window]
Fig. 3.
The interaction between
Hsp90 and viral proteins. A,
co-sedimentation of Hsp90 in RAF-1 fraction with vRNP upon glycerol
density gradient centrifugation. Aliquots of the RAF-1/Hsp90 fraction
(6 µl of phenyl-Superose eluate) were incubated in the absence
(lower panel) or presence of 1 µg of NP
equivalent vRNP (upper panel) in 100 µl of a
reaction buffer containing 50 mM HEPES-NaOH (pH 7.9), 50 mM KCl, 6 mM MgCl2, and 3 mM DTT. Subsequently, the mixtures were loaded onto 1.2 ml
of a 30-60% glycerol density gradient buffer (50 mM
HEPES-NaOH (pH 7.9), 50 mM KCl, 6 mM
MgCl2, 3 mM DTT, and 30-60% glycerol) and
centrifuged at 4 °C in a SW50.1 rotor at 45,000 rpm for 5 h.
Fractionation (200 µl) was carried out from the top of the gradient.
One hundred microliters of each fraction were precipitated with 10%
trichloroacetic acid and were loaded onto 7.5% SDS-PAGE. Proteins were
visualized by silver staining. Viral RNA polymerase subunits
(lane 1, 5 µl) and Hsp90 (lane
2, 0.5 µl) are shown as the control for gel mobility. A
portion of the 90-kDa polypeptide that sedimented with the vRNP
fractions (lanes 6 and 7) is indicated
by arrowheads. B, Hsp90 interacted with the
PB2 subunit. GST pull-down assays were carried out with recombinant
Hsp90 fused with GST at its carboxyl terminus (Hsp90-GST;
lane 3) or with GST (lane
2). Eluates and input vRNP (lane 1, 5 µl) were loaded onto 7.5% SDS-PAGE, and Western blot analysis was
carried out using rabbit anti-PB2, -PB1, and -PA polyclonal antisera.
C, Hsp90 also interacted with the PB2 subunit. GST
pull-down assays were carried out with GST (lane
2), recombinant Hsp90-GST (lanes 3 and 4), or Hsp90-GST (lanes 5 and
6) in the presence (lanes 2,
4, and 6) or absence (lanes
3 and 5) of vRNP, as described above. Western
blot analysis was carried out with anti-PB2 antiserum. Control vRNP
(1.3 µl) is also shown (lane 1).
(lanes 2-5). The low level of stimulatory
activity in the case of Hsp70 could be the result of its general
chaperone activity. Thus, the RAF stimulatory activity of RAF-1/Hsp90,
possibly through PB2, could not be replaced by Hsp70.
View larger version (64K):
[in a new window]
Fig. 4.
Effect of Hsp70 on viral RNA synthesis.
A, GST pull-down assays were carried out with GST
(lane 2), Hsp90 -GST (lanes
3 and 4), or GST-Hsp70 (lanes
5 and 6) in the presence (lanes
2, 4, and 6) or absence
(lanes 3 and 5) of vRNP (5 µl). The
affinity beads were washed with NETN buffer containing 50 mM (Low) or 1 M NaCl
(High). Western blot analysis was carried out with anti-PB2,
-PB1, and -PA antisera. Control vRNP (lane 1, 1.3 µl) is also shown. B, in vitro viral RNA
synthesis was carried out in the absence (lane 1)
or presence of recombinant Hsp90 (lanes 2-5;
6.3, 12.5, 25, and 50 ng, respectively) or Hsp70 (lanes
6-9; 6.3, 12.5, 25, and 50 ng, respectively). An RNA
product derived from 53-merVwt is indicated by an
arrowhead.
-PB2
Interaction--
Next, we tried to determine the domains involved in
the interaction between Hsp90 and PB2. Based on the reports (20-24)
concerning the domain organization of Hsp90, we divided Hsp90 into
three regions, a hydrophobic amino-terminal region (N), a highly acidic middle region (M), and a carboxyl-terminal homodimerization region (C),
as shown in Fig. 5A. GST
pull-down assays were carried out with three Hsp90 deletion mutants
and wild-type Hsp90 containing GST at their carboxyl termini in
binding buffers containing 50 mM (low salt) or 1.0 M (high salt) KCl. After the binding reaction was carried
out at 37 °C for 60 min, the affinity beads were washed twice with
NETN buffer containing 100 mM (low salt) or 1.0 M NaCl (high salt). Hsp90 mutants and wild-type Hsp90
interacted with the PB2 subunit (Fig. 5B), but not with the
PB1 and PA subunits (data not shown). Wild-type Hsp90 was able to
interact with PB2, irrespective of low or high salt binding and low or
high salt washing conditions (lane 9), although
the high salt wash slightly decreased the PB2-Hsp90 interaction. All
of the Hsp90 mutants were capable of interacting with PB2 under low
salt washing conditions (first and third
panels from the top), but with less efficiency than that observed in the case of wild-type Hsp90. Under the high salt
washing condition, the Hsp90 carboxyl-terminal mutant (C) released
PB2 (second and fourth panels,
lane 7). Thus, the N and M regions were capable
of binding to PB2 more strongly than was the C region. These results
indicate that each Hsp90 region was involved with a different affinity
in the Hsp90-PB2 interaction.
View larger version (41K):
[in a new window]
Fig. 5.
Domain analysis of Hsp90 .
A, schematic representation of Hsp90 consisting of 732 amino acids (aa) and its mutants. The chaperone domain (~1-210 aa),
highly acidic domain (~220-270 aa), and homo-dimerization domain
(~540-732 aa) are indicated by closed squares.
Three mutants, N (1-213 aa), M (214-432 aa), and C (433-732 aa), are
indicated by thick bars. B, the PB2
binding efficiency of Hsp90 mutants. GST pull-down assays were carried
out with N (lanes 2 and 3), M
(lanes 4 and 5), and C
(lanes 6 and 7) mutants, and with
wild-type Hsp90-GST (lanes 8 and 9) in
the absence (lanes 2, 4, 6,
and 8) or presence (lanes 3,
5, 7, and 9) of vRNP (5 µl). Western
blot analysis was carried out with anti-PB2 antiserum. Control vRNP
(lane 1, 1.3 µl) is also shown. C,
the stimulatory activities of Hsp90 mutants. In vitro
viral RNA synthesis was carried out in the absence (lane
2) or presence of equivalent moles (0.6 pmol) of the
purified RAF-1/Hsp90 (lane 1), recombinant
wild-type Hsp90 (lane 9), or mutants
(lanes 3-8).
deletion mutants (Fig. 5C). Although these three mutants
were able to interact with PB2 under the low salt condition, which was
also used in the RNA synthesis system, their stimulatory activities were significantly different. The N and C regions of Hsp90 showed a
quite low level of stimulatory activity (lanes 3 and 5), and their activities were much lower than that of
either purified RAF-1/Hsp90 (lane 1) or
full-length recombinant Hsp90 (lane 9). In
contrast, the specific activity of the M region was clearly higher than
that of the others (lane 4). Mutant proteins
containing the M region in connection with other regions, such as NM
(lane 8) and MC (lane 6)
regions, showed higher activity than did N or C alone. Densitometric
scanning analysis revealed that NM possessed slightly higher activity
than M. On the other hand, the NC mutant lacking the M region was as
inefficient at stimulation as was N or C mutant alone. Taken together
with results of GST pull-down assays (Fig. 5B), it is
suggested that Hsp90 interacts with PB2 through N, M, or NM; moreover,
the Hsp90 middle region, which contains a highly acidic region, is
responsible for the RAF-1 stimulatory activity of Hsp90.
NM region or wild-type Hsp90 tagged with GST at their
carboxyl termini (Fig. 6). The labeled
wild-type PB2 and PB2 fragments, N515 and N383 (Fig. 6A),
were produced with rabbit reticulocyte lysates,
[35S]methionine, and PB2 mRNAs encoding each PB2
protein, because recombinant wild-type and mutant PB2 proteins could
not be synthesized efficiently in E. coli cells. The binding
efficiencies of wild-type PB2 and N515 fragments to wild-type Hsp90
and the NM region were approximately equal. The N383 fragment showed
less binding activity toward the NM region. Therefore, it is possible
that the NM region, which is fully active as regards RAF-1 activity
(Fig. 5C), interacts with PB2 through the region between its
amino terminus and the amino acid position 515. N383 was able to bind
to wild-type Hsp90 more efficiently than to the NM region. Although
we do not know the exact reason for this at present, we assume that
wild-type Hsp90 binds to the N383 fragment as a chaperone as well as
RAF-1. The structure of the PB2 fragment would be much less stable than that of the wild-type protein, and thus would be targeted by the Hsp90
chaperone. Because dimerization and/or oligomerization of Hsp90 is
required for its chaperone activity (20, 22), Hsp90 NM lacking the
carboxyl-terminal region that is involved in dimerization and/or
oligomerization was unable to behave as a molecular chaperone.
View larger version (15K):
[in a new window]
Fig. 6.
Binding of Hsp90 to
PB2 fragments. A, schematic representation of the PB2
subunit (759 aa) and its fragments. The PB1 binding domain (51-259 aa)
and cap binding sequence (544-556 aa) are indicated by
closed squares (25). The nuclear localizing
signals (NLS; 449-495 and 736-739 aa) are indicated by
horizontal striped squares (25). PB2
fragments (258-401, 258-401 aa; N515, 1-515 aa; and N383, 1-383 aa)
are indicated by thick bars. B, the
binding efficiency of PB2 fragments to Hsp90. GST pull-down assay
was carried out with the GST-tagged Hsp90 MM region and wild-type
Hsp90 in the presence of in vitro translated PB2
fragments (3 µl). The amount of [35S]methionine-labeled
PB2 mutants was measured using an image analyzer (BAS2000, Fuji Film).
The binding efficiency is shown as ratios of the amounts of PB2 bound
to GST-tagged proteins to those of input PB2.
View larger version (30K):
[in a new window]
Fig. 7.
Localization of Hsp90 and effects of a PB2
fragment containing a Hsp90 binding site on the expression of viral
proteins. Indirect immunofluorescence analysis was carried
out with MDCK cells (A-D) or HeLa cells (E-P).
Cells were mock-infected (eight panels on the
left) or infected (eight panels on the
right) with influenza virus A/Puerto Rico/8/34 at a
multiplicity of infection of 3. At 9 h after infection, the cells
were fixed with acetone:methanol = 1:1
(A-D) or 3% paraformaldehyde solution
(E-P). Samples were incubated with rabbit polyclonal
antiserum reactive to the viral RNA polymerases and NP
(anti-pol/NP; A and C) or Hsp90
(anti-Hsp90; B and D). This anti-mouse
Hsp90 antiserum was able to cross-react with both human and canine
Hsp90 and Hsp90 (Ref. 46 and data not shown). In
panels E-P, HeLa cells had been transfected at
12 h before infection with myc-258-401 (E,
I, M, G, K, and
O), PB2-myc (F, J, and N),
NP-myc (H, L, and P), and
FLAG-Hsp90 (E, F, I, J,
M, and N) expression vectors that were
constructed from a mammalian expression vector, pCAGGS (47), such that
they contained the indicated epitope tag sequences. The cells
expressing myc-258-401 mutants or NP-myc are indicated by
arrowheads. Double staining was carried out with a
combination of rabbit anti-myc polyclonal antiserum (E and
F) and mouse anti-FLAG epitope monoclonal antibody
(I and J), or a combination of mouse anti-myc
epitope monoclonal antibody (G and H) and rabbit
anti-influenza A/Puerto Rico/8/34 HA polyclonal antiserum (K
and L). These fluorescence images are shown as merged images
(M-P). The fields of view differ among panels
A-D, but those of panels E,
I, and M and panels F,
J, and N, and those of panels
G, K, and O and panels H,
L, and P, correspond to each other.
Magnifications are indicated by the white bars in
panel A (10 µm, for A-D) and in
panel E (10 µm, for E-P).
(Fig. 6B). Based on this observation and the functional domain map of PB2 (Fig. 6A),
we prepared a PB2 deletion mutant spanning amino acid positions 258 and
401 with an additional myc tag sequence at its amino terminus (myc-258-401). This deletion mutant contained a putative Hsp90 binding domain, but not contained the functional domains already reported, such as the cap structure recognition sequence, PB1 binding
domain, and nuclear localizing signals (25). It is expected that in
infected cells, myc-258-401 disturbs the interaction between PB2 and
Hsp90 in a dominant negative-manner but does not inhibit the assembly
of viral RNA polymerase subunits, nor does it inhibit the cap structure
recognition activity of wild-type PB2. Furthermore, myc-258-401 may
inhibit the nuclear localization of Hsp90 if the nuclear localization
of Hsp90 depends on PB2-Hsp90 interaction, because myc-258-401 does
not contain the nuclear localizing signal sequence.
, encoding an
Hsp90-containing amino-terminal FLAG tag (Fig. 7J). On
the other hand, nuclear localization of FLAG-Hsp90 was less often
observed in cells co-expressing myc-258-401 (Fig. 7, I and
M). myc-258-401 was present around the outside periphery of
the nucleus and/or in spots in the cytoplasm (Fig. 7, E and
G) and it co-localized with FLAG-Hsp90 at the outside periphery of the nucleus (Fig. 7M). Cytoplasmic vacuole-like
spaces where both FLAG-Hsp90 and myc-258-401 are not present were
observed in a small percentage of the cells expressing the myc-258-401 (panel M, cell on the right).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin (36). The chimeric P
protein in which domain I is replaced with -tubulin is capable of
functioning as the viral RNA polymerase subunit (36). In the case of
influenza virus, RAF-2p48/UAP56/BAT1 containing an acidic region has
been found to function as a chaperone for NP (15). This has also been
demonstrated in the case of DNA virus. Template-activating factors I,
II, and III contain functional highly acidic regions and stimulate
adenovirus transcription and replication from viral DNA complexed with
basic core proteins or histones (37-39). Along these lines, we
hypothesized that Hsp90 may function as the acidic molecular chaperone
for PB2, a basic polymerase subunit.
and on the expression of influenza virus HA protein. In some cells expressing myc-258-401 PB2 fragment, vacuole-like spaces were generated (Fig. 7M), which have also been observed in cells
that were cultured in the presence of excess concentrations of
geldanamycin, a potent Hsp90 inhibitor2 (45). Thus, in some
myc-258-401-positive cells, not only PB2-Hsp90 interactions, but also
the essential function(s) of Hsp90, might have been suppressed as a
result of the interactions between myc-258-401 and Hsp90. Expression
of recombinant proteins such as NP (Fig. 7P) and GFP (data
not shown) did not affect viral gene expression. In contrast, the
expression of viral proteins such as HA (Fig. 7O) or nascent
PB2 (data not shown) was inhibited in cells expressing myc-258-401. We
have considered the possibility that myc-258-401 inhibits the
interaction between PB2 wild-type and Hsp90 proteins, such that Hsp90
can no longer function as a host factor. We have not yet examined
whether or not myc-258-401 inhibits the production of progeny virions,
because cells stably expressing myc-258-401 are not currently
available. Because Hsp90 is essential for living cells, the development
of knock-out models is impossible (26); we are now establishing cell
lines in which the level of Hsp90 is inducibly down-regulated. With
such cells in hand, we will examine the function of Hsp90 in the virus
multiplication cycle.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Ishihama, A.,
and Nagata, K.
(1988)
CRC Crit. Rev. Biochem.
23,
27-76[Medline]
[Order article via Infotrieve]
2.
Braam, J.,
Ulmanen, I.,
and Krug, R. M.
(1983)
Cell
34,
609-618[Medline]
[Order article via Infotrieve]
3.
Plotch, S. J.,
Bouloy, M.,
Ulmanen, I.,
and Krug, R. M.
(1981)
Cell
23,
847-858[CrossRef][Medline]
[Order article via Infotrieve]
4.
Li, M. L.,
Rao, P.,
and Krug, R. M.
(2001)
EMBO J.
20,
2078-2086[CrossRef][Medline]
[Order article via Infotrieve]
5.
Galarza, J. M.,
Peng, Q.,
Shi, L.,
and Summers, D. F.
(1996)
J. Virol.
70,
2360-2368[Abstract]
6.
Luo, G. X.,
Luytjes, W.,
Enami, M.,
and Palese, P.
(1991)
J. Virol.
65,
2861-2867 7.
Poon, L. L.,
Pritlove, D. C.,
Fodor, E.,
and Brownlee, G. G.
(1999)
J. Virol.
73,
3473-3476 8.
Krug, R. M.,
Ueda, M.,
and Palese, P.
(1975)
J. Virol.
16,
790-796 9.
Moyer, S. A.,
Baker, S. C.,
and Horikami, S. M.
(1990)
J. Gen. Virol.
71,
775-783 10.
Moyer, S. A.,
Baker, S. C.,
and Lessard, J. L.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
5405-5409 11.
Takagi, T.,
Iwama, M.,
Seta, K.,
Kanda, T.,
Tsukamoto, T.,
Tominaga, S.,
and Mizumoto, K.
(1996)
Arch. Virol.
141,
1623-1635[CrossRef][Medline]
[Order article via Infotrieve]
12.
Nagata, K.,
Momose, F.,
and Okuwaki, M.
(1999)
Recent Res. Dev. Virol.
1,
559-597
13.
Shimizu, K.,
Handa, H.,
Nakada, S.,
and Nagata, K.
(1994)
Nucleic Acids Res.
22,
5047-5053 14.
Momose, F.,
Handa, H.,
and Nagata, K.
(1996)
Biochimie (Paris)
78,
1103-1108
15.
Momose, F.,
Basler, C. F.,
O'Neill, R. E.,
Iwamatsu, A.,
Palese, P.,
and Nagata, K.
(2001)
J. Virol.
75,
1899-1908 16.
Hohfeld, J.,
Cyr, D. M.,
and Patterson, C.
(2001)
EMBO Rep.
2,
885-890[CrossRef][Medline]
[Order article via Infotrieve]
17.
Rutherford, S. L.,
and Lindquist, S.
(1998)
Nature
396,
336-342[CrossRef][Medline]
[Order article via Infotrieve]
18.
Queitsch, C.,
Sangster, T. A.,
and Lindquist, S.
(2002)
Nature
417,
618-624[CrossRef][Medline]
[Order article via Infotrieve]
19.
Yamanaka, K.,
Ishihama, A.,
and Nagata, K.
(1990)
J. Biol. Chem.
265,
11151-11155 20.
Minami, Y.,
Kimura, Y.,
Kawasaki, H.,
Suzuki, K.,
and Yahara, I.
(1994)
Mol. Cell. Biol.
14,
1459-1464 21.
Nemoto, T.,
Ohara-Nemoto, Y.,
Ota, M.,
Takagi, T.,
and Yokoyama, K.
(1995)
Eur. J. Biochem.
233,
1-8[Medline]
[Order article via Infotrieve]
22.
Nemoto, T.,
and Sato, N.
(1998)
Biochem. J.
330,
989-995[Medline]
[Order article via Infotrieve]
23.
Stebbins, C. E.,
Russo, A. A.,
Schneider, C.,
Rosen, N.,
Hartl, F. U.,
and Pavletich, N. P.
(1997)
Cell
89,
239-250[CrossRef][Medline]
[Order article via Infotrieve]
24.
Meng, X.,
Devin, J.,
Sullivan, W. P.,
Toft, D.,
Baulieu, E. E.,
and Catelli, M. G.
(1996)
J. Cell Sci.
109,
1677-1687[Abstract]
25.
Ohtsu, Y.,
Honda, Y.,
Sakata, Y.,
Kato, H.,
and Toyoda, T.
(2002)
Microbiol. Immunol.
46,
167-175[Medline]
[Order article via Infotrieve]
26.
Chang, H. C.,
and Lindquist, S.
(1994)
J. Biol. Chem.
269,
24983-24988 27.
Buchner, J.
(1999)
Trends Biochem. Sci.
24,
136-141[CrossRef][Medline]
[Order article via Infotrieve]
28.
Huang, Y. T.,
Romito, R. R., De, B. P.,
and Banerjee, A. K.
(1993)
Virology
193,
862-867[CrossRef][Medline]
[Order article via Infotrieve]
29.
De, B. P.,
Lesoon, A.,
and Banerjee, A. K.
(1991)
J. Virol.
65,
3268-3275 30.
Lai, M. M.
(1998)
Virology
244,
1-12[CrossRef][Medline]
[Order article via Infotrieve]
31.
Curran, J.,
Pelet, T.,
and Kolakofsky, D.
(1994)
Virology
202,
875-884[CrossRef][Medline]
[Order article via Infotrieve]
32.
Das, T.,
and Banerjee, A. K.
(1993)
Cell. Mol. Biol. Res.
39,
93-100[Medline]
[Order article via Infotrieve]
33.
Harty, R. N.,
and Palese, P.
(1995)
J. Gen. Virol.
76,
2863-2867 34.
Spriggs, M. K.,
and Collins, P. L.
(1986)
J. Gen. Virol.
67,
2705-2719 35.
Chattopadhyay, D.,
and Banerjee, A. K.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
8932-8936 36.
Chattopadhyay, D.,
and Banerjee, A. K.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
7977-7981 37.
Nagata, K.,
Kawase, H.,
Handa, H.,
Yano, K.,
Yamasaki, M.,
Ishimi, Y.,
Okuda, A.,
Kikuchi, A.,
and Matsumoto, K.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
4279-4283 38.
Okuwaki, M.,
Iwamatsu, A.,
Tsujimoto, M.,
and Nagata, K.
(2001)
J. Mol. Biol.
311,
41-55[CrossRef][Medline]
[Order article via Infotrieve]
39.
Okuwaki, M.,
and Nagata, K.
(1998)
J. Biol. Chem.
273,
34511-34518 40.
Hu, J.,
and Seeger, C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1060-1064 41.
Hu, J.,
Toft, D. O.,
and Seeger, C.
(1997)
EMBO J.
16,
59-68[CrossRef][Medline]
[Order article via Infotrieve]
42.
Kimura, N.,
Nishida, M.,
Nagata, K.,
Ishihama, A.,
Oda, K.,
and Nakada, S.
(1992)
J. Gen. Virol.
73,
1321-1328 43.
Nakagawa, Y.,
Kimura, N.,
Toyoda, T.,
Mizumoto, K.,
Ishihama, A.,
Oda, K.,
and Nakada, S.
(1995)
J. Virol.
69,
728-733[Abstract]
44.
Hung, J. J.,
Chung, C. S.,
and Chang, W.
(2002)
J. Virol.
76,
1379-1390 45.
Whitesell, L.,
Mimnaugh, E. G., De,
Costa, B.,
Myers, C. E.,
and Neckers, L. M.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
8324-8328 46.
Miyata, Y.,
and Yahara, I.
(1992)
J. Biol. Chem.
267,
7042-7047 47.
Niwa, H.,
Yamamura, K.,
and Miyazaki, J.
(1991)
Gene
108,
193-199[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Pastorino, E. Boucomont-Chapeaublanc, C. N. Peyrefitte, M. Belghazi, T. Fusai, C. Rogier, H. J. Tolou, and L. Almeras Identification of Cellular Proteome Modifications in Response to West Nile Virus Infection Mol. Cell. Proteomics, July 1, 2009; 8(7): 1623 - 1637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Y.-L. Wang, J. Stork, and P. D. Nagy A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes J. Virol., April 1, 2009; 83(7): 3276 - 3287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Newcomb, R.-L. Kuo, Q. Ye, Y. Jiang, Y. J. Tao, and R. M. Krug Interaction of the Influenza A Virus Nucleocapsid Protein with the Viral RNA Polymerase Potentiates Unprimed Viral RNA Replication J. Virol., January 1, 2009; 83(1): 29 - 36. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Pogany, J. Stork, Z. Li, and P. D. Nagy In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70 PNAS, December 16, 2008; 105(50): 19956 - 19961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Taguwa, T. Okamoto, T. Abe, Y. Mori, T. Suzuki, K. Moriishi, and Y. Matsuura Human Butyrate-Induced Transcript 1 Interacts with Hepatitis C Virus NS5A and Regulates Viral Replication J. Virol., March 15, 2008; 82(6): 2631 - 2641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Jorba, E. Area, and J. Ortin Oligomerization of the influenza virus polymerase complex in vivo J. Gen. Virol., February 1, 2008; 89(2): 520 - 524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Naito, Y. Kiyasu, K. Sugiyama, A. Kimura, R. Nakano, A. Matsukage, and K. Nagata An influenza virus replicon system in yeast identified Tat-SF1 as a stimulatory host factor for viral RNA synthesis PNAS, November 13, 2007; 104(46): 18235 - 18240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Castorena, S. A. Weeks, K. A. Stapleford, A. M. Cadwallader, and D. J. Miller A Functional Heat Shock Protein 90 Chaperone Is Essential for Efficient Flock House Virus RNA Polymerase Synthesis in Drosophila Cells J. Virol., August 15, 2007; 81(16): 8412 - 8420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-K. Shin, Q. Liu, S. K. Tikoo, L. A. Babiuk, and Y. Zhou Effect of the phosphatidylinositol 3-kinase/Akt pathway on influenza A virus propagation J. Gen. Virol., March 1, 2007; 88(3): 942 - 950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Naito, F. Momose, A. Kawaguchi, and K. Nagata Involvement of Hsp90 in Assembly and Nuclear Import of Influenza Virus RNA Polymerase Subunits J. Virol., February 1, 2007; 81(3): 1339 - 1349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Serva and P. D. Nagy Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication J. Virol., March 1, 2006; 80(5): 2162 - 2169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Deng, J. Sharps, E. Fodor, and G. G. Brownlee In Vitro Assembly of PB2 with a PB1-PA Dimer Supports a New Model of Assembly of Influenza A Virus Polymerase Subunits into a Functional Trimeric Complex J. Virol., July 1, 2005; 79(13): 8669 - 8674. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Kampmueller and D. J. Miller The Cellular Chaperone Heat Shock Protein 90 Facilitates Flock House Virus RNA Replication in Drosophila Cells J. Virol., June 1, 2005; 79(11): 6827 - 6837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. G. Engelhardt, M. Smith, and E. Fodor Association of the Influenza A Virus RNA-Dependent RNA Polymerase with Cellular RNA Polymerase II J. Virol., May 1, 2005; 79(9): 5812 - 5818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Bechtel, R. C. Winant, and D. Ganem Host and Viral Proteins in the Virion of Kaposi's Sarcoma-Associated Herpesvirus J. Virol., April 15, 2005; 79(8): 4952 - 4964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Yamashita, W. Kamitani, H. Yanai, N. Ohtaki, Y. Watanabe, B.-J. Lee, S. Tsuji, K. Ikuta, and K. Tomonaga Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress J. Virol., February 15, 2005; 79(4): 2033 - 2041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kawaguchi, T. Naito, and K. Nagata Involvement of Influenza Virus PA Subunit in Assembly of Functional RNA Polymerase Complexes J. Virol., January 15, 2005; 79(2): 732 - 744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Mullin, R. M. Dalton, M. J. Amorim, D. Elton, and P. Digard Increased amounts of the influenza virus nucleoprotein do not promote higher levels of viral genome replication J. Gen. Virol., December 1, 2004; 85(12): 3689 - 3698. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. T. Vreede, T. E. Jung, and G. G. Brownlee Model Suggesting that Replication of Influenza Virus Is Regulated by Stabilization of Replicative Intermediates J. Virol., September 1, 2004; 78(17): 9568 - 9572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. Qanungo, D. Shaji, M. Mathur, and A. K. Banerjee Two RNA polymerase complexes from vesicular stomatitis virus-infected cells that carry out transcription and replication of genome RNA PNAS, April 20, 2004; 101(16): 5952 - 5957. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Area, J. Martin-Benito, P. Gastaminza, E. Torreira, J. M. Valpuesta, J. L. Carrascosa, and J. Ortin 3D structure of the influenza virus polymerase complex: Localization of subunit domains PNAS, January 6, 2004; 101(1): 308 - 313. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |