From the Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
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ABSTRACT |
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Approximately 4 million Americans are infected
with the hepatitis C virus (HCV), making it a major cause of chronic
liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We
performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein
DBX. DBX has significant amino acid sequence identity to mouse PL10, an
ATP-dependent RNA helicase. The binding of DBX to HCV core
protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent. When expressed in
mammalian cells, HCV core protein and DBX were co-localized at the
endoplasmic reticulum. In a mutant strain of Saccharomyces
cerevisiae, DBX complemented the function of Ded1p, an essential
DEAD box RNA helicase. HCV core protein inhibited the growth of
DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core
protein also inhibited the in vitro translation of capped
but not uncapped RNA. These findings demonstrate an interaction between
HCV core protein and a host cell protein involved in RNA translation
and suggest a mechanism by which HCV may inhibit host cell mRNA translation.
Hepatitis C virus (HCV)1
was discovered by cDNA cloning in 1989 and shown to cause chronic
liver disease (1, 2). Approximately 4 million Americans and 150 million
individuals worldwide are infected with HCV and at risk for cirrhosis
and hepatocellular carcinoma (3-6). Because development of a robust
cell culture system for HCV infection has remained elusive (6),
extremely little is known about HCV-host cell interactions and how they influence cell physiology or viral replication.
HCV is a positive single-stranded RNA virus and a member of the
Flaviviridae family (1, 7-10). Once HCV infects cells, the
positive, single-stranded RNA genome is translated into a polyprotein
of 3010 to 3033 amino acids, depending upon the strain (7-10). The
viral RNA is not capped, and translation occurs via an internal
ribosome entry site at the 5' end of the viral RNA (11, 12). The
mechanism of translation of uncapped viral RNA therefore differs
from that used by virtually all cellular mRNAs that are capped at
their 5' ends. The HCV polyprotein is cleaved by both host cell and
viral proteases into several smaller polypeptides (7-10, 13). The
major structural proteins are a core protein and two envelope proteins
called E1 and E2. The core protein forms the nucleocapsid of the mature
virion, and E1 and E2 are present in the viral envelope. A small
polypeptide called P7 is also generated as a result of cleavage at the
E2-NS2 junction, but its function is not clear. Four major
nonstructural proteins called NS2, NS3, NS4, and NS5 are also
generated, two of which, NS4 and NS5, are further processed into
smaller polypeptides called NS4A, NS4B, NS5A, and NS5B. Most of the
nonstructural proteins have enzymatic activities that are critical for
viral replication.
After cells are infected with a virus, viral proteins can interact with
host cell proteins and influence cell physiology. In previous studies,
HCV core protein has been shown to bind to lymphotoxin- Yeast Two-Hybrid Screening--
The Matchmaker Two-Hybrid System
2 was used to screen human liver Matchmaker cDNA library HL4002AB
(CLONTECH) with the cytoplasmic domain (amino acids
1 to 123, which precede the first predicted transmembrane segment) of
HCV core protein as bait in the yeast two-hybrid assay (17). Library
screening was performed using previously described methods (18, 19). To
construct the bait plasmid, DNA encoding amino acids 1-123 of HCV core
protein (numbering as in Ref. 8) was amplified by PCR with pHCV-1 (13),
provided by M. Houghton (Chiron Corp.) as template. The HCV sequences
in pHCV-1 derive from a library made from the plasma of an infectious chimpanzee (13). The amplified DNA was cloned into the GAL4 DNA binding
domain fusion vector pAS2-1 (CLONTECH) to yield
pAS2-1-HCV-core1-123. Saccharomyces cerevisiae
strain Y190 was sequentially transformed with
pAS2-1-HCV·core1-123 and library recombinants in the GAL4 activation domain fusion vector pACT2
(CLONTECH). Positive pACT2-derived plasmids were
rescued and used to co-transform yeast with
pAS2-1-HCV·core1-123, pLAM5'-1
(CLONTECH), and pAS2-1 to confirm the specificity
of the reactions. For analysis of PL10 and Ded1p binding, cDNAs
encoding PL10 from amino acids 408 to 660 and Ded1p amino acids 368 to
604 (corresponding to the longest portion of DBX isolated in the
two-hybrid screen) were amplified by PCR from template plasmids (20),
provided by T.-H. Chang (Ohio State University). The amplified
cDNAs were cloned into pACT2 and used to co-transform yeast with
pAS2-1-HCV-core1-123. DNA sequencing of isolated library
plasmid inserts and the bait constructs was performed on a 373A
Sequencer (Applied Biosystems) at the Columbia University Cancer Center
DNA core facility. Sequence analysis was performed using the Wisconsin
Package (Genetics Computer Group) and applications available via the
internet at the National Center for Biotechnology Information World
Wide Web site.2
In Vitro Binding Assays--
A PCR product encoding the
cytoplasmic domain of HCV core protein (amino acids 1-123) was cloned
into pBFT4 for in vitro transcription-translation (21).
DBX cDNA encoding amino acids 409 to 662 was excised
from plasmid pACT2 by restriction endonuclease digestion and cloned into pGEX2T (Amersham Pharmacia Biotech) to yield
pGEX2T-DBX409-662, which expressed a glutathione
S-transferase (GST) fusion protein in Esherichia
coli. Plasmid construction was confirmed by DNA sequencing.
In vitro transcription-translation was performed with the
TNT T7 Coupled Reticulocyte Lysate System (Promega) using L-[35S]methionine (NEN Life Science
Products). Binding assays were performed as described previously
(21).
Cell Transfection and Confocal Immunofluorescence
Microscopy--
A PCR product encoding full-length HCV core protein
(amino acids 1-191) obtained using pHCV-1 (13) as template was cloned in-frame into pBFT4, which contains an initiation codon and FLAG tag 5'
to the cloning site. A DNA fragment was isolated by restriction endonuclease digestion at sites flanking the initiation and termination codons and cloned into pSVK3 (Amersham Pharmacia Biotech) to obtain pSVK3-FLAG-core for expression of HCV core protein with a FLAG tag at
its amino terminus. To obtain full-length DBX cDNA, PCR was performed using a Marathon-ready cDNA human liver library (CLONTECH) as template to amplify the first 1439 nucleotides of DBX cDNA, which was ligated in-frame into
pGEX2T-DBX409-662 to produce pGEX2T-DBX. The coding region
of pGEX2T-DBX was isolated by restriction endonuclease digestion and
cloned into pBluescript II SK Yeast Strains--
Yeast strain YTC83 [MATa
ded1::TRP1 ura3-52 lys2-801 ade2-101 trp1- Effects of HCV Core Protein on Growth of Yeast Strains--
The
coding region for full-length HCV core protein (amino acid 1-191) was
excised from pBFT4 by restriction endonuclease digestion and ligated
into p423GPD (ATCC) to produce p423GPD-core. The coding region for the
cytoplasmic domain of HCV core protein (amino acid 1-123) was also
ligated into p423GPD to yield p423GPD-core1-123. Constructs were confirmed by DNA sequencing. Yeast strains YTC83, YNM1DX, and YNM1DD were transformed with p423GPD, p423GPD-core, and
p423GPD-core1-123 using the lithium acetate-mediated method (24) and grown on histidine-leucine dropout plates for 7 days.
Plates were photographed to record colony growth.
Effect of HCV Core Protein on in Vitro Translation--
cDNA
encoding HCV core protein from amino acids 1 to 123 was ligated into
pGEX4T-3 (Amersham Pharmacia Biotech) to produce a GST fusion protein
(GST-core1-123) in E. coli. Plasmid construction was confirmed by DNA sequencing. pGEM-luc (Promega) was
linearlized with XhoI and used as a template for luciferase RNA transcription with the RiboMAX RNA Production System-SP6 (Promega). When capped RNA was synthesized, 3 mM 7mGpppG
(New England Biolabs) was included in the reaction mixture. The DNA
template was removed by digestion with DNase following the
transcription reaction, and synthesized mRNA was purified using the
RNeasy Mini Kit (Qiagen). For in vitro translation, 16.5 µl of Flexi Rabbit Reticulocyte Lysate (Promega) was used and
incubated for 1 h at 4 °C with 8.25 µl of
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) loaded with
either 300 ng of GST-core1-123 or GST followed by
centrifugation for 5 min at 2000 × g. Translation reactions were then performed according to the manufacturer's instructions, and luciferase activity was measured by luminescence emission using the Luciferase Assay System (Promega).
HCV Core Protein Binding to DBX--
Screening of 8 × 106 recombinants of a human liver cell cDNA library
with the cytoplasmic domain of HCV core protein as bait in the yeast
two-hybrid assay led to the isolation of 5 positive clones, 3 of which
encoded portions of DBX, the longest from amino acid 409 to amino acid
662. The 2 other positive clones encoded portions of epsilon 14-3-3, a
member of the 14-3-3 family of proteins that has numerous proposed
functions, including activities in signal transduction. DBX is the
human orthologue of the mouse DEAD box protein PL10 (25-27). PL10 is
the functional orthologue of S. cerevisiae Ded1p, an
ATP-dependent RNA helicase for capped mRNA (20). DBX is
95% identical in primary structure to PL10 and 54% identical to Ded1p
(Fig. 1A). In the yeast
two-hybrid assay, HCV core protein interacts with DBX and PL10 but not
with Ded1p (Fig. 1B).
We confirmed the interaction between HCV core protein and DBX in an
in vitro binding assay. The cytoplasmic domain of HCV core
protein was synthesized by in vitro translation and
incubated with GST or a GST fusion protein containing DBX from amino
acid 409 to amino acid 662. Proteins were precipitated with
glutathione-Sepharose, and HCV core protein binding was analyzed by
autoradiography. HCV core protein did not bind to GST but did bind to
GST-DBX fusion protein in buffers containing NaCl concentrations as
high as 1 M (Fig.
2A). Binding also occurred in
buffers containing 1% of the nonionic detergent Nonidet P-40 (Fig.
2B).
Co-localization of HCV Core Protein and DBX in Cells--
An
interaction between HCV core protein and DBX in mammalian cells was
further supported by their intracellular co-localization. Indirect
confocal immunofluorescence microscopy of transfected HeLa cells showed
that full-length HCV core protein, which contains the cytoplasmic
domain and a single transmembrane segment, was localized to the
endoplasmic reticulum in discrete foci (Fig. 3A). A similar localization in
the endoplasmic reticulum has been reported by others (13). Focal
aggregates of HCV core protein likely arise because this polypeptide
multimerizes (28). In cells not expressing HCV core protein, DBX had a
more diffuse cytoplasmic distribution (Fig. 3A). In cells
expressing HCV core protein, however, DBX was found in most instances
in discrete foci that co-localized with HCV core protein (Fig.
3B). The antibodies used to detect the respective epitope
tags of each protein did not cross-react significantly (Fig.
3B). HCV core protein therefore forms aggregates at the
endoplasmic reticulum membrane with which DBX apparently
associates.
DBX Rescues Ded1-deletion Yeast Mutants and Rescue Is Prevented by
HCV Core--
DBX likely functions as an ATP-dependent RNA
helicase for cellular mRNA, which can be inferred from its sequence
similarity to mouse PL10 and yeast Ded1p (20, 27). To examine the
effect of HCV core protein on DBX function, we took advantage of yeast genetics and the fact that S. cerevisiae has only one
essential DBX-like protein, Ded1p (20). When driven by a yeast GPD
promoter and carried on a centromere plasmid, mouse PL10
cDNA, as described previously (20), and DBX cDNA
rescued the lethality of cells with a chromosomal ded1
deletion. This indicates that DBX can likely function as a RNA helicase
as it can replace the function of the yeast DEAD box RNA helicase
Ded1p. Expression of full-length HCV core protein severely inhibited
the growth of DBX- and PL10- complemented
ded1-deletion yeast but not ded1-deletion yeast
complemented with DED1 cDNA driven by the same promoter
on a centromeric plasmid (Fig. 4). This
is consistent with the observation that DBX and PL10, but not Ded1p,
bind to HCV core protein. The cytoplasmic domain of HCV core protein
that binds to DBX, without a transmembrane segment, did not
significantly inhibit the growth of DBX- and PL10- complemented ded1-deletion yeast (data not
shown), suggesting that inhibition of function may result from trapping
of these proteins in aggregates at the endoplasmic reticulum membrane
(see Fig. 3).
Inhibition of in Vitro Translation of Capped mRNA by HCV Core
Protein--
We examined the effects of HCV core protein on the
translation of capped and uncapped luciferase RNA in an in
vitro reticulocyte lysate assay. If HCV inhibits the function of
DBX as a RNA helicase, it should theoretically decrease the translation
of capped RNA but not significantly affect the translation of uncapped
RNA. In the in vitro translation assay, the cytoplasmic
portion of HCV core protein significantly inhibited the in
vitro translation of luciferase from capped but not uncapped RNA
(Fig. 5). Capped RNA translation was
approximately 4-fold higher than uncapped RNA translation in this assay
(data not shown). This finding suggests that HCV core protein may
inhibit the translation of capped mRNA in cells, presumably by
inhibiting DBX function.
HCV core protein binds to the human DEAD box protein DBX. DBX
rescues the lethal phenotype of ded1-deletion, demonstrating that it can function as a RNA helicase for capped mRNA, replacing the essential yeast DEAD box RNA helicase Ded1p. Our findings that HCV
core protein prevents DBX from rescuing ded1-deletion yeast
and that it inhibits the translation of capped RNA in vitro strongly suggest that it may inhibit cellular mRNA translation in vivo. These results, however, cannot establish if
translation inhibition occurs as a result of HCV core protein
inhibiting DBX RNA helicase activity per se or by an
interaction that results in "trapping" DBX at a location near the
membrane of the endoplasmic reticulum where in cannot function
properly. Inhibition of host cell mRNA translation could
theoretically provide viral RNA molecules with enhanced access
to ribosomes and the rest of the protein synthesis machinery of the
cell, a phenomenon shared by several different viruses (29). A recent
report has shown that high levels of expression of HCV structural and
nonstructural proteins is toxic to mammalian cells (30); however, it is
not clear if this toxicity results from inhibition of host cell
translation. Because the development of a robust cell culture system to
study HCV has remained elusive, it would be extremely difficult to
directly investigate the effects of HCV infection on host cell mRNA
translation. Despite these methodological constraints limiting the
ability to directly test the hypothesis, our discovery that HCV core
binds to DBX and inhibits capped RNA translation in experimental assays suggests that it can similarly inhibit mRNA translation in infected human cells.
DEAD box RNA helicases unwind capped mRNA (20), and inhibition of
their function should decrease translation of cellular mRNA.
Inhibition of DBX function by HCV core protein may only partially
inhibit host mRNA translation in mammalian cells because they
contain other putative RNA helicases (31). In contrast, the translation
of HCV RNA, which is not capped, utilizes internal ribosome entry sites
(11, 12), and can be unwound by its own RNA helicase, which is part of
the HCV NS3 protein (32, 33), and may proceed without DBX. This
hypothetical mechanism is reminiscent of that used by poliovirus, which
inhibits translation factor eIF-4F (34, 35) and also has RNA with
internal ribosome entry sites (36). In cells, eIF-4F exists as a
complex with eIF-4B, which has RNA binding activity, and eIF-4A, which
is also a DEAD box RNA helicase (37). HCV and poliovirus infection may
both therefore cause a decrease in the unwinding of capped mRNA in host cells.
In addition to inhibiting capped mRNA translation in infected host
cells, the interaction between HCV core protein and DBX may play other
possible roles, including the recruitment of DBX to participate in HCV
replication itself. Recruitment of host cells proteins into virions to
enhance viral replication has been demonstrated in other systems. For
example, the principal structural protein of the human immunodeficiency
virus HIV-1 binds to cyclophilins and recruits cyclophilin A into viral
particles, which appears to be necessary for efficient viral
replication (38, 39). In a similar fashion, recruitment of DBX into HCV
particles by binding to core protein may enhance viral replication.
This could theoretically occur by DBX altering viral genomic RNA
structure in viral particles in newly infected cells. Testing of this
hypothesis is limited at the present time because of the lack of an
efficient cell culture system for HCV.
HCV core protein has also been shown to bind to lymphotoxin- Finally, it should be noted that the best current treatment regimens
for chronic hepatitis C are effective in only a minority of patients
(42). If interactions between HCV and host cell proteins alter cell
survival or enhance viral replication, they could be rational targets
for antiviral drug design. Regardless of the physiological
significance, the tight binding of any polypeptide to a structural or
nonstructural protein of HCV may potentially interfere with viral
replication. The identification of polypeptides such as DBX that bind
to HCV proteins therefore has implications for the design of compounds
which may be therapeutically useful in the treatment of patients with
chronic hepatitis C.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor and
other tumor necrosis factor receptor family members (14, 15). A
truncated form of HCV core protein also interacts with
ribonucleoprotein K in the nucleus (16). We now show that HCV core
protein binds to a cellular RNA helicase and, in experimental systems,
inhibits capped RNA translation. This provides a novel mechanism by
which HCV may inhibit mRNA translation in infected cells or recruit
a cellular protein to enhance its own replication.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Stratagene) to produce
pBluescript-DBX. A cDNA containing the 3' 668 nucleotides of DBX,
excluding the stop codon, was amplified by PCR and ligated into
pBluescript II-DBX to replace the corresponding nucleotides. The entire
DBX coding region was then excised by restriction endonuclease
digestion and ligated into pcDNA3.1 (-)/Myc-His A (Invitrogen) to
produce pcDNA3.1/His A-DBX-myc, that encoded full-length DBX with a
c-myc tag at its carboxyl terminus. All plasmid constructs were
confirmed by DNA sequencing. HeLa or COS-7 cells (ATCC) grown on glass
slides were transfected with pSVK3-FLAG-core, pcDNA3.1/HIS
A-DBX-myc or both using Tfx-20 (Promega), or DMRIE-C (Life
Technologies, Inc.). Cells were washed in phosphate-buffered saline
48 h after transfection and fixed with methanol for 5 min at
20 °C followed by acetone at
20 °C for 20 s. Indirect
immunofluorescence microscopy was performed as described (22). To
detect express FLAG-tagged proteins in double-labeling experiments,
FLAG-probe (Santa Cruz Biotechnology), a rabbit polyclonal antibody,
was used. To reduce background, FLAG-probe was incubated with COS-7 cells fixed with methanol/acetone at a 1:100 dilution for 12-16 h
before use. Anti-FLAG M2 monoclonal antibody (Eastman Kodak Co.) was
used in single-labeling experiments at a 1:200 dilution. Monoclonal
anti-c-myc antibody 9E10 (Babco) was used at a 1:1000 dilution.
Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and
rhodamine-conjugated goat anti-mouse IgG secondary antibodies were
obtained from Jackson Immuno Research Laboratories. Microscopy was
performed using a Zeiss LSM 410 confocal laser scanning system attached
to Zeiss Axiovert 100TV inverted microscope (Carl Zeiss). Images were
processed using Photoshop software (Adobe) on a Macintosh G3 computer
(Apple Computer).
1
his3-
200 leu2-
1 pPL1004 (PL10/CEN/LEU2)], which contains a chromosomal ded1 deletion complemented by
PL10 cDNA (20), was provided by T.-H. Chang. To obtain a
yeast strain with a chromosomal ded1 deletion complemented
by DBX cDNA, full-length DBX cDNA was
excised from pGEX2T-DBX by restriction endonuclease digestion and
ligated into pRS315pG1 (provided by T.-H. Chang). This plasmid was used
to transform yeast strain YTC75 [MATa ded1::TRP1
ura3-52 lys2-801 ade2-101 trp1-
1 his3-
200 leu2-
1 pDED1008 (DED1/CEN/URA3)] (20), provided by T.-H. Chang,
which was then grown on leucine dropout plates. Transformants were
replica-plated onto 5-fluoroorotic acid plates as described (23) to
yield strain YNM1DX. To obtain a yeast strain with a chromosomal
ded1 deletion complemented by DED1 cDNA
driven by a glyceraldehyde-3-phosphate (GPD) promoter on a centromeric
plasmid, the native promoter, 5'-untranslated region, and part of the
5'-coding region were excised by restriction endonuclease digestion
from pDED1009 (DED1/CEN/LEU2) (provided by T.-H. Chang). The
GPD promoter, isolated from pRS315pG1 by restriction endonuclease
digestion, and 477 5'-coding nucleotides of DED1, amplified
by PCR, were then sequentially ligated into this pDED1009-derived
plasmid to yield pDEDGPD. Yeast strain YTC75 was then
transformed with pDEDGPD, and 5-fluoroorotic acid
counter-selection was performed to obtain strain YNM1DD [MAT a
ded1::TRP1 ura3-52 lys2-801 ade2-101 trp1-
1
his3-
200 leu2-
1 pDEDGPD
(DED1/CEN/LEU2)]. All constructs were confirmed by DNA sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primary structures of DBX, PL10, and Ded1 and
their interactions with HCV core protein in the yeast two-hybrid
assay. A, alignment of deduced amino acid
sequences of DBX (GenBankTM accession number AF000982), PL10
(GenBankTM accession number J04847), and Ded1p (GenBankTM accession
number X57278) is shown. Identical amino acids are shown as
white on cyan. Conserved substitutions are shown
as black on magenta. Dots represent
gaps to optimize alignments, which were obtained using the Pileup
program. B, two-hybrid assays showing interaction of HCV
core protein with DBX and PL10 but not with Ded1p. Yeast strain Y190
was co-transformed with a plasmid expressing the cytoplasmic domain of
HCV fused to the GAL4 DNA binding domain and plasmids expressing either
a portion of DBX or the corresponding portions of PL10 or Ded1p fused
to the GAL4 transcriptional activation domain. Transformants giving
-galactosidase activity (positive interactions) are blue.
Control reactions of DBX, PL10, and Dep1p GAL 4 activation domain
fusion proteins with GAL4 DNA binding domain alone were negative (data
not shown).
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Fig. 2.
Binding of DBX to HCV core protein in
vitro. A, a standard amount of
35S-HCV core protein (amino acids 1-123), 10% of which is
shown in the autoradiogram (lane 1), was used in each
binding assay. 35S-HCV core protein was incubated with
glutathione-Sepharose (lane 2): 20 µg of GST coupled to
glutathione-Sepharose (lane 3) in binding buffer containing
0.15 M NaCl and 0.2 µg of GST-DBX fusion protein coupled
to glutathione-Sepharose in buffers containing the NaCl concentrations
indicated above each lane (lanes 4-8).
Glutathione-Sepharose was then washed with buffer containing the
indicated NaCl concentration, and the bound proteins were eluted with
4% SDS, subjected to SDS-polyacrylamide gel electrophoresis, and
detected by autoradiography of dried slabs gels. B, binding
assay similar to that shown in panel A in which GST-DBX
fusion protein was incubated with 35S-HCV core protein in
buffers containing 0.15 M NaCl and 0.05 to 1.0% of Nonidet
P-40 (lanes 4-6). Migrations of molecular mass standards
are indicated in kilodaltons at the left of each
panel.
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Fig. 3.
Immunofluorescence localization of DBX and
HCV core protein in mammalian cells. A, HeLa cells were
transiently transfected with cDNA encoding FLAG-tagged HCV core
protein (left panel) or with cDNA encoding myc-tagged
DBX (right panel). Cells were incubated with monoclonal
anti-FLAG or anti-myc (9E10) antibody followed by rhodamine-conjugated
secondary antibody. HCV core protein appears primarily in large,
discrete foci at the endoplasmic reticulum membrane, whereas DBX has a
more diffuse cytoplasmic localization. B, co-localization of
DBX and HCV core protein in COS-7 cells transiently transfected to
express both FLAG-tagged HCV core protein and myc-tagged DBX. All cells
were fixed and incubated with the same combination of rabbit anti-FLAG
polyclonal antibody and mouse anti-myc monoclonal (9E10) antibody
followed by both fluorescein isothiocyante-conjugated goat anti-rabbit
and rhodamine-conjugated goat anti-mouse antibodies. Cells transfected
to express FLAG-tagged HCV core protein alone (left column)
showed essentially only green fluorescence, resulting from
anti-FLAG and fluorescein isothiocyanate-conjugated antibody labeling
(row G). Cells transfected to express myc-tagged DBX alone
(middle column) showed essentially only red
fluorescence, resulting from anti-myc and rhodamine-conjugated antibody
labeling (row R). The right column shows COS-7
cells co-transfected to express both FLAG-tagged HCV core protein
(row G) and myc-tagged DBX (row R). Merged images
(row M) appear yellow where green
fluorescence corresponding to HCV core protein localization and
red fluorescence corresponding to DBX localization
overlap.
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Fig. 4.
Inhibition of DBX and PL10 but not Ded1p by
HCV core protein. Yeast strains with chromosomal ded1
deletion complemented with either DBX, PL10, or
DED1 cDNAs driven by the yeast GPD promoter on
centromeric plasmids were transformed with a plasmid that expressed
full-length HCV core protein (top) or control plasmid
p423GPD (bottom). The resulting transformants were spread on
histidine, leucine drop-out plates and incubated at 30 °C for 7 days, and photographs (negatives are shown) were taken of each plate.
Note colony growth of all yeast strains transfected with control
plasmid (bottom panels). In contrast,
DBX- and PL10- complemented
ded1-deletion strains do not demonstrate significant colony
growth when HCV core protein is expressed, whereas growth of the
DED1-complemented strain is unaffected (top
panels).
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Fig. 5.
Inhibition of translation of capped mRNA
in vitro by HCV core protein. Rabbit reticulocyte
lysates were incubated with glutathione-Sepharose beads loaded with
either 300 ng of a GST-HCV core fusion protein or GST. In
vitro synthesized capped or noncapped luciferase mRNAs were
translated at 30 °C for 90 min, and luciferase activity was
measured. Results are expressed as the relative luciferase activities
produced in reticulocyte lysate-treated equal concentrations of GST-HCV
core fusion protein (shaded bars) or GST (open
bars, arbitrarily assigned 100% activity). Values shown are means
±S.E. (n = 6). *p < 0.0001;
, no significant difference.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor and other tumor necrosis factor receptor family members (14,
15) as well as ribonucleoprotein K (16). In our yeast two-hybrid
screen, we did not isolate clones for these proteins, possibly because
of subtle differences in our bait construct and the different cDNA
library we used. The demonstration that other proteins interact with
HCV core protein suggests that its expression in cells may have myriad
consequences. Other groups (40, 41) have also reported that HCV core
protein represses transcription from the p53 promoter and other
eukaryotic promoters. The overall effect of HCV core protein on cell
physiology under natural conditions of infection is, however, difficult
to assess at the present time because of lack of a cell culture system
for HCV.
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ACKNOWLEDGEMENTS |
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We thank T.-H. Chang (Ohio State University) for providing reagents and invaluable advice, S. P. Goff (Columbia University) for helpful discussions, M. Houghton (Chiron Corp.) for providing pHCV-1, and P. J. Mustacchia (Columbia University) and T. W. Chun (State University of New York) for reviewing the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Blowitz-Ridgeway Foundation. The confocal microscopy facility used for this work was established by National Institutes of Health (NIH) Grant 1S10 RR10506 and supported by NIH Grant 5 P30 CA13696 as part of the Herbert Irving Cancer Center at Columbia University.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.
Present address: Dept. of Internal Medicine, Nagoya National
Hospital, 4-1-1 sannomaru naka-ku, Nagoya aichi, Japan 460-0001.
§ To whom correspondence should be addressed: Dept. of Medicine, College of Physicians and Surgeons, Columbia University, 630 West 168th St., 10th Floor, Rm. 508, New York, NY 10032. Tel.: 212-305-8156; Fax: 212-305-6443; E-mail: hjw14{at}columbia.edu.
2 http://www.ncbi.nlm.nih.gov/.
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ABBREVIATIONS |
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The abbreviations used are: HCV, hepatitis C virus; GST, glutathione S-transferase; GPD, glyceraldehyde-3-phosphate; PCR, polymerase chain reaction.
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