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(Received for publication, July 10,
1995; and in revised form, November 16, 1995) From the
Myristoylation of human immunodeficiency virus (HIV) Gag protein
is essential for virus particle budding. Two reactions are involved;
activation of free myristate to myristoyl-CoA and transfer of the
myristoyl residue to the Gag N-terminal glycine. We have investigated
the effects of triacsin C, an inhibitor of long chain acyl-CoA
synthetase, on release of HIV Gag virus-like particle (VLP) produced
using the recombinant baculovirus system. First, inhibition of acyl-CoA
formation by triacsin C was confirmed using the membrane fractions of
insect Sf9 cells as an enzyme source. Second, when HIV Gag
protein was expressed in the presence of triacsin C (0-48
µM), Gag myristoylation was inhibited in a dose-dependent
manner. Budding of Gag VLP, however, did not follow similar inhibition
kinetics but appeared unaffected up to 24 µM, yet was
completely abolished at 48 µM when the myristoylation of
Gag protein was also completely inhibited. The
``all-or-none'' inhibition of Gag VLP budding suggests that
although inhibition of acyl-CoA synthetase blocks the production of
myristoylated Gag protein, only complete inhibition of Gag
myristoylation prevents VLP budding. Thus, relatively few myristoylated
Gag molecules are sufficient for plasma membrane targeting and VLP
budding.
Human immunodeficiency virus (HIV), ( Gag is synthesized
initially in the cytosol as a precursor protein, Pr55, and is targeted
to the plasma membrane where particle assembly and packaging of viral
genomic RNA occur(5) . During synthesis, Pr55 Gag is acylated
at the N-terminal glycine residue exclusively with myristic
acid(6, 7) , a modification which, when combined with
an N-terminal basic region(8, 9) , is essential for
Pr55 targeting to the plasma membrane, since non-myristoylated Pr55
obtained by amino acid substitution at the N-terminal glycine fails to
bud from the cell
surface(4, 10, 11, 12, 13) .
However, this technology is inappropriate for a study of the level of
myristoylation required for efficient virus-like particle (VLP)
budding. Such studies are necessary as myristoylation has been
suggested to be a suitable target for therapeutic drug development
against HIV(14) , and yet the consequence of partial
myristoylation has not been addressed. Gag myristoylation consists
of two reactions; activation of myristic acid to myristoyl-CoA by
acyl-CoA synthetase and transfer of the myristoyl group from
myristoyl-CoA to the N-terminal glycine of Pr55 by N-myristoyltransferase. Heteroatom-substituted analogues of
myristic acid(15) , phospholipid containing such a myristic
acid analogue(16) , and analogues of N-myristoyl
glycine(17, 18, 19) have been reported to
inhibit HIV replication, and most of these inhibitors are expected to
inhibit N-myristoyltransferase activity. However, the
biochemical characterization of these compounds in relation to their
effect on HIV remains poorly understood, and precise correlation
between Gag myristoylation and HIV particle formation remains unclear. We have previously reported a series of compounds, termed triacsin A
to D, isolated from the culture filtrate of Streptomyces sp.
SK-1894, which contain 11 carbon alkenyl chains with an N-hydroxytriazene moiety at the termini(20) . Although
all triacsins inhibit acyl-CoA synthetase from a wide variety of
sources, triacsin C was shown to be the most potent inhibitor in a
large number of studies in which triacsins were utilized to investigate
the function of acyl-CoA synthetase in lipid and related
metabolisms(21, 22, 23, 24, 25, 26) .
Here, to investigate the function of HIV Gag myristoyl moiety in the
process of HIV assembly and particle budding, triacsin C was employed
to inhibit the expression of myristoylated Pr55 in the recombinant
baculovirus system. We show that the inhibition of Gag myristoylation
by triacsin C follows dose-dependent kinetics but that the inhibition
of VLP budding exhibits sudden shutoff kinetics. These data suggest
that only a relatively small proportion of total Gag molecules need to
be myristoylated for efficient VLP budding and indicate that total
inhibition of myristoylation will be required for effective anti-viral
therapy.
Spodoptera frugiperda (Sf9) cells and a
recombinant Autographa californica nuclear polyhedrosis virus
(baculovirus) containing the full-length HIV-1 gag gene used
in this study were kindly supplied by Dr. Ian Jones (Institute of
Virology and Environmental Microbiology, Oxford, UK). This gag recombinant virus, which does not include HIV pol gene-encoding protease, produces doughnut-like VLPs (composed of
uncleaved Gag precursor, Pr55) corresponding to immature HIV
particles(12, 13, 27, 28) .
Figure 1:
Inhibition of
acyl-CoA synthetase activity in the Sf9 cell membrane fraction
by triacsin C. Sf9 cells (1.5
Figure 2:
Effect of triacsin C on release of HIV Gag
protein. Monolayers of 2
Figure 3:
Electron microscopic examination of Sf9 cells infected with a recombinant baculovirus-expressing
HIV Gag protein in the presence of triacsin C. Sf9 cells were
infected with the recombinant baculovirus and cultured as described in
the legend for Fig. 2. The cells were harvested for electron
microscopic examination at 36 h postinfection prior to appreciable cell
lysis. A single arrowhead shows an HIV Gag VLP containing an
electron-dense fringe (that is a ``doughnut-like'' or
``immature-type'' HIV), a double arrowhead indicates
a baculovirus particle containing a rod-shaped core, and an arrow indicates an electron-dense ring structure. A, the cells
cultured in the absence of triacsin C; B, the cells cultured
in the presence of 24 µM triacsin C; C and D, the cells cultured in the presence of 48 µM triacsin C.
The expression system used here, like the gag expression system using vaccinia virus
vectors(37, 38) , produces baculoviruses as well as
HIV Gag VLPs. Since HIV Gag protein is myristoylated and, in contrast,
there are no acylated proteins reported in baculovirus(39) ,
the titer of baculoviruses grown in triacsin C-treated cells was used
as a general measure of the side effects of triacsin C on
non-myristoylated proteins. Equal levels of infectious baculovirus were
present in the supernatants from the cells treated with all the doses
of triacsin C used in this study (Fig. 4), suggesting that
triacsin C treatment had little effect on the synthesis of
non-myristoylated proteins or their folding and incorporation into
baculovirus particle.
Figure 4:
Effect of triacsin C on baculovirus
growth. The culture media in Fig. 1were used for assessment of
produced baculoviruses.
Figure 5:
Inhibition of HIV Gag myristoylation in Sf9 cells expressing HIV Gag protein in the presence of
triacsin C. Infection of the recombinant baculovirus and incubation
with triacsin C were performed as described in the legend to Fig. 1. At 36 h postinfection, the cells were metabolically
labeled with [9,10 (n)-
Figure 6:
Incorporation of myristoylated Gag
proteins into HIV Gag VLPs. At 36 h postinfection, the cells were
metabolically labeled with [9,10 (n)-
It is well established that HIV Gag myristoylation is
essential for plasma membrane targeting of Gag protein, as when the
myristoylation acceptor glycine located at the N terminus of Gag
protein is mutated, the budding of HIV Gag VLP is completely abolished,
and their assembly is restricted to intracellular
locations(4, 10, 11, 12, 13) .
However, it has also been reported that the non-myristoylated Gag
proteins obtained by site-directed mutagenesis can be rescued into Gag
VLPs by co-expression with myristoylated Gag(40, 41) .
Similar findings have been reported for studies with Rous sarcoma
virus(42) , although not for murine leukemia
virus(43) . One would assume, therefore, partial reduction in
the level of Gag myristoylation might reduce the number of Gag
molecules located to plasma membrane and result in a reduction in the
level of Gag VLP release proportional to the level of myristoylation
inhibition. In this paper, we used triacsin C, a well defined inhibitor
of acyl-CoA synthetase, and found that activation of myristic acid to
myristoyl-CoA catalyzed by acyl-CoA synthetase was dose dependently
inhibited by triacsin C (Fig. 1), and correspondingly,
dose-dependent inhibition of Gag myristoylation occurred in the
Gag-expressing cells (Fig. 5). In contrast to the
dose-dependent inhibition of Gag myristoylation (Fig. 5), the
budding of Gag VLP from the plasma membrane of infected cells exhibited
an ``all-or-none'' phenotype (Fig. 3). The aspect was
much more clearly highlighted by analyzing the ratio of total to The assembly of Gag proteins is believed to occur after Gag
proteins reach the plasma membrane, since electron microscopic
observation shows that an electron-dense half-ring corresponding to the
gathering of Gag proteins is present underneath locally extruded plasma
membrane(2, 44) . Nonetheless, electron-dense ring
structures in cytoplasmic VLP have also been documented in the case of
non-myristoylated Gag protein (12, 45) , and we found
similar structures in a perinuclear area of the cells expressing Gag
proteins in the presence of 48 µM triacsin C. Pre-assembly
in the cytoplasm has been traditionally thought of as the morphological
pathway for D-type retroviruses and plasma membrane assembly the
pathway for C-type (as for HIV) retroviruses(1) . Our data
suggest this distinction is not as clear cut as is commonly thought,
especially when non-myristoylated form of Gag protein is concerned. We consider that myristoylation of retrovirus Gag protein is a
potential target for development of new anti-viral agents. We showed
here that Gag VLP was produced up to and including 24 µM of triacsin C and composed of mixture of non-myristoylated and
myristoylated Gag proteins. In this system, we were unable to assess
whether or not HIV particles containing predominantly non-myristoylated
Gag proteins are as stable or as infectious as authentic HIV particles
whose Gag molecules are believed to be fully myristoylated. It is
possible that, although budding of VLP is not prevented because of
partial myristoylation, there is an effect on processing or uncoating.
As non-processed Gag protein always results in an uninfectious state (46, 47, 48) , anti-myristoylation therapy
may prove effective even if particles continue to be produced.
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2868-2873
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)the causative
agent of acquired immunodeficiency syndrome in humans, is classified
into the lentiviruses family of retroviridae(1) . The life
cycle of HIV has been studied extensively, and evidence has accumulated
to suggest a mechanism of viral assembly and particle formation (see (2) for review). The main structure components of HIV particles
are encoded by the gag gene, and expression of Gag protein
alone in a number of expression systems produces HIV-like particles
analogous to the immature type of authentic
HIV(2, 3, 4) .
Materials
Triacsin C was purified from a culture
broth of Streptomyces sp. SK-1894 as reported
previously(20) . Triacsin C was usually added to every assay
described below as an ethanol solution, and the final volume of ethanol
never exceeded 2.5%. Equivalent concentrations of ethanol alone showed
no effects on the assays. Myristic, palmitic, and oleic acids were
purchased from Funakoshi, Japan. [
C]Myristic
acid (58.0 Ci/mol), [C]palmitic acid (56.0
Ci/mol), and [C]oleic acid (56.0 Ci/mol) were
purchased from DuPont NEN, and [C]rainbow
protein molecular weight markers and
[9,10(n)-
H]myristic acid were from
Amersham. Anti-HIV-1 p17 peptide serum (D7327) and anti-HIV-1 p24
peptide serum (D7320) were purchased from Aalto Bio Reagents Ltd.
(Ireland), and anti-sheep IgG alkaline phosphatase conjugate was from
Zymed Laboratories, Inc. Prestained protein molecular weight markers
(low range) were obtained from Bio-Rad. Other reagents unless otherwise
specified were commercially available of analytical grade. Cells and Virus
Sf9 cells were propagated
at 27 °C in Grace's insect cell culture medium (Life
Technologies, Inc.) supplemented with 10% fetal calf serum (Life
Technologies, Inc.) and 50 µg/ml gentamycin (Life Technologies,
Inc.).Measurement of Cell Viability
Sf9 cell
viability in the presence of triacsin C was measured by trypan blue
exclusion method(29) .Preparation of a Membrane Fraction of Sf9
Cells
1.5 
10
Sf9 cells were
collected and suspended on ice in 20 ml of 0.1 M potassium
phosphate buffer (pH 7.4) containing 1 mM dithiothreitol
(DTT). After brief sonication for 10 s at 100 watts six times (model
W-225R, Heat System, Ultrasonics Inc.), a membrane fraction was
pelleted by centrifugation at 100,000 g for 1 h and
resuspended in 2 ml of the buffer described above. Protein in the
membrane fraction was quantitated by the method of Bradford (30) using Protein Assay Kit (Bio-Rad).Assay for Acyl-CoA Synthetase Activity
The isotope
method for long chain acyl-CoA synthetase activity (31) was
employed with slight modification. An assay mixture containing 0.1 M Tris-HCl (pH 8.0), 5 mM DTT, 150 mM KCl,
15 mM MgCl
, 10 mM ATP, 1 mM CoA,
1 mMC-fatty acid (0.02 µCi, diluted with
cold fatty acid), triacsin C (added as a 2.5-µl ethanol solution to
make a final concentration of 0-48 µM), and Sf9 cell membrane fraction (15 µg of protein) in a total
volume of 100 µl was incubated at 27 °C for 20 min. The
reaction was stopped by adding an isopropanol:heptane:1 M sulfuric acid mixture (40:10:1, v/v), and the radioactive free
fatty acid was extracted with heptane twice. The aqueous layer
containing synthesized C-labeled acyl-CoA was counted with
a liquid scintillation spectrometer (Aloka).Virus Infection and Treatment of Triacsin
C
Monolayers of 2 10
Sf9 cells in
35-mm tissue culture dishes (Costar) were inoculated with the
recombinant virus containing HIV-1 gag gene at a multiplicity
of 10 plaque-forming units/cell. After adsorption at 27 °C for 1 h,
the cells were washed twice and cultured with 0.5 ml of serum-free
Grace's medium in the presence of triacsin C at 27 °C.Baculovirus Titration
Titers of recombinant
baculoviruses were determined by the plaque assay as described
elsewhere(32) .Western Blotting
At 48 h postinfection, cells and
culture media were harvested separately and analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE) on 14% acrylamide
gels(33) . Western blotting was carried out as described
elsewhere(34) . After transfer, nitrocellulose membrane
(Amersham) was incubated with anti-HIV-1 p24 peptide serum or
anti-HIV-1 p17 peptide serum (see text and legends for figures) and
anti-sheep IgG alkaline phosphatase conjugate. The immunocomplexes were
visualized using nitro blue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate (Promega).In Vivo Labeling
At 36 h postinfection, monolayer
cells in 35-mm tissue culture dishes were metabolically labeled with
250 µCi of [9,10(n)-H]myristic acid
in 0.5 ml of serum-free Grace's medium with triacsin C either for
3 h in the case of immunoprecipitation or overnight for purification of
Gag VLPs.Immunoprecipitation
Labeled cells were rinsed with
cold phosphate-buffered saline and lysed in 0.5 ml of
radioimmunoprecipitation assay buffer(35) . After clarification
at 18,500 g for 10 min, 100 µl of the supernatant
was incubated with 10 µl of anti-HIV-1 p17 peptide serum on ice for
1 h, and the immuno complexes were precipitated with 20 µl of
protein G-Sepharose 4B (Zymed Laboratories, Inc.) on ice for 1 h. The
immunoprecipitates were washed four times with washing buffer (0.5%
Nonidet P-40, 100 mM Tris-HCl (pH 7.4), 150 mM NaCl),
and either analyzed by SDS-PAGE or measured by a liquid scintillation
spectrometer. After electrophoresis, the gels were subjected to
fluorography in Amplify (Amersham) for 30 min and exposed with an x-ray
film (Hyperfilm-ECL, Amersham) at -80 °C.Purification of HIV Gag VLPs
Infected culture
media were clarified at 4 °C at 18,500 g for 10
min, and the supernatants were centrifuged through 20% (w/v) sucrose
cushions in SW55 tubes at 4 °C at 147,000 g for 2
h. The virus pellets were resuspended in 100 µl of
phosphate-buffered saline and layered onto 20-60% (w/v) sucrose
gradients in SW55 tubes. After centrifugation at 4 °C at 147,000
g overnight, the gradient was fractionated by 300
µl, and 40-50% sucrose fractions were pooled and subjected to
SDS-PAGE. Fluorography and autoradiography were carried out as
described above.Electron Microscopy
The procedure for electron
microscopic examination was described previously(36) . Cells
were collected at 36 h postinfection before appreciable cell lysis and
washed with 0.05 M cacodylate buffer (pH 7.2). The cells were
fixed with 2% glutaraldehyde for 2 h at 4 °C prior to treatment
with 1% osmium tetroxide for 2 h at 4 °C. After dehydration in
ethanol, the cells were embedded in epoxy resin. Ultrathin sections
were stained with uranyl acetate and lead citrate and examined in a
Hitachi H-800 electron microscopy.
Inhibition of Myristoyl-CoA Synthesis by Triacsin
C
Since acyl-CoA synthetase activity from insect cell had not
been previously reported, the characteristics of the enzyme present in
the membrane fraction of Sf9 cells was determined. ATP, CoA,
and Mg were required to elicit full activity (Table 1), similar to the characteristics of the enzyme activity
present in rat liver and other animal
cells(21, 23, 24) . Furthermore, myristic
acid appeared to be activated by the same synthetase as oleic acid (Table 1). Triacsin C inhibited the activation of both myristic
and palmitic acids in a dose-dependent fashion with the IC
values calculated to be 27 and 21 µM, respectively (Fig. 1). In contrast, inhibition of oleic acid activation
required a higher IC
value of about 50 µM.
These studies established that triacsin C inhibited myristoyl-CoA
formation by acyl-CoA synthetase in Sf9 cell membrane
fractions.
10 cells) in
20 µl of 0.1 M potassium phosphate buffer containing 1
mM DTT were sonicated (at 100 watts for 10 s six times) on
ice. A membrane fraction precipitated by centrifugation (at 100,000
g for 1 h) was used as an enzyme source. Acyl-CoA
synthetase activity was assayed in a 100-µl mixture containing 0.1 M Tris-HCl (pH 8.0), 5 mM DTT, 150 mM KCl,
15 mM MgCl, 10 mM ATP, 1 mM CoA,
1 mMC-fatty acid (0.02 µCi) (,
myristic acid;
, palmitic acid; , oleic acid), triacsin C
(0-48 µM), and the membrane fraction (15 µg of
protein). After a 20-min incubation at 27 °C, produced
[
C]acyl-CoA and C-fatty acids were
separated, and the radioactivity of [C]acyl-CoA
was counted by a liquid scintillation
spectrometer.
Effect of Triacsin C on Sf9 Cells
To investigate
the effect of triacsin C on Sf9 cell viability, confluent
monolayers of Sf9 cells were cultured in serum-free
Grace's medium with increasing levels of triacsin C for 48 h at
27 °C, and the cell viabilities were determined by the trypan blue
staining. No significant difference was observed compared to untreated
control with the drug levels of up to 48 µM, although Sf9 cell viability was severely affected at 96 µM (data not shown). Accordingly, levels of 0-48 µM triacsin C were used for further experiments.Inhibition of HIV Gag VLP Budding by Triacsin
C
The effect of triacsin C on HIV Gag protein synthesis and
subsequent VLP budding was examined using Sf9 cells infected
with a recombinant baculovirus-expressing Gag
protein(12, 13, 27, 28) . Infected
cells were cultured in the presence of a range of concentrations of
triacsin C, and the culture media and cells were harvested for Western
blotting and electron microscopic examination. Western blotting was
carried out using an anti-HIV-1 p24 peptide serum, which recognizes a
major antigen located in the central region of Gag protein. Near
identical levels of Pr55 were detected in all the cytoplasmic
fractions, indicating minimum effect of triacsin C on Gag protein
synthesis (Fig. 2). The previously reported Gag proteolytic
products, p47 (p17+p24+p9), p39 (p24+p9+p6), and
p24, which occur to some degree by cell-directed proteolysis (discussed
in (12) ), were also visible, and, as these were unaffected by
drug treatment, Gag protein processing also appears largely unaffected
by triacsin C treatment, although there was a lower level of
proteolytic conversion to p24 in the 48 µM sample. In the
culture supernatants, however, in contrast to almost identical levels
of Gag antigens in 0-24 µM treatments, no detectable
antigen was present in the 48 µM sample (Fig. 2).
Electron microscopic examination confirmed these findings. Infected
cells treated with 24 µM triacsin C showed abundant HIV
Gag VLPs budding from the cell surface indistinguishable from
non-treated cells (Fig. 3, A and B). In
contrast, in the cells treated with 48 µM of the drug,
there were no detectable HIV Gag VLPs produced from the cell surface
nor any electron-dense structure underneath the plasma membrane (Fig. 3C). However, electron-dense ring structures were
frequently observed in the perinuclear area of the cell (Fig. 3D) and resembled those reported in the case of
non-myristoylated Gag protein obtained by site-directed
mutagenesis(12) . We conclude, therefore, 48 µM triacsin C inhibits budding of HIV Gag VLP with little effect on
Gag protein synthesis.
10Sf9 cells in
35-mm tissue culture dishes were infected with the recombinant
baculovirus-containing HIV-1 gag gene. After adsorption, the
cells were washed and cultured in serum-free Grace's medium in
the presence of various concentrations of triacsin C (0-48
µM) as indicated. At 48 h postinfection, the cells and
culture media were harvested separately and analyzed by Western
blotting using anti-HIV-1 p24 peptide serum. Pr55 and its proteolytic
products, p47 (p17+p24+p9), p39 (p24+p17), and p24, were
detected. Lane 1, prestained molecular weight marker; lanes 2-6, cell lysates; lanes 7-11,
culture media.
Inhibition of Myristoylation of HIV Gag Protein by
Triacsin C
To examine inhibition of myristoylation of HIV Gag
protein in vivo, Sf9 cells infected with the
recombinant baculovirus-expressing Gag protein were metabolically
labeled with [H]myristic acid for 3 h at 36 h
postinfection in the presence of triacsin C. The cell lysates were
immunoprecipitated with anti-HIV-1 p17 peptide serum, which recognizes
the N-terminal third of the Gag polyprotein, and subjected to SDS-PAGE.
From the Western blot analysis of the resulting supernatant fractions
and the electron microscopic observation of the infected cells
described above, an all-or-none effect of triacsin C on the Gag
myristoylation was expected. Surprisingly, however, triacsin C
inhibited the Gag myristoylation in a dose-dependent manner (Fig. 5A). Quantitation of H label
associated with the immunoprecipitates by liquid scintillation counting
confirmed the dose-dependent inhibition of Gag myristoylation by
triacsin C with an IC value of 6.7 µM (Fig. 5B).
H]myristic acid
for 3 h and lysed with radioimmunoprecipitation assay buffer, and then
Gag proteins were immunoprecipitated with anti-HIV-1 p17 peptide serum. A, the immunoprecipitates resolved by SDS-PAGE and visualized
by fluorography. Pr55 and its proteolytic products, p47
(p17+p24+p9) and p17, are highlighted. Lane 1, C-rainbow protein molecular weight markers; lanes
2-6, immunoprecipitates of the cells treated with triacsin C
(0-48 µM) as indicated. B, radioactivity of
immunoprecipitates measured by a liquid scintillation
counter.
Incorporation of Non-myristoylated Gag Protein with
Myristoylated Gag Protein into HIV Gag VLP
Our data showed an
obvious discrepancy between the kinetics of inhibition of Gag VLP
budding and that of Gag myristoylation. To further understand this
phenomenon, we metabolically labeled infected cells overnight to allow
labeled Gag proteins to be incorporated into VLPs, fractionated VLPs
from the culture media by sucrose density gradient centrifugation, and
analyzed them on SDS-PAGE. Western blotting revealed equivalent levels
of Pr55 antigens in the VLP fractions from the cells treated with
0-24 µM triacsin C but no antigens of an equal
density fraction from the cells with 48 µM (Fig. 6A). Levels of H-myristoylated
Pr55 molecules in the VLPs, however, decreased linearly with an
increasing dose of triacsin C (Fig. 6B). These results
demonstrate that inhibition of myristoylation of almost all Gag
molecules is required for the prevention of budding of Gag VLP. Partial
inhibition leads to a reduction in the relative level of myristoylated
Gag but not the level of assembled VLP. This suggests that
non-myristoylated and myristoylated Gag molecules co-assemble in
various ratios to form complexes that are efficiently targeted to the
plasma membrane to produce Gag VLP.
H]myristic acid for 16 h (to allow
labeled Gag molecules to be incorporated into VLPs) in the presence of
triacsin C, and the VLPs released to the culture media were purified by
sucrose density gradient centrifugation. The VLP fraction was subjected
to SDS-PAGE and then analyzed by Western blotting using anti-HIV-1 p17
peptide serum (A) and by direct fluoro-autoradiography (B). Lane 1, C-rainbow protein molecular
weight markers; lanes 2-6, HIV Gag VLP fractions from
the cells cultured in the presence of triacsin C (0-48
µM) as indicated.
H-myristoylated Gag protein in the budded VLP in the
presence of triacsin C (Fig. 6). This observation suggests that
non-myristoylated and myristoylated Gag proteins co-assemble
efficiently to form Gag VLP, presumably because the addition of the
myristoyl group does not affect the conformation of the putative
assembly domains of Gag proteins. It has been shown previously that
myristoylated Gag proteins can co-assemble with non-myristoylated
Gag-Pol proteins and lead to release as part of an assembled particle (40, 41) . However, it is difficult to assess the
exact level of Gag myristoylation necessary for Gag VLP budding, as the
level of Gag-Pol incorporation found in mature HIV particles was
typically 5% of total Gag antigen. Semi-quantitative measures of the
relative levels of myristoylated and non-myristoylated Gag in our
experiments (Fig. 6) suggest that as little as 25% of the total
Gag molecules needs to be derived from myristoylated Gag to ensure VLP
release.
)
-We thank Dr. Ian Jones (Institute of Virology
and Environmental Microbiology, Oxford, UK) for a recombinant
baculovirus containing the full-length HIV-1 gag gene as well
as discussions on the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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S. Cen, M. Niu, J. Saadatmand, F. Guo, Y. Huang, G. J. Nabel, and L. Kleiman Incorporation of Pol into Human Immunodeficiency Virus Type 1 Gag Virus-Like Particles Occurs Independently of the Upstream Gag Domain in Gag-Pol J. Virol., January 15, 2004; 78(2): 1042 - 1049. [Abstract] [Full Text] [PDF]
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M. Andrawiss, Y. Takeuchi, L. Hewlett, and M. Collins Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association J. Virol., November 1, 2003; 77(21): 11651 - 11660. [Abstract] [Full Text] [PDF]
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 D. R. Larson, Y. M. Ma, V. M. Vogt, and W. W. Webb Direct measurement of Gag-Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy J. Cell Biol., September 29, 2003; 162(7): 1233 - 1244. [Abstract] [Full Text] [PDF]
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R. Halwani, A. Khorchid, S. Cen, and L. Kleiman Rapid Localization of Gag/GagPol Complexes to Detergent-Resistant Membrane during the Assembly of Human Immunodeficiency Virus Type 1 J. Virol., April 1, 2003; 77(7): 3973 - 3984. [Abstract] [Full Text] [PDF]
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 S. Sakuragi, T. Goto, K. Sano, and Y. Morikawa HIV type 1 Gag virus-like particle budding from spheroplasts of Saccharomycescerevisiae PNAS, June 11, 2002; 99(12): 7956 - 7961. [Abstract] [Full Text] [PDF]
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A. Khorchid, R. Halwani, M. A. Wainberg, and L. Kleiman Role of RNA in Facilitating Gag/Gag-Pol Interaction J. Virol., March 19, 2002; 76(8): 4131 - 4137. [Abstract] [Full Text] [PDF]
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Y. Morikawa, D. J. Hockley, M. V. Nermut, and I. M. Jones Roles of Matrix, p2, and N-Terminal Myristoylation in Human Immunodeficiency Virus Type 1 Gag Assembly J. Virol., January 1, 2000; 74(1): 16 - 23. [Abstract] [Full Text]
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 Y. Morikawa, T. Goto, and K. Sano In Vitro Assembly of Human Immunodeficiency Virus Type 1 Gag Protein J. Biol. Chem., September 24, 1999; 274(39): 27997 - 28002. [Abstract] [Full Text] [PDF]
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Y.-M. Lee, B. Liu, and X.-F. Yu Formation of Virus Assembly Intermediate Complexes in the Cytoplasm by Wild-Type and Assembly-Defective Mutant Human Immunodeficiency Virus Type 1 and Their Association with Membranes J. Virol., July 1, 1999; 73(7): 5654 - 5662. [Abstract] [Full Text]
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J.-C. Paillart and H. G. Göttlinger Opposing Effects of Human Immunodeficiency Virus Type 1 Matrix Mutations Support a Myristyl Switch Model of Gag Membrane Targeting J. Virol., April 1, 1999; 73(4): 2604 - 2612. [Abstract] [Full Text]
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S. Campbell and A. Rein In Vitro Assembly Properties of Human Immunodeficiency Virus Type 1 Gag Protein Lacking the p6 Domain J. Virol., March 1, 1999; 73(3): 2270 - 2279. [Abstract] [Full Text]
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J. B. Bowzard, R. P. Bennett, N. K. Krishna, S. M. Ernst, A. Rein, and J. W. Wills Importance of Basic Residues in the Nucleocapsid Sequence for Retrovirus Gag Assembly and Complementation Rescue J. Virol., November 1, 1998; 72(11): 9034 - 9044. [Abstract] [Full Text] [PDF]
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Y.-M. Lee, C.-J. Tian, and X.-F. Yu A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner J. Virol., November 1, 1998; 72(11): 9061 - 9068. [Abstract] [Full Text] [PDF]
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M. Yeager, E. M. Wilson-Kubalek, S. G. Weiner, P. O. Brown, and A. Rein Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: Implications for retroviral assembly mechanisms PNAS, June 23, 1998; 95(13): 7299 - 7304. [Abstract] [Full Text] [PDF]
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