(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), ()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) .
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 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 mM
C-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.
Figure 2:
Effect of triacsin C on release of HIV Gag
protein. Monolayers of 2 10
Sf9 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.
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)-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.
Figure 6:
Incorporation of myristoylated Gag
proteins into HIV Gag VLPs. At 36 h postinfection, the cells were
metabolically labeled with [9,10 (n)-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.
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 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.
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.