Previous Article | Next Article 
Journal of Virology, November 1998, p. 9061-9068, Vol. 72, No. 11
Department of Molecular Microbiology and
Immunology, Johns Hopkins University School of Hygiene and Public
Health, Baltimore, Maryland 21205
Received 11 June 1998/Accepted 13 July 1998
It is unclear whether proteolytic processing of the human
immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus
assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to
block virus assembly and release from the plasma membrane of COS cells
but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as
virus production. We have now addressed this issue with CD4+ T cells, which are natural target cells
of HIV-1. In these cells, myristylation of Gag proteins
was required for proteolytic processing of Gag proteins and production
of extracellular viral particles. This result was not due to a lack of
expression of the viral protease in the form of a Gag-Pol precursor or
a lack of interaction between unmyristylated Gag and Gag-Pol
precursors. The processing defect of unmyristylated Gag was partially
rescued ex vivo by coexpression with wild-type
myristylated Gag proteins in HeLa cells. The cell type-dependent
processing of HIV-1 Gag precursors was also observed when another part
of the plasma membrane binding signal, a polybasic region in the matrix
protein, was mutated. The processing of unmyristylated Gag precursors
was inhibited in COS cells by HIV-1 protease inhibitors. Altogether,
our findings demonstrate that the processing of HIV-1 Gag precursors in
CD4+ T cells occurs normally at the plasma membrane during
viral morphogenesis. The intracellular environment of COS
cells presumably allows activation of the viral protease and
proteolytic processing of HIV-1 Gag proteins in the absence of plasma
membrane binding.
The infectious virions of
human immunodeficiency virus type 1 (HIV-1), like those of other
retroviruses, are produced by the envelopment of the retroviral capsid
core. This core structure, consisting of the gag and
pol gene products, is surrounded by the lipid membrane of
the host cell, which contains viral glycoproteins encoded by the
env gene (7, 26). The gag gene
of HIV-1 encodes the major structural proteins of the viral core, which
is initially synthesized as a 55-kDa polyprotein precursor
(Pr55Gag) and subsequently cleaved to yield the matrix
(MAp17), capsid (CAp24), nucleocapsid (NCp7), and p6 proteins by the
viral protease encoded by the pol gene region
(6). The pol gene encodes three enzymatic
components, including the protease (PRp11), reverse transcriptase
(RTp66/51), and integrase (INp34). The HIV-1 Pol protein is synthesized
as a precursor 160-kDa Gag-Pol fusion polyprotein (Pr160Gag-Pol) by a The newly synthesized Gag and Gag-Pol precursors are
transported from the cytoplasm to the inner face of the plasma
membrane, where viral budding takes place (7, 13, 26). In
type C retroviruses, including HIV-1, assembly of the Gag and
Gag-Pol precursors into capsids is thought to occur at the plasma
membrane simultaneously with virus budding. In type B/D
retroviruses, however, immature capsids are first
formed within the cytoplasm and then transported to the plasma
membrane for budding. In both type C and type B/D retroviruses, the
matrix domain of the Gag protein has been shown to contain a targeting
signal for intracellular transport of Gag and Gag-Pol precursors to
the plasma membrane (7, 13, 26).
In most retroviruses, myristic acid is cotranslationally and
covalently attached to the N-terminal glycine residue of the Gag
protein (21). This myristic acid modification of Gag
proteins has been shown to be essential for their intracellular
transport to the plasma membrane (7, 13, 26). In type C
retroviruses, mutations blocking this modification lead to a
failure of extracellular viral particle production (1, 3-5, 16,
17, 19, 22, 25, 30). In type B/D retroviruses, immature capsids
preassemble within the cytoplasm in the absence of
myristylation, but the capsids are not transported to
the plasma membrane and are instead accumulated in the cytoplasm
(20).
It is well established that activation of the viral protease is
required for the formation of infectious retroviral particles (24). However, when and how viral protease is activated
during viral morphogenesis remains largely unknown. In general,
mutations that prevent transport to or stable association of Gag and
Gag-Pol precursors with the plasma membrane block protease
activation (24). This observation has been well supported by
experiments in which myristic acid modification of the Gag
protein has been prevented in type C retroviruses (1, 19,
25) or in type D retroviruses (20). In retroviruses
such as Rous sarcoma virus, in which the Gag molecule is not modified
by myristic acid, mutations in the matrix protein that block
plasma membrane targeting also dramatically block protease activation
(27).
It has been suggested that HIV-1 may be an important exception to this
rule (5, 10, 24). In HIV-1, proteolytically processed mature
Gag proteins can be detected in the cytosolic fraction of infected
cells, suggesting activation of the viral protease before targeting to
the plasma membrane (10). Furthermore, unmyristylated Pr55Gag precursors in
HIV-1, unlike those in other retroviruses, are proteolytically
processed into mature viral proteins in transfected COS cells (5,
17, 30) despite the presence of defects in intracellular
transport (30) and virus assembly at the plasma membrane
(5, 17, 30). However, proteolytic processing of unmyristylated HIV-1 Gag proteins has not been observed
in transfected HeLa cells (1). Since neither COS cells
nor HeLa cells are natural target cells for HIV-1, it remains to be
determined whether myristylation of HIV-1 Gag proteins
is required for proteolytic processing of Gag precursors in clinically
relevant CD4+ T cells.
In this study, we have investigated whether the plasma membrane binding
signal in the matrix domain of the HIV-1 Gag protein is required for
proteolytic processing of the Gag precursors. We found that mutation of
the membrane binding signal resulted in a defect in the proteolytic
processing of the Gag precursors in HeLa cells but not in COS cells.
Furthermore, the Gag processing defect in the membrane binding mutants
was also observed in CD4+ T cells. Neither lack of
expression of the Pr160Gag-Pol precursor containing
the HIV-1 viral protease nor of
Pr55Gag-Pr160Gag-Pol precursor
interaction could explain the processing defect in the
unmyristylated Gag precursor. Proteolytic
processing of the unmyristylated Gag precursors in COS
cells could be inhibited by HIV-1 protease inhibitors, indicating that
HIV-1 protease may be activated prior to plasma membrane association in
COS cells. Our findings suggest that processing of HIV-1 Gag precursors
occurs normally at the plasma membrane during viral morphogenesis, by a
pathway similar to that described for other retroviruses.
Construction of mutants.
The wild-type infectious proviral
plasmid HXB2Hygro (Myr+) was constructed by replacing the neomycin
resistance gene in the infectious proviral plasmid HXB2Neo
(14) with the hygromycin resistance gene. The hygromycin
resistance gene was amplified from pCEP4 (Invitrogen) by PCR with two
primers containing ClaI and XhoI sites at the 5'
and 3' ends, respectively. The amplified and
ClaI-XhoI-digested fragments were cloned into the
infectious HXB2Neo proviral plasmid, which was digested with the
same enzymes. The isogenic myristylation-negative
mutant proviral plasmid (Myr
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 ribosomal
frameshifting mechanism (8, 28). The Pr160Gag-Pol precursor is eventually processed into
mature proteins.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) was constructed by swapping the
ApaI-ApaI fragment from the MGA plasmid
(30) into HXB2Hygro.
(18) containing the HIV-1 viral protease active-site
substitution (aspartic acid to alanine) was subcloned into the pGEM3Z
vector. The subcloned pGEM3Z vector was used to change the wild-type
nucleotide sequence 5'-AAT TTT TTA GGG-3' to 5'-AAC TTC TTA AGG G-3' at
the 1 frameshifting site. The insertion of one adenosine induces
100% fusion of the pol gene with the upstream
gag gene, and the two thymidine substitutions were
introduced to achieve expression of the
Pr160Gag-Pol fusion polyprotein without
any frameshifting into the 1 open reading frame. None of these
mutations changed the amino acid sequence of the Gag or Gag-Pol
proteins.
Cells and transfection.
COS-7 cells (an African green monkey
kidney cell line) were obtained from the American Type Culture
Collection (ATCC). HeLa CD4+ 
-galactosidase
(-Gal) cells (a derivative of a human cervical carcinoma cell line)
were obtained from the AIDS Research and Reference Reagent Program,
National Institutes of Health, Bethesda, Md. HeLa CD4+
-Gal cells were used instead of the original HeLa cells in this study because HeLa CD4+ -Gal cells have a high
transfection efficiency that results in a level of viral protein
expression comparable to that of COS-7 cells. Hereafter, HeLa
CD4+ -Gal cells are referred to as HeLa cells. Both
COS-7 and HeLa cells were maintained in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum and antibiotics. The
CD4+ T-lymphoid cell line SupT-1 was also obtained from the
American Type Culture Collection and was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics.
/SupT-1, and Pr160/SupT-1 cell
lines, uninfected SupT-1 CD4+ T cells were transfected with
Myr+, Myr, and Pr160 proviral plasmids, respectively, using
Lipofectin as suggested by the manufacturer (Gibco BRL). At 48 h
after transfection, cells were selected with 0.8 mg of hygromycin B per
ml (for Myr+/SupT-1 and Myr/SupT-1) or 1.2 mg of G418 per ml (for
Pr160/SupT-1) for 2 to 3 weeks until stable CD4+ T
cells were generated. Myr+/H9 and Myr/H9 cell lines were also generated, as described above for Myr+/SupT-1 and Myr/SupT-1 cells.
The established CD4+ T-cell lines were batch selected and
maintained in RPMI 1640 with 10% fetal bovine serum, antibiotics, and
0.2 mg of hygromycin B or G418 per ml.
Protein analysis and sera. Immunoblotting of cell lysates or extracellular viral particles was performed as previously described (14). An HIV-1-positive human serum was obtained from an HIV-1-infected patient from Baltimore, Md. Goat anti-HIV-1 p6 antiserum has been previously described (29). Rabbit anti-CAp24 antiserum was obtained from the AIDS Research and Reference Reagent Program, National Institutes of Health. Mouse monoclonal anti-RTp66/51 antibody was purchased from BTI, Columbia, Md. Alkaline phosphatase (AP)-conjugated goat anti-rabbit immunoglobulin G (IgG) and AP-conjugated rabbit anti-goat IgG antibodies were purchased from Sigma ImmunoResearch, and AP-conjugated goat anti-mouse IgG antibody was purchased from Jackson ImmunoResearch Laboratories, Inc.
Coimmunoprecipitation. The SupT-1-derived T-cell lines were lysed by incubation at room temperature for 5 min in phosphate-buffered saline (PBS) containing 1% Triton X-100, and the cell lysates were clarified by centrifugation at 14,000 rpm for 20 min. The precleared cell lysates were incubated overnight at 4°C with protein A-Sepharose beads that had been preincubated with polyclonal anti-HIV-1 p6 antiserum and then washed with PBS containing 1% Triton X-100. After incubation, the immunoprecipitated materials were obtained by centrifugation and six washes with PBS containing 1% Triton X-100. To release the immunoprecipitates, the Sepharose beads were boiled in sample loading buffer (0.08 M Tris-HCl [pH 6.8], 2.0% sodium dodecyl sulfate [SDS], 10% glycerol, 0.1 M dithiothreitol, 0.2% bromophenol blue) for 5 min and then briefly centrifuged at 12,000 × g. The immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and viral proteins were visualized by immunoblotting.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Myristic acid modification of the HIV-1 Gag proteins is essential for proteolytic processing of Gag precursors in a cell type-dependent manner. It appears that myristic acid modification of HIV-1 Gag protein is not required for proteolytic processing of Pr55Gag precursors in the African green monkey kidney cell line COS (5, 17, 30). On the other hand, myristylation is required for processing of Pr55Gag precursors in the human cervical carcinoma cell line HeLa (1). The discrepancy between these previous studies has not yet been resolved and could be attributed to the use of different plasmid constructs, transfection methods, levels of protein expression, or cell types to express the HIV-1 viral proteins. We examined all of these possibilities by transfecting these two cell types with the same plasmid construct by the same transfection method. The expressions of the viral proteins after transfection were comparable in the COS and HeLa cells.
In COS cells transfected with mutant Myr proviruses, immunoblotting
with an HIV-1-positive human serum clearly showed the presence of the
mature CAp24 and cleavage-intermediate p25 proteins, containing CAp24
and the p2 spacer peptide (Fig. 1A). The
mature CAp24 and cleavage-intermediate p25 proteins were confirmed by immunoblotting with a polyclonal anti-CAp24 antiserum (Fig. 1B). As
expected, the Pr55Gag precursors in COS cells
transfected with wild-type Myr+ proviruses were also processed into
mature viral proteins (Fig. 1A and B). It is noteworthy that the p25
cleavage intermediate was relatively more abundant in the Myr
transfected COS cells than in the Myr+ transfected COS
cells (compare lanes 2 and 3 in Fig. 1A and B).
|
transfected HeLa cells (Fig. 1C, lane 2), although the
Pr55Gag precursors were processed into mature
viral proteins in Myr+ transfected HeLa cells (Fig. 1C, lane 3).
It is unlikely that the Pr55Gag precursors in the Myr
transfected HeLa cells are being processed and the cleavage products
are simply being degraded very rapidly, because there was
more uncleaved Pr55Gag in Myr transfected
HeLa cells (Fig. 1C, lane 2) than in Myr+ transfected HeLa cells (Fig.
1C, lane 3).
The difference in HIV-1 Gag processing between COS and HeLa cells was
not simply due to the concentration of Gag precursor molecules. When
cell lysates from equal numbers of transfected COS and HeLa cells were
used, the amount of Gag protein detected in the HeLa cells was not
lower than in the COS cells (compare Fig. 1A and C). Comparable
expression of Gag proteins in transfected COS and HeLa cells was
further demonstrated in other experiments (see Fig. 2). Therefore, the
differences in proteolytic processing of unmyristylated
Pr55Gag precursors that we observed in the two cell
lines probably reflect differences in cell type rather than in
other experimental conditions.
A polybasic domain at the N terminus of the HIV-1 Gag proteins is also required for proteolytic processing of Gag precursors in a cell type-dependent manner. It is not clear whether myristylation of HIV-1 Gag per se or plasma membrane binding of Gag and Gag-Pol molecules is required for Gag processing in HeLa cells but not in COS cells. In addition to the importance of the myristic acid modification, a polybasic domain at the N terminus of HIV-1 Gag protein has also been shown to play an important role in membrane binding (30, 31). To test the role of the polybasic domain in proteolytic processing of HIV-1 Gag proteins, we took advantage of a mutant, B5, in which substitutions have been made in five basic residues in the polybasic domain at the N terminus of the HIV-1 Gag protein (30). The B5 mutant has been shown to have a defect in intracellular transport of the Gag precursors and production of extracellular viral particles (30).
In this experiment, COS cells or HeLa cells were mock transfected or transfected with either the wild-type or the mutant B5 plasmid. At 3 days after transfection, the cells were lysed and analyzed by SDS-PAGE and immunoblotting. Immunoblotting with an HIV-1-positive human serum (Fig. 2A) and the polyclonal anti-CAp24 antiserum (Fig. 2B) demonstrated that the mutant Pr55Gag precursors from COS cells transfected with the B5 plasmid were processed into the mature CAp24 and p25 viral proteins, as seen in COS cells transfected with the Myr mutant (Fig.
1). As expected, the wild-type Pr55Gag
precursors from COS cells transfected with wild-type plasmid were
also efficiently processed into mature CAp24 proteins.
|
Myristic acid modification of the HIV-1 Gag proteins is
essential for proteolytic processing of Gag proteins in
CD4+ T cells.
To examine the role of
myristic acid modification of the N terminus of the Gag protein
in processing of Pr55Gag precursors in
CD4+ T cells, we generated SupT-1 CD4+
T-cell lines expressing unmyristylated
Pr55Gag and Pr160Gag-Pol
proteins (Myr/SupT-1) and wild-type myristylated
Pr55Gag and Pr160Gag-Pol
proteins (Myr+/SupT-1).
/SupT-1 cells were
expressed at levels comparable to those in the Myr+/SupT-1 cells
(Fig. 3A, lanes 2 and 3). The
myristylated Pr55Gag precursors in the
Myr+/SupT-1 cells were efficiently processed into mature viral proteins
such as CAp24 (Fig. 3A, lane 3). In the mutant Myr/SupT-1 cells,
however, processing of unmyristylated Pr55Gag proteins into CAp24 was not detected (Fig. 3A,
lane 2).
|
/SupT-1 cells (Fig. 3B, lane 2) was comparable to
that in the wild-type Myr+/SupT-1 cells (Fig. 3B, lane 3).
Therefore, the defect in the processing of
Pr55Gag precursors could not be explained by a lack of
Pr160Gag-Pol precursor expression.
Immunoblotting with HIV-1-positive human serum also
demonstrated that in SupT-1 CD4+ T cells, the
myristic acid modification of HIV-1 Gag proteins is essential
for production of extracellular viral particles (Fig. 3C). This finding
is consistent with results of previous studies conducted with COS and
HeLa cells (1, 5, 17, 30).
In addition to our findings with SupT-1 cells, the processing defect in
the HIV-1 Gag precursors in the absence of myristic acid
modification was also observed in H9 CD4+ T cells.
Immunoblotting with HIV-1-positive human serum demonstrated that
the Pr55Gag precursors in the Myr/H9 cells were
expressed at levels comparable to those in the Myr+/H9 cells
(Fig. 3D, lanes 2 and 3). The myristylated Pr55Gag precursors in the Myr+/H9 cells were
efficiently processed into mature viral proteins such as CAp24 (Fig.
3D, lane 3). In the mutant Myr/H9 cells, however, processing of
unmyristylated Pr55Gag proteins into
CAp24 was not detected (Fig. 3D, lane 2).
Stable T-cell lines were used for these studies because it was
difficult to detect viral Gag proteins in CD4+ T cells
after transient transfection. It is unlikely that the results would be
substantially different in transiently transfected T cells, since we
observed similar Gag processing defects in the myristylation mutants in transiently and
stably transfected HeLa cells (data not shown). However, the
possibility that unmyristylated Pr55Gag
could be processed in transiently transfected T cells has not been formally excluded.
Pr55Gag precursors are associated with
Pr160Gag-Pol precursors in
myristylation-negative mutant CD4+ T
cells.
It is possible that the lack of
Pr55Gag processing in mutant
Myr/ SupT-1 CD4+ T cells was caused
by a lack of interaction between
unmyristylated Pr55Gag and
unmyristylated Pr160Gag-Pol
precursors without targeting to the plasma membrane. This
possibility was tested by coimmunoprecipitation analysis. A polyclonal
anti-p6 antiserum (29) recognizing only the
Pr55Gag precursor was used to coprecipitate the
Pr160Gag-Pol precursor. The p6 domain is present at
the C terminus of the Pr55Gag precursor but not the
Pr160Gag-Pol precursor. If an interaction occurred
between the Pr55Gag and
Pr160Gag-Pol precursors, the polyclonal anti-p6
antiserum would coimmunoprecipitate the
Pr160Gag-Pol precursor in the presence of the
Pr55Gag precursor but not in its absence.
/SupT-1, Myr+/SupT-1, and Pr160/SupT-1 cells with an HIV-1-positive human serum showed that the Pr55Gag precursors were detected in the
Myr/SupT-1 and Myr+/SupT-1 cells (Fig.
4A, lanes 2 and 3) but not in the
Pr160/SupT-1 cells (Fig. 4A, lane 4). A monoclonal
anti-RTp66/51 antibody showed that comparable amounts of
Pr160Gag-Pol precursors were expressed in all three
cell lines (Fig. 4B, lanes 2 to 4).
|
/SupT-1, Myr+/SupT-1, and Pr160/SupT-1
cells were first immunoprecipitated with anti-p6 antiserum.
The immunoprecipitated materials were then separated by
SDS-PAGE and visualized by immunoblotting. Immunoblotting with
HIV-1-positive human serum revealed comparable quantities of
Pr55Gag precursors in the immunoprecipitated materials
from the mutant Myr/SupT-1 and wild-type Myr+/SupT-1 cells but, as
expected, not from the Pr160/SupT-1 cells (Fig. 4C). The
immunoglobulin heavy chain from the goat anti-p6 antiserum which
was used for immunoprecipitation was detected in lanes 1 and 4 in Fig.
4C, presumably because of a weak cross-reactivity of the alkaline phosphatase-conjugated rabbit anti-human IgG with the immunoglobulin heavy chain in the goat anti-p6 antiserum. These bands were not detected when the goat anti-p6 antiserum was not used during
immunoprecipitation (Fig. 4C, lanes 5 and 6).
Upon longer exposure of the same immunoblots, comparable quantities of
Pr160Gag-Pol precursors were detected in the
lysates from the Myr/SupT-1 and Myr+/SupT-1 cells (Fig. 4D, lanes 2 and 3) but not in those from the Pr160/SupT-1 cells (Fig. 4D, lane
4). The coimmunoprecipitation of Pr160Gag-Pol
with either Myr+ or Myr Pr55Gag was further confirmed
by immunoblotting with a monoclonal anti-RTp66/51 antibody (Fig.
4E, lanes 2 and 3). The Pr160Gag-Pol precursors
were not detected by the anti-RTp66/51 antibody when the cell lysate of
Pr160/SupT-1 cells was immunoprecipitated with p6 antiserum (Fig.
4E, lane 4), demonstrating that the coimmunoprecipitation of
Pr160Gag-Pol precursors from the Myr/SupT-1 and
Myr+/SupT-1 cell lysates could not be explained by cross-reactivity of
p6 antiserum to Pr160Gag-Pol. In the absence of p6
antiserum, neither the Pr55Gag nor the
Pr160Gag-Pol precursors of the viral proteins were
precipitated by protein A-Sepharose beads (Fig. 4C to E, lanes 5 and 6), indicating that the
Pr160Gag-Pol precursors were specifically
coimmunoprecipitated with the Pr55Gag precursors by
p6 antiserum.
In addition to the coimmunoprecipitation analysis presented here, we
have recently demonstrated that unmyristylated Gag and Gag-Pol proteins of HIV-1 form an assembly-intermediate
complex, which is characterized as a large oligomer that has a
density of 1.10 to 1.13 g/ml and is primarily composed of
Pr55Gag and Pr160Gag-Pol precursors
in infected CD4+ T cells (15). All of these
findings are consistent with the idea that the interaction between
unmyristylated Gag and Gag-Pol precursors may occur
before their association with the plasma membrane.
The processing defect in the Pr55Gag precursors in the myristylation-negative mutant is rescued ex vivo by coexpression with the wild-type Pr55Gag proteins. Myristic acid modification of HIV-1 Gag proteins may alter the conformation of the Pr55Gag and Pr160Gag-Pol precursors; in the absence of this modification, it is possible that misfolded Pr55Gag precursors cannot be processed into mature viral proteins. However, this explanation is unlikely, since previous experiments have demonstrated that the lack of myristylation does not alter the conformation of Gag molecules (16) and that unmyristylated Pr55Gag precursors can be processed efficiently in vitro by HIV-1 protease (1). Another possibility is that misfolding of unmyristylated Pr160Gag-Pol precursors generates a defective protease that cannot be activated. This possibility is also less likely, because it has been suggested that the lack of myristylation does not alter the conformation of the Gag-Pol molecules (18). The other possibility is that the defect in processing of unmyristylated Pr55Gag precursors might result from a lack of targeting of the Pr160Gag-Pol precursors to the plasma membrane, a prerequisite for activation of the viral protease. If this explanation is the case, providing a plasma membrane targeting signal in trans by coexpression with wild-type myristylated Pr55Gag precursors should activate the viral protease activity.
To distinguish among these possibilities, we examined whether expression of myristylated Pr55Gag precursors in trans could induce processing of Pr55Gag precursors by HIV-1 protease of the unmyristylated Pr160Gag-Pol precursors. The myristylated Pr55Gag precursors were provided by the
Pol plasmid, which contains a
complete deletion of the HIV-1 pol gene including the viral protease. Transfection of this construct into HeLa cells resulted in
expression of unprocessed Pr55Gag precursors alone, as
detected by the HIV-1-positive human serum (Fig.
5A, lane 4). Furthermore, CAp24 proteins
were not detected in HeLa cells transfected with the mutant Myr
construct alone (Fig. 5A, lane 3); however, mature CAp24 proteins
were detected in the cells cotransfected with the mutant Myr and
Pol plasmids (Fig. 5A, lane 6). A larger quantity of mature CAp24
proteins was detected in cells transfected with the wild-type Myr+
plasmid (Fig. 5A, lane 2) or cotransfected with the Myr+ and Pol
plasmids (Fig. 5A, lane 5), as determined by reactivity with the
HIV-1-positive human serum. The specificity of CAp24 production
was verified by reactivity with the polyclonal anti-CAp24
antiserum (Fig. 5B). This finding demonstrated that the
HIV-1 viral protease of the unmyristylated
Pr160Gag-Pol precursors could be activated when
a plasma membrane targeting signal was provided by
myristylated Pr55Gag
precursors in trans. This finding is consistent with a
previous report that unmyristylated HIV-1 Gag proteins
can interact with myristylated Gag proteins and are
incorporated into released viral particles (16).
|
and Pol plasmids than in those cells transfected with
the Myr+ and Pol plasmids (compare lanes 5 and 6 in Fig. 5),
suggesting that the interaction between
myristylated Pr55Gag precursors and
unmyristylated Pr55Gag or
Pr160Gag-Pol precursors expressed from two separate
mRNAs may be less efficient than that expressed from a single mRNA.
Processing of unmyristylated
Pr55Gag precursors in COS cells was inhibited by HIV-1
protease inhibitors.
It is possible that the HIV-1 protease in the
myristylation mutant virus is activated in COS cells.
Alternatively, unmyristylated Pr55Gag
precursors could be cleaved by a cellular protease in COS cells. To
address this issue, COS cells were mock transfected or transfected with
the wild-type, the myristylation mutant, or the HIV-1
protease mutant Pr plasmid (15). After transfection,
half of the cells were treated with the HIV-1 protease inhibitor and
the other half were used as an untreated control. At 3 days after
transfection, the cells were lysed and analyzed by SDS-PAGE and
immunoblotting.
(Fig. 6, lanes 3 and 7). These data suggest that
processing of unmyristylated Pr55Gag
precursors in COS cells was due to activation of the viral protease and
not the activity of a cellular protease.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
In this study, we have demonstrated that myristylation of the HIV-1 Gag protein is essential for the proteolytic processing of Pr55Gag precursors as well as for virus production in CD4+ T cells. In HIV-1, the role of myristic acid modification of Gag proteins in the proteolytic processing of Gag precursors has been controversial. Some experiments have demonstrated that myristic acid modification of the HIV-1 Gag protein is required for the proteolytic processing of Gag precursors in HeLa cells (1). Other experiments have shown that processing of unmyristylated HIV-1 Gag protein can occur in COS cells, suggesting that activation of the viral protease occurs prior to plasma membrane association (5, 17, 30). Our data suggest that the normal pathway for HIV-1 viral protease activation in CD4+ T cells requires plasma membrane targeting and association.
Although the expression of the unmyristylated Pr160Gag-Pol precursors containing the viral protease and the interaction between the unmyristylated Pr160Gag-Pol and the unmyristylated Pr55Gag precursors were not affected in CD4+ T cells and HeLa cells (data not shown), the processing of unmyristylated Pr55Gag precursors into mature viral proteins was not observed in these cells. When the plasma membrane targeting signal was provided by wild-type Gag molecules in trans, the HIV-1 viral protease of the unmyristylated Pr160Gag-Pol precursors was able to process the Pr55Gag precursors into mature viral proteins in HeLa cells.
In addition to myristic acid modification, it has been shown that a polybasic domain at the N terminus of HIV-1 Gag proteins plays an important role in the intracellular transport and plasma membrane association of the Pr55Gag and Pr160Gag-Pol precursors (30, 31). Mutations substituting five basic-charge residues in the polybasic domain dramatically abolished the proteolytic processing of HIV-1 Gag precursors into mature viral proteins in HeLa cells and CD4+ T cells (data not shown) but not in COS cells. Since processing of membrane binding mutant Gag precursors was observed in nonhuman COS cells but not in human HeLa or CD4+ T cells, these findings demonstrate that the two elements of the bipartite plasma membrane binding signal at the N terminus of the HIV-1 Gag protein, myristic acid modification and a polybasic domain, are simultaneously required for productive processing of HIV-1 Gag precursors. Altogether, these results reinforce the idea that productive processing of Gag precursors in all retroviruses, including HIV-1 (11), is preceded by the targeting of Gag and Gag-Pol precursors to the plasma membrane (24).
Assembly, budding, and maturation of retroviruses are highly dynamic and tightly regulated processes (7, 24, 26). Since assembly is achieved by uncleaved Gag and Gag-Pol precursors, premature activation of the viral protease may be detrimental to virus assembly (2, 12). In general, activation of the retroviral protease is dependent on virus assembly and budding at the plasma membrane (24). At present, the mechanism(s) by which retroviral protease activity is regulated is largely unknown, but several mechanisms have been proposed (24).
It is possible that retroviral proteases, including the HIV-1 protease, can be activated by autoprocessing, through a mechanism similar to that seen for pepsin (9). Dimerization of HIV-1 viral protease is known to be a prerequisite for its function (24). In this regard, dimerization of Pr160Gag-Pol precursors, which is required for autoprocessing, may not occur until virus assembly and budding are initiated at the plasma membrane.
Autoprocessing of HIV-1 Pr160Gag-Pol precursors may also be dependent upon conformational changes in the molecules that can be influenced by conditions in the cellular environment such as pH, lipid composition, or salt concentration. These conditions may differ between the cytoplasm and the plasma membrane, where virus budding occurs. Conformational changes in Pr160Gag-Pol precursors could also be induced by protein modifications such as phosphorylation or dephosphorylation, which are accomplished by cellular enzymes at the plasma membrane.
Another possibility is that the HIV-1 protease is activated from the Pr160Gag-Pol precursors by processing with a cellular protease, through a process similar to that seen for trypsin and other serine proteases (23). One can imagine that a cellular protease, normally localized only on the inner face of the plasma membrane, could partially cleave Pr160Gag-Pol precursors and liberate the viral protease. This event would subsequently trigger a cascade processing of viral Pr160Gag-Pol and Pr55Gag precursors by the released viral protease.
In addition to regulation of HIV-1 viral protease activity by activation, it is also possible that the viral protease is regulated by inhibition. During the late stages of the viral life cycle, the activity of HIV-1 viral protease can be suppressed by a cellular factor(s) until virus assembly and budding take place at the plasma membrane. At the plasma membrane, the inhibitory cellular factors are removed from the budding particles, allowing subsequent activation of the viral protease and production of infectious virions.
Taken together, our data and other previously published observations suggest that cellular factors, which normally are restricted to the plasma membrane, are required for activation of the HIV-1 protease in CD4+ T cells. It remains to be determined what triggers HIV-1 protease activation in COS cells in view of the apparent lack of association of Pr55Gag and Pr160Gag-Pol precursors with the plasma membrane. Further study will be required to elucidate the mechanism of HIV-1 protease activation and yield data that may lead to the development of effective antiviral agents.
| |
ACKNOWLEDGMENTS |
|---|
We thank Casey Morrow for the pGPpr construct, Richard
Markham and David Schwartz for comments on the manuscript, and Liza Dawson for helpful discussions on the project.
This work was supported by Public Health Service grants AI-35525 and DA-09541 from the National Institutes of Health.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Room E4012, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-3768. Fax: (410) 614-8263. E-mail: xfyu{at}jhsph.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. |
Bryant, M., and L. Ratner.
1990.
Myristoylation-dependent replication and assembly of human immunodeficiency virus 1.
Proc. Natl. Acad. Sci. USA
87:523-527 |
| 2. |
Burstein, H.,
D. Bizub, and A. M. Skalka.
1991.
Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers.
J. Virol.
65:6165-6172 |
| 3. |
Freed, E. O.,
J. M. Orenstein,
A. J. Buckler-White, and M. A. Martin.
1994.
Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production.
J. Virol.
68:5311-5320 |
| 4. | Gheysen, D., E. Jacobs, F. de Foresta, C. Thiriart, M. Francotte, D. Thines, and M. de Wilde. 1989. Assembly and release of HIV-1 precursor Pr55 Gag virus-like particles from recombinant baculovirus-infected insect cells. Cell 59:103-112[Medline]. |
| 5. |
Gottlinger, H. G.,
J. G. Sodroski, and W. A. Haseltine.
1989.
Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus 1.
Proc. Natl. Acad. Sci. USA
86:5781-5785 |
| 6. | Henderson, L. E., T. D. Copeland, R. C. Sowder, A. M. Schultz, and S. Oroszlan. 1988. Analysis of proteins and peptides from sucrose gradient banded HTLV-III, p. 135-147. In D. Bolognesi (ed.), Human retroviruses, cancer and AIDS: approaches to prevention and therapy. Wiley Interscience, New York, N.Y. |
| 7. | Hunter, E. 1994. Macromolecular interactions in the assembly of HIV and other retroviruses. Semin. Virol. 5:71-83. |
| 8. | Jacks, T., M. D. Power, F. R. Masiarz, P. A. Luciw, P. J. Barr, and H. E. Varmus. 1988. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature (London) 331:280-283[Medline]. |
| 9. | James, M. N. G., and A. R. Sielecki. 1986. Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8 A resolution. Nature (London) 319:33-38[Medline]. |
| 10. |
Kaplan, A. H., and R. Swanstrom.
1991.
Human immunodeficiency virus type 1 Gag proteins are processed in two cellular compartments.
Proc. Natl. Acad. Sci. USA
88:4528-4532 |
| 11. |
Kaplan, A. H.,
M. Manchester, and R. Swanstrom.
1994.
The activity of the protease of human immunodeficiency virus type 1 is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum efficiency.
J. Virol.
68:6782-6786 |
| 12. |
Krausslich, H. G.
1991.
Human immunodeficiency virus proteinase dimer as a compartment of the viral polyprotein prevents particle assembly and viral infectivity.
Proc. Natl. Acad. Sci. USA
88:3213-3217 |
| 13. | Krausslich, H. G., and R. Welker. 1996. Intracellular transport of retroviral capsid components. Curr. Top. Microbiol. Immunol. 214:25-63[Medline]. |
| 14. | Lee, Y.-M., X.-B. Tang, L. M. Cimakasky, J. E. K. Hildreth, and X.-F. Yu. 1997. Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes. J. Virol. 71:1443-1452[Abstract]. |
| 15. | Lee, Y.-M., and X.-F. Yu. 1998. Identification and characterization of virus assembly intermediate complexes in HIV-1-infected CD4+ T cells. Virology 243:78-93[Medline]. |
| 16. |
Morikawa, Y.,
S. Hinata,
H. Tomoda,
T. Goto,
M. Nakai,
C. Aizawa,
H. Tanaka, and S. Omura.
1996.
Complete inhibition of human immunodeficiency virus Gag myristoylation is necessary for inhibition of particle budding.
J. Biol. Chem.
271:2868-2873 |
| 17. | Pal, R., M. S. Reitz, Jr., E. Tschachler, R. C. Gallo, M. G. Sarngadharan, and F. D. Veronese. 1990. Myristoylation of Gag proteins of HIV-1 plays an important role in virus assembly. AIDS Res. Hum. Retroviruses. 6:721-730[Medline]. |
| 18. |
Park, J., and C. D. Morrow.
1992.
The nonmyristylated Pr160Gag-Pol polyprotein of human immunodeficiency virus type 1 interacts with Pr55Gag and is incorporated into viruslike particles.
J. Virol.
66:6304-6313 |
| 19. |
Rein, A.,
M. R. McClure,
N. R. Rice,
R. B. Luftig, and A. M. Schultz.
1986.
Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.
Proc. Natl. Acad. Sci.
83:7246-7250 |
| 20. |
Rhee, S. S., and E. Hunter.
1987.
Myristylation is required for intracellular transport but not for assembly of D-type retrovirus capsids.
J. Virol.
61:1045-1053 |
| 21. | Schultz, A. M., L. E. Henderson, and S. Oroszlan. 1988. Fatty acylation of proteins. Annu. Rev. Cell Biol. 4:611-647. |
| 22. |
Spearman, P.,
J.-J. Wang,
N. Vander Heyden, and L. Ratner.
1994.
Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly.
J. Virol.
68:3232-3242 |
| 23. | Stroud, R. M., A. A. Kossiakoff, and J. L. Chambers. 1977. Mechanisms of zymogen activation. Annu. Rev. Biophys. Bioeng. 6:177-193[Medline]. |
| 24. | Vogt, V. M. 1996. Proteolytic processing and particle maturation. Curr. Top. Microbiol. Immunol. 214:95-131[Medline]. |
| 25. |
Weaver, T. A., and A. T. Panganiban.
1990.
N myristoylation of the spleen necrosis virus matrix protein is required for correct association of the Gag polyprotein with intracellular membranes and for particle formation.
J. Virol.
64:3995-4001 |
| 26. | Wills, J. W., and R. C. Craven. 1991. Form, function, use of retroviral Gag proteins. AIDS (Philadelphia) 5:639-654. |
| 27. |
Wills, J. W.,
R. C. Craven,
R. A. Weldon, Jr.,
T. D. Nelle, and C. R. Erdie.
1991.
Suppression of retroviral MA deletions by the amino-terminal membrane-binding domain of p60src.
J. Virol.
65:3804-3812 |
| 28. | Wilson, W., M. Braddock, S. E. Adams, P. D. Rathjen, S. M. Kingsman, and A. J. Kingsman. 1988. HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems. Cell 55:1159-1169[Medline]. |
| 29. |
Yu, X.-F.,
Z. Matsuda,
Q.-C. Yu,
T.-H. Lee, and M. Essex.
1995.
Role of the C terminus Gag protein in human immunodeficiency virus type 1 virion assembly and maturation.
J. Gen. Virol.
76:3171-3179 |
| 30. |
Yuan, X.,
X.-F. Yu,
T.-H. Lee, and M. Essex.
1993.
Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor.
J. Virol.
67:6387-6394 |
| 31. |
Zhou, W.,
L. J. Parent,
J. W. Wills, and M. D. Resh.
1994.
Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids.
J. Virol.
68:2556-2569 |
Journal of Virology, November 1998, p. 9061-9068, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Lehmann, M., Milev, M. P., Abrahamyan, L., Yao, X.-J., Pante, N., Mouland, A. J.
(2009). Intracellular Transport of Human Immunodeficiency Virus Type 1 Genomic RNA and Viral Production Are Dependent on Dynein Motor Function and Late Endosome Positioning. J. Biol. Chem.
284: 14572-14585
[Abstract] [Full Text] -
Abdurahman, S., Hoglund, S., Goobar-Larsson, L., Vahlne, A.
(2004). Selected amino acid substitutions in the C-terminal region of human immunodeficiency virus type 1 capsid protein affect virus assembly and release. J. Gen. Virol.
85: 2903-2913
[Abstract] [Full Text] -
Fackler, O. T., d'Aloja, P., Baur, A. S., Federico, M., Peterlin, B. M.
(2001). Nef from Human Immunodeficiency Virus Type 1F12 Inhibits Viral Production and Infectivity. J. Virol.
75: 6601-6608
[Abstract] [Full Text] -
Qiu, J.-T., Liu, B., Tian, C., Pavlakis, G. N., Yu, X.-F.
(2000). Enhancement of Primary and Secondary Cellular Immune Responses against Human Immunodeficiency Virus Type 1 Gag by Using DNA Expression Vectors That Target Gag Antigen to the Secretory Pathway. J. Virol.
74: 5997-6005
[Abstract] [Full Text] -
Sei, S., Yang, Q.-e., O'Neill, D., Yoshimura, K., Nagashima, K., Mitsuya, H.
(2000). Identification of a Key Target Sequence To Block Human Immunodeficiency Virus Type 1 Replication within the gag-pol Transframe Domain. J. Virol.
74: 4621-4633
[Abstract] [Full Text] -
Nguyen, D. H., Hildreth, J. E. K.
(2000). Evidence for Budding of Human Immunodeficiency Virus Type 1 Selectively from Glycolipid-Enriched Membrane Lipid Rafts. J. Virol.
74: 3264-3272
[Abstract] [Full Text] -
Qiu, J.-T., Song, R., Dettenhofer, M., Tian, C., August, T., Felber, B. K., Pavlakis, G. N., Yu, X.-F.
(1999). Evaluation of Novel Human Immunodeficiency Virus Type 1 Gag DNA Vaccines for Protein Expression in Mammalian Cells and Induction of Immune Responses. J. Virol.
73: 9145-9152
[Abstract] [Full Text] -
Buchschacher, G. L. Jr., Yu, L., Murai, F., Friedmann, T., Miyanohara, A.
(1999). Association of Murine Leukemia Virus Pol with Virions, Independent of Gag-Pol Expression. J. Virol.
73: 9632-9637
[Abstract] [Full Text] -
Lee, Y.-M., Liu, B., Yu, X.-F.
(1999). 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.
73: 5654-5662
[Abstract] [Full Text] -
Ono, A., Freed, E. O.
(1999). Binding of Human Immunodeficiency Virus Type 1 Gag to Membrane: Role of the Matrix Amino Terminus. J. Virol.
73: 4136-4144
[Abstract] [Full Text] -
Liu, B., Dai, R., Tian, C.-J., Dawson, L., Gorelick, R., Yu, X.-F.
(1999). Interaction of the Human Immunodeficiency Virus Type 1 Nucleocapsid with Actin. J. Virol.
73: 2901-2908
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»