(Received for publication, December 5, 1994)
From the
The human immunodeficiency virus type 1 internal structural
protein precursor, p55, and its corresponding matrix proteolytic
fragment, p17, are phosphorylated at Ser by protein
kinase C. COS-7 cells transfected with plasmids encoding either the
wild-type or Ser
Ala mutated human
immunodeficiency virus type 1 gag gene matrix domain proteins
were treated with phorbol 12-myristate 13-acetate (PMA), and the
phosphorylation of the expressed p17 proteins was examined by
radioimmunoprecipitation, SDS-polyacrylamide gel electrophoresis, and
autoradiography. PMA treatment of transfected cells resulted in a
4-5-fold increase in wild-type p17 (but not mutated p17)
phosphorylation; however, mutated p17 exhibited a low basal level of
phosphorylation that was not affected by PMA, suggesting that
additional sites were phosphorylated. PMA treatment of cells expressing
wild-type p17 produced a dramatic shift in the localization of p17 from
the cytosol to the membrane fraction within 8-15 min, followed by
a slow quantitative dissociation of p17 back into the cytosol by 90
min. The cytosol-to-membrane translocation was dependent on N-myristoylated p17 since cells expressing p17 with a
Gly
Ala mutation did not localize to the membrane.
PMA also failed to induce the translocation of fully N-myristoylated Ser
Ala p17, suggesting
that p17 phosphorylation at Ser
was responsible for
membrane association. This conclusion was confirmed by the finding of
phosphorylated wild-type p17 in the membrane fraction only after PMA
treatment. These results suggest that a ``myristoyl-protein
switch'' regulates the reversible membrane targeting of p17 by
protein kinase C-mediated phosphorylation. This signal may provide a
mechanism for the cellular regulation of virus development through
modulation of gag protein-related developmental steps such as capsid
targeting, assembly, encapsidation, budding, and maturation.
The internal structural proteins of HIV-1 ()are
central to its replication and may play an important role in both the
early and late phases of the virus life
cycle(1, 2, 3, 4, 5, 6) .
The internal structural proteins are initially synthesized by the HIV-1 gag gene as a precursor polypeptide, p55, which contains all
of the signals necessary for membrane targeting, viral particle
assembly, and budding from the host cell(5) . Subsequent
morphogenesis of the immature virus involves the proteolytic processing
of p55 into gag fragments that direct the maturation of the virus into
infectious virions (7, 8, 9) and that, upon
reinfection, participate in the nuclear targeting and integration of
the provirus into the host's
genome(6, 10, 11, 12) .
Of specific importance to each of these phases of the virus life cycle is the N-terminal 17-kDa MA domain of p55 or its corresponding proteolytic fragment, p17. The MA protein exhibits unique structural and functional properties depending upon the phase of the virus life cycle. For example, the MA domain provides the signal for the cotranslational N-myristoylation of p55 on its N-terminal glycine(13, 14, 15) . N-Myristoylation and gag polypeptide sequence(s) outside of the matrix domain (16) have been shown to be essential for p55 membrane association and for the ensuing events involved in viral particle assembly(14) . During this assembly process, sequences within the N-terminal 100 residues of the MA domain are also required for recruitment of the viral envelope glycoprotein into the assembled virion(10, 12) . Subsequent maturation involves release of the MA domain by a virally encoded protease and association of the resulting p17 fragment with the inner virus envelope by an unknown mechanism(7, 8, 9) . With each new round of infection, the karyophilic properties of the prerequisite viral preintegration complex have been ascribed to a basic domain covering amino acids 25-33 of p17(6, 12, 17, 18) . Thus, the participation of the MA protein in several different aspects of virus development is driven by primary sequence and tertiary structural features that sequentially become manifest at successive stages of virus formation. The functional consequences of these MA protein-driven processes also depend upon the timely presence of both viral and cellular components, which provide a necessary structural environment for the progress of virus development; and it is likely that one or more of these steps are susceptible to independent regulation by the host cell.
We have previously demonstrated that HIV gag proteins are
phosphorylated in vivo by PKC and have identified a
prospective PKC phosphorylation site motif at Ser within
the MA domain of p55 that is highly conserved among all strains of
HIV-1(19, 20) . The presence of this sequence implies
that PKC phosphorylation may play a role in regulating the function of
p55 and p17. In this report, we have expressed the N-terminal 17-kDa MA
domain of HIV-1 p55 (i.e. p17) to determine the functional
consequences of gag protein phosphorylation. Activation of PKC with PMA
in cells expressing p17 resulted in the reversible translocation of p17
from the cytosol to the membrane fraction. In addition, p17 membrane
association was specifically mediated by both the phosphorylation of
Ser
by PKC and N-myristoylation. The
identification of this new membrane targeting signal provides an
interesting clue into cellular mechanisms that may regulate the
function of HIV gag proteins during virus replication.
The p17
coding sequence used for all constructs consisted of the 396-base pair
5`-sequence of HXB2 encoding the MA domain of the gag gene
(nucleotides 333-729)(21) . The sequence was obtained by
polymerase chain reaction with Taq polymerase (22) by
introducing a TGA stop codon after codon 132 using the forward
(GCGCGATGGGTGCGAGAGCGTCA) and reverse (GCGCGCTCAGTAATTTTGGCTGAC)
oligodeoxynucleotide primers. Expression vector pcDL17 was constructed
by cloning the 396-base pair p17 sequence into the BamHI site
of pcDL-SR396 containing the SV40 promoter and human T
lymphotropic virus type 1 long terminal repeat(23) . Plasmid
pcDL17ala2 containing the p17 Gly
Ala mutation was
produced by polymerase chain reaction using a 24-mer synthetic
oligonucleotide (GGGCCCATGGCTGCGAGAGCGTCA) as the forward primer.
Plasmid pcDL17ala111 was constructed similarly, except that the
Ser
Ala mutation was introduced using an 84-mer
synthetic oligonucleotide
(GCGCGCTCAGTAATTTTGGCTGACCTGATTGCTGTGTCCTGTGTCAGCTGCTGCTTGCTGTGCTTTTTTCTTAGCTTTGTTTTG)
as the reverse primer. The coding sequences of all three constructs
were confirmed by the dideoxynucleotide sequencing procedure of Sanger et al.(24) .
Figure 1:
PMA stimulation of p17
phosphorylation. COS-7 cells were transfected with pcDL17wt expressing
p17wt or with pcDL17ala111 expressing p17ala111, and after 48 h, cells
were metabolically labeled for 4 h with
[P]phosphoric acid and treated with PMA for 8
min. A, p17 phosphorylation was detected by
immunoprecipitation with a p17-specific pAb, followed by SDS-PAGE and
autoradiography (lanes 1 and 2, exposure for 5 days; lanes 3 and 4, exposure for 11 days); B, the
level of p17 expression was determined in a parallel experiment by
SDS-PAGE and immunoblotting with a second p17-specific mAb. Lane
1, pcDL17wt and treated with 100 nM PMA (+PMA); lane 2, pcDL17wt and not treated with
PMA (-PMA); lane 3, pcDL17ala111 and treated
with 100 nM PMA (+PMA); lane 4,
pcDL17ala111 and not treated with PMA (-PMA); The arrow indicates the position of
p17.
Because of the recognized
involvement of N-myristoylation in the membrane association of
a number of different cellular and viral
proteins(27, 28, 29) , the N-myristoylation of p17wt and p17ala111 was first
investigated. Cells were transfected with pcDL17wt or pcDL17ala111 for
48 h, followed by metabolic labeling with
[9,10-H]myristate for 4 h, and p17 was
immunoprecipitated with a p17-specific pAb (Fig. 2A, lanes 1-3). To establish that the incorporation of
radiolabel into p17 reflected actual N-acylation and was not
the result of metabolic recirculation of radiolabel into incorporated
amino acids, the same experiment was carried out by transfecting cells
with pcDL17ala2 containing a Gly
Ala point mutation,
a mutation known to specifically block N-myristoylation(30) . Radiolabeling of p17 was seen
only in immunoprecipitates from cells transfected with p17wt or
p17ala111 and not with p17ala2, while similar levels of all three
proteins were expressed as shown by immunoblotting (Fig. 2B, lanes 1-3). These experiments
establish that the expressed p17wt and p17ala111 mutants are N-myristoylated as expected.
Figure 2:
N-Myristoylation of p17. COS-7
cells were transfected with pcDL17wt, pcDL17ala2, or pcDL17ala111, and
after 48 h, cells were metabolically labeled for 4 h with
[9,10-H]myristic acid. A, radiolabeled
p17 was detected by immunoprecipitation with a p17-specific pAb,
followed by SDS-PAGE and fluorography; B, the level of p17
expression was determined in a parallel experiment by SDS-PAGE and
immunoblotting with a second p17-specific mAb. Lane 1,
pcDL17wt; lane 2, pcDL17ala2; lane 3, pcDLala111. The arrow indicates the position of
p17.
The subcellular localization of the three p17 proteins was next examined by transfecting cells with pcDL17wt, pcDL17ala2, or pcDL17ala111. After 48 h, transfected cells were homogenized in an isotonic buffer and fractionated into nuclear, membrane, and cytosolic fractions by differential centrifugation. All three p17 proteins were found exclusively in the cytosolic fraction (Fig. 3, Control, C lane). Since the cytosolic distribution of p17wt and p17ala111 cannot be attributed to a block in N-myristoylation or to an unexpected post-translational deacylation (Fig. 2), it most likely reflects an intrinsic property of normally N-myristoylated p17 to be localized in the cytosol.
Figure 3: Effect of PMA stimulation on subcellular distribution of p17. COS-7 cells were transfected with pcDL17wt, pcDL17ala2, or pcDL17ala111 and treated with 100 nM PMA for 8, 30, and 90 min. Control cells were not treated with PMA. Cell homogenates were separated into membrane (M), cytosolic (C), and nuclear (N) fractions by differential centrifugation. Each fraction was solubilized, separated by SDS-PAGE, and visualized by immunoblotting with a p17-specific mAb. The arrows indicate the positions of p17.
Confirmation of
this conclusion was obtained by labeling cells expressing p17wt for 4 h
with [P]phosphoric acid followed by treatment
with PMA for 8 min and identifying phosphorylated p17wt in the
cytosolic and membrane fractions by immunoprecipitation (Fig. 4). A prominent labeled 17-kDa band was observed in the
membrane fraction from cells treated with PMA (Fig. 4, lane5), but not in the membrane fraction from untreated cells (lane3). In contrast, the cytosolic fractions from
untreated and PMA-treated cells both contained a minor radiolabeled
17-kDa band of equal intensity (Fig. 4, lanes4 and 6), while immunoprecipitates from cells transfected
with the control vector did not contain a labeled 17-kDa band (lanes1 and 2).
Figure 4:
Effect
of PMA on membrane localization of phosphorylated p17. COS-7 cells were
transfected with the pcDL-SR396 control vector or pcDL17wt, and
after 48 h, transfected cells were metabolically labeled with
[
P]phosphoric acid. Cells were then treated with
100 nM PMA (+PMA) for 8 min or not treated (-PMA), and cell extracts were separated into membrane (M) and cytosolic (C) fractions by differential
centrifugation. Phosphorylated p17 was isolated by immunoprecipitation
with a p17-specific pAb and visualized by SDS-PAGE and autoradiography. Lanes 1 and 2, cells transfected with control vector; lanes 3 and 4, cells transfected with pcDL17wt, but
not treated with PMA; lanes 5 and 6, cells
transfected with pcDL17wt and with PMA. The arrow indicates
the position of p17.
The current model for replication of HIV envisions a central role for p55 in capsid assembly(5, 10, 12) . Of particular interest is the MA domain, which has specific roles in the targeting of gag to the plasma membrane and in the recruitment of viral envelope glycoprotein into the assembling virion. Following particle release and proteolytic processing of p55, the resulting p17 subsequently accounts for the inner MA coat of the lipid envelope of the mature infectious virus (6, 7, 8, 9) . p17 is also known to play an essential role early in the infection process including the nuclear targeting of a specific viral preintegration complex that is responsible for the reverse transcription of viral genomic RNA and integration of the resulting cDNA into the host genome(6, 10, 11, 12) . The involvement of MA proteins in each of these phases of the virus life cycle is driven by unique structural features encoded into p17 that express themselves successively in functionally distinct ways depending upon proteolytic processing, the timely availability of specific viral and cellular components, and regulatory signals received from the cellular host. An understanding of the specific molecular signals regulating these processes may reveal new opportunities for interfering with virus development.
Activation of HIV-1-infected T-cells is known to stimulate the transcription of early spliced viral RNA transcripts encoding regulatory proteins essential for the subsequent production of the structural and enzymatic proteins required for virus assembly(1) . The pleiotropic response to general cellular activation could also have post-transcriptional consequences affecting the targeting and/or assembly of the newly synthesized gag proteins into the assembling virus. Such an expectation is consistent with the observation that treatment of HIV-1infected monocytic cells with interleukin-6 increases virus replication without increasing viral RNA synthesis (31) . Furthermore, this post-transcriptional induction of HIV-1 proteins mediated by interleukin-6 is blocked by a specific PKC inhibitor(32) . These observations are compatible with the model proposed in this study that one of the consequences of cellular activation may be a post-translational role for PKC in regulating the function(s) of gag proteins in the virus life cycle.
We have previously found that p55 and p17 are phosphorylated by PKC
when cells are activated by PMA(20) . In the present study, we
have shown by site-directed mutagenesis that a PKC phosphorylation site
motif is located at Ser within the MA domain of p55. This
domain is highly conserved among different strains of
HIV-1(19) , thus raising the possibility that PKC-mediated
phosphorylation is of fundamental importance in gag protein assembly or
post-assembly events. We have also found that the activation of cells
with PMA promotes the rapid translocation of p17 from the cytosol to
the membrane fraction and that this cytosol-to-membrane shift is
dependent upon N-myristoylation. A direct relationship between
phosphorylation of p17 at Ser
and p17 membrane
association is further indicated by our finding that when cells
expressing p17 are treated with PMA, (i) p17 is quantitatively
translocated to the membrane fraction coincident with the appearance of
newly phosphorylated p17 in the same fraction and (ii) membrane
association of phosphorylated p17 is blocked by a Ser
Ala point mutation. We speculate that cytokine-induced
phosphorylation of gag proteins may stimulate HIV replication by
promoting or accelerating the intracellular targeting and assembly of
p55 into viral capsids. This effect could also extend to post-assembly
virus maturation and influence the course of subsequent reinfection.
The regulation of p17 membrane association by PKC is reminiscent of
a similar relationship between the membrane association of the N-myristoylated alanine-rich protein kinase C substrate
(MARCKS) and PKC(28) . However, in the case of MARCKS,
phosphorylation has the opposite effect of promoting the dissociation
of the membrane-bound MARCKS into the cytosol. Therefore, HIV-1 p17
appears to represent a new category of phosphorylation-dependent
``myristoyl-protein switches'' (33) in which PKC
promotes the membrane association of N-myristoylated p17.
Other examples of myristoyl-protein switches include the promotion of
membrane association of recoverin by calcium (33) and
ADP-ribosylation factor by ADP(34) . One mechanism that might
account for the specific membrane association of phosphorylated p17 is
suggested by epitope mapping of p17 with a mAb that recognizes an
epitope that includes both the N and C termini of p17(35) .
This suggests that the native conformation of p17 involves the
juxtapositioning of the N-terminal myristoylated domain of p17 near to
its C-terminal Ser. If such is indeed the case, we
hypothesize that phosphorylation of Ser
might facilitate
a conformational change involving the covalently bound myristate. The
consequence of such a ``switch'' could result in the exposure
of a domain that promotes membrane association. However, since the
membrane fraction used in our analysis includes various cellular
components in addition to lipid membranes, the specificity of this
membrane targeting signal remains undefined.
While the physiological significance of gag protein phosphorylation is not yet known, we suggest that cellular activation could affect the subcellular targeting of either the mature MA protein or its p55 precursor. In the case of p55, such a signal could promote the rapid assembly of viral particles at the plasma membrane from the pool of accumulating gag precursors in the cytoplasm. In the case of p17, cytokine-induced phosphorylation could provide an independent targeting signal that is important for its association with the inner lipid virus envelope of the mature virion or for regulating the involvement of p17 in preintegration complex formation or nuclear targeting. It might also facilitate the interaction of gag proteins with viral envelope glycoprotein during capsid assembly. In any event, inhibition of such a post-translational activation signal could account in part for the anti-HIV-1 activity reported for specific PKC inhibitors(32) .
Based on analogy
with other retroviral MA proteins, one would predict that the membrane
targeting determinants of p55 would be located within the MA
domain(36, 37, 38, 39) . Indeed, the
dependence of p55 membrane binding on N-myristoylation and the
subsequent developmental events (14) , the close association of
the p17 fragment with the inner lipid envelope of the mature
virus(7, 8, 9) , and the recognized role for
involvement of N-myristoylation in the membrane association of
a number of cellular proteins (27, 28, 29) would predict that the mature MA
protein is also localized to the membrane fraction. So it was somewhat
surprising to find that p17 is expressed exclusively in the cytosol.
Nevertheless, this finding is consistent with the report that small
nonoverlapping deletions covering most of the MA domain have no
noticeable effect on p55 processing or on virus formation (10) and that truncations, deletions, and point mutations
C-terminal to the MA domain block the membrane association of the
mutated gag proteins (16) . ()Such a membrane
targeting potential for the MA domain is also incompatible with the
known role for mature p17 in providing the karyotypic nuclear targeting
signal of the post-infection preintegration
complex(11, 12) . Our findings therefore confirm and
extend earlier reports that while N-myristoylation may be
necessary for p55 membrane association, other structural signals are
also required(14, 16) .