©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of HIV-1 gag Protein Subcellular Targeting by Protein Kinase C (*)

(Received for publication, December 5, 1994)

Gang Yu (§) Fu Sheng Shen Stacey Sturch Angelo Aquino (¶) Robert I. Glazer (**) Ronald L. Felsted (§§)

From the Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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^2 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.


INTRODUCTION

The internal structural proteins of HIV-1 (^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.


EXPERIMENTAL PROCEDURES

Materials

[P]Phosphoric acid (8500-9120 Ci/mmol), [-P]ATP (3000 Ci/mmol), [9,10-^3H]myristic acid (10-60 Ci/mmol), and mAb NEA-9282 specific to HIV p17 were purchased from DuPont NEN. PMA was obtained from Sigma. p17-specific pAb was purchased from International Enzymes, Inc. (Fallbrook, CA). The Lipofectin transfection system was from Life Technologies, Inc.

Cells and Vectors

COS-7 cells were obtained from the American Type Culture Collection (Rockville, MD) and were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 40 mM Hepes (pH 7.4), and 0.1 mM sodium pyruvate.

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-SRalpha396 containing the SV40 promoter and human T lymphotropic virus type 1 long terminal repeat(23) . Plasmid pcDL17ala2 containing the p17 Gly^2 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) .

In Vivo Expression, Phosphorylation, and N-Myristoylation of p17

COS-7 cells on 100-mm dishes were grown to 80% confluency and transfected with 10 µg of plasmid by Lipofectin (Life Technologies, Inc.) following the manufacturer's instructions. At 48 h post-transfection, cells were washed twice with Hanks' balanced salt solution and incubated for 30 min with 10 ml of phosphate-free Iscove's modified Dulbecco's medium (Life Technologies, Inc.) before adding 0.25 mCi/ml [P]phosphoric acid for 4 h. In some experiments, 100 nM PMA was added during the last 10 min of radiolabeling. Total cell extracts were prepared by scraping and washing cells in ice-cold Hanks' balanced salt solution, extracting with lysis buffer (10 mM Tris-HCl (pH 7.4) containing 0.15 NaCl, 1% Triton X-100, 0.5% sodium deoxycholate 1.5 mM MgCl(2), 1 mM EDTA, 2 mM sodium vanadate, 10 mM NaF, and protease inhibitors (2 µg/ml aprotinin, 1 µg/ml pepstatin A, 28 µg/ml phenylmethanesulfonyl fluoride, and 70 µg/ml L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone)) by five passages through a 21-gauge needle, and clarifying by centrifugation at 13,000 times g for 15 min at 4 °C. Myristoylation was determined by metabolic radiolabeling of cells for 4 h with 250 µCi/ml [9,10-^3H]myristic acid 48 h after transfection.

Subcellular Fractionation

48 h after transfection, COS-7 cells were washed, scraped, and suspended in hypotonic buffer (10 mM Tris-HCl (pH 7.9) containing 1.5 mM MgCl(2), 10 mM KCl, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, and protease inhibitors); incubated on ice for 10 min; homogenized with 20 strokes of a glass Dounce homogenizer; and centrifuged at 500 times g for 5 min at 4 °C to yield the nuclear fraction. The nuclear fraction was then suspended in 200 µl of extraction buffer (20 mM Tris-HCl (pH 7.9) containing 20% glycerol, 1.5 mM MgCl(2), 0.5 mM dithiothreitol, and protease inhibitors), and 4 M KCl was added to a final concentration of 0.3 M(25) . The final suspension was rocked for 30 min at 4 °C and centrifuged at 13,000 times g for 15 min to yield the nuclear fraction. The 500 times g post-nuclear supernatant fraction was further fractionated by centrifugation at 100,000 times g for 1 h at 4 °C. The resulting pellet was dissolved in 20 mM Tris-HCl (pH 7.9) containing 0.5 M NaCl, 1% Triton X-100, 0.5 mM dithiothreitol, and 1 mM EDTA; clarified by centrifugation at 13,000 times g for 15 min at 4 °C; and designated as the membrane fraction. The corresponding 100,000 times g supernatant fraction was designated as the cytosolic fraction.

Immunoprecipitation of p17, SDS-PAGE, and Immunoblotting

Immunoprecipitation was carried out on the membrane and cytosolic fractions or cell extracts from [P]phosphoric acid- or [9,10-^3H]myristic acid-labeled cells by adding 10 µg of anti-p17 pAb and incubating overnight at 4 °C. Immunoprecipitates were recovered by the addition of 50 µl of protein G-agarose (Life Technologies, Inc.) and incubation at 4 °C for 1-2 h. The agarose beads were then pelleted and washed five times with lysis buffer. Immunoprecipitates were suspended in SDS sample buffer and separated by SDS-PAGE using Daiichi 10-20% gradient gels (Integrated Separation Systems, Natick, MA) (26) . Gels were treated with ENHANCE (DuPont NEN) before drying to detect tritium by fluorography. Radiophosphorylated proteins were detected on dried gels by autoradiography and were quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). For immunoblotting, total nuclear, membrane, and cytosolic fractions were separated by SDS-PAGE, immunoblotted using the p17-specific mAb in 5% milk, and visualized using the ECL Western blotting detection system (Amersham Corp.).


RESULTS

Cellular Activation Stimulates Phosphorylation of HIV-1 p17

We have previously reported that p55 and p17 are phosphorylated in vitro and in vivo by PKC(20) . However, since those earlier experiments were performed with a recombinant vaccinia vector containing the HIV gag-pol genes and expressing both p55 and its mature proteolytic products, we could not be certain whether p17 was directly phosphorylated by PKC or that p17 phosphorylation arose indirectly via the proteolytic processing of phosphorylated p55. To determine if pre-existing p17 is directly phosphorylated by PKC, COS-7 cells were transfected with a plasmid (pcDL17wt) expressing the MA domain of p55, and after 48 h, the transfected cells were radiolabeled with [P]phosphoric acid for 4 h and treated with PMA for 8 min. Following PMA treatment, cell extracts were prepared from untreated and PMA-treated cells, and p17 was radioimmunoprecipitated (Fig. 1). PMA treatment resulted in a 4-5-fold increase in the phosphorylation of p17wt immunoprecipitated from stimulated cells (Fig. 1A, lanes1 and 2). This difference was observed even though equal amounts of the p17 proteins were expressed in both lanes as revealed by immunoblotting (Fig. 1B, lanes1 and 2). These results confirm our earlier report that activation of PKC in cells after PMA treatment resulted in a stimulation of gag protein phosphorylation (20) and also extend this observation by demonstrating a direct stimulation of p17 phosphorylation even in the absence of p55. Therefore, these data raise the possibility that external cellular activation signals could have post-translational stimulatory effects on HIV replication though phosphorylation of p55 and p17.


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.



Identification of Ser as the Site of HIV-1 gag Protein Phosphorylation by PKC

We previously identified a putative PKC phosphorylation site motif at Ser within the MA domain of p55 that we proposed as the site for PKC-mediated gag protein phosphorylation(20) . To assess this possibility, cells were transfected with the plasmid pcDL17ala111 expressing p17 with the point mutation Ser Ala and treated with PMA (Fig. 1). Phosphorylation of mutated p17 was dramatically lowered in comparison with p17wt and was not stimulated after PMA treatment (Fig. 1A, lanes3 and 4) despite the expression of similar levels of wild-type and mutated proteins (Fig. 1B, lanes3 and 4). The low basal level of p17ala111 phosphorylation was only observed after radiography for 11 days rather than after exposure for 5 days as for p17wt. The marked reduction in p17ala111 phosphorylation compared with p17wt confirms our earlier prediction that Ser is the site of gag protein phosphorylation by PKC; however, the presence of a low basal level of p17ala111 phosphorylation that is not affected by PMA suggests that p17 may also be phosphorylated at sites other than Ser by other protein kinases.

Subcellular Distribution of p17

Because of the involvement of the MA domain in several different phases of the HIV life cycle, we speculated that gag protein phosphorylation might have a critical role in regulating the participation of p55 and/or p17 in some stage of HIV development. For example, p55 plays a central role in directing the assembly of HIV at the plasma membrane, and p17 is differentially targeted either to the inner virus lipid envelope during virus maturation (7, 8, 9) or to the nucleus after infection as part of a preintegration complex(6, 10, 11, 12) . Therefore, we hypothesized that PKC might have an effect on gag protein subcellular targeting, an event that might be manifested in the subcellular distribution 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-^3H]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^2 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-^3H]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.



Effect of PKC Activation on p17 Subcellular Localization

The effect of cellular activation on the subcellular distribution of p17 was then examined in cells transfected with pcDL17wt, pcDL17ala2, and pcDL17ala111. After 48 h, transfected cells were treated with PMA and subfractionated into nuclear, membrane, and cytosolic fractions (Fig. 3). PMA treatment resulted in a time-dependent shift of p17wt from the cytosolic fraction (Fig. 3, p17wt) into the membrane fraction within 8 min, and by 90 min, a complete reversal of membrane-associated p17 back into the cytosolic fraction was observed. A similar analysis of p17ala2 revealed that PMA treatment had no effect on its cytosolic distribution (Fig. 3, p17ala2), thus establishing that PMA-induced membrane association of p17 is dependent upon N-myristoylation. Similarly, transfection of cells with pcDL17ala111 resulted in cytosolic distribution exclusively throughout the treatment interval (Fig. 3, p17ala111). Since p17ala111 is known to be N-myristoylated (Fig. 2A, lane3), the simplest explanation for the absence of a PMA-induced membrane association is the absence of phosphorylation of Ser.

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-SRalpha396 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.




DISCUSSION

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) . (^2)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) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Clinical Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892.

Present address: Universita Degli Studi di Roma Tor Vergata, Dip. Med. Sper. Sci. Biochem., Cattedra di Farmacologia, 00133 Roma, Italy.

**
Present address: Dept. of Pharmacology, Georgetown University Medical Center, Washington, D. C. 20007.

§§
To whom correspondence should be addressed: NIH, Bldg. 37, Rm. 5D02, Bethesda, MD 20892-4255. Tel.: 301-496-4691; Fax: 301-496-5839.

(^1)
The abbreviations used are: HIV-1, human immunodeficiency virus type 1; gag, internal structural protein(s); MA domain, matrix domain of the HIV-1 gag gene; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; pAb, polyclonal antibody; PAGE, polyacrylamide gel electrophoresis; MARCKS, N-myristoylated alanine-rich protein kinase C substrate.

(^2)
G. Yu and R. L. Felsted, manuscript in preparation.


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