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Originally published In Press as doi:10.1074/jbc.M313137200 on May 21, 2004

J. Biol. Chem., Vol. 279, Issue 31, 32426-32434, July 30, 2004
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The Host Cell MAP Kinase ERK-2 Regulates Viral Assembly and Release by Phosphorylating the p6gag Protein of HIV-1*

Bénédicte Hemonnot{ddagger}§, Christine Cartier{ddagger}§||, Bernard Gay{ddagger}, Sandra Rebuffat{ddagger}, Martine Bardy{ddagger}, Christian Devaux{ddagger}, Véronique Boyer**, and Laurence Briant{ddagger}{ddagger}{ddagger}

From the {ddagger}Laboratoire Infections Rétrovirales et Signalisation Cellulaire, Centre National pour la Recherche Scientifique, UMR 5121-Université Montpellier 1, Institut de Biologie, 4 Boulevard Henri IV, CS89508, 34960 Montpellier cedex 2, France and the **Neurodégénérescence et Plasticité, INSERM EMI0108, CHU La Tronche, BP217, 38043 Grenoble, France

Received for publication, December 2, 2003 , and in revised form, May 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The host cell MAP kinase ERK-2 incorporated within human immunodeficiency virus type 1 particles plays a critical role in virus infectivity by phosphorylating viral proteins. Recently, a fraction of the virus incorporated late (L) domain-containing p6gag protein, which has an essential function in the release of viral particles from the cell surface, was reported to be phosphorylated by an unknown virus-associated cellular protein kinase (Muller, B., Patschinsky, T., and Krausslich, H. G. (2002) J. Virol. 76, 1015–1024). The present study demonstrates the contribution of the MAP kinase ERK-2 in p6gag phosphorylation. According to mutational analysis, a single ERK-2-phosphorylated threonine residue, belonging to a highly conserved phosphorylation MAP kinase consensus site, was identified at position 23 within p6gag. Substitution by an alanine of the Thr23 phosphorylable residue within the pNL4.3 molecular clone was found to decrease viral release from various cell types. As observed from electron microscopy experiments, most virions produced from this molecular clone remained incompletely separated from the host cell membrane with an immature morphology and displayed a reduced infectivity in single round infection experiments. Analysis of protein processing by Western blotting experiments revealed an incomplete Pr55gag maturation and a reduction in the virion-associated reverse transcriptase proteins was observed that was not related to differences in intracellular viral protein expression. Altogether, these data suggest that phosphorylation of p6gag protein by virus-associated ERK-2 is involved in the budding stage of HIV-1 life cycle.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As other intracellular parasites, the human immunodeficiency virus type 1 (HIV-1)1 is necessarily dependent upon cell factors for replication. Participation of host cell components is specially required for any of the many HIV-1 gag-encoded functions. Indeed, a variety of Pr55gag-interacting factors have been identified including actin (1–4), calmodulin (5), the motor protein KIF-4 (6), the nuclear transporter karyopherin {alpha} (7, 8), the human nuclear shuttling protein VAN (9), the translation elongation factor 1-{alpha} (10), the translation initiation factor 2 (11), the HO3 hystidyl-tRNA synthase (12), the heat shock protein 70 (13), and a number of polycomb group proteins (14). As a consequence, a number of these host cell proteins has been found to be incorporated within HIV-1 virions in addition to virus-encoded components (15, 16). A specific implication of host cell factors was reported to occur during the late budding stage. Indeed, the Pr55gag precursor protein is transported to the budding site by an unknown mechanism that most likely utilizes host cell factors and associates with the plasma membrane through cotranslational modifications by the cellular myristyl-S-transferase (17, 18). The accumulation of the Pr55gag protein at the plasma membrane then allows its multimerization and is followed by the projection outward of the cell of a spherical budding particle. Recently, the recruitment at the budding site of class E Vps proteins including AIP1/ALIX (19, 20) and the ubiquitin-conjugating enzyme homologue Tsg101 (21–24) through interactions with the Pr55gag polyprotein, was reported to be required for the complete release of the viral particle. Interactions with Tsg101, mediated via the PTAP motif identified as the L-domain contained within the p6gag region, were demonstrated to be enhanced by ubiquitination of the Pr55gag precursor, suggesting a possible interaction with unknown ubiquitin-conjugating enzymes (22, 25).

Soon after the viral particle pinches off and is released in the extracellular compartment, the viral protease becomes activated and the Pr55gag polyprotein is cleaved into MAp17gag, CAp24gag, NCp7gag, and p6gag proteins and p1, p2 spacer peptides. Most of these mature products have also been shown to interact with cellular proteins. As an example, interactions occurring with CAp24gag have been documented as the mechanism directing incorporation into virions of cyclophilin A (26, 27), a host cell compound that acts as an uncoating factor and participates to the initial uptake of HIV-1 by target cells (28, 29). In addition, a number of these Pr55gag processing products was also suggested to interact with host cell protein kinases as they were found to be phosphorylated. Phosphorylation of the viral MAp17gag by host cell kinase(s) was found to occur at various steps of the HIV-1 replicative cycle. Indeed, the C-terminal phosphorylation of MAp17gag protein during and immediately after virus production was proposed to facilitate the dissociation of the viral matrix protein from the cellular membrane (30). Additional phosphorylation of MAp17gag by the MAP kinase (MAPK) ERK-2 was proposed to promote membrane dissociation of the reverse transcription complex from the cell membrane at the site of entry, allowing its nuclear translocation (31, 32). For CAp24gag, three distinct phosphorylation sites on serine residues were identified (33). As mutations at these sites were observed to block HIV-1 replicative cycle at an early reverse transcription step, CAp24gag phosphorylation was proposed to contribute to the early post-entry step by promoting the dissociation of the viral core. Recently, we observed that CAp24gag interacts with and is phosphorylated by the PKA-C{alpha} subunit that we found to be incorporated within HIV-1 particles (34).

More recently, the proline-rich peptide of 52 amino acids termed p6gag, derived from the C terminus of the Pr55gag precursor, was reported to be the predominant phosphoprotein of HIV-1 and the contribution of one or several virus-associated kinase(s) was hypothesized (35). Having demonstrated that the cellular MAPK ERK-2 is specifically incorporated within HIV-1 viral particles (36), it remained to investigate its role in p6gag phosphorylation. The present study was thus designed to define the precise contribution of ERK-2 in the phosphorylation of the p6gag viral protein, to determine critical residues phosphorylated within p6gag and to elucidate the involvement of p6gag phosphorylation in the HIV-1 life cycle. As observed from in vitro phosphorylation assays, the p6gag protein and its p15gag precursor were found to be phosphorylated by ERK-2 at the level of a single threonine residue (Thr23) located within a conserved MAPK canonical consensus sequence. By analyzing point mutation of the p6gag protein within a pNL4.3 molecular clone, we observed that substitution of the ERK-2 phosphorylable residue within p6gag leads to the production of immature viruses unable to separate from the host cell membrane. A faint number of particles released from the producing cells was isolated that were found to display altered morphology and size. Such particles were characterized by incomplete maturation of Pr55gag precursors accompanied by modifications in the incorporation of reverse transcriptase subunits. As defined from single round infectivity assays performed in the MAGI indicator cell line, Thr23 mutated virions displayed a reduced infectivity as compared with wild type viruses. Altogether, our data indicate that phosphorylation of a unique site of the p6gag domain by ERK-2 plays a critical role in the late stage of the HIV-1 life cycle, by contributing to the regulation of viral assembly and/or release.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfections—Human embryonic kidney 293T cells and MAGI cells (37) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine. In transfection experiments, cells (3 x 105) were plated in 6-well culture plates and incubated in the presence of viral DNA diluted in ExGen 500 transfection reagent (Euromedex) at a ratio of 6 units/µg of DNA.

Western Blot Analysis—Proteins were separated on a 14% ProSieve® 50 polyacrylamide gel (FMC), then transferred to polyvinylidene difluoride membrane (Immobilon P, Millipore) and revealed by immunoblotting using an anti-CAp24gag mouse monoclonal antibody (mAb) (ICN), an anti-RT rabbit polyclonal serum (kindly provided by J. L. Darlix, ENS, Lyon, France), or an anti-p6gag polyclonal serum obtained after immunization of rabbits with GST-p6gag fusion protein. Anti-MAp17gag mAb was obtained from Fitzgerald Industries International, Inc., anti-ERK-2 from Santa Cruz Biotechnology, Inc., and anti-GST from Amersham Biosciences. Secondary antibodies conjugated to horseradish peroxidase were revealed by enhanced chemiluminescent detection.

Virion Production and Purification—Viral stocks were obtained by transfection of the pNL4.3 HIV-1 molecular clone into 293T cells. Briefly, culture supernatants collected 40 h after transfection were cleared from cellular debris by low-speed centrifugation and filtered on 0.45-µm pore size membranes (Millipore). Virions were sedimented by ultracentrifugation through 20% sucrose cushions, at 25,000 rpm for 2 h 30 min at 4 °C (SW28 rotor), resuspended in 200 µl of phosphate-buffered saline and layered onto the top of an Optiprep velocity gradient (6–20% (w/v) iodixanol; Abcys SA). Centrifugation was run for 3 h at 26,000 x g at 4 °C. Virions were recovered from virus-containing fractions by additional ultracentrifugation at 95,000 rpm for 5 min at 4 °C (TLA100.2 rotor) and lysed. Additional preparation of a highly purified virus (HIV strain HZ321, from clade A) obtained by multiple density gradient purification from infected HUT78 supernatant (38) was kindly provided by R. Trauger (Immune Response Corp., Carlsbad, CA).

GST Fusion Proteins, Site-directed Mutagenesis, and Plasmid Production—p6gag and p15gag sequences were amplified by PCR experiments from the pNL4.3 proviral DNA with 5'-GCGTGGATCCCAGAGCAGAC, 5'-CGGGAATTCTCATTGTGAC and 5'-GCGGATCCCAAAGAAAGACTGTTAAG, 5'-CCGAATTCTTATTGTGACGAGGGGTC oligonucleotide primer pairs, respectively, and cloned in-frame into the pGEX 5X.1 expression vector (Amersham Biosciences). Vectors encoding GST-p6gag mutants, where codons encoding each serine/threonine residue were replaced by sequences encoding an alanine, and the pNL4.3T23A plasmid were obtained by site-directed mutagenesis using the Gene Editor mutagenesis kit (Promega) and primers are listed in Table I. All mutant sequences were confirmed by sequencing. GST-NCp7gag, GST-MAp17gag, and GST-CAp24gag expression vectors were kindly provided by J. Darlix (ENS, Lyon) and vector encoding GST-Elk was a gift of D. A. Brenner (Department of Pharmacology, University of North Carolina, Chapel Hill).


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  		TABLE I
Serine/threonine residues in the p6gag sequence encoded by the GST-p6gag plasmid or the pNL4.3T23A molecular clone were replaced by an alanine by site-directed mutagenesis using the Gene Editor mutagenesis kit (Promega) and the corresponding primers

 
In Vitro Phosphorylation Assays—Viral lysate phosphorylation assays were performed in the presence of 2 µCi of [{gamma}-32P]ATP and kinase buffer containing 50 mM Hepes (pH 7.5), 5 mM MnCl2 at 30 °C for 30 min. When phosphorylation of peptides or GST-fused proteins was assayed, 20 ng of recombinant activated ERK-2 (Stratagene) (39) was added to the reaction mixture. Phosphorylated products were separated by SDS-PAGE and detected by autoradiography.

Two-dimensional Gel Electrophoresis—Purified cell-free virions were analyzed by two-dimensional gel electrophoresis as follows. Virions were solubilized in buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, 65 mM dithioerythritol, 40 mM Tris, and protease inhibitor for 2 h on a wheel. Viral lysates were clarified by centrifugation for 30 min at 20,000 x g. Each sample was added to a rehydratation solution containing 8 M urea, 2 M thiourea, 4% CHAPS, 65 mM dithioerythritol, bromphenol blue, and 1% Pharmalytes pH 3–10 solution (Amersham Biosciences). Immobiline DryStrips, 18 cm, covering a pH range of 3–10 were allowed to rehydrate in the above solution for 4 h without current and then for 12 h under a 30-V current under low viscosity paraffin oil. Isoelectrofocusing was then performed according to the following voltage/time profile: 500 V for 1 h, 1,000 V for 1 h, linearly increasing the gradient to 8,000 V for 2 h, and a final phase of 8,000 V for 40,000 V/h up to a total amount of 50,000 V/h. After the first dimensional run, the individual strip was equilibrated, and then put on the top of the second dimensional gel (15%) with the Hofer Dalt System (Amersham Biosciences) at 150 V/10 gels. Proteins were then transferred onto polyvinylidene difluoride, hybridized with anti-p6gag serum and horseradish peroxidase-conjugated secondary antibodies, and revealed with ECL reagents.

Viral Infectivity Assays—MAGI indicator cells, which stably express the {beta}-galactosidase reporter gene cloned downstream of the HIV-1 LTR promoter, were platted at 8 x 104 cells per well, in 24-well plates and exposed to HIV-1 stock solutions normalized according to the CAp24gag antigen content by enzyme-linked immunosorbent assay (Beckman-Coulter) or to RT activity determined as previously reported (40). Forty-eight hours post-infection, viral infectivity was monitored by quantification of o-nitrophenyl {beta}-D-galactopyranoside hydrolysis from the cell lysates as previously described (41). {beta}-Galactosidase activity evaluated by measuring absorbance at 410 nm was normalized according to total protein content in the cell lysate.

Electron Microscopy Analysis—Transfected 293T cells were processed for thin-layer electron microscopy as follows: 40 h post-transfection, cells were fixed in situ with 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) for 60 min at 4 °C. Cells were then post-fixed with 2% osmium tetroxide, washed in cacodylate buffer containing 0.5% tanic acid, and embedded in epon (Embed-812, Electron Microscopy Sciences Inc.). Cell-free virions sedimented by ultracentrifugation for 10 min at 95,000 x g were fixed for 2 h at 4 °C in 2.5% glutaraldehyde in cacodylate buffer (pH 7.2). After extensive washes in 0.1 M Sorensen phosphate buffer (pH 7.2), viruses were included in a fibrin clot as described by Charret and Fauré-Fremiet (42). Viruses were then post-fixed with 2% osmium tetroxide and 0.5% tannic acid, dehydrated, and embedded in epon. Sections were counterstained with uranyl acetate and lead citrate and examined with an Hitachi H7100 transmission electron microscope.

ERK-2 Knock-out Experiments—ERK-2 knock-out was performed by transfection of specific siRNA using the MAPK1(ERK-2) siRNA/siAb assay kit (Dharmacon RNA Technologies). Briefly, 100 pmol of pooled siRNA duplexes specific for the erk-2 sequence or control siRNA duplexes were transfected into 293T cells using the OligofectAMINE transfection reagent at a ratio of 1 µl for 20 pmol of siRNA. Inhibition of ERK-2 expression was assayed by immunoblotting of the total cell extracts using anti-ERK-2 mAbs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
p6gag Protein and Its p15gag Precursor Are Phosphorylated by ERK-2 Virus-associated Kinase—The p6gag protein was recently shown to be phosphorylated in vivo and the possible contribution of virus-associated kinase(s) in these phosphorylation events was evoked (35). To investigate such hypothesis, a lysate of highly purified NL4.3 virions was incubated in the presence of [{gamma}-32P]ATP and appropriate kinase buffer. Phosphorylated proteins were separated by SDS-PAGE and revealed by autoradiography. As shown in Fig. 1A, several phosphoproteins evidenced and their nature were investigated by probing the membrane with antibodies raised against HIV-1 proteins. In these experimental conditions, phosphorylated products at 55 and 6 kDa were revealed by using anti-p6gag serum. This observation indicates that the p6gag protein and its Pr55gag precursor are both phosphorylated by host cell protein kinase(s) incorporated within HIV-1 particles. The contribution of host cell contaminating constituents was excluded as similar data were obtained using immunogen grade preparations of HIV strain HZ321 as a substrate (38) (data not shown). In addition, 24- and 17-kDa phosphorylated proteins were recognized, respectively, with anti-CAp24gag and anti-MAp17gag mAbs, specific for processed forms of these proteins. This observation corroborates previous literature data indicating that MAp17gag and CAp24gag were phosphorylated by virus-associated kinases (32, 34). In agreement with our published observations (36), an additional 42-kDa phosphorylated protein, which we previously identified as the host cell ERK-2 protein kinase incorporated within HIV-1 particles, was also observed from these phosphorylation experiments.



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  		FIG. 1.
In vitro analysis of HIV-1 proteins phosphorylation. A, highly purified virus lysate was incubated in the presence of [{gamma}-32P]ATP in kinase buffer. Phosphorylated products were revealed by autoradiography and identified by probing the membrane with various antibodies as indicated. The positions of relative molecular weight (Mr) markers are indicated on the left. B, immobilized GST viral fusion proteins were incubated in the presence of activated ERK-2 and [{gamma}-32P]ATP in kinase buffer. Proteins were then separated on SDS-PAGE and revealed by autoradiography (upper panel). GST and GST-Elk proteins are shown as controls. Levels of GST fused proteins loaded in each lane were ascertained in the Western blotting experiment by incubation of the membrane with horseradish peroxidase-conjugated anti-GST mAb (lower panel). C, schematic representation of the Pr55gag precursor, processing intermediates, and final products observed during sequential processing of the Pr55gag precursor.

 
We next investigated the contribution of ERK-2 in p6gag phosphorylation. GST fusion proteins containing the sequences encoding the p6gag protein or its p15gag precursor (see Fig. 1C, lower panel for its composition) were generated and used in an in vitro phosphorylation experiment in the presence of recombinant activated ERK-2 protein kinase (i.e. dually phosphorylated on threonine 183 and tyrosine 185 residues). As shown in Fig. 1B, the GST-p15gag precursor and GST-p6gag proteins were found to be phosphorylated. Interestingly, a stronger phosphorylation signal was detected with GST-p15gag compared with GST-p6gag. In contrast, no phosphorylation was observed using GST-NCp7gag or GST-CAp24gag as a substrate. As previously reported by others (32), ERK-2-dependent phosphorylation of GST-MAp17gag was observed in our experimental conditions. GST alone and GST-Elk, a natural substrate of ERK-2, are shown as negative and positive controls, respectively. The amount of GST fusion proteins loaded in each lane was ascertained by Western blotting of the membrane with anti-GST mAb. Altogether, our results indicate that the p6gag protein packaged into virions is phosphorylated and suggest the contribution of ERK-2 virus-associated protein kinase as demonstrated by in vitro experiments. These phosphorylations are susceptible to occur before the complete processing of p6gag as suggested by the observation of phosphorylated forms of Pr55gag and p15gag precursors.

A Single ERK-2 Consensus Sequence within the p6gag Protein Is Targeted by ERK-2—To define which amino acid residue(s) of p6gag is/are phosphorylated by the ERK-2 recombinant protein, the presence of the ERK-2 consensus motives was scanned in the total p6gag sequence. A single canonical ERK-2 motif represented by the (Ser/Thr)-Pro minimal sequence was identified at position 23 of the p6gag protein encoded by the pNL4.3 molecular clone. According to the compilation of HIV-1 sequences listed in the NIAID data base (43), this motif was found to be conserved in most HIV-1 consensus sequences with a duplication observed in the consensus from clades M, F, and H (see Table II). In some cases (clades A and G), the Thr-Pro motif was absent but an equivalent Ser-Pro motif was found immediately beside, suggesting that a strong selection pressure leads to the conservation of an ERK-2 consensus at this site. ERK-2-dependent phosphorylation of the Thr23 residue was then analyzed in an in vitro phosphorylation assay using synthetic peptides mimicking either wild type (WT) p6gag spanning residues 12–39, or an equivalent peptide with an alanine replacing the threonine residue at position 23 (T23A) (Fig. 2A) incubated in the presence of recombinant activated ERK-2. As shown in Fig. 2B, phosphorylation of the WT peptide was evidenced from 0.3 to 15 nmol of peptide. In contrast, no phosphorylation signal was observed with equivalent amounts of T23A mutated peptide. These data indicate that residue Thr23 accounts for ERK-2-dependent phosphorylation of a p6gag peptide spanning residues 12 to 39. The existence of additional ERK-2 phosphorylation site(s) in p6gag was next analyzed. A systematic mutational analysis was performed to modify each serine or threonine residue within the p6gag sequence into an alanine residue. WT or mutated p6gag proteins were expressed in-frame with the GST sequence and used in an in vitro kinase experiment. As shown in Fig. 2C, p6gag protein was found to be phosphorylated when incubated with catalytically active ERK-2. ERK-2-dependent phosphorylation of p6gag was totally abolished only when the Thr23 residue was mutated into an alanine. No modification of GST-p6gag phosphorylation was observed when other serine or threonine residues within p6gag were substituted. Thus, the threonine residue at position 23 within p6gag appears to be the unique residue of this protein targeted by recombinant activated ERK-2 in vitro.


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  		TABLE II
Consequences of p6gag variability on Pol sequence

ERK-2 consensus sites represented by the T-P or the S-P minimal sequence are underlined.

 



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  		FIG. 2.
In vitro phosphorylation analysis of WT and mutated p6gag peptides and GST-fused p6gag protein. A, amino acid sequence of p6gag from HIV-1NL4.3 and NL4.3T23A viruses. The sequences of synthetic WT and T23A peptides spanning residues 12 to 39 of p6gag are boxed. B, in vitro phosphorylation of WT and T23A synthetic peptides were analyzed by incubating increasing amounts of peptides with recombinant activated ERK-2 protein and [{gamma}-32P]ATP. Peptides were then separated on SDS-PAGE and revealed by autoradiography. C, immobilized WT or mutated GST-p6gag fusion proteins were incubated in the presence of activated ERK-2 and [{gamma}-32P]ATP in kinase buffer. Phosphorylated proteins are revealed by autoradiography (upper panel). The amount of GST-fused proteins loaded in each lane was ascertained by incubating the membrane with an anti-GST mAb (lower panel). D, phosphorylation of p6gag protein incorporated within NL4.3 and NL4.3T23A cell-free virions was analyzed by two-dimensional gel electrophoresis and hybridization with an anti-p6gag serum. Spots corresponding to phosphorylated forms of p6gag are indicated.

 
We next attempted to define the contribution of the Thr23 residue in in vivo phosphorylation of p6gag. To this end, a molecular clone of HIV-1 with residue Thr23 within p6gag substituted by an alanine (referred as pNL4.3T23A) was derived from the pNL4.3 proviral DNA by site-directed mutagenesis as described by Ottman et al. (44). This mutation induces an asparagine to serine modification in the Pol protein, a substitution that occurs spontaneously in primary HIV-1 isolates, mainly from clades A and G (see the corresponding consensus sequences in Table II). Purified preparations of NL4.3 and NL4.3T23A particles were first subjected to a in vitro phosphorylation assay. When the NL4.3T23A virus lysate was incubated in the presence of [{gamma}-32P]ATP, a weak decrease (less that 30%) of p6gag phosphorylation was observed (data not shown), suggesting that p6gag might be phosphorylated on multiple residues by kinase distinct from ERK-2. To further investigate this hypothesis, the NL4.3 and NL4.3T23A samples were then subjected to two-dimensional gel electrophoresis experiments, and p6gag isoforms were detected by an anti-p6gag serum. As shown in Fig. 2D, three spots were assigned to wild type forms of virion-associated p6gag protein that were proposed to correspond to various phosphorylation levels of this protein. In contrast, only two spots were detected for the T23A mutated forms of p6gag protein indicating that substitution of the Thr23 residue within p6gag affects its phosphorylation in vivo. Altogether, our data indicate that the p6gag protein is phosphorylated in vitro and in vivo on residue Thr23 and suggest the contribution of ERK-2 in these phosphorylation events.

Contribution of Thr23 Residue Phosphorylation of p6gag Protein in HIV-1 Release and Morphology—We next investigated the functional relevance of p6gag phosphorylation by ERK-2 in the life cycle of the virus. The ability of the pNL4.3T23A mutant construct to direct virus particle release was studied in transient transfection experiments. Forty hours after transfection of 293T cells, culture supernatant were collected and RT activity was measured. As shown in Fig. 3A, RT levels produced from the pNL4.3T23A molecular clone were found to be reproducibly 2.5–3.5 times lower than those observed from the parental pNL4.3 plasmid. This difference was observed for any amount of viral DNA transfected. Similar results were obtained using other cell lines, including the promonocytic THP1 cell line, the H9 CD4+ lymphoblastoid cell line, or HeLa cells (data not shown). These data indicate that substitution by alanine of the ERK-2 phosphorylable Thr23 residue within p6gag results in a marked decrease in viral particle release from transfected cells. This decrease is independent of the host cell type.



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  		FIG. 3.
Contribution of ERK-2-dependent phosphorylation of p6gag on viral release. A, 293T cells were transfected with increasing amounts of pNL4.3 or pNL4.3T23A plasmids. Forty hours after transfection, viral particles release was monitored by quantification of RT activity in the culture supernatant. Transfection efficiency was ascertained by co-transfecting a {beta}-galactosidase expression vector and RT values were normalized according to the total protein content. Each value is the mean of six separate experiments and error bars indicate ± S.D. (pNL4.3, gray bars; pNL4.3T23A, black bars). B, 293T cells were transfected with the pNL4.3 molecular clone alone or cotransfected with pNL4.3 and ERK-2-specific siRNA. Seventy-two hours after transfection, inhibition of ERK-2 expression was monitored by immunoblotting total cell extracts with anti-ERK-2 mAbs. Expression of HIV-1 proteins was ascertained by immunoblotting the cell lysates with anti-p6gag serum and anti-CAp24gag mAbs. C, viral release was measured by quantification of the CAp24gag antigen concentration in the culture supernatants. RT values were normalized according to the number of viable cells. Values obtained from 293T mock transfected cells are shown as control. Each value is the mean of two separate experiments performed in duplicate. Values are expressed as mean ± S.D.

 
Interestingly, similar data were obtained when viral particle release was measured from cells with knocked-out ERK-2. Indeed, when viral particle release was assayed from 293T cells expressing the pNL4.3 plasmid transfected with ERK-2-specific siRNA, a significant decrease in viral production, measured as CAp24gag antigen production (Fig. 3C) or RT levels in culture supernatant (data not shown) was found to be concomitant to the inhibition of ERK-2 expression (Fig. 3B). Only faint amounts of viral particles are observed that are related to the residual pool of ERK-2 in the host cells (Fig. 3B, right lane). The observation of similar phenotypes from cells expressing the pNL4.3T23A molecular clone or knocked-out for ERK-2 expression corroborates in vitro experiments and supports the contribution of ERK-2 in p6gag phosphorylation in vivo.

Release of virions produced from 293T cells transfected with pNL4.3 or pNL4.3T23A plasmids was next analyzed in thin layer electron microscopy (EM) experiments. A reduced number of viral particles was observed with pNL4.3T23A-transfected cells, the majority remained attached to the host cell membrane (see Fig. 4A). Clusters of two or more particles that failed to detach from each other were also observed to be released from these cells. Rare cell-free viral particles were visualized. Among them, a majority displayed a layer of electron dense material underlying the inner leaflet of the membrane and an absence of cone-shaped cores at the center of the particle characteristic of immature virions (Fig. 4B), whereas a faint proportion of virions with condensed cores corresponding to mature particles was observed. A careful examination of a large number of images indicated that the particle size of NL4.3T23A cell-free virions differed from that of NL4.3 particles, as plotted in Fig. 4C. The diameter of NL4.3T23A particles ranged from 87 to 129 nm with a mean diameter of 105.1 ± 10.9 nm and was significantly higher than that of wild type virions (average particle diameter = 81.7 ± 7.4 nm, range = 64–102 nm). In our experimental conditions, WT particles appear smaller than the expected size of 100 nm because of technical approaches used to embed cell-free virions prior to EM observations. Additional analysis of NL4.3 and NL4.3T23A virion density was performed by iodixanol velocity gradient centrifugation. Particle sedimentation analysis, assessed by revelation of the CAp24gag antigen content of each fraction by immunoblotting experiments, revealed that the T23A-mutated particles migrate at a lower density than WT virions (1.087 and 1.099, respectively) with a density shift of 0.012 g/ml from the mutant to the wild type (data not shown). Furthermore, the density distribution of the NL4.3T23A particles was found to be wider than that of WT virions, ranging from 1.079 to 1.133 and 1.080 to 1.110 g/ml, respectively. The NL4.3T23A particles thus appeared to be relatively less dense and more heterogeneous than those of NL4.3. Altogether these data indicate that mutation of the Thr23 phosphorylable residue within the p6gag protein of HIV-1 interferes with viral particle release from the host cell membrane, leading to the production of low amounts of cell-free virions that differ in size, shape, and maturation from WT viruses.



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  		FIG. 4.
Analysis of protein expression and maturation in NL4.3 and NL4.3T23A viruses. A, transfected cells were processed for EM as described under "Materials and Methods." Arrows indicate particles tethered to the plasma membrane. Characteristic images observed for WT and T23A viruses are enlarged. Morphologic characterization of NL4.3 and NL4.3T23A virions (B) and diameter of WT and T23A mutated cell-free particles (C) were defined from EM observations. The frequency of occurrence in the total population (%) is represented according to the particle size in nanometers. Gray bars, WT viruses; black bars, T23A viruses. Mean values for each population are indicated.

 
Consequences of ERK-2-dependent Phosphorylation of p6gag on HIV-1 Protein Maturation and Packaging—Viral proteins content was further analyzed in immunoblotting assays using lysates of cell-free NL4.3 and NL4.3T23A particles normalized according to their gp41env envelope glycoprotein content. In agreement with mature phenotypes observed from microscopy experiments, only a faint proportion of unprocessed Pr55gag precursor was detected from cell-free NL4.3 particles (Fig. 5A) with a corresponding accumulation of processed CAp24gag and MAp17gag proteins. In contrast, a high level of unprocessed Pr55gag polyprotein remained associated with NL4.3T23A virions. Concomitantly a moderate reduction in processed CAp24gag accompanied by a significant reduction in levels of virus-associated MAp17gag was observed in the NL4.3T23A particles as compared with NL4.3 viruses. This observation indicates that the substitution of Thr23 by an alanine results in Pr55gag processing defects. However, the presence of a substantial amount of cleaved CAp24gag protein in the T23A particles suggests that in the T23A-mutated particles a proportion of Gag precursor undergoes complete maturation. Unlike most processing defects, there was no detectable increase in Pr55gag cleavage intermediates, suggesting that Pr55gag was either mostly processed or not cleaved at all. In addition, a significant decrease in p66RT and p51RT incorporation was observed in NL4.3T23A particles as compared with NL4.3 virions.



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  		FIG. 5.
Analysis of protein expression and maturation in NL4.3 and NL4.3T23A viruses. A, supernatants from mock transfected 293T cells, or cells expressing pNL4.3, pNL4.3T23A, or cell-associated viral proteins were processed for Western blotting experiments as described under "Materials and Methods." Proteins were revealed with an anti-CAp24gag, an anti-MAp17gag mAb, or with an antisera directed to p6gag and RT proteins. Position of viral proteins is indicated. B, supernatants prepared from mock transfected 293T cells, or cells expressing pNL4.3 or pNL4.3T23A maintained in the presence of inhibitory amounts of indinavir sulfate were processed for Western blotting experiments and revealed with an anti-p6gag serum.

 
To determine whether these variations were generated at the level of protein synthesis or incorporation, we next evaluated polyprotein precursors and viral proteins expression in lysates of cells transfected with pNL4.3 or pNL4.3T23A molecular clones. As shown in Fig. 5A (right panel) similar amounts of uncleaved polyprotein precursors (including Pr160gag-pol, Pr55gag, and gp160env) were detected from the total extracts of cells expressing wild type or mutated viral genomes. In addition, similar expression levels of mature CAp24gag, MAp17gag, and RT subunits were found in both cell extracts. Thus, variations in viral protein content of the T23A-mutated virions do not appear to be related to the modification of viral precursor expression in infected cells. Altogether our data indicate that substitution of the single ERK-2 phosphorylable amino acid within the p6gag domain results in the decrease of Pr55gag processing as well as in the misincorporation of RT subunits.

It was previously reported that virus phenotypes including alterations in the pattern of Pr55gag processing, defects in RT protein incorporation, and immature morphology of viral particles may correlate with an absence or defects of the viral protease activity (37, 45–47). To understand the mechanism by which the p6gag mutation interferes with the viral phenotype, and to determine the precise contribution of the viral protease, we examined the NL4.3T23A budding in the absence of protease activity. 293T cells were transfected with pNL4.3 and pNL4.3T23A DNAs and cultured for 24 h in the presence of indinavir sulfate (5 µM) (kindly provided by Merck). Cell-free virions were then processed for immunoblot analysis. As shown in Fig. 5B, patterns revealed by an anti-p6gag serum are similar for NL4.3- and NL4.3T23A-mutated viruses. Moreover, an accumulation of unprocessed Pr160gag-pol and Pr55gag precursors and processing intermediates as p41b associated to viral particles was observed, indicating that the viral processing was abolished. In these experimental conditions, the NL4.3T23A virion sample produced from protease inhibitor-treated cells contained less Pr55gag than did the NL4.3 virions produced in similar conditions. As the elimination of protease activity did not allow to recover comparable expression levels of viral precursor proteins for wild type and T23A-mutated virions, our result suggests that differences in viral particle maturation might be linked to a defect in virus release rather than an altered protease activity. In conclusion, the substitution of the Thr23 residue within p6gag results in alterations of viral protein maturation and packaging as well as in alteration of virions release.

Contribution of Thr23 Residue Phosphorylation in HIV-1 Infectivity—Infectivity of NL4.3 and NL4.3T23A particles was next assessed on the MAGI indicator cell line on the basis of a single round of replication (48). Virions were purified from supernatants of transfected 293T cells, normalized either for CAp24gag content or RT activity, and incubated at different infectious concentrations with MAGI indicator cells. Forty-eight h post-infection, viral infection of the cells was monitored by quantification of 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal) hydrolysis measured from the cell lysates. As presented in Fig. 6A, infection level of cells exposed to NL4.3T23A virions was found to be 2.5 to 4 times lower that observed from cells exposed to similar amounts of NL4.3 particles. Such differences were observed at any infectious input tested. Similar data were obtained with infectious supernatants normalized for RT activity (Fig. 6B). Thus, NL4.3T23A particles appear to display reduced infectivity as compared with NL4.3 particles. This observation is in agreement with assembly and maturation defects observed from NL4.3T23A particles. Altogether these data indicate that phosphorylation of the Thr23 residue within p6gag contributes to maintain HIV-1 infectivity.



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  		FIG. 6.
Consequences of p6gag phosphorylation by ERK-2 on viral infectivity. Infectivity of WT and T23A mutated particles was monitored using the MAGI indicator cell line. Supernatants obtained from transfected 293T cells expressing either pNL4.3 or pNL4.3T23A plasmids were normalized for CAp24gag content (A) or RT activity (B) and increasing amounts of virus were added to MAGI cells. Forty-eight hours post-infection, {beta}-galactosidase activities were determined from the cell lysates and results were normalized according to the total protein content. Each value is the mean of 6 separate experiments and errors bars indicate ± S.D. NL4.3, gray bars; NL4.3T23A, black bars.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In addition to their own viral gene products, HIV-1 particles incorporate a vast array of host proteins while budding from the infected cells. Some of these host cell components have been identified as protein kinases and their implication in virus life cycle by the means of viral protein phosphorylation was clearly established (see Ref. 49 for review). In addition to MAp17gag and CAp24gag that have previously been reported to be phosphorylated by protein kinases packaged within viral particles (32, 33), we report here that the p6gag protein and its p15gag and Pr55gag polyprotein precursors are phosphorylated by protein kinase(s) incorporated within HIV-1 virions. Using GST-fused proteins, p6gag was found to be phosphorylated by ERK-2, a host cell MAPK that we previously found to be specifically incorporated within HIV-1 particles (36). This finding further extends the recent observations published by Muller et al. (35), indicating that within HIV-1 particles, the p6gag protein is detected under a phosphorylated form and suggesting the contribution of an unknown host cell kinase belonging to the cyclin-dependent/MAPK family.

We show here, from a systematic mutational analysis, that p6gag phosphorylation by ERK-2 occurs at a unique consensus Thr-Pro phosphorylation site surrounding the Thr23 residue. This sequence was found to be conserved in most HIV-1 consensus sequences, supporting a possible physiological function. Substitution within the complete pNL4.3 DNA of the sequence encoding the phosphorylable threonine residue at position 23 in p6gag by an alanine was found to generate viral particles with drastically reduced infectivity. As observed from EM experiments, a majority of NL4.3T23A virions, that were controlled to display an alteration in virus-associated p6gag phosphorylation, remained attached to the host cell membrane and frequent clusters of two or more particles that failed to detach from each other were observed. Most of the particles released in the cell supernatant were found to display an immature morphology and showed a significant increase in size as compared with WT virions. Analyzing viral maturation of NL4.3T23A virions, the persistence of the unprocessed Pr55gag precursor and misincorporation of viral proteins were observed. Indeed, when the overall levels of gp41env proteins were normalized, an ~8-fold lower level of RT subunits was found in the p6gag mutant virus compared with the NL4.3 virions. Altogether, such modifications may account for the observed reduction of viral infectious capacity.

Beside the major contribution of ERK-2 in p6gag phosphorylation, our data highlight the fact that p6gag is targeted by several host cell kinases. Indeed, in vitro phosphorylation assays performed using preparations of NL4.3T23A virions as a substrate demonstrated only a 25% inhibition of p6gag phosphorylation. Furthermore, two-dimensional analysis showed the persistence of phosphorylated p6gag proteins when the ERK-2-targeted Thr23 residue was mutated. This result corroborates previous observations by Muller et al. (35) indicating that synthetic inhibitors of MAP kinase only reduced p6gag phosphorylation by 40%. The precise role of additional phosphorylation(s) of p6gag by kinases distinct from ERK-2 remains to be investigated.

In our study, several arguments suggest that the phenotype observed for the NL4.3T23A mutant particles is not linked to changes in the pol reading frame in a region upstream of the protease. Indeed, the substitution of the Thr23 residue by an alanine in the pol frame generates a mutation that is frequently encountered in infectious primary isolates of HIV-1. In addition, phenotypes observed are very similar to those generated by p6gag substitutions that were demonstrated to be silent in Pol (47). Finally, the fact that comparable levels of cell-associated polyprotein precursors have been detected from cells producing NL4.3 and NL4.3T23A virions indicates that protein expression and synthesis were not compromised and may rather suggest a defect in the protein incorporation and/or precursors processing. This hypothesis is supported by the fact that, in the context of an inactive protease, a reduced release of NL4.3T23A particles was still noticed, indicating that the T23A mutation interferes at the level of virus assembly rather than in Pol protein processing.

Interestingly, our findings are reminiscent of phenotypes observed from deletion mutants of the p6gag L-domain containing protein or when proline residues within the PTAP motif were mutated, resulting in the accumulation of virions at the cell surface in intermediate stages of budding (50–53) and in the release of a very reduced number of particles that are extremely large in size (54, 55). However, in contrast to p6gag mutants previously described in the literature, the T23A mutation failed to produce a complete inhibition of virion separation from the cell membrane. Yet, the proportion of particles released from the cells was significantly reduced as compared with WT virions.

Previous literature reports have proposed that the p6gag functional role relies both on the presence of the L-domain sequence and on additional determinants located outside of this region within p6gag, which remain to be identified (56). We propose from our data that ERK-2-dependent phosphorylation of p6gag at the level of a single threonine residue is required for proper cell membrane separation, virion-virion detachment, and correct core maturation in this context. Thus, phosphorylation events might contribute to the p6gag protein late-budding activities.

The precise mechanism by which mutation of ERK-2 phosphorylable residue modifies viral budding remains to be determined. It has been suggested that phosphorylation of p6gag is likely to occur concomitantly with or after the release of p6gag at the site of budding at the plasma membrane (35). Existence of a more appropriate conformation for the phosphorylation of the p6gag domain in the context of p15gag or Pr55gag precursors, as demonstrated from the present study, is in agreement with this hypothesis. Recent evidences have been obtained that the p6gag domain of the Pr55gag plays a predominant role in HIV-1 budding by facilitating efficient virus release through interactions with various cellular proteins, mainly the Tsg101 tumor suppressor protein (21, 22, 24). This interaction is optimized by p6gag monoubiquitination as proteasomal blockade profoundly interferes with viral release, morphology, and infectivity of secreted virions by altering the processing of Pr55gag polyprotein (22, 57). As approaching phenotypes were observed when the Thr23 ERK-2-phosphorylable residue of p6gag was mutated, a relationship may exist between p6gag phosphorylation, recruitment of ubiquitin, and viral maturation. This hypothesis is supported by the fact that group IV WW domain-containing proteins (a protein family including the E2 ubiquitin ligases) preferentially bind to short sequences with Ser-Pro or Thr-Pro sequences in a phosphorylation-dependent manner (58, 59). Thus, phosphorylation events may act by facilitating the cooperation between p6gag and host cell proteins involved in endocytic internalization and endosomal sorting by regulating the recruitment of WW domains of ubiquitin-ligases and consequently the ubiquitin-dependent release of the assembled virions.

In conclusion, we propose that phosphorylation of the p6gag protein by ERK-2 may contribute in regulating the dynamic of HIV-1 budding/maturation process. The precise machinery of this mechanism remains to be explored. It has been known for years that L-domain containing proteins from several viruses including pp12 protein of Moloney murine leukemia virus, pp24 of Mason-Pfizer monkey virus, and vesicular stomatitis virus M protein are phosphorylated (60–64). Although a functional role has been proposed for some of these phosphorylation events, their functional relevance in the viral replicative cycle still remains unclear. We demonstrate here, for the first time, that the phosphorylation of L-domain containing proteins of HIV-1 participates in viral life cycle. As common retroviral budding mechanisms have been suggested, similar processes may exist for viruses other than HIV-1.


   FOOTNOTES
 
* This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), the French agency against AIDS (ANRS), and the association "Ensemble contre le SIDA." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Contributed equally to the results of this work. Back

Fellow from the French agency against AIDS (ANRS). Back

|| Supported by a grant from the association "Ensemble contre le SIDA." Back

{ddagger}{ddagger} To whom correspondence should be addressed: UM1-CNRS UMR 5121, Institut de Biologie, 4 Bd Henri IV, CS89508, 34960 Montpellier cedex 2, France. Tel.: 33-4-67-60-86-60; Fax: 33-4-67-60-44-20; E-mail: laurence.briant{at}univ-montp1.fr.

1 The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; PR, protease; WT, wild type; mAb, monoclonal antibody; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E2, ubiquitin carrier protein. Back


   ACKNOWLEDGMENTS
 
We thank R. Trauger for providing us with highly purified virus, and Jean-Luc Darlix, Valérie Tanchou et Didier Décimo (ENS, Lyon), for providing us with reagents and helpful discussions. We are indebted to Laurence Molina (IBPH, Montpellier) for technical assistance in bidimensional gel electrophoresis and Robert Z. Mamoun for critical reading of the manuscript.



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