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J Biol Chem, Vol. 274, Issue 27, 19434-19440, July 2, 1999
From We previously reported the presence of two
cellular serine/threonine protein kinases incorporated in human
immunodeficiency virus type 1 (HIV-1) particles. One protein kinase is
MAPK ERK2 (mitogen-activated protein kinase), whereas the other one, a
53-kDa protein, still needs to be identified. Furthermore, we
demonstrated that the capsid protein CAp24 is phosphorylated by one of
those two virion-associated protein kinases (Cartier, C., Deckert, M., Grangeasse, C., Trauger, R., Jensen, F., Bernard, A., Cozzone, A.,
Desgranges, C., and Boyer, V. (1997) J. Virol. 71, 4832-4837). In this study, we showed that CAp24 is not a direct
substrate of MAPK ERK2. Moreover, using site-directed mutagenesis of
each of the 9 serine residues of CAp24, we demonstrated the
phosphorylation of 3 serine residues (Ser-109, Ser-149, and Ser-178) in
the CAp24. Substitution of each serine residue did not affect viral
budding, nor viral structure. By contrast, substitution of Ser-109,
Ser-149, or Ser-178 affects viral infectivity by preventing the reverse transcription process to be completely achieved. Our results suggest that CAp24 serine phosphorylation is essential for viral uncoating process.
The retroviral gag gene encodes the major structural
proteins of HIV-11 particles.
The p55gag precursor is cleaved by the viral protease in
different proteins: the matrix MAp17, the capsid CAp24, the
p6gag, the nucleocapsid NCp7 associated with viral RNA, and
finally, two small spacer peptides p2 and p1 (1). The Gag protein
expression in a cellular context is sufficient to generate
retroviral-like particles that bud at the plasma membrane (for review,
see Ref. 2). Moreover, Gag proteins are able to produce immature
capsids that lack a complete lipid envelope in a cell-free reaction
(3). However, although proteolytic processing is not a prerequisite for
the formation of particles, it is absolutely essential for infectivity
(4).
Phosphorylation of viral proteins plays an important role in regulation
of viral cycle (for review, see Ref. 5). Several HIV-1 proteins,
including structural and accessory proteins, have been shown to be
phosphorylated. The cytoplasmic domain of Vpu is phosphorylated by
casein kinase 2 at 2 serine residues, and this phosphorylation is
necessary for the degradation of CD4 in endoplasmic reticulum (6-9).
Nef is phosphorylated by the protein kinase C in vitro
(10-12) and in vivo (13), and this phosphorylation leads to
an increased down-regulation of CD4 from the cell surface (14). Rev is
phosphorylated in vitro by casein kinase 2 and mitogen-activated protein kinase (MAPK) (15-17), promoting rapid formation of an efficient RNA binding state (18). Vif is phosphorylated in vitro and in vivo on 5 serine residues, 2 of
those are phosphorylated by MAPK and the 3 others by another
unidentified protein kinase. The phosphorylation of at least two of
these sites seems to be essential for Vif function and HIV-1
replication (19, 20). Two forms of Tat are synthesized during HIV-1
infection, and only the two-exon form (Tat 86) can be phosphorylated by
the preactivated double-stranded RNA-dependent kinase.
Whereas the functional significance of this phosphorylation is still
unclear, it has been suggested that it could promote the release of the
protein from the substrate-binding region of the preactivated
double-stranded RNA-dependent kinase (21).
Structural proteins, CAp24 and MAp17, have been shown to be
phosphorylated. Phosphorylation of MAp17 is well documented. Indeed, MAp17 is phosphorylated on serine (22, 23) and tyrosine residues (24).
Controversial results have been published concerning the function of
those phosphorylations. It has been shown that the phosphorylation of
serine residues of MAp17 would be essential to the association of the
protein with the preintegration complex (25). Phosphorylation of the
COOH-terminal Tyr residue (Tyr-131) of MAp17 would be implicated in the
nuclear translocation of the preintegration complex and, thus, would be
essential for the infection of non dividing cells (26, 27). However,
participation of MAp17 during the infection of nondividing cells and
the putative role of the phosphorylation of Tyr-131 in this process
have been discussed by other authors (28-31). Although several studies
have been published attempting to identify the cellular
serine/threonine protein kinase implicated in MAp17 phosphorylation,
results are still controversial. MAp17 has been identified as a
substrate of protein kinase C (32), and this result is consistent with the identification of a highly conserved consensus protein kinase C
phosphorylation motif in the HIV-1 Gag protein. Recently, the ERK/MAPK
has been proposed to phosphorylate MAp17, although there are no
consensus ERK/MAPK recognition sites within MAp17 (33). Compared with
phosphorylation of MAp17, phosphorylation of CAp24 has not been studied
in detail. It has only been shown that CAp24 is phosphorylated on
serine residues (22, 23). However, the phosphorylation sites of CAp24
are still unknown.
We previously reported the presence of two cellular serine/threonine
protein kinases incorporated in HIV-1 particles (34). We have
identified one of those protein kinases as MAPK ERK2, and this result
has been recently confirmed by others (33). The identity of the other
protein kinase is still unknown. We also showed that CAp24 is
phosphorylated on serine residues by one of those protein kinases.
In this study, we showed that MAPK ERK2 does not phosphorylate CAp24.
We demonstrated by site-directed mutagenesis that the three major
phosphorylation sites of CAp24 were Ser-109, Ser-149, and Ser-178.
Furthermore, by investigating the function of this phosphorylation
during viral infection, we showed that this phosphorylation was not
implicated in viral assembly, budding, or Gag protein maturation
processes. By contrast, we demonstrated that phosphorylation of CAp24
on these residues was directly implicated at the early steps of
infection, during the reverse transcription process. These results
reveal a new regulation level of HIV-1 infection.
Plasmid DNAs and Site-directed Mutagenesis--
The parental
viral DNA clone used in these studies is the biologically active
plasmid pNL4-3 (35) and was propagated in Escherichia coli
HB101 (clone 1035). The Unique Site Elimination mutagenesis system
(Amersham Pharmacia Biotech) was used for in vitro
mutagenesis of the CAp24 protein as described by the supplier. The
1.3-kilobase pair BssHII-ApaI fragment encoding
for the Gag proteins was removed from pNL4-3 plasmid containing a
complete infectious clone of HIV-1 and subcloned into the
BssHII and ApaI sites of pET-11c (New England
Biolabs, Inc.). The resulting construct pET-11c/gag was used for
site-directed mutagenesis. Each of the 9 serine residues of CAp24 was
substituted by an alanine residue using
the target mutagenic primers listed in Table I. The Unique Site
Elimination selection primer used to substitute the ScaI site of pET-11c in MluI site was
5'-CTGTGACTGGTGACGCGTCAACCAAGT-3'. All mutant sequences were confirmed
by sequencing using the T7 SequencingTM kit (Amersham
Pharmacia Biotech). Each mutant (SP) was subcloned back in pNL4-3 using
BssHII and ApaI sites. pET-11c/gag and its derivatives were propagated in E. coli DH5 Solid-state Phosphorylation Assay--
Equivalent amounts (1 µg) of GST, GST-p24 (kindly provided by V. Tanchou, Ecole Normale
Supérieure, Lyon, France) and GST-Elk fusion proteins (kindly
provided by D. A. Brenner, University of North Carolina, Chapel
Hill, NC), produced in E. coli DH5 Cell Culture, Transfections, and Virion Production--
Human
kidney 293 cells (ATCC CRL-1573) and HeLa P4 cells (36) were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum (Life Technologies, Inc.), and 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine (and 500 µg/ml Geneticin for HeLa P4 cells). Human CD4+ T lymphocyte cell
lines (H9, SupT1, and C8166) were maintained in RPMI 1640 (Life
Technologies, Inc.) supplemented with 10% fetal calf serum and 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM
glutamine. Peripheral blood mononuclear cells (PBMCs) were isolated
from whole blood of two seronegative individuals using a Ficoll-Hypaque
gradient (Amersham Pharmacia Biotech). For viral infection assay,
a mix of PBMCs from the two donors was stimulated with 10 µg/ml of
phytohemagglutinin (Life Technologies, Inc.) for 72 h and then
maintained in RPMI 1640 as described above supplemented with 20 units/ml interleukin 2 (Roche Molecular Biochemicals).
Viral proteins were expressed transiently by polyethyleneimine
transfection (2 units, ExGen 500, Euromedex) of 1 µg of viral DNA
into 293 cells (4 × 105 cells). Supernatants from
transfected cells were renewed 3 days after transfection and collected
24 h later (day 4). At the end of the experiments, the samples
were analyzed for reverse transcriptase (RT) activity, and 0.45-µm
pore size-filtered viral stocks were stored at
For electron microscopy analysis, DNA transfections were performed on
HeLa P4 cells by calcium phosphate precipitation method on cell
suspension (37) using 10 µg of DNA for 3 × 106 cells.
Viral Infectivity Assays--
All viral stocks used in a given
experiment were normalized for RT activity. Virus was tested for the
induction of cytopathogenic effects by infecting 5 × 104 C8166 cells with 100 µl of virus (at 105
cpm/ml RT activity) or 106 SupT1 cells with 2 ml of virus
(at 2.105 cpm/ml RT activity). The appearance of
cytopathogenic effects on C8166 cells and SupT1 cells was scored,
respectively, 3 and 6 days later.
Two × 106 PBMC and 105 H9 cells were
exposed to 200 µl of the 293 cell-derived viruses at RT activity of
5.104 cpm/ml for 1 h at 37 °C, washed with Hanks'
balanced solution, and plated in 2 ml of fresh medium in 24-well and
6-well plates, respectively. Experiments were realized in duplicates
with PBMCs and in triplicates with H9 cells. Virus infectivity was
monitored by measurement of virion-associated RT activity from cell
culture supernatants every 4 days, starting from 5 days
post-infection.
Reverse Transcriptase Assay and p24 Antigen Enzyme-linked
Immunosorbent Assay--
Supernatants from transfected or infected
cells were harvested and assessed for virion-associated RT activity and
p24 antigen content. Cell culture supernatants were cleared of cells
and cellular debris by centrifugation. For each sample, 100 µl of
culture supernatant were tested for RT activity as described elsewhere
(38). The p24 antigen was determined by enzyme-linked immunosorbent
assay as follow: microplates (Nunc Maxisorb) were coated overnight at room temperature with 1 µg/well in 0.05 M carbonate
buffer, pH 9.5, of an anti-p24 mouse monoclonal antibody from hybridoma
183-H12-5C (39). Plates were saturated for 2 h at 37 °C with
PBS, 10% bovine serum albumin (Sigma). After washing with PBS, 0.1%
Tween 20, 100 µl of supernatants or known quantities of recombinant
CAp24 protein (p24His6 kindly provided by B. Mandran, BioMerieux, Lyon, France) diluted in PBS, 0.1% Tween 20, 10% bovine serum albumin were
incubated for 2 h at 37 °C. Biotinylated rabbit antiserum to
p24 (500 ng/ml) (kindly provided by B. Mandran) were added and
incubated for 2 h at 37 °C. After washing, bound antibodies were detected with streptavidin conjugated with peroxidase for 30 min
at 37 °C. After washing, the enzymatic reaction was developed with
O-phenylenediamine (Sigma) and H2O2.
The color development was stopped with 2 N HCl, and the
absorbancy values were read at 492 nm.
Viral Protein Analysis--
293 cells were transfected with WT
or mutant pNL4-3 DNA. At 72 h post-transfection, 20 ml of
supernatant were collected, filtered through a 0.45-µm pore size
filter, and centrifuged through a 4-ml cushion of 20% sucrose at
35,000 rpm for 2 h at 4 °C in a 50.2 Ti rotor (Beckman).
Virions were dissolved in TEN (0.1 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA) and lysed with
2 × SDS gel-loading buffer (40). Virion-associated proteins,
normalized for RT activity, were separated by electrophoresis on a 10%
ProSieve®50 polyacrylamide gel (FMC), then transferred to PVDF
membrane (Immobilon-P, Millipore). Proteins were analyzed by
immunoblotting using either anti-RT rabbit polyclonal antibodies
(kindly provided by J. L. Darlix, Ecole Normale Supérieure,
Lyon, France) or anti-CAp24 human monoclonal antibody (kindly provided
by D. Bourel, Establissement de Transfusion Sanguine, Lille, France)
and an enhanced chemiluminescence detection kit (Pierce).
Electron Microscopy Analysis--
Transfected HeLa P4 cells were
processed for thin-layer electron microscopy as follows.
Seventy-two hours post-transfection, cells were washed once with medium
and fixed in situ with 2.5% glutaraldehyde in PBS for 30 min at 4 °C. Cells were then post-fixed with 1% osmium tetroxide
and embedded in epon. Sections were counterstained with uranyl acetate
and lead citrate and examined with a Philips CM 120 transmission
electron microscope.
In Vitro Phosphorylation Assay and Immunoprecipitation of
CAp24--
293 cells were transfected with WT or mutant pNL4-3
plasmid. Forty-eight hours post-transfection, medium was renewed, and supernatants were harvested 24 h later. Sixteen ml of 0.45-µm pore size-filtered supernatant were centrifuged through a 4-ml cushion
of 20% sucrose as described above. Viral pellets were dissolved with
50 µl of lysis buffer and submitted to an in vitro phosphorylation assay as described elsewhere (34). Lysates were precleared by incubation in 500 µl of TLB buffer (1 M
Tris, pH 7.4, 1% Triton X-100, 10% glycerol, 140 mM NaCl,
2 mM EDTA) containing 15 µl of protein A/G plus agarose
(Santa Cruz) and 5 µl of heat-inactivated normal human serum for
1 h at 4 °C. After centrifugation for 5 min at 10,000 × g, supernatants were incubated for 1 h at 4 °C with
1 µg of anti-CAp24 human monoclonal antibody, and the CAp24 immunocomplexes were isolated after overnight incubation at 4 °C
using 20 µl of protein A/G plus agarose, pelleted, and washed five
times with 1 ml of TLB buffer. Twenty µl of 2 × SDS gel-loading buffer were added to pellet, boiled for 3 min, loaded, and separated on
a SDS-15% PAGE. Phosphorylated proteins were transferred to PVDF
membrane (Immobilon-P, Millipore), autoradiographed, and then
immunodetected with an anti-CAp24 mouse monoclonal antibody (kindly
provided by B. Verrier, Ecole Normale Supérieure, Lyon, France)
revealed by chemiluminescence.
Viral RNA Analysis--
Viral stocks from 293 transfected cells
were concentrated through a 20% sucrose cushion. Pellets were
resuspended in 200 µl of viral buffer (50 mM Tris, pH
7.4, 100 mM NaCl, 10 mM MgCl2, and
6 mM CaCl2), an aliquot was taken for
measurement of RT activity, and the remainder was lysed in 1% SDS, 100 µg of proteinase K/ml, 10 units of DNase I RNase-free (Promega) in
the presence of 8.5 units of RNase inhibitor (Promega) for 1 h at
37 °C. Nucleic acids were extracted twice with phenol-chloroform and
once with chloroform, ethanol-precipitated, and resuspended in water.
RNA from equivalent quantities of virions, as assessed by RT activity,
was denatured for 15 min at 68 °C in 50% formamide and then blotted
onto a Duralon membrane (Stratagene) using a slot blot apparatus. Blots
were probed with a PCR Analysis of Viral DNA in Infected Cells--
Viral stocks
from 293 transfected cells were normalized for the RT content
(2.105 cpm/ml) and treated with 100 units of DNase I
(Promega)/ml of virus in the presence of 10 mM
MgCl2 and 6 mM CaCl2, for 1 h
30 min at 37 °C, prior to infection. The infection was allowed to proceed for 2, 6, or 24 h using 106 SupT1 cells
incubated in 2 ml of a DNase-treated virion preparation. Total DNA was
extracted by standard procedures (40) and resuspended in 50 µl of TE
(10 mM Tris, pH 8.0, 1 mM EDTA). Seven-hundred ng of each DNA preparation were used as a template in PCR. All reactions were carried out in a total volume of 50 µl in a solution containing 1 µM oligonucleotide primer, 250 µM each deoxynucleotide, 1.5 mM
MgCl2, 50 mM KCl, 10 mM Tris, pH
8.3, and 1 unit of Taq DNA polymerase (Roche Molecular
Biochemicals, Mannheim, Germany). The standard program for
amplification of viral sequences was 30 cycles of a 30 s
denaturation step (94 °C), a 30-s annealing step (56 °C), and a
30-s extension step (72 °C).
All samples were first screened for contaminating pNL4-3 DNA carried
over from transfection by amplification with a forward primer and a
reverse primer corresponding to pUC18 sequences from pNL4-3 (41). Any
samples containing contaminating plasmid DNA detectable by PCR
amplification were discarded.
To normalize the quantity of total cellular DNA present in each sample,
human GAPDH DNA was amplified under the conditions described above with
a forward primer corresponding to nucleotides 70 to 93 (5'-GTGAAGGTCGGAGTCAACGGATTT-3') and a reverse primer corresponding to
nucleotides 430 to 406 (5'-GGGCAGAGATGATGACCCTTTTGG-3').
Sequences of the primer pairs used to amplify viral DNA at the
different steps of reverse transcription; i.e. 5'-R and
3'-U5 to generate the R-U5 DNA, 5'-R and MA to study the second strand transfer, U3pos.1 (5'-R-CTGGAAGGGCTAATTCACTCC-3') and 3'-ASPBS to
detect the full-length cDNA; are described elsewhere (35). To
detect the double LTR circular form (2-LTR), a nested PCR on 1/100 of the initial reaction using primers LTR-U5:
5'-GTCTGTTGTGTGACTCTGGT-3' (nucleotides 566-585) and LTR-R:
5'-GAGGCTTAAGCAGTGGGTTC-3' (nucleotides 9601-9582) was carried out
with primers N-LTR-U5: 5'-GTCAGTGTGGAAAATCTCTAGCA-3' (nucleotides
612-634) and N-LTR-R: 5'-CAGATCTGGTCTAACCAGAG-3' (nucleotides
9554-9535) for a further 30 cycles. Amplified DNA was analyzed by 1%
agarose gel electrophoresis.
The CAp24 Is Not a Substrate of ERK2 MAPK--
We have previously
showed that CAp24 is phosphorylated by one of the two cellular protein
kinases incorporated in viral particles. Moreover, we have identified
one of those serine/threonine kinases as the ERK2 MAPK. The other
protein kinase (53 kDa apparent molecular mass) is still unidentified.
In order to determine whether CAp24 could interact with ERK2 MAPK and
could be phosphorylated, a solid-state kinase assay was performed.
Activated ERK2 MAPK was incubated with either GST-p24, GST, or GST-Elk
fusion proteins immobilized on GSH-Sepharose beads in the presence of
[
These results demonstrate that CAp24 is not a direct substrate for ERK2
MAPK.
Construction of CAp24 Substitution Mutants--
It has been shown
that CAp24 is phosphorylated on serine residues. The CAp24 protein
contains 9 highly conserved serine residues. In order to study which
serine residues were phosphorylated, we generated by site-directed
mutagenesis 9-mutant proviral clones in which each serine residue was
substituted by an alanine residue (Fig.
2). The presence of mutated codon has
been checked by sequencing each proviral clone.
Effects of CAp24 Mutations on Particle Formation and
Release--
In order to test the ability of the CAp24 mutants to
assemble and release virions, 293 cells were transfected with WT or
mutant proviral DNAs. At day 4 post-transfection, supernatants were
harvested and analyzed for RT content. As shown in Fig.
3A, RT activities of mutant
particles were comprised between 131 and 79% of WT particles. Regarding to the standard deviations, those values are not
significantly different. Similar results were obtained by measuring p24
antigen content in cell culture supernatants (data not shown).
Moreover, the transfection efficiency was similar as measured by CAT
activity after co-transfection of each proviral DNA with pCMV-CAT (data not shown). Thus, production of all mutant viruses from the transfected 293 cells was not affected, suggesting that assembly and release were
normal in those mutants.
It has been shown that the p55gag precursor is sufficient for
budding and production of immature particles. In order to verify if the
maturation process of p55gag has been completed in mutant
particles, we performed a Western blot analysis of viral proteins
contained in these particles. The maturation process of p55gag
was detected with an anti-CAp24 antibody that also reacts with the
p55gag and the intermediate processing product p41gag.
No significant difference between WT and mutant viruses was observed
(Fig. 3B, lower panel). Similar amounts of CAp24
and no accumulation of Gag precursor were detected for each mutant compared with WT. To assess whether similar amounts of viral lysates were loaded, the blot was reprobed with an anti-RT polyclonal antibody
(Fig. 3B, upper panel). Again, no significant
difference was observed for the RT content of each viral particle.
These results show that the maturation process has been properly
achieved in all mutant viruses.
Thus, the CAp24 mutations did not appear to affect the late steps of
the virus life cycle, such as protein processing, virus assembly, and release.
State of Phosphorylation of Mutated CAp24--
In order to
determine whether the mutations introduced in CAp24 have modified the
state of phosphorylation of the protein, we performed a phosphorylation
assay followed by an immunoprecipitation of CAp24. Virus stocks
produced from 293 transfected cells were lysed and incubated with
[
Thus, each mutation introduced at Ser-109, Ser-149, and Ser-178 reduced
the phosphorylation level of CAp24.
Infectivity of Virions Containing CAp24 Mutations--
To
determine whether the mutant particles were infectious, H9 and PBMCs
were incubated with WT or mutant virions that had been harvested from
supernatants of 293 transfected cells. Culture medium was harvested
every 4 days and tested for virion-associated RT activity.
As shown in Fig. 4A, viral
replication peaked at day 16 for the WT, SP1, SP3, SP10, SP12, and SP13
viruses. A delay was observed with the SP2 mutant virus. By contrast,
no replication could be detected when SP11, SP14, and SP15 mutant
viruses were tested, even after 40 days of culture. Cytopathic effect
of all mutant viruses was also examined with C8166 or SupT1 cells,
showing that SP11, SP14, and SP15 mutant viruses were unable to induce
syncytia formation in these cells (data not shown). Thus, SP11, SP14,
and SP15 viruses were not infectious in three different T cell
lines.
When PBMCs were used as target cells, viral replication peaked at day
12 for the WT, SP1, SP2, SP3, SP12, and SP13 viruses (Fig.
4B). SP10 and SP11 showed a delay in replication, whereas SP14 and SP15 mutant viruses did not replicate in those cells. Thus,
mutant viruses SP14 and SP15, containing Ser-109 or Ser-149 mutation,
were noninfectious in T cell lines and primary lymphocytes. A
cell-dependent phenotype for the replication of SP11 virus
could be obtained when Ser-178 was mutated.
Morphological Characteristics and Viral RNA Contents of SP11, SP14,
and SP15 Mutant Viruses--
In order to assess whether the lack of
infectivity of SP11, SP14, and SP15 mutant viruses might be due to
structural default, morphology of those viruses has been studied by
electronic microscopy. SP11, SP14, and SP15 viruses harvested 72 h
post-transfection presented the same morphological characteristics as
the WT virus (data not shown). This result indicates that alanine
substitution of Ser-109, Ser-149, or Ser-178 did not affect the
ultrastructure of viral particles.
To determine whether the lack of infectivity of these mutants was due
to a default in RNA content of the viral particles, virion RNA was
quantified. WT, SP11, SP14, and SP15 viruses were obtained from 293 transfected cells. Viral RNA was extracted from the viral stocks
normalized for RT activity, blotted, and probed with an
PCR Analysis of Viral DNA Synthesis--
We have shown that
alanine substitution of Ser-109, Ser-149, or Ser-178 affects the
infectious ability of virions without modifying significantly the viral
ultrastructure or the RNA packaging. We then studied the steps of
reverse transcription process by PCR after entry of viral particles
into target cells.
SP11, SP14, and SP15 mutant viruses were produced from 293 transfected
cells, treated by DNase I to remove any plasmid contaminant, normalized
for RT activity, and used to infect SupT1 cells. DNA was extracted from
SupT1 cells 2, 6, and 24 h post-infection and used as a template
in PCRs with primer pairs designed to amplify the products of reverse
transcription. In order to check the absence of plasmid DNA
contaminating samples, all samples were tested with a primer pair that
specifically amplify the pUC vector (41), and all samples found
positives were discarded.
The primer pair 5'-R/3'-U5 annealed with the early intermediate
products of reverse transcription: the minus- and the plus-strand strong stop DNA; primer pair 5'-R/MA detected the second strand transfer and the elongated positive-strand DNA; primer pair
U3pos.1/3'-ASPBS enabled to amplify the completed double-stranded viral
DNA. Finally, the nuclear double LTR circular form was detected by PCR
with the primer pair LTR-U5/LTR-R followed by a second PCR with the internal primer pair N-LTR-U5/N-LTR-R.
As shown in Fig. 5B, the first two steps of reverse
transcription tested were detected for all three mutant viruses at
24 h post-infection. Same results were obtained at the 2- and 6-h post-infection points (data not shown). No signal detection for the
full-length cDNA could be observed when SP14 and SP15 viruses were
used to infect SupT1 cells. By contrast, this DNA intermediate could be
amplified after infection of cells with the SP11 mutant or the WT
viruses. The next reverse transcription step is characterized by the
translocation of the double-stranded cDNA from the cytoplasm to the
nucleus. As expected, no signal was observed for SP14 and SP15 mutant
viruses, whereas no nested PCR product could be detected with SP11
mutant virus.
These results show that substitution of Ser-109, Ser-149, and Ser-178
blocks the infection process during the early steps of the viral life
cycle at the reverse transcription level.
In this report, we have shown that CAp24 is not a direct substrate
of the virion-associated cellular protein kinase ERK2 MAPK and that
Ser-109, Ser-149, and Ser-178 are essential amino acids required during
the early steps of cellular infection.
It has been shown that CAp24 could be phosphorylated by a
virion-associated protein kinase. Two cellular protein kinases are incorporated in viral particles: the ERK2 MAPK and a 53-kDa protein kinase (34). In this study, we have demonstrated that ERK2 MAPK cannot
phosphorylate directly CAp24. This result is consistent with the one
from Jacqué et al. (33) showing that activators and
inhibitors of the ERK2 MAPK pathway have no effect on the phosphorylation of CAp24. Taken together, these findings suggest that
CAp24 could be phosphorylated by the 53-kDa protein kinase. Identification of this protein kinase could allow us to confirm this
hypothesis. Further experiments need to be done to isolate and sequence
this protein before any assessment could be achieved on its role during
the phosphorylation process of the viral protein.
We then focused our study on the identification of the phosphorylation
sites of CAp24 and their putative role in the HIV infection cycle.
Generation of mutant viruses allowed us to emphasize the essential
function of three serine residues: Ser-109, Ser-149, and Ser-178.
Substitution of one of those serine residues significantly reduces the
phosphorylation of the mutated CAp24. Difference in the state of
phosphorylation of the WT and the mutated CAp24 would be due to the
substitution of one serine residue, suggesting that Ser-109, Ser-149,
and Ser-178 were phosphorylated in the WT CAp24. This is the first
evidence concerning the identification of the phosphorylation sites of
CAp24. Indeed, phosphorylation of CAp24 has been demonstrated 10 years
ago (22, 23), and since then, nothing new has been published on this
topic. The fact that the phosphorylation of mutated CAp24 is only
reduced implicates that the two other nonsubstituted serine residues
are still phosphorylated. Thus, it is likely that the conformation of
the mutated CAp24 was not significantly modified by the substitution
since, for each mutant virus SP11, SP14, and SP15, the two other
phosphorylation sites are still reachable to the protein kinase.
Our results allowed us to clarify the role of CAp24 phosphorylation
during the viral life cycle. Indeed, whereas the three mutant viruses
SP11, SP14, and SP15 were able to assemble, bud, and presented a
cone-shape capsid, they were unable to infect T cell lines. In fact,
these viruses were able to enter the cells as shown by the presence of
the first intermediate products of reverse transcription (Fig.
5B), but the reverse transcription process cannot be
completely achieved. The difference in infectivity observed between
SP11 and the two other mutants SP14 and SP15 is apparently
cell-dependent. Indeed, the ability of SP11 mutant virus to
infect cells depends on the target cells used (Fig. 4). This suggests
that a cellular function is able to make up for the phosphorylation of
Ser-178. Furthermore, PCR analysis of different reverse transcription
steps showed a difference between SP11 and SP14 or SP15 viruses. In the
case of SP11 mutant virus, the reverse transcription process is able to
progress till the synthesis of the full-length cDNA, whereas SP14
and SP15 mutant viruses were not able to perform reverse transcription
till this point (Fig. 5B). This could be explained by the
fact that phosphorylation of Ser-109 and Ser-149 (SP14 and SP15,
respectively) was required before phosphorylation of Ser-178 (SP11).
Phosphorylation of those 3 residues might occur during the reverse
transcription process. Without the phosphorylation of Ser-109 and
Ser-149, reverse transcription could not progress after the second
strand transfer, whereas phosphorylation of Ser-178 would be necessary
after the synthesis of the full-length cDNA and thus before the
translocation of the viral DNA into the nucleus. Taken together, these
results suggest that phosphorylation of CAp24 takes place at a very
early step of the cellular infection.
Several reports have used mutagenesis to study the functional role of
the different domains of CAp24, but none have modified the Ser-109,
Ser-149, or Ser-178 residues. Regarding the localization of these three
serine residues, none is implicated in a secondary structure such as
Many studies have demonstrated the phosphorylation of viral proteins,
but little is known about the phosphorylation of structural proteins.
For example, the core protein of hepatitis B virus, that constitutes
the capsid of the virus, is phosphorylated on serine residues (46, 47),
and although the phosphorylated residues have been identified and
several protein kinases have been proposed as candidate for performing
this phosphorylation (48-51), the function of this phosphorylation is
still unclear. In the case of the HIV capsid, we have shown that
phosphorylation of CAp24 is necessary just after the entry of the virus
in the target cell. At this point, two processes occur: the reverse
transcription of viral RNA and the translocation of the viral DNA into
the nucleus. We have demonstrated that with the phosphorylation mutant
viruses, the reverse transcription process could not be completely
achieved. This suggests that the phosphorylation could be implicated in the disassembly of the viral capsid. The detection of the first intermediate reverse transcripts is consistent with the fact that the
reverse transcription has been shown to take place prior to or during
the formation of the mature viral particle (52, 53). Thus, the reverse
transcription process does not depend on the dissociation of the core
structure, and phosphorylation of Ser-109, Ser-149, and Ser-178 could
be necessary to release the reverse transcripts before their
translocation to the nucleus. The addition of negatively charged
phosphate group in a structure such as the viral capsid could generate
some repulsion powers and destabilize the capsid. Implication of
phosphorylation in such a case has already been demonstrated for the
tegument proteins of herpes simplex virus. Morrison et al.
(54) showed that phosphorylation of structural proteins can promote the
dissociation of the viral tegument. If the phosphorylation of CAp24 by
a virion-associated kinase is necessary for the disassembly of viral
core, then, there is certainly a third component to the system. Indeed,
the presence of the two components in a mature viral particle should
destabilize the structure before the infection. Nevertheless, we showed
that CAp24 could be phosphorylated by a virion-associated kinase inside the virion. This could be due to a low level of activation of this
kinase, and when the viral particle attaches to the target cell, a
stimulus would be created to completely activate the kinase and thus
trigger the total disassembly of the viral capsid. This could allow the
phosphorylation process to start at the proper time. For example, it
has been shown that the interaction between gp120 and CD4 is able to
activate cellular kinases implicated in signal transduction (55-59).
One of these kinases could be the stimulus useful to trigger the
phosphorylation of CAp24 and thus the disassembly of viral core.
The assembly process has been well documented for the past few years
(60), whereas little is known about the uncoating process and the very
early events of cellular infection. It has been proposed that
cyclophilin A could play a role in dissociation of CAp24 dimers (61,
62). It could be interesting to study the putative interaction between
cyclophilin A and the phosphorylated CAp24. Does cyclophilin A interact
with the cellular protein kinase that phosphorylates CAp24? Nothing is
known about the putative phosphorylation of cyclophilin A. The
phosphorylation status of the CAp24 and/or the cyclophilin A could
modify the stability of the core structure. Answering these questions
could clarify the first steps of the cellular infection and suggest a
possible level of regulation to block the infection.
We thank J.-L. Darlix, V. Tanchou, and Y. Goldberg for helpful discussions. We thank S. Peyrol and I. Raccurt for
technical assistance for electron microscopy analysis.
*
This work was supported by grants from INSERM, the Agence
Nationale de Recherche sur le SIDA, and the SIDACTION program.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a doctoral fellowship from Fondation Marcel
Mérieux and the Eurodoc program (Conseil Regional
Rhône/Alpes). To whom correspondence should be addressed. Tel.:
33-4-67-86-60-86; Fax: 33-4-67-60-44-20; E-mail:
cartier{at}lyon151.inserm.fr.
The abbreviations used are:
HIV, human
immunodeficiency virus;
ERK, extracellular signal-regulated kinase;
MAPK, mitogen-activated protein kinase;
PBMCs, peripheral blood
mononuclear cells;
PVDF, polyvinylidene difluoride;
PAGE, polyacrylamide gel electrophoresis;
RT, reverse transcriptase;
GST, glutathione S-transferase;
CAT, chloramphenicol
acetyltransferase;
PBS, phosphate-buffered saline;
WT, wild-type;
PCR, polymerase chain reaction;
LTR, long terminal repeat.
Identification of Three Major Phosphorylation Sites within
HIV-1 Capsid
ROLE OF PHOSPHORYLATION DURING THE EARLY STEPS OF INFECTION*
§,,,, and
Virus des Hépatites, Rétrovirus
Humains et Pathologies Associées, INSERM U271, 151 Cours. A. Thomas, 69 424 Lyon Cedex 03, France and the ¶ LaboRetro,
Unité de Virologie Humaine, INSERM U412, Ecole Normale
Supérieure de Lyon, 46 allée d'Italie, 69 364 Lyon Cedex
07, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, pNL4-3, and
its SP derivatives were propagated in HB101 (clone 1035) to prevent
recombinations. Supercoiled plasmids were purified with the Plasmid
Mega kit (Qiagen).
Mutagenesis primers used in site-directed mutagenesis
, were immobilized on
glutathione-Sepharose beads (Amersham Pharmacia Biotech). Beads were
then incubated with 20 ng of activated MAPK ERK2 (Stratagene) for
2 h at 4 °C and washed five times in HBB buffer (20 mM Hepes, pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.05%
Triton X-100, and CompleteTM, a protease inhibitor mixture
from Roche Molecular Biochemicals). Phosphorylation assay was performed
for 20 min at 20 °C by resuspending the beads in kinase buffer (50 mM Hepes, pH 7.5, 5 mM MnCl2)
containing 2 µCi of [
-32P]ATP. The reaction was
stopped by washing five times in cold HBB buffer. Proteins were then
separated by SDS-15% PAGE, transferred to PVDF membrane (Immobilon-P,
Millipore), immunoblotted with a polyclonal anti-ERK2 antibody (Santa
Cruz), and autoradiographed to visualize phosphorylated products, as
described elsewhere (34).
80 °C until used.
All experiments were done in triplicate. The transfection efficiency
was assessed 72 h after co-transfection of pCMV-CAT (Roche
Molecular Biochemicals) and of each proviral DNA using a CAT
enzyme-linked immunosorbent assay (Roche Molecular Biochemicals).
-32P-labeled 115-base pair fragment
from the gag region (nucleotides 1544-1659). Relative RNA
contents of WT and mutant virions were determined either by a 24-h
autoradiography or by using a Molecular Dynamics PhosphorImager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]-ATP. As shown in Fig.
1, no direct interaction, and thus no phosphorylation, could be detected when activated ERK2 MAPK was incubated with GST-p24 or GST proteins. By contrast, GST-Elk, one of
the known substrates of ERK2 MAPK, indeed interacted and was
phosphorylated as expected.
View larger version (49K):
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Fig. 1.
In vitro phosphorylation assay
between GST-p24 and MAPK ERK2. Recombinant activated MAPK ERK2 was
incubated with either GST or the GST-p24 or GST-Elk fusion proteins, in
presence of [ -32P]-ATP, subjected to SDS-15% PAGE,
and blotted onto a PVDF as described under "Experimental
Procedures." Membrane was subjected to an autoradiography
(32P) and then processed for immunodetection with an
anti-ERK2 MAPK polyclonal antibody (anti-MAPK).
View larger version (17K):
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Fig. 2.
Schematic representation of CAp24
substitution mutants. The specific serine residue (Ser) modified
in each mutant and the name of corresponding plasmid construct are
shown on the left (A, alanine; L,
leucine; P, proline; S, serine). MHR,
major homology region.
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[in a new window]
Fig. 3.
Viral particle formation and release.
A, release of RT-containing viral particles from 293 cells
transfected with CAp24 WT and mutant viruses. The virion-associated RT
activity of each mutant is presented as a percentage of WT activity.
S.D. values are calculated based on five independent transfections.
B, analysis of viral protein content of CAp24 mutant
particles by Western blot. Viral protein lysates, normalized to contain
comparable RT activity, were subjected to SDS-10% PAGE and immunoblot
analysis. Blots were sequentially probed with rabbit anti-RT and human
anti-CAp24 antibodies, and protein bands were visualized using
chemiluminescent detection. The WT viral construct and respective
mutants are indicated above each lane. The position of migration of RT
heterodimer (p51pol and p66pol), Gag precursor
(p55gag), intermediate processing product (p41gag), and
cleaved protein (CAp24) are indicated on the right.
C, immunoprecipitation of radiophosphorylated mutated CAp24.
Radiophosphorylated viral lysates obtained after transfection of 293 cells with the indicated mutant virus were immunoprecipitated with an
anti-CAp24 human monoclonal antibody, separated by SDS-15% PAGE,
transferred to a PVDF membrane, autoradiographed (32P), and
revealed with an anti-CAp24 mouse monoclonal antibody
(anti-CAp24).
-32P]ATP. Phosphorylated CAp24 was then
immunoprecipitated and visualized by autoradiography as described
elsewhere (34). As shown in Fig. 3C, upper panel,
all the capsid proteins were phosphorylated, with a significant
decrease in signal for the SP11, SP14, and SP15 mutant viruses. Similar
amounts of CAp24 were immunoprecipitated in each virus as shown in Fig.
3C, lower panel.
View larger version (34K):
[in a new window]
Fig. 4.
Replication kinetics of CAp24 mutants.
Virus stocks obtained by transfection of 293 cells with the indicated
molecular clones were normalized for RT activity and used to infect the
H9 T-cell line (A) or PBMCs (B). The infectivity
was monitored by RT activity in the cell supernatant over time. All
data presented are representative of three independent experiments with
similar results. 
, WT; 
, SP1; ×, SP2; 
, SP3; 
, SP10; 
,
SP11; 
, SP12; 
, SP13; *, SP14; +, SP15.
-32P-labeled fragment from the Gag region. Known
quantities of RNA from WT virus were used to establish a standard curve
and to determine RNA quantity within mutant particles (Fig.
5A). Virion RNA incorporated in SP11, SP14, and SP15 mutant viruses were at 47.9, 59.5, and 42% of
the WT virus, respectively. Thus, mutant particles were able to
incorporate viral RNA with an efficiency slightly lower than the WT
virus. However, it is unlikely that such a reduction in RNA
incorporation could generate a total lack of infectivity as observed in
H9, C8166, and SupT1 cell lines or PBMCs.
View larger version (55K):
[in a new window]
Fig. 5.
Analysis of phosphorylation-defective mutant
viruses. A, analysis of viral RNA content. Right
panel, slot blot analysis of virion RNA extracted from viruses
produced from transfection of 293 cells with the indicated clone. Each
virus was normalized for the RT content corresponding to 100 ng of RNA
in the WT. Left panel, PhosphorImager quantification of
bands shown in right panel. B, PCR analysis of
viral DNA synthesis. Infection of SupT1 cells was performed with WT,
SP11, SP14, or SP15 mutant viruses normalized for RT activity and
treated with DNase I. Twenty-four hours post-infection, cells were
lysed, and synthesis intermediates of viral DNA were amplified by PCR.
Amplified fragments were resolved on 1% agarose gel and visualized
with ethidium bromide. The intermediates of reverse transcription
process is indicated on the left. The position of expected
fragment is indicated on the right. To normalize the
quantity of total cellular DNA, human GAPDH DNA was amplified from the
same set of samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helix or 
hairpin (42, 43). It is admitted that CAp24 is
constituted by two domains: an amino-terminal domain (1-146 residues),
whose refolding is implicated in the formation of the cone-shaped core
(44, 45), and a carboxyl-terminal domain (151 to 231 residues), which
is implicated in the dimerization of the CAp24. The several amino acids
located between this two domains (146-151 residues) are necessary for
forming the high-affinity capsid dimer interface (43). Modification of
one of those two functions leads to a structural default of the viral
particles as shown by studies using mutagenesis. SP11, SP14, and SP15
viruses do not present any structural defaults. Furthermore, Ser-109 is located in the amino-terminal domain of the CAp24 and is likely to be
exposed to the surface of the capsid (44), Ser-149 is located in the
linker previously cited, and Ser-178 is located in the
carboxyl-terminal domain, on the inner side of the cone-shaped structure (45). Surprisingly, although those 3 serine residues were
located in different functional domains of the CAp24, their substitution led to similar phenotypes regarding to the late steps of
viral life cycle and the morphogenesis of the virion. This observation
suggests that phosphorylation of these residues is necessary for a
mechanism different from the ones already described.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: UJF-LAPSEN, INSERM U318, CHU de Grenoble, 38 043 Grenoble Cedex 9, France.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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