Full Peptide Synthesis, Purification, and Characterization of
Six Tat Variants
DIFFERENCES OBSERVED BETWEEN HIV-1 ISOLATES FROM AFRICA AND
OTHER CONTINENTS*
Jean-Marie
Péloponèse Jr.
§,
Yves
Collette¶,
Catherine
Grégoire
,
Christian
Bailly**,
Daniel
Campèse
,
Eliane F.
Meurs
,
Daniel
Olive¶, and
Erwann P.
Loret
§§
From the
Laboratoire d'Ingénierie des
Systèmes Macromoléculaires, Institut de Biologie
Structurale et Microbiologie, CNRS UPR 9027, 31 Chemin Joseph Aiguier,
13402 Marseille, France, the ¶ Unité de Cancerologie
Expérimentale, INSERM U 119, 27 Boulevard Leî Roure,
13009 Marseille, France, the ** Institut de Recherche sur le Cancer,
INSERM U 124, Place de Verdun, 59045 Lille, France, and the

Unité de Virologie et Immunologie
Cellulaire, Institut Pasteur, 28 rue du Dr. Roux,
75015 Paris, France
 |
ABSTRACT |
AIDS in Africa is characterized by the equal
distribution of mortality between the two genders because of highly
virulent human immunodeficiency virus type 1 (HIV-1) strains. The viral protein Tat trans-activates viral gene expression and is essential for
HIV-1 replication. We chemically synthesized six different Tat
proteins, with sizes ranging from 86 to 101 residues, from HIV-1
isolates located in different parts of the world including highly
virulent African strains. Protein purification, mass spectroscopy, and
amino acid analysis showed that the synthesis was successful in each
case but with different yields. We show that all have the ability to
bind the HIV long terminal repeat (LTR) RNA trans-activation response
element (TAR) region, involved in Tat-mediated trans-activation, but
structural heterogeneities are revealed by circular dichroism. These
Tat synthetic proteins cross membranes but differ in their ability to
trans-activate an HIV LTR-reporter gene in stably transfected HeLa
cells. Two Tat proteins from virulent African HIV-1 strains were much
more active than those from Europe and the United States. The
interferon-induced kinase (PKR), involved in cell antiviral defense,
phosphorylates only Tat variants corresponding to less or nonvirulent
HIV-1 isolates. Our results indicate that the high virulence of some
African HIV-1 strains could be related to Tat activity.
 |
INTRODUCTION |
The HIV1 encodes
regulatory proteins such as Tat, which profoundly affect the course of
viral gene expression in infected cells. Tat stimulates the production
of full-length viral transcripts by RNA polymerase II (1, 2). This
function is generally associated with the ability of Tat to bind the
nascent leader RNA hairpin TAR located at the 5'-end of all HIV-1
mRNAs (3). Tat presents a highly conserved basic region that can
adopt an extended structure to fit into the TAR major groove (4).
Furthermore, this basic region provides for Tat the capacity to cross
membranes (5, 6).
However, the role of Tat in the HIV cycle is more than to facilitate
the elongation of HIV mRNA. Tat is required for efficient HIV-1
reverse transcription (7). Activities of different cellular kinase is
modulated by Tat, which results in the activation of viral
transcription in infected cells (8, 9). Tat protein is released from
HIV-1-infected cells and can be detected in sera from HIV-1-infected
patients (10). Extracellular Tat may account for the decrease of the
host immune response and cellular disorders connected to AIDS
pathology. Tat in synergy with a basic fibroblast growth factor (bFGF)
is involved in the induction of Kaposi's sarcoma lesions (11). Tat is
able to repress the major histocompatibility complex (MHC) class I
genes and provides the virus with a mechanism to evade the host immune
response (12). Finally, Tat participates in the induction of apoptosis
in lymphocytes and contributes to the depletion of the CD4+ T cells in
AIDS (10, 13).
One of the primary cellular responses to viral infection is the
production of interferon. The double-stranded RNA-dependent protein kinase (PKR) is induced by interferon and can be activated by
double-stranded RNA such as the HIV RNA (14). Following its activation,
PKR in turn inactivates the initiation factor eIF2
, leading to the
inhibition of protein synthesis, which can be detrimental for the viral
life cycle. Overexpression of PKR in HIV-1-infected cells was recently
shown to inhibit the replication of the virus (15). However, interferon
does not have a strong antiviral action during the course of natural
infection. Tat was shown to inhibit the PKR activity in
vitro and therefore Tat may contribute to the HIV resistance to
interferon (16). Interestingly, once activated, PKR is able to
phosphorylate Tat but the physiological significance of this
phosphorylation is not known (17).
Tat is encoded by two exons and its second exon can be variable in size
depending on the nature of the HIV-1 isolates (18). Most structural
studies on Tat are made with a Tat protein 86 residues long,
corresponding to the Bru HIV-1 isolate (19) or a closely related
sequence from the HXB2 HIV-1 isolate (20). However, the analysis of Tat
protein sequences allowed us to identify six structural groups
(21).
In this study, we have chemically synthesized six Tat proteins
representative of each structural group. We show that all have the
ability to bind the HIV LTR TAR region but structural heterogeneities appeared when analyzed by circular dichroism. Interestingly, these synthetic proteins cross membranes but differ in their ability to
trans-activate an HIV LTR-reporter gene in stable transfected HeLa
cells. We show a correlation between the rate of trans-activation, the
Tat-PKR interaction, and HIV-1 virulence.
 |
MATERIALS AND METHODS |
Protein Synthesis, Purification, and
Characterization--
Peptides were assembled according to the method
of Barany and Merrifield (22) on HMP preloaded resin (0.5-0.65 mmol)
(Perkin Elmer, Applied Biosystem Inc., Forster City, CA) on an
automated synthesizer (ABI 433A, Perkin Elmer, Applied Biosystem Inc.). To avoid derivatives with deletion, the N-terminal extremities without
Fmoc were capped with a 4.75% acetic anhydre (Merck), 6.25% DIEA 2.0 M, 1.5% 1-hydroxybenzotriazol 1 M (HOBt)
(Perkin Elmer, Applied Biosystem Inc., Warrington, UK), and 87.5%
N-methylpirrolidone (Perkin Elmer). Each deprotection step
was monitored with a conductivity device. The peptides were deprotected
and removed from the resin with trifluoroacetic acid (TFA) complemented
with 10% methylphenyl sulfide (Merck) and 5% ethanedithiol (Merck) as
scavenger. Purification was done with Beckman high pressure liquid
chromatography (HPLC) apparatus and a Merck C8 reverse-phase column
(10 × 250 mm). Buffer A was water with 0.1% TFA and buffer B was
acetonitrile with 0.1% TFA. Gradient was buffer B from 20 to 40% in
40 min with a 2 ml/min flow rate. HPLC analysis was done with a Merck
C8 reverse phase column (4 × 125 mm) with similar buffers but a
gradient from 10 to 50% B in 40 min and a 0.8 ml/min flow rate.
Electrospray mass spectrometry was carried out with a Perkin Elmer
single quad PE-SCIEX API 150ex. Amino acid analyses were performed on a
model 6300 Beckman analyzer.
Electrophoretic Mobility Shift Assays--
The 59-nucleotides
TAR RNA containing the essential UUU pyrimidine bulge was prepared by
in vitro T3 RNA polymerase transcription. Binding reaction
mixtures (20 µl) contained 0.2 nmol of radiolabeled TAR RNA, 0-100
ng of Tat in TK buffer (50 mM Tris, pH 7.4, 20 mM KCl, 0.1% Triton X-100). Complexes were separated from
unbound RNA by electrophoresis in nondenaturing 8% polyacrylamide gels containing 0.1% Triton X-100. The gel was pre-run for 30 min before loading the sample (25 µl). The electrophoresis was continued for 90 min at about 200 V. The relative amounts of free and/or bound RNA were
determined by phosphoimaging.
Circular Dichroism--
CD spectra were measured with a 50-µm
path length cell from 260 to 178 nm on a Jobin-Yvon (Long-Jumeau,
France) UV CD spectrophotometer (Mark VI). The instrument was
calibrated with (+)-10-camphorsulfonic acid. A ratio of 2.1 was found
between the positive CD band at 290.5 nm and the negative band at 192.5 nm. Data were collected at 0.5 nm intervals with a scan rate of 1 nm
per min. CD spectra are reported as 
per amide. The samples were
prepared in 20 mM phosphate buffer, pH 4.5, or in 50%
trifluoroethanol (TFE). Protein concentrations were in a range from 0.5 to 1 mg/ml.
Trans-activation with HIV LTR Transfected Cells--
Functional
trans-activation by synthetic Tat was assessed using P4 cells. These
HeLa-CD4 cells carry the bacterial lacZ gene under the control of the
HIV LTR, and cytoplasmic accumulation of
-galactosidase is strictly
dependent on the presence of Tat. 80% confluent cells plated in a
12-well plate were incubated for 24 h at 37 °C, 5%
CO2, with Tat protein contained in DMEM medium supplemented
with 0.1% BSA. Following this incubation period, cells were washed
with phosphate-buffered saline, proteins were extracted and analyzed
for
-galactosidase using the commercial antigen capture
enzyme-linked immunosorbent assay (
-galactosidase ELISA, Boehringer
Mannheim, France), according to the manufacturer instructions. Values
were normalized using the concentration of total proteins of the
various cell lysates as determined by the Bradford method.
In Vitro Phosphorylation--
Monoclonal antibodies directed
against PKR were first bound to protein A-agarose for 60 min at room
temperature (1 µl Mab/100 µl agarose) in buffer I (20 mM Tris-HCl, pH 7.6, 40 mM NaCl, 50 mM KCl, 1% Triton X-100, 1 mM EDTA, 0.05%
aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM 2-mercaptoethanol, 25% glycerol). The beads were washed
three times in buffer I and incubated with cytoplasmic extracts from
interferon-treated Daudi cells at 4 °C for 20 h (extracts
equivalent to 106 cells/100 µl of beads). They were then
washed three times in buffer I, further washed three times with buffer
II (20 mM Tris-HCl, pH 7.6, 100 mM KCl, 0.1 mM EDTA, 0.05% aprotinin, 20% glycerol), and resuspended
in buffer III (buffer II supplemented with 2 mM MgCl2). For the phosphorylation reaction, each sample (30 µl of beads) was incubated with 40 µl of buffer III supplemented
with 2 mM MnCl2 and 10 µl of
[32P-
]ATP solution (1.25 mCi/ml
[32P-
]ATP (3000 Ci/mmol; ICN), 10 µM
ATP, 1 mM MgCl2 in buffer II). The reaction was
performed in the absence or in the presence of 1 µg/ml of
poly(I)·poly(C) (Amersham Pharmacia Biotech). After incubation for 15 min at 30 °C, eIF2
(2 µl of a purified preparation; gift of
Christopher Proud) or the different Tat proteins (0.2 to 0.4 µg) were
added, and the reaction was continued for another 15 min. All Tat
proteins come from 100 µg of lyophilized proteins resuspended in 100 mM phosphate buffer, pH 4.5, containing 5% bovine serum
albumin. For PKR phosphorylation assay, they were diluted 10 times in
buffer II supplemented with 2 mM MgCl2 and 2 mM MnCl2 and added immediately to the
incubation mix. At the end of the incubation, an equal volume of 2×
protein electrophoresis buffer was added, and the products were
analyzed by SDS-polyacrylamide gel electrophoresis on 17.5%
polyacrylamide gels.
 |
RESULTS AND DISCUSSION |
The six Tat proteins representative of each structural group were
synthesized with a Fast Fmoc chemistry in solid phase synthesis (22).
Tat Bru (86 residues) and Tat Jr (101 residues) have similar sequences
and are representative of HIV-1 isolates found in Europe and
North-America (20, 23). Tat Oyi (101 residues) also has similarities
with European and North American Tat sequences but was identified from
a healthy Gabones man bearing a defective HIV-1 strain (24). Tat Z2 (86 residues) came from a Zairian HIV-1 isolate and is closely related to
an ancestral HIV-1 isolate (ZR59) identified recently (25). Tat Z2 is
the closest of the ancient forms of Tat proteins. In contrast, Tat Mal
(87 residues) and Tat Eli (99 residues) appeared more recently and came
from highly virulent HIV-1 isolates recovered, respectively, from a 24-year old woman and a 7-year old boy in Zaire (now Democratic Republic of Congo) following a dramatic increase of AIDS in central Africa during the 1980s (26).
Each chemical synthesis was done in a single run using a fast Fmoc
chemistry. We use an HBTU activator in each case, and deprotection was
monitored by the measurement of the conductivity. The first sequence to
be synthesized was Tat Bru (Fig. 1) with
carboxymethylated cysteines (Cm-Cys Tat Bru). Previous peptide
syntheses were carried out with Tat HXB2 (86 residues), which has a
sequence very close to Tat Bru (27, 28). After purification of Cm-Cys
Tat Bru (data not shown), our synthetic protein showed that it could
bind TAR RNA (Fig. 3) but could not trans-activate in our HeLa cell assay. The position of the seven cysteine residues is strictly conserved among Tat variants and the importance of cysteine residues for trans-activation is well described in the literature (18). This
explains why Cm-Cys Tat Bru does not trans-activate in our HeLa cells
assay. Another synthesis of Tat Bru was done with free cysteines this
time. This synthetic protein turned out to be able to bind TAR (Fig. 3)
and trans-activates in our HeLa cell assay (Fig. 5). To avoid
precipitation that occurred at neutral pH, the Tat proteins were
dissolved in phosphate buffer at pH 4.5 and then directly mixed to the
cell medium that has a neutral pH. Probably the different ions present
in cell medium neutralized the cysteines. In these conditions, which
were rather close to those encountered by Tat in the blood of HIV
infected patients, we observed a much higher trans-activation activity
(Fig. 5) compared with what was previously reported with synthetic Tat
proteins. Therefore, all the other synthetic Tat variants were done
with free cysteines.

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Fig. 1.
Amino acid sequences of the six Tat variants
synthesized. The sequences are compared with Tat Bru which
represents the sequence most used in laboratories and comes from an
HIV-1 isolated in France (19), whereas Tat Jr come from an American
HIV-1 isolate (23). Tat Oyi is closely related to Tat Jr and Tat Bru
but comes from an HIV-1 identified in a healthy patient in Gabon (24).
Tat Z2 comes from an HIV-1 isolate close to ancestral strains of the
virus (25). Tat Mal and Tat Eli comes from HIV-1 strains isolated
during the 1980s in central Africa following a burst of HIV
heterosexual infection (26).
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Peptide synthesis starts from the C-terminal extremity and the yield of
synthesis for the first 50 residues was from 90 to 98% as a function
of the Tat sequence. This yield decreased significantly beyond this
level for Tat Bru, Tat Jr, and Tat Oyi and are revealed by the HPLC
analysis of the synthesis before purification (Fig. 2). The conductimetry analysis of the
deprotection steps revealed that the sequence just after the basic
region was critical (data not shown). Tat Eli is the sequence that gave
the best synthesis yield and a significant decrease in conductimetry
was observed only from step 88. For Tat Mal, the final deprotection was
not carried out, and half of the crude peptide was cleaved from the resin with an Fmoc on at the N terminus. This gave a 10-min shift for
the major fraction in the analytical HPLC and could have been helpful
for the purification (Fig. 2E). Unfortunately it was not possible to have soluble Tat after the removal of the Fmoc even in the
presence of a solution containing 6 M guanidine and
dithiothreitol (data not shown). The Fmoc was removed before cleavage
for the other Tat and in the crude peptide remaining from Tat Mal (data not shown). The final yield after synthesis and purification varied from 5% for Tat Jr to 16% for Tat Eli. The purity and
characterization of the Tat proteins were determined by analytical HPLC
(Fig. 2), mass spectroscopy, and amino acid analysis (data not shown).
In each case, the results indicated that we obtained a pure protein with the correct molecular weight and the expected amino acid composition.

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Fig. 2.
HPLC analyses. HPLC was
carried out with a C8 reverse phase column at 280 nm (see "Materials
and Methods" for the experimental procedure). For each
panel, the lower run shows the result of the
peptide synthesis before purification, whereas the upper run
shows the result after the last purification step. Tat Bru
(A), Tat Jr (B), Tat Z2 (C), Tat Oyi
(D), Tat Mal (E), and Tat Eli (F) were
cleaved from the resin with TFA. After precipitation with butylmethyl
ether, the proteins were dissolved in 0.1% TFA buffer (lower
runs in each panel). For each protein, purification was
done with two successive semipreparative HPLC runs, and then the pure
fractions were analyzed (upper runs in each
panel) and identified by mass spectroscopy and amino acid
analysis (data not shown). In each case, the major HPLC peak turned out
to be the full sequence. Peaks between 10 and 15 min are derivatives of
50 residues, whereas peaks close to the major fraction are derivatives
with one to fifteen deletions from the N terminus. Highly hydrophobic
fractions were derivatives with a high molecular weight probably
because of side chain incomplete deprotections.
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The six synthetic Tat proteins can bind to TAR RNA (Fig.
3) and can inhibit PKR (data not shown).
Nevertheless, structural changes exist among these proteins and are
revealed by circular dichroism in two different solvents (Fig.
4), and table I shows the results of the
CD data analysis (31). In aqueous buffer (Fig. 4A), it can
be deduced from the intensity of the negative 200 nm band that
nonorganized structures are predominant (29). Table
I reveals that there is no
helix in
aqueous buffer excepted for Tat Mal and Tat Z2, which could have a
short helix of eight to nine residues. In the presence of
trifluoroethanol that mimic the membrane environment (30), three CD
bands characteristic of
helix (29) appeared in all spectra (Fig.
4B). Table I shows that 19 to 30 residues are able to adopt
an
helix structure as a function of the Tat variants. It is
possible that this conformational heterogeneity helps Tat to cross the
membrane barrier because most membrane proteins contain amphipathic
helix. It was shown that peptides derived from Tat can adopt such a
conformation (32). Interestingly, the caboxymethylation of cysteines
leads to an increase in
helix (Table I) and shows that the
cysteines regions probably do not favor this secondary structure.

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Fig. 3.
Electrophoretic mobility shift analysis for
the synthetic Tat proteins. The protein concentration (ng/µl) is
indicated at the top of each gel lane. Complexes and free RNA are
identified as c and f, respectively. The same RNA
preparation was used for the titration with the six proteins. A Tat Bru
derivative with carboxymethylated cysteines (Bru Cm-Cys) was also
tested. The equilibrium dissociation constants (Kd)
were measured directly from these electrophoretic mobility shift
assays. The Kd values vary from approximately 50 nM for both Tat Eli and Tat Mal to about 140 nM
for Tat Oyi and Tat JR. In addition we noted that, beyond the variation
of the Kd values, the binding profiles with the
various proteins were somewhat different. For example, low
concentrations of Tat Bru produced a well resolved single complex and
only with rather high concentrations (>4.5 ng/µl) aggregates which
were formed and which failed to enter the acrylamide gels. In contrast,
multimeric complexes were easily identified with Tat Eli even when
using low concentrations. Up to three retarded bands could be seen with
Tat Eli and to a lower extent with Tat Jr. No such effects were
observed with the shorter proteins such as Tat Bru and Tat Mal, for
example, as well as with the longer protein Tat Oyi.
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Fig. 4.
Circular dichroism. CD spectra of Tat Z2
(open triangle), Tat Oyi (solid triangle), Tat
Bru (open circle), Tat Bru Cm-Cys (no mark), Tat Jr
(solid circle), Tat Mal (open square), and Tat
Eli (solid square) were measured from 260 to 178 nm with a
50 µM path length, either in phosphate buffer 20 mM (pH 4.5) (A) or in 50% TFE complemented with
water (B). The differences observed in the CD spectra
revealed a structural heterogeneity of Tat variants whatever the size.
It is not possible to gather CD spectra in two categories constituted
of short Tat (open mark) and long Tat (solid
mark). In phosphate buffer, CD spectra are characterized by a
negative band near 200 nm, typical of nonorganized structure. The
intense magnitude of the 200 nm band observed with Tat Bru Cm-Cys show
that cysteines play a structural role in Tat. In TFE buffer, CD spectra
are characterized by three CD bands typical of helix. It was
reported that Tat Jr had its C-terminal extremity able to fit into a
groove between the N-terminal and the basic regions (21). This
particularity makes the Tat Jr C terminus more accessible to solvent
and explains the intense magnitude of the helix CD bands.
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Table I
Secondary structure analyses
The percentage of secondary structures for each peptide was determined
from the CD data analysis according to the method of Manavalan & Johnson (31). H, -helix; B, -sheet and/or extended structures; T,
-turn; O, other structures.
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The ability of our synthetic Tat to cross the membrane and
trans-activate the HIV LTR was tested with transfected HeLa cells using
two independent transfection assays. The gene reporter under the
control of HIV-1 LTR was either
-galactosidase (Fig.
5) or luciferase (data not shown). Tat
Oyi was unable to trans-activate in both assays. All Tat proteins
contain a cluster of seven cysteine residues (region 22-37), which are
important for trans-activation because the mutation of one of them
abolishes Tat trans-activation (18). Tat Oyi differs from all other
variants by a substitution of Cys-22 to Ser-22. Tat Oyi has a sequence
very close to Tat Jr, which does trans-activate in our test (Fig. 5).
In support of the importance of the cysteines in the LTR
trans-activation, Tat Bru Cm-Cys cannot trans-activate the HIV LTR
(data not shown) but can bind TAR (Fig. 3).

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Fig. 5.
Trans-activation assay with HeLa cells
transfected with a LTR HIV-1 -gal. The
LTR contains the up- and downstream DNA sequences required for the HIV
transcription, and TAR is present at the beginning of the mRNA
(33). The cellular cofactors required for HIV trans-activation are
present in HeLa cells. Without Tat, there is a basal expression of
-galactosidase that is indicated as a control (column C).
The histograms show the trans-activation observed with the different
Tat variants using two concentrations: 1 µM (light
gray box) and 5 µM (gray box). In each
case, Tat was added to the cell buffer, and therefore expression of
-gal up to the control means that the synthetic Tat was able to
cross the cytoplasmic and nucleus membranes, bind TAR, and interact
with cellular cofactors. Only Tat Bru Cm-Cys (data not shown) and Tat
Oyi failed in this experiment although they bind TAR (Fig. 1). Tat Mal
and Tat Eli show a level of trans-activation three to four times higher
to that of Tat Bru. Tat Z2, the closest of ancestral Tat proteins, has
a low trans-activation level, and evolution could favor HIV-1 isolates
with more efficient Tat. Similar results were obtained with another
transfection assay using luciferase as gene reporter, and Tat Mal and
Tat Eli at 1 µM did trans-activate the LTR to a level
comparable with that obtained with a transfected pCMV-Tat (data not
shown). The range of concentrations used in the different experiments
were from 0.2 to 10 µM (data not shown). At 0.2 µM, only Tat Eli was showing a level of trans-activation
significantly higher than the base level. At 10 µM, the
six Tat were showing levels of trans-activation so high that it was
impossible to see differences between them because there was
saturation.
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The major result of trans-activation tests was the high activity of Tat
Mal and Tat Eli, which correlates with the high virulence of these two
HIV-1 isolates. Interestingly, the long C-terminal extremity of Tat Eli
seems to improve the trans-activation compared with Tat Mal (Fig. 5).
However, the long C-terminal effect was not always observed with Tat Jr
compared with Tat Bru as a function of the concentration (Fig. 5). In a
different assay, the ability of Tat to be phosphorylated by PKR was
verified (Fig. 6). Previous studies have
shown only the phosphorylation to Tat Bru by PKR. Here we have further
analyzed the six synthetic Tat variants with regard to their relation
to PKR. When analyzed for their ability to serve as substrates for PKR,
they proved to be different (Fig. 6). The Tat residues which are
important for phosphorylation by PKR are Ser-62, Thr-64, and Ser-68
(17). The position of these three residues is well conserved in Tat
corresponding to poorly virulent or nonvirulent HIV-1 isolates, such as
Bru, Oyi, and Jr. In contrast to this, the virulent Tat variant Mal and
Eli do not have these residues in similar positions. Fig. 6A
shows that when PKR is activated with a synthetic RNA (poly(I)-poly(C), Tat Bru and Tat Oyi can be clearly phosphorylated but not Tat Mal and
Tat Eli. Phosphorylation level is low for Tat Z2, which also differs
from the STS consensus (Gly-62, Pro-68) but has kept the central
Thr-64. Tat Jr also has a low phosphorylation level although it has the
STS consensus. In a previous study, we mentioned that the C-terminal
extremity of Tat Jr was unable to fit in a groove made by the
N-terminal and the basic region as the C-terminal extremity of other
long Tat variants (21). This structural particularity may disturb the
interaction of Tat Jr with PKR. Fig. 6B shows that if PKR
and Tat concentrations are increased, there is still no phosphorylation
for Tat Mal. This experiment shows the high specificity of the PKR
phosphorylation for Tat Bru compared with Tat Mal.

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Fig. 6.
Activated PKR phosphorylates preferentially
Tat variants from nonvirulent HIV isolates. A,
monoclonal antibody-bound PKR on protein A-agarose (34) was first
incubated for 15 min in the absence or presence of 1 µg/ml of
poly(I)-poly(C) and further incubated for 15 min either as such (PKR
only), in presence of eIF2 , or 0.2 µg of the different Tat. After
incubation, the 32P-labeled proteins were analyzed by
SDS-polyacrylamide gel electrophoresis. The arrows indicate
the position of eIF2 (35 kDa) and Tat (14 kDa), respectively.
Whereas the ability of Tat BRU to be phosphorylated by PKR could be
confirmed, only another Tat variant, TAT Oyi, was also clearly
phosphorylated. A Tat Bru derivative with carboxymethylated cysteines
(Bru Cm-Cys) was also tested. B, the phosphorylation
reaction was performed as in panel A except that the
concentrations of Tat and of bound PKR were increased (0.4 µg of Tat
and Daudi cell extracts equivalent to 3 × 106
interferon-treated cells, respectively). An increase in the
concentration of PKR or Tat could not lead to Tat Mal phosphorylation,
whereas the phosphorylation of Tat Bru (with free cysteines) was
clearly increased.
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The trans-activation assays and the PKR-mediated phosphorylation assays
demonstrate that our Tat variants behave as natural Tat proteins. Tat
Bru can trans-activate HIV-1 LTR whereas Tat Oyi cannot, but they are
both phosphorylated by PKR. Tat Mal and Tat Eli have a high
trans-activation activity but are not phosphorylated by PKR. This
demonstrates for the first time that the PKR-mediated phosphorylation
of Tat is not related to the ability of Tat to trans-activate HIV-1
LTR. However, Tat Z2 and Tat Jr are poorly phosphorylated but donot
have a high trans-activation activity. Only a low phosphorylation is
perhaps enough to modify the Tat trans-activation activity, but it is
possible that the correlation that we observed between the high
trans-activation activity and virulent HIV strains could be also
attributed to differences in uptake into the cell and/or
phosphorylation by PKR. These studies show that few mutations can
induce more efficient Tat proteins, which did correlate with the
emergence of highly virulent HIV-1 strains in Africa. This result
emphasizes the essential role played by Tat in the HIV infection and
all the benefit that a Tat inhibitor could bring to AIDS therapies.
 |
ACKNOWLEDGEMENT |
We are greatly indebted to Pierre Parouto for
fruitful discussion.
 |
FOOTNOTES |
*
This work was supported by l'Agence Nationale pour la
Recherche sur le SIDA (ANRS), INSERM, the Pasteur Institute, and CNRS.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 fellowship cofinanced from la
Société Berroise de Raffinage (Shell Oil Company) and la
région Provence Alpes Côtes-d'Azur.
Recipient of a fellowship from la Fondation pour la Recherche
Médicale (FRM Sidaction).
§§
To whom correspondence should be addressed. E-mail:
loret{at}ibsm.cnrs-mrs.fr.
 |
ABBREVIATIONS |
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
Tat, HIV-1 trans-acting transcriptional
activator;
LTR, long terminal repeated noncoding sequences located at
the two extremities of the HIV-1 provirus;
TAR, RNA trans-activation
response element;
HPLC, high pressure liquid chromatography;
TFA, trifluoroacetic acid;
TFE, 2,2,2-trifluoroethanol;
CD, circular
dichroism;
HMP, 4-hydroxymethyl-phenoxymethyl-copolystyrene-1%
divinylbenzene;
Fmoc, N-(9-fluorenyl)methoxycarbonyl.
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REFERENCES |
-
Fisher, A. G.,
Feinberg, M. B.,
Josephs, S. F.,
Harper, M. E.,
Marselle, L. M.,
Reyes, G.,
Gonda, M. M.,
Aldovini, A.,
Debouk, C.,
Gallo, R. C.,
and Wong-staal, F.
(1986)
Nature
320,
367-371[Medline]
[Order article via Infotrieve]
-
Peterlin, B. M.,
Luciw, P. A.,
Barr, P. J.,
and Walker, M. D.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
9734-9738[Abstract]
-
Berkhout, B.,
Gatignol, A.,
Rabson, A. B.,
and Jeang, K. T.
(1990)
Cell
62,
7257-7267
-
Loret, E. P.,
Georgel, P.,
Johnson, W. C.,
and Ho, P. S.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
9734-9738[Abstract]
-
Vivès, E.,
Brodin, P.,
and Lebleu, B.
(1997)
J. Biol. Chem.
272,
16010-16017[Abstract/Free Full Text]
-
Efthymiadas, A.,
Briggs, L. J.,
and Jans, D. A.
(1998)
J. Biol. Chem.
273,
1623-1628[Abstract/Free Full Text]
-
Harrich, D.,
Ulich, C.,
Garcia-Martinez, L. F.,
and Gaynor, R. B.
(1997)
EMBO J.
6,
1224-1235[CrossRef]
-
Garcia-Martinez, L. F.,
Mavankal, G.,
Neveu, J. M.,
Lane, W. S.,
Ivanov, D.,
and Gaynor, R. B.
(1997)
EMBO J.
10,
2836-2850[CrossRef]
-
Yang, X.,
Gold, M. O.,
Tang, D. N.,
Lewis, D. E.,
Aguilar-Cordova, E.,
Rice, A. P.,
and Herrmann, C. H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12331-12336[Abstract/Free Full Text]
-
Westendorp, M. O.,
Frank, R.,
Ochsenbauer, C.,
Stricker, K.,
Dhein, J.,
Walczak, H.,
Debatin, K.-M.,
and Krammer, P. H.
(1995)
Nature
375,
497-500[CrossRef][Medline]
[Order article via Infotrieve]
-
Ensoli, B.,
Gendelman, R.,
Markham, P.,
Fiorelli, V.,
Colombini, S.,
Raffeld, M.,
Cafaro, A.,
Chang, H. K.,
Brady, J. N.,
and Gallo, R. C.
(1994)
Nature
371,
674-680[CrossRef][Medline]
[Order article via Infotrieve]
-
Howcroft, T. K.,
Strebel, K.,
Martin, M. A.,
and Singer, D. S.
(1993)
Science
260,
1320-1323[Medline]
[Order article via Infotrieve]
-
Li, C. J.,
Friedman, D. J.,
Wang, C.,
Metelev, V.,
and Pardee, A. B.
(1995)
Science
268,
429-431[Medline]
[Order article via Infotrieve]
-
Roy, S.,
Katze, M. G.,
Parkin, N. T.,
Edery, I.,
Hovanessian, A. G.,
and Sonenberg, N.
(1990)
Science
247,
1216-1219[Medline]
[Order article via Infotrieve]
-
Benkirane, M.,
Neuveut, C.,
Chun, R. F.,
Smith, S. M.,
Samuel, C. E.,
Gatignol, A.,
and Jeang, K. T.
(1997)
EMBO
16,
611-624[Abstract/Free Full Text]
-
McMillan, N. A.,
Chun, R. F.,
Siderovski, D. P.,
Galabru, J.,
Toone, W. M.,
Samuel, C. E.,
Mak, T. W.,
Hovanessian, A. G.,
Jeang, K. T.,
and Williams, B. R.
(1995)
Virology
213,
413-424[CrossRef][Medline]
[Order article via Infotrieve]
-
Brand, S.,
Kobayashi, R.,
and Mathews, M. B.
(1997)
J. Biol. Chem.
272,
8388-8395[Abstract/Free Full Text]
-
Jeang, K. T.
(1996)
HIV-1 Tat: Structure & Function, pp. 3-18, Los Alamos National Laboratory (Ed) Human Retroviruses & AIDS Compendium III
-
Barre-Sinoussi, F.,
Chermann, J. C.,
Rey, F.,
Nugeyre, M. T.,
Chamaret, S.,
Gruest, J.,
Dauguet, C.,
Axler-Blin, C.,
Vezinet-Brun, F.,
Rouzioux, C.,
Rozenbaum, W.,
and Montagnier, L.
(1983)
Science
220,
868-871[Medline]
[Order article via Infotrieve]
-
Rosen, C. A.,
Sodroski, J. G.,
Goh, W. C,
Dayton, A. I.,
Lippke, J.,
and Haseltine, W. A.
(1986)
Nature
319,
555-559[Medline]
[Order article via Infotrieve]
-
Grégoire, C. J.,
and Loret, E. P.
(1996)
J. Biol. Chem.
271,
22641-22646[Abstract/Free Full Text]
-
Barany, G.,
and Merrifield, R. B.
(1980)
in
The Peptide: Analysis, Synthesis, Biology (Gross, E., and Meinhofer, J., eds), Vol. 2, pp. 1-284, Academic Press, New York
-
O'Brien, W. A.,
Koyanagi, Y.,
Namazie, A.,
Zhao, J. Q.,
Diagne, A.,
Idler, K.,
Zack, J. A.,
and Chen, I. S.
(1990)
Nature
348,
69-73[CrossRef][Medline]
[Order article via Infotrieve]
-
Huet, T.,
Dazza, M. C.,
Brun-Vezinet, F.,
Roelants, G. E.,
and Wain-Hobson, S.
(1989)
AIDS
3,
707-715[Medline]
[Order article via Infotrieve]
-
Zhu, T.,
Korber, B. T.,
Nahmias, A. J.,
Hooper, E.,
Sharp, P. M.,
and Ho, D. D.
(1998)
Nature
391,
594-597[CrossRef][Medline]
[Order article via Infotrieve]
-
Alizon, M.,
Wain-Hobson, S.,
Montagnier, L.,
and Sonigo, P.
(1986)
Cell
46,
63-74[Medline]
[Order article via Infotrieve]
-
Chun, R.,
Glabe, C. G.,
and Fan, H.
(1990)
J. Virol.
64,
3074-3077[Medline]
[Order article via Infotrieve]
-
Jeyapaul, J.,
Reddy, M. R.,
and Khan, S. A.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
7030-7034[Abstract]
-
Johnson, W. C., Jr.
(1985)
Methods Biochem. Anal.
31,
61-163[Medline]
[Order article via Infotrieve]
-
Urry, D. W.
(1972)
Biochim. Biophys. Acta
265,
115-168[Medline]
[Order article via Infotrieve]
-
Manavalan, P.,
and Johnson, W. C., Jr.
(1987)
Anal. Biochem.
167,
76-85[Medline]
[Order article via Infotrieve]
-
Loret, E. P.,
Vives, E.,
Ho, P. S.,
Rochat, H.,
van Rietschoten, J.,
and Johnson, W. C., Jr.
(1991)
Biochemistry
30,
6013-6023[Medline]
[Order article via Infotrieve]
-
Clavel, F.,
and Charneau, P.
(1994)
J. Virol
68,
1179-1185[Abstract]
-
Laurent, A. G.,
Krust, B.,
Galabru, J.,
Svab, J.,
and Hovanessian, A. G.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
4341-4345[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.