HEED, the Product of the Human Homolog of the Murine
eed Gene, Binds to the Matrix Protein of HIV-1*
Régis
Peytavi
§,
Saw See
Hong
¶,
Bernard
Gay
,
Arnaud Dupuy
d'Angeac
,
Luc
Selig
,
Serge
Bénichou
,
Richard
Benarous
, and
Pierre
Boulanger
**
From the
Laboratoire de Virologie Moléculaire
and Pathogénèse Virale, CNRS UMR-5812, Faculté de
Médecine, 2, Boulevard Henri IV, 34060 Montpellier and the
Laboratoire des Interactions Moléculaires
Hôte-Pathogène, INSERM CJF-97-03, Faculté de
Médecine Cochin-Port Royal, 24 Rue du Faubourg Saint-Jacques,
75014 Paris, France
 |
ABSTRACT |
heed, the human homolog of mouse
eed and Drosophila esc, two members of the
trithorax (trx) and Polycomb group
(Pc-G) of genes, was isolated by screening an activated
lymphocyte cDNA library versus the immunodeficiency
virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system
in yeast. The human EED protein (HEED) had 99.5% identity with the
mouse EED protein and contained seven WD repeats. Two heed
gene transcripts were identified, with a putative 407-nucleotide-long
intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively. The shorter HEED isoform, originated
from the unspliced message, lacked the seventh WD repeat. HEED was
found to bind to MA protein in vitro, as efficiently as
in vivo in yeast cells. Site-directed mutagenesis and phage
biopanning suggested that the interaction between HEED and MA involved
the N-terminal region of the MA protein, including the first polybasic
signal, in a MA conformation-dependent manner. In the HEED
protein, however, two discrete linear MA-binding motifs were identified
within residues 388-403, overlapping the origin of the fifth WD
repeat. Deletion of the C-terminal 41 residues of HEED, spanning the
seventh WD repeat, as in the 494-residue HEED protein, was
detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a
conformational role of the HEED C-terminal domain in the MA-HEED interaction. MA and HEED proteins co-localized within the nucleus of
co-transfected human cells and of recombinant baculovirus co-infected insect cells. This and the failure of HEED to bind to uncleaved GAG
precursor suggested a role of HEED at the early stages of virus
infection, rather than late in the virus life cycle.
 |
INTRODUCTION |
Multiple structural and physiological functions have been assigned
to the matrix (MA)1 of the
human immunodeficiency virus type 1 (HIV-1), the protein that
constitutes the N-terminal domain (132 amino acids from the initiator
methionine) of both GAG (Pr55GAG) and GAG-POL (Pr160GAG-POL) polyprotein precursors (reviewed in Refs. 1-3). At late stages of the
virus life cycle, the MA protein is a key factor of virion morphogenesis, as it is required for intracellular transport (4, 5) and
plasma membrane targeting of Pr55GAG and Pr160GAG-POL and extracellular
budding of the virions (6-13). At early stages of the infectious
cycle, the MA seems to be involved in the infectivity and efficacy of
cell entry of the virus (5, 14-17). The MA has also been found to be
associated with both reverse transcriptase and integrase within the
pre-integration complex (18, 19), and its nuclear localization has been
reported (20).
We have generated a variety of mutations in the gag gene of
HIV-1, consisting of insertions, substitutions, or deletions in the
different structural domains of the Pr55GAG and expressed the
corresponding protein mutants in recombinant baculovirus-infected insect cells (21-28). One of our C-truncated GAG mutants, carrying an
amber mutation at codon 143 in the N-terminal portion of the capsid
(CA) domain (GAGamb143; see Ref. 23), corresponds to the MA
domain, extended at its C terminus of the first 11 amino acids from the
CA. Under its unmyristoylated form (myr
), the recombinant protein of
GAGamb143myr(
) showed a trans-dominant negative
effect on the plasma membrane targeting and extracellular budding of
GAG particles assembled by recombinant wild type Pr55GAG co-expressed
in trans in the same Sf9 cells; membrane-enveloped GAG particles were found to accumulate within intracytoplasmic vesicles
(23). The same trans-dominant negative phenotype on wild
type Pr55GAG was displayed by the N-myristoylated form of the C-terminally deleted MA, GAGamb120myr(+) (23). This
suggested that recombinant MA proteins expressed by
GAGamb143myr(
) and GAG amb120myr(+)
competed with some cellular protein partner(s), participating in the
GAG precursor transport and/or secretion.
In the aim to identify and characterize the cell protein(s) involved in
this process, the recombinant MA protein expressed by
GAGamb143myr(
), referred to as MA143 in the present study, was used as a molecular bait to screen a cDNA library from
activated human peripheral blood lymphocytes in the two-hybrid system
in yeast (29). One of the most frequently isolated clones showed a high
sequence homology with the mouse gene eed (embryonic
ectoderm development; see Ref. 30). Murine eed is a highly
conserved homolog of the Drosophila esc (extra sex
combs) gene, a member of the trithorax (trx)
and Polycomb group (Pc-G) of genes, whose products act as silencers of homeotic genes responsible for
anterior-posterior patterning during embryogenesis (30, 31). The
product of murine eed has been found to possess a
transcriptional repressor activity (32). Phage biopanning and
site-directed mutagenesis were used to map the interacting sites of the
two molecular partners, HIV-1 MA protein on one hand and human
eed (heed) gene product, the HEED protein, on the
other hand. Co-localization of MA and HEED proteins within the nucleus
of co-expressing cells, and the absence of detectable interaction
between HEED and uncleaved GAG precursor in vivo and
in vitro, suggested that HEED and MA would play a role at
early stages of the HIV-1 infectious cycle, rather than at late steps,
possibly in the process of integration of the provirus into the
host-cell genome or of transcriptional regulation of cellular genes.
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MATERIALS AND METHODS |
Yeast Two-hybrid System and cDNA Library Screening
Generation of the LexA-MA Bait--
The portion of the
HIV-1LAI gag gene coding for the MA and the
first 11 residues from the CA domain (MA143), wild type MA132 or
MA-derived deletion mutants (MA120 and 132D1 to 132D9), was cloned in
frame with the DNA-binding lexA gene into the pBTM116 vector
(29). The sequence of the construct was verified by DNA sequencing, and
the expression of recombinant fusion protein in yeast was confirmed by
gel electrophoresis and immunoblot analysis using anti-MA antibody, as
described below.
Generation of the pGAD-cDNA Library--
The cDNA
library was generated by reverse transcription of poly(A)-containing
mRNAs isolated from phytohemagglutinin-activated (PHA, 5 µg/ml)
peripheral blood lymphocytes, using a 5'
NotI/(dT)18 primer and the TimeSaver cDNA
synthesis kit (Amersham Pharmacia Biotech). EcoRI single
strand oligonucleotide adapters were then ligated to the cDNAs, and
the library was inserted into the EcoRI and NotI
sites of the Gal4 transcription activation domain vector pGAD3S2X, a
modified version of the pGAD GH (CLONTECH)
containing a NotI site in its polylinker. This resulted in a
Gal4 activation domain (AD)-cDNA hybrid.
Screening in Yeast--
Two-hybrid screens were performed as
described in detail in previous studies (33, 34).
Generation of HEED Mutants
HEED deletion mutants HEEDNt120-535, HEEDNt65-494, and
HEEDNt96-494 were three natural clones isolated from human cells by our two-hybrid screen (refer to Fig. 2b). The murine
eed clone was kindly provided by O. Denisenko, and a
chimeric full-length m/HEED protein was genetically reconstituted by
fusion of the N-terminal 64-residue coding sequence from mouse EED to
coding sequence 65-535 from human EED. Other deletions and
substitutions were constructed in the HEEDNt120-535 backbone (Fig.
2c) as follows: internal deletion mutant HEED
264-416,
double-truncated mutant HEEDNt120-415, and substitution mutants
HEED394 and HEED399. Site-directed mutagenesis was performed on DNA
coding for HEEDNt120-535, subcloned into the pBluescript II KS(
)
phagemid (Stratagene), using the polymerase chain reaction technique
and two complementary mutagenic oligonucleotides (see Ref. 35), whose
sequence and position in the different genes will be communicated upon
request. Mutations were verified by DNA sequencing.
Screening of a Lambda gt11 Phage Library
A human spleen cDNA library (5'-Stretch Plus cDNA
library; CLONTECH) was probed with
heedNt120-535 DNA isolated from the pGAD-cDNA hybrid
plasmid by EcoRI-NotI digestion and labeling by
random priming (Random Primers DNA Labeling System, Life Technologies, Inc.), using [
-32P]dCTP (Amersham Pharmacia Biotech).
Northern Blot Analysis--
The tissue distribution of the
heed transcripts was analyzed by hybridization of blotted
mRNAS from various human organs and tissues (Human MultiTissue
Northern blot IV, CLONTECH) with
[32P]dCTP-labeled heedNt120-535 DNA probe.
In Vitro Transcription-Translation
The HEEDNt120-535 and gag-derived DNAs were inserted
in pBluescript RN3 plasmid (36) and HEED and GAG proteins obtained by
combined in vitro transcription-translation reaction, using a commercial kit (TNT kit, Promega) and the manufacturer's protocol. Protein labeling was performed using
L-[35S]methionine (in vivo cell
labeling grade, specific activity
37 TBq/mmol, Amersham Pharmacia Biotech).
GST Fusion Constructs and in Vitro Binding Assays
HEEDNt120-535 protein and different GAG proteins were expressed
in Escherichia coli as glutathione S-transferase
(GST) fusion proteins, using the pGEX-KG plasmid (37). Fusion proteins
were purified by affinity on glutathione-Sepharose gel and recovered by
thrombin cleavage of the GST linker, using a commercial kit (Bulk GST
Purification Module, Amersham Pharmacia Biotech). For in
vitro interactions, 20-µl aliquots of glutathione-Sepharose bead
suspension were mixed with 400-µl aliquots of sonicated bacterial cell lysate (corresponding to 100 ml of E. coli culture) in
binding buffer (BB: phosphate-buffered saline, containing 0.2% Nonidet P-40 and a mixture of protease inhibitors (Protease Inhibitor Mixture,
Boehringer Mannheim), at 1 tablet per 10 ml of buffer) and incubated
for 30 min at 4 °C. After extensive rinsing in BB, 20-µl aliquots
of the affinity beads were mixed with 50-100 µl (100 µg) of
affinity purified HEED or GAG proteins and 900 µl of BB and incubated
for 2 h at room temperature. The beads were then washed three
times with 500 µl of BB, resuspended, and boiled in 50 µl of SDS
sample buffer. Eluted proteins were analyzed by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblotting. Alternatively, 5-µl
aliquots of [35S]methionine-labeled cell-free translated
proteins (HEED or GAG proteins) were mixed with 200 µl of BB and 20 µl of glutathione-Sepharose beads pre-adsorbed with GST-fused HEED or
GAG proteins. After 2 h incubation at room temperature, the
samples were processed for SDS-PAGE analysis as above, and gels were
dried and autoradiographed.
Gel Electrophoresis and Immunological Analyses
Polyacrylamide gel electrophoresis of SDS-denatured protein
samples and immunoblotting techniques have been described in detail in
previous studies (21, 23, 24). GAG proteins containing the MA domain
were detected on blots using monoclonal antibody anti-MAp17 (Epiclone
5003, mapped to the motif DTGHSSQVSQNY, within residues 121-132 in the
MA; see Ref. 23). In certain cases, polyclonal anti-GAG antibody
(laboratory-made; see Ref. 25) was used. Antiserum against HEED was
prepared in rabbit, by injection of purified HEEDNt120-535 protein
obtained by affinity purification of GST-fused protein as described
above. Anti-HEED serum was used at a working dilution of 1:2,000 in
immunoblotting reactions.
Phage-displayed Hexapeptide Library and Biopanning
A filamentous phage-displayed hexapeptide library (kindly
provided by G. Smith; see Ref. 38) was used for biopanning onto immobilized protein ligate. Specific ligand elution of phages has been
described in previous studies (25, 39, 40). Purified MA143 was
immobilized on enzyme-linked immunosorbent assay plates used as the
solid support. Elution of phages was carried out using HEEDNt120-535
protein as soluble competing ligand. In the reverse biopanning
experiment, purified HEEDNt120-535 was coated onto the plate, and
MA143 was used as the soluble competing ligand. The hexapeptide
phagotopes were identified by manual DNA sequencing of the recombinant
fUSE5 pIII protein, using the dideoxynucleotide chain termination
method (41), oligonucleotide 5'-TGAATTTTCTGTATGAGG-3' as the primer,
and Sequenase kit version 2.0 (Amersham Pharmacia Biotech). Multiple
sequence alignment was performed using the W(1.4) version of the
Clustal program (42).
HEED Expression in Baculovirus-infected Insect Cells
HEEDNt120-535 gene was cloned under the polyhedrin promoter of
Autographa californica multiple nuclear polyhedrosis virus and expressed in recombinant A. californica multiple nuclear
polyhedrosis virus-infected Sf9 cells. The intermediate
baculoviral vectors and cloning strategies have been reported in
previous studies (21-23, 26, 27). In double infection experiments,
Sf9 cells were infected with equal multiplicity of infection of
two recombinant baculoviruses (5 plaque-forming units/cell), one
expressing the HEED protein, the other expressing the MA143.
Co-localization of HEED and MA proteins was analyzed using
immunoelectron microscopy with double labeling.
Fluorescent Tagging and Cellular Localization of HEED
The cDNA of full-length m/HEED chimera was cloned into the
pEGFP-C1 vector (CLONTECH), and HEED protein was
expressed in fusion to the C terminus of the green fluorescent protein
variant EGFP (GFPmut1; see Ref. 43). HeLa or 293 cells were transfected
with the pEGFP-C1-heed vector, using a conventional calcium
phosphate method, with or without cotransfection with a
MA143-expressing vector (pcDNA3.1, Invitrogen). Cellular
localization of HEED and MA proteins was analyzed by confocal laser
system microscopy, using a Bio-Rad 1024 apparatus. The Bio-Rad 1024 was
interfaced with an argon/krypton ion laser, equipped with fluorescence
filters and detectors allowing detection of fluorescein isothiocyanate (or EGFP) and rhodamine markers. Images were acquired sequentially for
each fluorescence, to avoid fluorescence cross-talk between the two
channels. 0.35-µm spaced sections were taken in the horizontal plane
and reintegrated into a single image. Co-localization of fluorescent
proteins was visualized by superimposition of EGFP and rhodamine images.
Electron and Immunoelectron Microscopy (EM and IEM)
Cell specimens were included, sectioned, and processed for
conventional EM or IEM, according to previously described methods (21,
25). Mouse monoclonal anti-MA antibody and rabbit anti-HEED antibody
were used for IEM, with the corresponding colloidal gold-labeled complementary antibodies, 5-nm gold-tagged anti-mouse Ig antibody, and
10-nm gold-tagged anti-rabbit Ig antibody, respectively. The specimens
were observed under the Hitachi HU7100 electron microscope.
The nucleotide sequence of the human eed clone analyzed in
this study has been registered in GenBankTM and EMBL Data
Bank under the accession numbers U90651 and AF099032.
 |
RESULTS |
Nomenclature of GAG and HEED Proteins and Their Mutants--
The
different gag-derived gene products used in the present
study, and called under the general term of GAG proteins, were abbreviated according to their major structural domains and amino acid
sequence characteristics (Fig.
1a). Thus, the wild type
recombinant MA protein, from the N-myristoylated N-terminal
glycine until tyrosine at position 132, was designated by MA132, and
the wild type capsid p24 protein (spanning residues 133-363 in
Pr55GAG), was abbreviated CA. The C-truncated GAG precursor mutants
carrying an amber or ocher stop codon that
terminates the GAG reading frame at position 120 within the
MA domain (GAGamb120), at position 143 within the CA domain
(GAGamb143) or at position 180 within the CA domain
(GAGoch180), have been described in previous studies (23).
These gag gene products were designated by MA120, MA143, and
MA180, respectively. MA120 is a matrix protein deleted of 12 residues
from its C-terminal end; MA143 consisted of a matrix protein with a
C-terminal addition of 11 residues belonging to the CA domain, and
MA180 is a matrix with a C-terminal addition of 49 residues from the
CA. MA13-CA was a construct corresponding to the entire CA domain
(p24CA) whose N terminus was fused to the last 13 residues from the MA
domain. The construction of the MA deletion mutants D1 to D9 (17) in
recombinant full-length precursor Pr55GAG has already been published
(23). For the purpose of the present study, each MA deletion was
reintroduced into the MA132 backbone. Substitution mutant MA132F
carried a phenylalanine residue and MA132EE a dipeptide
glutamate-glutamate in lieu of the C-terminal tyrosine. The different
GAG protein constructs are depicted in Fig. 1 (a and
b).

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Fig. 1.
Genetic constructs of HIV-1
GAG-derived proteins. a, GAG
gene constructs, aligned with the different structural domains of
Pr55GAGLAI, i.e. the MA (residues 1-132),
CA-(133-363), spacer peptide sp1-(364-377), NC-(378-433),
sp2-(434-447), and p6-(448-500). The CA domain is represented by a
solid box, the MA domain, NC, p6, and spacer peptides by
open boxes. b, mutants D1 to D9 (10- to
15-residue deletions), scanning the MA domain, were generated in the
MA132 backbone. Figures at the mutation positions represent the newly
adjacent amino acid residues at the deletion junctures.
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The different HEED proteins and HEED mutants are sketched in Fig.
2. The amino acid sequence (amino acids
1-535) of the murine eed gene product (32) was taken as the
reference for numbering the residues in the human EED homolog. The
product of the unspliced heed mRNA contained a stop
codon at position 495, whereas the HEED protein translated from the
spliced transcript ended its open reading frame at arginine 535, as in
mouse (Fig. 2a). Our original HEED clone isolated by
two-hybrid screen, HEEDNt120-535, was an N-terminal truncated (Nt)
form, starting its open reading frame at position Asp-120 and ending at
residue Arg-535 (Fig. 2b). The largest HEED protein isolated
from human cells was called HEEDNt65-535, as it lacked the N-terminal
64 residues of the corresponding mouse sequence. Its shorter
equivalent, translated from the unspliced heed mRNA, was
termed HEEDNt65-494. Full-length m/HEED protein was a chimeric
eed gene product consisting of the N-terminal 64 residues
from mouse EED, fused to sequence 65-535 of human EED protein.
N-terminal deletion mutant HEEDNt96-494 started its sequence at serine
96. Other HEED constructs used were internal deletion (
) mutant
HEED
264-416, double-truncated mutant HEEDNt120-415, and
substitution mutants HEED394 and HEED399 (Fig. 2c).

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Fig. 2.
eed gene products. a,
postulated structure of the human eed transcripts, with the
position of the 411-nucleotide-long intron. The 5' end of the
eed mRNAs, undetermined for human eed
(question mark), was deduced from the mouse eed
mRNA (32). b, the three human EED proteins Nt120-535,
Nt65-494, and Nt96-494 (indicated by hatched boxes),
isolated by the yeast two-hybrid and gt11 screens, are aligned with
mouse EED protein (represented by a stippled box), with its
seven WD repeats indicated by roman numerals. The figures
correspond to the amino acid numbers of the mouse EED protein sequence,
as shown in Fig. 3a. c, HEED protein constructs
and mutants. Full-length chimeric mouse-human EED protein (m/HEED) and
N-truncated (Nt) protein Nt65-535 were reconstructed from
the natural clones isolated; HEED mutants were generated in the
Nt120-535 backbone (our original MA-binding clone). The symbols (+) or
( ) on the right refer to the capability of HEED constructs
to bind to MA, as determined by the two-hybrid assay in yeast.
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Isolation of a Human Homolog of Murine EED (HEED) by Interaction
with HIV-1 MA in the Yeast Two-hybrid Screen--
The
HIVILAI MA143 was fused to the DNA-binding LexA protein and
used as a molecular bait for screening a cDNA library from phytohemagglutinin (PHA)-activated human lymphocytes, cloned in fusion
with the Gal4-activating domain. Out of 3 × 106
double transformants obtained, 58 positive clones were selected and
arranged into nine groups of cDNAs. The most represented group (29.4% of the isolated clones) consisted of a unique cDNA sequence of 1,600 base pairs in length. Comparison with DNA sequences in data
banks revealed that it presented 91% identity, at the nucleotide level, with the murine (Mus musculus) gene eed
(embryonic ectoderm development; see Refs. 30 and 32), a highly
conserved homolog of the Drosophila Polycomb group
(Pc-G) of gene member esc (extra sex combs; see
Ref. 31). This human homolog of the murine eed was called
heed. The heed gene product, the HEED protein,
was found to interact with the three forms of MA proteins assayed by
the two-hybrid system in yeast, the C-terminally extended MA143, wild
type MA132, and C-terminally deleted MA120 (Fig.
3, lanes 1, 2 and
6), implying that the interacting domain was localized in
the N-terminal 120 residues of the MA domain. The amino acid sequence
of the HEED protein is shown in Fig.
4a. Our original heed gene product isolated (referred to as HEEDNt120-535)
showed an open reading frame of 416 residues, starting at aspartic acid 120 and ending at the same C-terminal arginine residue 535 as the
murine EED protein (Figs. 2b and 4a).

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Fig. 3.
Interaction of HEED and HIV-1 GAG products
in vivo. Affinity of HEED for MA and GAG products was
analyzed in yeast, using the two-hybrid assay with -galactosidase
activity detection. MA and other GAG-derived products were cloned in
fusion with LexA protein (BD hybrid) and HEED (clone
Nt120-535) or Raf cloned in fusion with the Gal4 activation domain
(AD hybrid). Shown are yeast transformants plated on medium
with histidine (left column), or without histidine
(middle column), and replica-plated on Whatman filters. The
right column shows the test for -galactosidase
( -gal) activity. Growth in the absence of His and
blue color in the -galactosidase assay are indicative of
interaction between hybrid proteins in yeast cells. Negative controls
involved Ras, Raf, or lamin as one of the two-hybrid partners
(horizontal lanes 3-5).
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Fig. 4.
a, amino acid sequence of the human EED
protein (HEED), deduced from DNA sequencing. The N-terminal mouse EED
protein sequence, from the putative N-terminal residue valine 1 to
serine 64 (32), is in italics. Identical residues in the mouse sequence
are shown as a dashed line under the human EED amino acid
sequence, written in capital letters. Amino acid changes are in
boldface. The seven putative WD repeats are shown by
solid underlines and are indicated by roman numerals
in parentheses. The open reading frame of HEED stopped
at position Arg-535, preceding an ocher termination codon.
b, nucleotide sequence of heed at the boundaries
of the putative intron. Unspliced 2.2-kb HEED mRNA would be
translated into a protein of 494 residues. Splicing of the HEED
mRNA (1.7 kb) would excise the TAA stop codon downstream to Lys-494
and restore the reading frame down to arginine residue 535. nt, nucleotide.
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The partial heedNt120-535 cDNA was then used to screen
a
gt11 human cDNA library from human spleen tissue. Two positive
clones of 2.2 kb in length were thus isolated and sequenced. Both
contained an extra 5' sequence but showed a shorter open reading frame, as compared with our original clone of 1.6 kb, ending at lysine residue
494. Their extra 5' sequence coded for additional N-terminal domains,
starting at phenylalanine residue 65 of the mouse EED protein for clone
HEEDNt65-494, and starting at serine 96 for clone HEEDNt96-494 (Figs
2b and 4a). The N-terminal sequence spanning residues 65-119 was then reintroduced into our original clone HEEDNt120-535 to generate the human protein HEEDNt65-535 (Figs 2b and 4a). Further attempts to recover longer 5'
sequences by reverse transcriptase- polymerase chain reaction
amplification using the appropriate primers and to isolate cDNA
clones longer than 2.2 kb were all unsuccessful, likely due to the
exceptionally GC-rich content of the eed gene upstream
sequence (32). Assuming a high level of sequence homology between the N
termini of murine and human gene products, as in the rest of the
sequence, a chimeric mouse/human EED protein (termed m/HEED) was
constructed by genetic fusion of the mouse N-terminal 64-residue coding
sequence to human Nt65-535 clone (Figs. 2b and
4a). The resulting full-length m/HEED protein migrated as a
polypeptide of 58 kDa in apparent molecular mass in SDS-PAGE (Fig.
6a).
Characterization and Tissue Distribution of HEED
Transcripts--
Northern blot analysis of the heed
mRNAs in a variety of human tissues was performed using
heedNt120-535 cDNA as a probe. As shown in Fig.
5a, two discrete bands of
heed transcript, migrating as 1.7- and 2.2-kb species,
respectively, were seen in all tissues analyzed. The same two mRNA
species have been found for mouse eed, and alternative
splicing events have been suggested to account for the smaller size
species (32). The difference in migration of the two transcripts from
human tissues in gel electophoresis was compatible with an intron of
about 400-500 nucleotides in length (Fig. 4b). Indeed, the
nucleotide sequence at the 3'-terminal end of clones HEEDNt65-494 and
Nt96-494, within codons 494-905, was strongly suggestive of a
splicing event, based on (i) the finding of consensus nucleotides at
canonical positions of the donor and acceptor sites of the putative
intron, and (ii) the respect of the GT-AG rule (Fig. 4b).
Splicing of this 407-nucleotide-long intron would result in excising
the ocher stop codon at position 495 and extending the HEED
open reading frame downstream to lysine 494, until arginine 535, corresponding to codon 535 in mouse (Fig. 4, a and
b). Thus, the shorter transcript of 1.7 kb would be
translated into a protein isoform of 535 residues, whereas the
unspliced transcript of 2.2 kb would encode a shorter protein isoform
of 494 residues (refer to Fig. 2a).

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Fig. 5.
a, Northern blot analysis of the tissue
distribution of HEED mRNA. b, Comparison of the
heed mRNA level in PHA-activated (pha-L) and
non-activated (rest-L) human peripheral blood lymphocytes.
Lower panels, control hybridization with actin cDNA
probe.
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The two heed transcripts were found to be much more abundant
in thymus, spleen, testis, uterus, and small intestine than in colon
and peripheral blood leukocytes (Fig. 5a) as well as resting human lymphocytes (Fig. 5b). However, PHA activation of
peripheral blood lymphocytes resulted in a significant enhancement of
both transcripts, compared with control, non-activated cells (Fig. 5b).
Sequence Characteristics of the HEED Protein--
HEEDNt65-535
showed a 99.5% identity at the protein level, with the corresponding
sequence of the mouse EED protein (Fig. 4a). One
conservative change (Glu to Gln substitution) was observed at position
431, and two nonconservative changes involved G68S and N360Y
substitutions (Fig. 4a). Another HEED clone has been recently isolated by using Pc-G protein Enx1 as the bait
(44, 45). This HEED protein would have a 100% identity with the mouse EED amino acid sequence and would start its coding sequence by a GUG
initiation codon specifying a valine. Such an unusual GUG initiation
codon has been reported for viruses, Drosophila, and mouse
but never for human so far. The reason for the few sequence differences
between our human EED and the mouse EED protein, and for the total
identity with mouse EED reported for the other human clone, is not
known but could reflect some tissue variability; adult peripheral blood
lymphocytes was the source of our HEED versus fetal brain
tissue for the other HEED clone.
As already reported for mouse EED, a specific feature of the HEED
protein was the occurrence of several repeats of the module GH-(30-40
residue spacer)-WD (46), now simply referred to as WD repeat motif
(47). In HEED (Fig. 4a), seven WD repeat motifs could be
distinguished GD129-WK167 (I), CH220-WT246 (II), KH278-WN312 (III), and
GH328-WR357 (IV). However, WD repeat (V) was ambiguous and could be
considered as two overlapping WD repeat motifs, one starting from IH388
and ending at WL408, the other from IH398 and ending at WK425, with a
18- or 25-residue spacer, alternatively. These two IH motifs were found
to be included in the MA-binding region, as shown below. WD repeat (VI)
was also ambiguous; it starts at the consensus GR(F)448 signal but
could end at WQ467 or WD483, with a 17- or 33-residue spacer,
respectively. Repeat VI was the last one found in HEED product from the
unspliced message, whereas the spliced mRNA product contained an
extra repeat, TH498-WD530 (VII).
Interaction of HEED with MA in Vitro--
The fact that the
original HEED protein isolated by our two-hybrid screen
versus MA143 was the N-truncated form HEEDNt120-535 implied
that the MA-binding domain was not located within the N-terminal 119 residues of the putative full-length HEED sequence and was different
from the transcriptional repression and K protein-binding domain
recently assigned to this location in mouse EED (32). Since the
N-terminal domain of HEED was dispensable for MA recognition and
interaction, most of our binding experiments and biological assays with HEED protein were performed, unless otherwise stated, using
the original N-truncated clone HEEDNt120-535 and
HEEDNt120-535-derived mutants (Fig. 2, b and
c).
GST-HEEDNt120-535 fusion protein was incubated with bacterially
expressed and affinity purified MA protein, and the complex was
isolated by affinity to glutathione-Sepharose beads. Both MA143 and
MA132 were found to bind to GST-fused HEEDNt120-535, with a higher
affinity for MA143 than for MA132 (Fig.
6b). This confirmed the data
from yeast two-hybrid test in vivo (Fig. 3) and suggested
that MA and HEED proteins could directly interact with each other and
that this interaction had occurred in yeast without the participation
of a third partner provided by the yeast cell. No significant binding
of GST-HEEDNt120-535 was observed with GAG-derived proteins MA180,
MA13-CA, and CA, and to full-length Pr55GAG precursor (data not shown).
MA120 could not be tested in our GST pull-down assay, since the epitope
recognized by our anti-MA monoclonal antibody has been mapped to the C
terminus of the MA domain and is deleted in MA120 (23). However, the results obtained with MA13-CA and CA suggested that the interaction in vitro between GST-HEED and MA protein did not directly
involve the MA-CA junction but that a short peptide sequence from the CA domain, as present in MA143, could participate in the stability of
the HEED·MA complex.

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Fig. 6.
Western blot analyses.
a, bacterially expressed, affinity purified,
full-length chimeric m/HEED (58 kDa), analyzed by SDS-PAGE and
immunoblotting using anti-HEED polyclonal antibody
(laboratory-made). b, interaction of HEED and
HIV-1 MA proteins in vitro in the GST pull-down assay.
GST-HEEDNt120-535 fusion protein was reacted with equal protein
amounts of bacterially expressed, affinity purified MA143, MA132,
and bound fractions were analyzed by SDS-PAGE and immunoblotting with
anti-MA monoclonal antibody. m, prestained molecular mass
markers. Lanes 1 and 3, control GST; lanes
2 and 4, GST-HEED.
|
|
Mapping of the HEED-interacting Region(s) in the MA Domain of GAG
Using the Two-hybrid Screen--
The next experiments were designed to
determine which specific region(s) of the MA domain were involved in
the interaction with HEED protein. Different mutants of the MA domain,
as well as other domains from Pr55GAG precursor, were constructed in
fusion with the LexA protein and assayed in yeast by the two-hybrid
test versus HEEDNt120-535 fused to the Gal4 transcription
activation domain. The results of the
-galactosidase activity
obtained with the different MA- and GAG-derived constructs are shown in
Fig. 3. No significant interaction was detected in yeast cells
co-expressing HEED with Pr55GAG, MA180, CA, and the MA13-CA construct.
Along with the results from in vitro binding tests, this
suggested that HEED-GAG interaction only involved the MA domain and not
the MA-CA junction or the CA domain. However, it seemed that the
occurrence of a longer CA sequence at the C-terminal extremity of the
MA domain, as in MA180 or in the full-length Pr55GAG, was detrimental to the MA-HEED interaction in yeast (compare the pattern of MA143 and
MA180 in lanes 1 and 9 of Fig. 3, respectively),
implying a conformation dependence of the MA binding to HEED. These
results also suggested that the intracellular interaction between
GAG-derived proteins and HEED would not take place at the late stage of
the virus cycle, when intact Pr55GAG self-assemble into capsids, but rather at early steps, when infectious virions containing cleaved GAG
precursors infect a new host cell.
Nine deletion mutants (10-15-residue deletions) scanning the MA domain
(17, 23) were generated in the MA132 protein (Fig. 1b), and
their interaction with HEED was assayed by the two-hybrid screen in
yeast (Fig. 7). As already observed in
in vitro binding assays, MA143 bound to HEED with a
consistently higher efficiency than MA132 (3-4-fold). This confirmed
the results of GST pull-down experiments and suggested that the
addition of a short stretch of 11 amino acid residues from the CA
domain to the MA C terminus enhanced the binding between MA and HEED
proteins, or the stability of the complex, or both. All the MA
deletions, except 132D8, provoked a significant decrease in the
-galactosidase activity, down to background levels. The interaction
observed with HEED and mutant 132D8 (residues 90-104), however, was
significantly higher than with wild type MA132 and almost similar to
that obtained with MA143.

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Fig. 7.
Affinity of HEED for wild type MA and MA
mutants. Positive control was given by the lacZ
expression of the Ras-Raf samples (not shown) and negative control
(shown on the left side of the panel) by MA132
versus Raf protein. 132D1 to 132D9 refer to the D1 to D9 MA
deletion mutants described in Fig. 1b. Shown is the average
of three separate experiments (± S.D.). -Gal,
-galactosidase.
|
|
Since it has been previously reported that the MA C-terminal residue
Tyr-132 is involved in the nuclear translocation of the MA protein
(15), MA mutants were generated in this region and assayed for their
binding to HEED. Mutant MA132F, carrying a Tyr-to-Phe hydrophobic
substitution at the MA C terminus, and MA132EE, carrying a substitution
by a pair of glutamic acid residues at position 132, which was designed
to mimic the phosphotyrosine acidic side group, showed the same HEED
binding efficiency as wild type MA132 (data not shown), suggesting that
a simple post-translational modification of the MA C terminus would not
account for significant variations in the affinity of the MA for HEED.
Taken altogether, the absence of HEED-MA180 interaction, the higher
efficiency of HEED interaction with MA143, compared with wild type
MA132, and the observation that 132D8, a mutant deleted of a small
region upstream to the second polybasic signal, bound to HEED as
strongly as the C-terminally extended MA143 suggested a significant
degree of conformation dependence of the MA binding to HEED. This was
also confirmed by the following experiments.
Identification of MA-HEED Interacting Domains by Biopanning of a
Phage-displayed Peptide Library--
A hexapeptide library was panned
on immobilized HEEDNt65-535, and specific ligand elution was performed
using an excess of MA143, acting as a competitor for MA-binding sites
on HEED. The hexapeptide phagotopes isolated, which theoretically
mimicked motifs of the MA domain (39, 40), are presented in Fig.
8a. Several phagotopes
contained hydroxylic, leucine, and glycine residues, reminiscent of the
RASVLSGGEL motif found within residues 4-13, near the N terminus of
the MA. The basic aromatic hexapeptide RSKYLV was in consensus with the
KKKYKL basic motif at position 26-31, and other phagotopes, such as
SLTTWA, YGAHRW, GFYPWS and TTYWRR, showed a certain degree of homology
with the tryptophan-containing motif YKLKHIVWASR within residues
29-39. The high level of degeneration and scatter of these peptides
suggested that the HEED-binding region(s) of the MA consisted of highly
structured domain(s) and were composed of
conformation-dependent motifs and/or discontinuous peptide
regions on the MA linear sequence (e.g. SRA-GIN and MSV-GNL, tentatively positioned within residues 6-13). However, the composition of phage hexapeptides strongly suggested that the HEED-interacting site(s) involved the N-terminal domain of the MA protein, including the
two
-strands carrying the first polybasic signal and possibly the
N-terminal half of helix II (Fig. 8a).

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Fig. 8.
Phage biopanning of MA and HEED
proteins. a, hexapeptides eluted from immobilized
HEED by ligand displacement using soluble MA143. 14 phagotopes out of
20 isolated and sequenced, which would theoretically be mimotopes of MA
motifs, are shown in tentative alignment with two domains of the MA,
within residues 4-13 and 20-40, respectively. Positions of
-strands and -helices are from Hill et al. (50) and
Kräusslich and Welker (8). b, hexapeptides phagotopes
isolated from immobilized MA143 and recovered by elution with
HEEDNt120-535 protein used as a competing ligand. 21 phagotopes are
shown in alignment with the HEED sequence, within residues 388-403
(mouse EED protein residue numbering; see Ref. 32). They could be
arranged in two families displaying two discrete motifs found in HEED
(V,I)HFPDF(S,T) and D(I,L,V,Y)H(R,K)NYV. Identical or conserved
residues are in boldface.
|
|
Mapping of the MA Interacting Regions in HEED--
In order to
identify the MA-binding site in HEED, deletions were generated in
heed cDNA and the different mutants (Fig. 2c) cloned into pGAD and co-expressed with the LexA-MA143 fusion protein in
yeast. Only full-length chimeric m/HEED and clones HEEDNt65-535 and
HEEDNt120-535 showed MA binding activity. The shorter HEED proteins
HEEDNt65-494 and Nt96-494, double-truncated mutant Nt120-415 and
single deletion mutant HEED
264-416, whose internal deletion removed
the three central WD repeats III, IV, and V, failed to interact with
the MA protein (data not shown; refer to Fig. 2, (+) and (
) on the
right). The level of expression of HEED-GAD fusion proteins
in yeast was analyzed by SDS-PAGE and immunoblot, using our anti-HEED
polyclonal antiserum. All HEED proteins, except HEEDNt96-494 and
Nt120-415, were found to be stable in yeast cells. Thus, the
absence of MA interaction with stably expressed HEED
264-416 and
HEEDNt65-494 proteins suggested that the integrity of the central
domain and of the C-terminal region of HEED, encompassing the
last WD repeat motif, was a key factor in HEED-MA interaction in yeast.
We next performed experiments of reverse biopanning, using MA143
protein as the immobilized ligate and excess of HEED as the specific
competitor for HEED-binding sites in MA. The phagotopes thus isolated
are presented in Fig. 8b. Twenty one phagotopes, out of 24 isolated and sequenced, could be arranged into two groups. The first
group displayed a sequence that had homology with a linear sequence of
HEED within residues 388-396 (site I, IHFPDFSTR); the second group was
highly homologous to an heptapeptide motif spanning residues 397-403
(site II, DIHRNYV). Some phagotopes (e.g. FGGSRR and ESSRYH)
seemed to overlap sites I and II. The MA-binding region was included in
a domain with a high probability of accessibility and immunogenicity,
according to conventional prediction program (data not shown). This
MA-binding domain overlapped the two canonical IH dipeptides at the
beginning of the fifth putative WD repeat, as shown in Figs.
2b and 4a.
Substitutions were then generated in the above-defined MA-binding site.
In mutant HEED394, the hydroxylic dipeptide Ser-394
Thr-395 was
changed into hydrophobic Ala-394
Ile-395, and in mutant HEED399, the
basic aromatic tetrapeptide motif HRNY within residues 399-402 was
mutated into the alanine stretch AAAA. The two mutations were replaced
into the HEEDNt120-535 backbone fused to pGAD, and HEED point
mutants HEED394 and HEED399 were assayed by the two-hybrid screen
in co-expression with LexA-MA143. The absence of detectable
-galactosidase signal (Fig. 2c) confirmed the mapping of
the MA-binding site to residues 388-403 in HEED.
Cellular Co-localization of HEED and MA-143--
Human cells were
co-transfected with plasmid vectors expressing the GFP-fused
HEEDNt120-535 and unmyristoylated MA143, and the co-expressing cells
were analyzed in double fluorescence microscopy using a confocal laser
system. This approach was preferred to co-immunoprecipitation of MA and
HEED proteins from cell lysates for the following reasons. (i) The step
of fixation preserves the integrity of cellular structures and proteins
that interact within the nucleus would not be dissociated from their
complex, as it could happen upon cell lysis and nuclear extraction.
(ii) A protein could be displaced from its partner by an antibody
molecule that competes for the same interacting site or introduces
subtle changes in the conformational structure of the binding site.
(iii) Tagging the proteins at one of their extremities to circumvent the latter inconvenience could also result in a change in their conformation and binding affinity. (iv) In our case, the HEED-binding site in the MA protein consisted of discontinuous,
conformation-dependent motifs, as suggested by the data of
Figs. 7 and 8a. In HeLa or HEK-293 cells, HEED and MA
proteins were visible in both nuclear and cytoplasmic compartments.
However, HEED mainly showed a nuclear localization (Fig.
9a), whereas the MA protein,
detected by anti-MA antibody and rhodamine-labeled conjugate, was
essentially localized in the cytoplasm (Fig. 9b). The
superimposition of the GFP and rhodamine fluorescence signals showed a
pattern of co-localization of the two proteins within the nucleus (Fig.
9c).

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Fig. 9.
Cellular co-localization of HEED and MA
analyzed by fluorescence microscopy of human cells co-expressing HEED
and MA proteins. a, subcellular localization of
chimeric m/HEED, detected by the fused green fluorescent
protein (GFP; see Ref. 49) signal in 293 cells. b,
fluorescence pattern of unmyristoylated MA143, detected by mouse
monoclonal anti-MA antibody and rhodamine-labeled anti-mouse IgG.
c, merging of the two fluorescence signals.
|
|
To analyze further this cellular co-localization, full-length chimeric
m/HEED and HEEDNt120-535 were expressed in recombinant baculovirus-infected Sf9 cells. In single infected cells, IEM analysis using anti-HEED antibody showed that HEED protein accumulated within the nucleus and was often found as inclusions at the periphery of the nucleoplasm, in the vicinity of, or even in close contact to,
the nuclear membrane (Fig. 10,
a and b). In cells co-expressing HEED protein and
MA143, the nuclear rim localization of HEED did not change
significantly, and double immunogold labeling revealed that HEED and
MA143 co-localized within an electron dense inclusion (Fig. 10,
c and d). The same pattern was observed with
full-length m/HEED and HEEDNt120-535, which implied that the
N-terminal 119 residues of HEED were not involved in nuclear targeting
and nuclear co-localization with MA143.

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Fig. 10.
Immunoelectron microscopic analysis of the
co-localization of HEED and MA in insect cells. a and
b, single infection of Sf9 cells by a
recombinant baculovirus expressing HEEDNt120-535. The HEED protein
was labeled with anti-HEED rabbit antibody and 5-nm colloidal
gold-tagged anti-rabbit IgG. Note the nuclear rim localization of
the HEED protein inclusions. c and d,
co-infection of Sf9 cells by two recombinant baculoviruses, one
expressing HEEDNt120-535 and the other expressing the unmyristoylated
MA143. The HEED protein was detected by anti-HEED rabbit antibody
and 10-nm colloidal gold-tagged anti-rabbit IgG and the MA protein by
mouse monoclonal anti-MA antibody and 5-nm colloidal gold-tagged
anti-mouse IgG.
|
|
 |
DISCUSSION |
By using an extended form of HIV-1 MA protein (MA143) as a bait in
a two-hybrid screen, we have isolated several gene products from
activated human lymphocytes that were potential cellular partners of
the viral MA protein. Out of the three most frequently encountered
clones, one corresponded to a glutaminyl-tRNA synthetase and the second
one to the elongation factor I
. A histidyl-tRNA synthetase and the
same elongation factor I
have been isolated by screening with wild
type MA132 in two other laboratories
(48).2 The functions of these
two potential MA partners in the virus life cycle are not known. The
third one isolated by our screen was HEED (Figs. 3 and 4). Although
HEED was found to interact to both full-length MA132 and C-truncated
MA120 in vitro and in yeast (Figs. 3, 6, and 7), HEED bound
with a significantly higher efficiency to both C-terminally extended
MA143 and deletion mutant 132D8 (Figs. 6 and 7). This likely reflected
a higher stability of the HEED·MA binary complex involving MA143 or
132D8 in yeast and in vitro, explaining why HEED was the
most abundant clone fished out of our pGAD library using the
pLexA-MA143 bait. The influence of the conformational structure of the
MA on the MA-HEED interaction was thus based on the following
observations: (i) no discrete MA region could be assigned to HEED
binding function by deletion scanning of the MA domain; (ii) both MA
deletion 132D8 and MA C-terminal extension MA143 had a positive effect
on HEED binding (Fig. 7); and (iii) phagotopes isolated by phage
biopanning did not strictly align with a single linear sequence in the
MA but rather to multiple peptides belonging to noncontiguous domains within the N terminus. The HEED-binding domain apparently involved the
N-terminal region of the MA protein, including the unstructured N
terminus, the adjacent portion of helix I, and the two
-strands carrying the first polybasic signal (Fig. 8a). These
multiple discrete regions are spatially contiguous in the MA
three-dimensional structure (1, 8, 50). The importance of the MA
N-terminal basic signal in early and late steps of the HIV-1 infectious
cycle has already been demonstrated, using substitution mutants and MA-derived peptide competitors (5, 51-54).
It has been previously suggested that the HIV-1 MA protein must be
phosphorylated at its C terminus to undergo dissociation from the
plasma membrane and be able to further participate in the virus life
cycle (15, 55, 56), an hypothesis that has recently raised some
controversy (57). We first tested whether the C-terminal tyrosine
(Tyr-132) was crucial for HEED interaction. The Y132F substitution
showed no effect, as mutant MA132F bound to HEED with the same
efficiency as wild type MA132. Then we checked whether the introduction
of two negative charges at the C terminus (mutant MA132EE) to mimic the
possible C-terminal phospho-tyrosine residue reported by Gallay
et al. (15, 18) resulted in a stronger HEED binding. This
was not the case either. Altogether, these results suggested that the
phosphorylated status of Tyr-132 was not required for MA binding to HEED.
The sequence of HEED showed seven putative WD repeat motifs (Fig.
4a). Members of the WD repeat protein family have been found to be involved in different but major cellular processes as transport, signaling, regulation of gene expression, and cell division (30, 32,
34, 46, 47, 58). In HEED protein, it is noteworthy that the fifth WD
repeat starts by two possible IH dipeptides. Since dipeptide IH is
homologous to the canonical GH start of WD repeats, the two IH could
serve as recognition signals, simultaneously or alternatively,
resulting in a short or a long fifth WD repeat. The region of the MA
binding (388-403; Figs. 4a and 8b) overlapped a
16-residue-long sequence including the two IH dipeptides on the
N-terminal side of the fifth WD repeat. It is thus conceivable that the
binding of MA to HEED could result in the silencing of the two
neighboring IH signals, and the blockage of the biological function(s)
associated with the fifth WD repeat.
The eed and esc homologs, as members of the
Pc-G gene family, have been reported to function as
transcriptional repressors and gene silencers (30-32, 47).
Retroviruses in general and HIV-1 in particular do not integrate
randomly into the host cell genome (reviewed in Ref. 59), but the
cellular and viral factors that control the reaction and the site(s) of
integration remain elusive (60). The integration process is better
understood at the molecular level for retrotransposons Ty1, Ty2, Ty3,
and Ty4, which have been shown to specifically integrate upstream to
genes that are transcribed by RNA polymerase III (61-63), and for Ty5,
which integrates into regions of silent chromatin via yeast proteins
Sir (64, 65). Like Sir proteins in yeast, the products of
Pc-G genes in upper eukaryotes are involved in the
maintenance of the silent state of chromatin, possibly by recruitment
of histone deacetylase enzymes (66, 67). After its integration, the
provirus can either remain latent in the context of the silent
chromatin or alternatively be activated. In the latter case, it has
been reported that the second exon of Tat (68) and histone acetylation
are critical factors influencing the transcriptional activation of proviral DNA (69-72). Thus, on the basis of the physical interaction between MA and HEED in vivo and in vitro, it
could be hypothesized that HIV-1 infection might deregulate silent
cellular genes or, as an alternate and not exclusive hypothesis, that
the product of heed might play a role in the docking of the
HIV-1 preintegration complex, which contains both MA and integrase, to
specific host DNA insertion sites, via binding to the MA (18, 55, 73, 74). Such a DNA targeting function has been assigned to the product of
INI1 gene, a cellular integrase interactor identified by the
Gal4 two-hybrid screen versus HIV-1 integrase (75). In preliminary experiments in vitro using an integrase assay
(76), a significant stimulation of both homologous and heterologous integration events was observed in the presence of MA and HEED proteins.3 As a potential
partner of or target for PIC, the HEED protein represents an
interesting clue to investigate further the molecular mechanisms of
provirus integration into the host cell genome in relation to the
chromatin structural state and remodeling and also constitutes a
possible anti-AIDS therapeutic target in the future.
 |
ACKNOWLEDGEMENTS |
We are grateful to Jeannette Tournier for EM
specimen processing and to Oleg Denisenko and Carol Bomsztyk for the
mouse eed clone. We thank our rotating students Florence
Ottonès, Maxime Gualtieri, and Cédric Bes for significant
contributions to this work. We also thank Roger Monier, Jean-Paul
`Czar' Levy, and Marthe-Elisabeth Eladari for their constant
moral support, and Liliane Cournud for expert secretarial aid.
 |
FOOTNOTES |
*
This work was supported in part by ANRS Grant AC14-2.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U90651 and AF099032.
§
Recipient of fellowships from the ANRS, SIDACTION, and AFM.
¶
Recipient of fellowship from the French Cystic Fibrosis
Association (AFLM).
**
To whom correspondence should be addressed: Laboratoire de
Virologie Moléculaire and Pathogénèse Virale,
Institut de Biologie, Faculté de Médecine, 2, Boulevard
Henri IV, 34060 Montpellier Cedex, France. Tel.: 33 (0)4 67 60 57 38;
Fax: 33 (0)4 67 54 23 78; E-mail: pboulang{at}infobiogen.fr.
The abbreviations used are:
MA, matrix; HIV-1, human immunodeficiency virus type 1; GST, glutathione
S-transferase; CA, capsid; PAGE, polyacrylamide gel
electrophoresis; GFP, green fluorescent protein; EM, electron
microscopy; IEM, immunoelectron microscopy; AD, activation domain; kb, kilobase pair(s); PHA, phytohemagglutinin.
2
A. Cimarelli and J. Luban, personal communication.
3
J. F. Mouscadet, personal communication.
 |
REFERENCES |
-
Conte, M. R.,
and Matthews, S.
(1998)
Virology
246,
191-198[CrossRef][Medline]
[Order article via Infotrieve]
-
Craven, R. C.,
and Parent, L. J.
(1996)
Curr. Top. Microbiol. Immunol
214,
65-94[Medline]
[Order article via Infotrieve]
-
Wills, J. W.,
and Craven, R. C.
(1991)
AIDS
5,
639-654[Medline]
[Order article via Infotrieve]
-
Fäcke, M.,
Janetzko, A.,
Shoeman, R. L.,
and Kräusslich, H. G.
(1993)
J. Virol.
67,
4972-4980[Abstract]
-
Yuan, X., Yu, X.,
Lee, T. H.,
and Essex, M.
(1993)
J. Virol.
67,
6387-6394[Abstract]
-
Boulanger, P.,
and Jones, I.
(1996)
Curr. Top. Microbiol. Immunol.
214,
237-259[Medline]
[Order article via Infotrieve]
-
Freed, E.,
Orenstein, J. M.,
Buckler-White, A. J.,
and Martin, M. A.
(1994)
J. Virol.
68,
5311-5320[Abstract]
-
Kräusslich, H.-G.,
and Welker, R.
(1996)
Curr. Top. Microbiol. Immunol
214,
25-63[Medline]
[Order article via Infotrieve]
-
Pal, R.,
Reitz, M. S.,
Tschachler, E.,
Gallo, R. C.,
Sarngadharan, M. G.,
and Veronese, F. D. M.
(1990)
AIDS Res. Hum. Retroviruses
6,
721-730[Medline]
[Order article via Infotrieve]
-
Rhee, S. S.,
and Hunter, E.
(1990)
Cell
63,
77-86[Medline]
[Order article via Infotrieve]
-
Rhee, S. S.,
and Hunter, E.
(1991)
EMBO J.
10,
535-546[Abstract]
-
Spearman, P.,
Wang, J. J.,
van der Heyden, N.,
and Ratner, L.
(1994)
J. Virol.
68,
3232-3242[Abstract]
-
Zhou, X.,
Parent, L. J.,
Wills, J. W.,
and Resh, M. D.
(1994)
J. Virol.
68,
2556-2569[Abstract]
-
Crawford, S.,
and Goff, S. P.
(1984)
J. Virol.
49,
909-917[Medline]
[Order article via Infotrieve]
-
Gallay, P.,
Swingler, S.,
Aiken, C.,
and Trono, D.
(1995)
Cell
80,
379-388[Medline]
[Order article via Infotrieve]
-
Wang, C.,
Zhang, Y.,
McDermott, J.,
and Barklis, E.
(1993)
J. Virol.
67,
7067-7076[Abstract]
-
Yu, X.,
Yuan, X.,
Matsuda, Z.,
Lee, T. H.,
and Essex, M.
(1992)
J. Virol.
66,
4966-4971[Abstract]
-
Gallay, P.,
Swingler, S.,
Song, J.,
Bushman, F.,
and Trono, D.
(1995)
Cell
83,
569-576[Medline]
[Order article via Infotrieve]
-
Miller, M. D.,
Farnet, C. M.,
and Bushman, F. D.
(1997)
J. Virol.
71,
5382-5390[Abstract]
-
Bian, J.,
Lin, X.,
and Tang, J.
(1995)
FASEB J.
9,
132
-
Carrière, C.,
Gay, B.,
Chazal, N.,
Morin, N.,
and Boulanger, P.
(1995)
J. Virol.
69,
2366-2377[Abstract]
-
Chazal, N.,
Carrière, C.,
Gay, B.,
and Boulanger, P.
(1994)
J. Virol.
68,
111-122[Abstract]
-
Chazal, N.,
Gay, B.,
Carrière, C.,
Tournier, J.,
and Boulanger, P.
(1995)
J. Virol.
69,
365-375[Abstract]
-
Hong, S. S.,
and Boulanger, P.
(1993)
J. Virol.
67,
2787-2798[Abstract]
-
Huvent, I.,
Hong, S. S.,
Fournier, C.,
Gay, B.,
Tournier, J.,
Carrière, C.,
Courcoul, M.,
Vigne, R.,
Spire, B.,
and Boulanger, P.
(1998)
J. Gen. Virol.
79,
1069-1081[Abstract]
-
Royer, M.,
Bardy, M.,
Gay, B.,
Tournier, J.,
and Boulanger, P.
(1997)
J. Gen. Virol.
78,
131-142[Abstract]
-
Royer, M.,
Hong, S. S.,
Gay, B.,
Cerutti, M.,
and Boulanger, P.
(1992)
J. Virol.
66,
3230-3235[Abstract]
-
Valverde, V.,
Lemay, P.,
Masson, J. M.,
Gay, B.,
and Boulanger, P.
(1992)
J. Gen. Virol.
73,
639-665[Abstract]
-
Fields, S.,
and Song, O.
(1989)
Nature
340,
245-247[CrossRef][Medline]
[Order article via Infotrieve]
-
Shumacher, A.,
Faust, C.,
and Magnusson, T.
(1996)
Nature
383,
250-253[CrossRef][Medline]
[Order article via Infotrieve]
-
Simon, J.
(1995)
Curr. Opin. Cell Biol.
7,
376-385[CrossRef][Medline]
[Order article via Infotrieve]
-
Denisenko, O.,
and Bomsztyk, C.
(1997)
Mol. Cell. Biol.
17,
4707-4717[Abstract]
-
Bouhamdan, M.,
Benichou, S.,
Rey, F.,
Navarro, J.-M.,
Agostini, I.,
Spire, B.,
Camonis, J.,
Slupphaug, G.,
Vigne, R.,
Benarous, R.,
and Sire, J.
(1998)
J. Virol.
70,
697-704[Abstract]
-
Margottin, F.,
Bour, S. P.,
Durand, H.,
Selig, L.,
Benichou, S.,
Richard, V.,
Thomas, D.,
Strebel, K.,
and Benarous, R.
(1998)
Mol. Cell
1,
565-574[Medline]
[Order article via Infotrieve]
-
Higuchi, R.,
Krummel, B.,
and Saiki, R. K.
(1988)
Nucleic Acids Res.
16,
7351-7367[Abstract]
-
Lemaire, P.,
Garrett, N.,
and Gurdon, J. B.
(1995)
Cell
81,
85-94[Medline]
[Order article via Infotrieve]
-
Guan, K. L.,
and Dixon, J. E.
(1991)
Anal. Biochem.
192,
262-267[Medline]
[Order article via Infotrieve]
-
Smith, G. P.,
and Scott, J. K.
(1993)
Methods Enzymol.
217,
228-257[Medline]
[Order article via Infotrieve]
-
Hong, S. S.,
and Boulanger, P.
(1995)
EMBO J.
14,
4714-4727[Abstract]
-
Hong, S. S.,
Karayan, L.,
Tournier, J.,
Curiel, D. T.,
and Boulanger, P.
(1997)
EMBO J.
16,
2294-2306[Abstract/Free Full Text]
-
Sanger, F.,
Nicklen, S.,
and Coulson, A. R.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
5463-5467[Abstract]
-
Higgins, D. G.,
and Sharp, P. M.
(1988)
Gene (Amst.)
73,
237-244[CrossRef][Medline]
[Order article via Infotrieve]
-
Yang, T. T.,
Kain, S. R.,
Kitts, P.,
Kondepudi, A.,
Yang, M. M.,
and Youvan, D. C.
(1996)
Gene (Amst.)
173,
19-23[CrossRef][Medline]
[Order article via Infotrieve]
-
Jones, C. A.,
Ng, J.,
Peterson, A. J.,
Morgan, K.,
Simon, J.,
and Jones, R. S.
(1998)
Mol. Cell. Biol.
18,
2825-2834[Abstract/Free Full Text]
-
Seewalt, R. G. A. B.,
van der Vlag, J.,
Gunster, M. J.,
Hamer, K. M.,
den Blaauwen, J. L.,
Satijn, D. P. E.,
Hendrix, T.,
van Driel, R.,
and Otte, A. P.
(1998)
Mol. Cell. Biol.
18,
3586-3595[Abstract/Free Full Text]
-
Neer, E. J.,
Schimdt, C. J.,
Nambudripad, R.,
and Smith, T. F.
(1994)
Nature
371,
297-300[CrossRef][Medline]
[Order article via Infotrieve]
-
Gutjarhr, T.,
Frei, E.,
Spicer, C.,
Baumgatner, S.,
White, A. H.,
and Noll, M.
(1995)
EMBO J.
14,
4296-4306[Abstract]
-
Lama, J.,
and Trono, D.
(1998)
J. Virol.
72,
1671-1676[Abstract/Free Full Text]
-
Kain, S. R.,
Adams, M.,
Kondepudi, A.,
Yang, T. T.,
Ward, W. W.,
and Kitts, P.
(1995)
BioTechniques
19,
650-655[Medline]
[Order article via Infotrieve]
-
Hill, C. P.,
Worthylake, D.,
Bancroft, D.,
Cristensen, M.,
and Sunquist, W. I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
3099-3104[Abstract/Free Full Text]
-
Cannon, P. M.,
Matthews, S.,
Clark, N.,
Byles, E. D.,
Iourin, O.,
Hockley, D. J.,
Kingsman, S. M.,
and Kingsman, A. J.
(1997)
J. Virol.
71,
3474-3483[Abstract]
-
Casella, C. R.,
Raffini, L. J.,
and Panganiban, A. T.
(1997)
Virology
228,
294-306[CrossRef][Medline]
[Order article via Infotrieve]
-
Kiernan, R. E.,
Ona, A.,
Englund, G.,
and Freed, E. O.
(1998)
J. Virol.
72,
4116-4126[Abstract/Free Full Text]
-
Fouchier, R. A. M.,
Meyer, B. E.,
Simon, J. H. M.,
Fischer, U.,
and Malim, M. H.
(1997)
EMBO J.
16,
4531-4539[Abstract/Free Full Text]
-
Bukrinskaya, A. G.,
Ghorpade, A.,
Heinzinger, N. K.,
Smithgall, T. E.,
Lewis, R. E.,
and Stevenson, M.
(1996)
Proc. Natl. Acad Sci. U. S. A.
93,
367-371[Abstract/Free Full Text]
-
Trono, D.,
and Gallay, P.
(1997)
Cell
88,
173-174
-
Freed, E.,
Englund, G.,
Maldarelli, F.,
and Martin, M. A.
(1997)
Cell
88,
171-173[Medline]
[Order article via Infotrieve]
-
Wall, M. A.,
Coleman, D. E.,
Lee, J. A.,
Iniguez-Lluhi, J. A.,
Posner, B. A.,
Gilman, A. G.,
and Sprang, S. R.
(1995)
Cell
83,
1047-1058[Medline]
[Order article via Infotrieve]
-
Sandmeyer, S. B.,
Hansen, L. J.,
and Chalker, D. L.
(1990)
Annu. Rev. Genet.
24,
491-518[CrossRef][Medline]
[Order article via Infotrieve]
-
Craigie, R.
(1992)
Trends Genet.
8,
187-190[CrossRef][Medline]
[Order article via Infotrieve]
-
Chalker, D. L.,
and Sandmeyer, S. B.
(1992)
Genes Dev.
6,
117-128[Abstract]
-
Devine, S. E.,
and Boeke, J. D.
(1996)
Genes Dev.
10,
620-633[Abstract]
-
Moore, J. H.,
Blomberg, M. A.,
Braiterman, L. T.,
Voytas, D. F.,
Natsoulis, G.,
and Boeke, J. D.
(1993)
Cell
73,
1007-1018[Medline]
[Order article via Infotrieve]
-
Zou, S.,
Wright, D. A.,
and Voytas, D. F.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
920-924[Abstract]
-
Zou, S.,
and Voytas, D. F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7412-7416[Abstract/Free Full Text]
-
Pirrotta, V.
(1997)
Curr. Opin. Genet. Dev.
7,
249-258[CrossRef][Medline]
[Order article via Infotrieve]
-
Pirrotta, V.
(1998)
Cell
93,
333-336[Medline]
[Order article via Infotrieve]
-
Jeang, K. T.,
Berkhout, B.,
and Dropulic, B.
(1993)
J. Biol. Chem.
268,
24940-24949[Abstract/Free Full Text]
-
El Kharroubi, A.,
Piras, G.,
Zensen, R.,
and Martin, M. A.
(1998)
Mol. Cell. Biol.
18,
2535-2544[Abstract/Free Full Text]
-
Van Lint, C.,
Emiliani, S.,
Ott, M.,
and Verdin, E.
(1996)
EMBO J.
15,
1112-1120[Abstract]
-
Verdin, E.,
Paras, P.,
and Van Lint, C.
(1993)
EMBO J.
12,
3249-3259[Abstract]
-
Benkirane, M.,
Chun, R. F.,
Xiao, H.,
Ogryzko, V. V.,
Howard, B. H.,
Nakatani, Y.,
and Jeang, K. T.
(1998)
J. Biol. Chem.
273,
24898-24905[Abstract/Free Full Text]
-
Bukrinsky, M. I.,
Sharova, N.,
Dempsey, M. P.,
Stanwick, T. L.,
Bukrinskaya, A. G.,
Haggerty, S.,
and Stevenson, M.
(1992)
Proc. Natl. Acad Sci. U. S. A.
89,
6580-6584[Abstract]
-
Bukrinsky, M. I.,
Haggerty, S.,
Dempsey, M. P.,
Sharova, N.,
Adzhubei, A.,
Spitz, L.,
Lewis, P.,
Goldfarb, D.,
Emerman, M.,
and Stevenson, M.
(1993)
Nature
365,
666-669[CrossRef][Medline]
[Order article via Infotrieve]
-
Kalpana, G. V.,
Marmon, S.,
Wang, W.,
Crabtree, G. R.,
and Goff, S. P.
(1994)
Science
266,
2002-2006[Medline]
[Order article via Infotrieve]
-
Carteau, S.,
Batson, S.,
Poljak, L.,
Mouscadet, J. F.,
de Rocquigny, H.,
Darlix, J.-L.,
Roques, B. P.,
Käs, E.,
and Auclair, C.
(1997)
J. Virol.
71,
6225-6229[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.