Previous Article | Next Article 
Journal of Virology, August 2001, p. 7131-7141, Vol. 75, No. 15
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7131-7141.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Functional Analysis of Adenovirus Protein IX
Identifies Domains Involved in Capsid Stability, Transcriptional
Activity, and Nuclear Reorganization
Manuel
Rosa-Calatrava,1
Linda
Grave,2
Francine
Puvion-Dutilleul,3
Bruno
Chatton,1 and
Claude
Kedinger1,*
Institut de Génétique et de
Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/ULP,
67404 Illkirch Cedex, C.U. de Strasbourg,1
Transgene S.A., 67000 Strasbourg,2 and
Laboratoire Organisation Fonctionnelle du Noyau, UPR 1983 CNRS,
94801 Villejuif Cedex,3 France
Received 5 January 2001/Accepted 22 April 2001
 |
ABSTRACT |
The product of adenovirus (Ad) type 5 gene IX (pIX) is known to
actively participate in the stability of the viral icosahedron, acting
as a capsid cement. We have previously demonstrated that pIX is also a
transcriptional activator of several viral and cellular TATA-containing
promoters, likely contributing to the transactivation of the Ad
expression program. By extensive mutagenesis, we have now delineated
the functional domains involved in each of the pIX properties: residues
22 to 26 of the highly conserved N-terminal domain are crucial for
incorporation of the protein into the virion; specific residues of the
C-terminal leucine repeat are responsible for pIX interactions with
itself and possibly other proteins, a property that is critical for pIX
transcriptional activity. We also show that pIX takes part in the
virus-induced nuclear reorganization of late infected cells: the
protein induces, most likely through self-assembly, the formation of
specific nuclear structures which appear as dispersed nuclear globules
by immunofluorescence staining and as clear amorphous spherical
inclusions by electron microscopy. The integrity of the leucine repeat
appears to be essential for the formation and nuclear retention of
these inclusions. Together, our results demonstrate the multifunctional
nature of pIX and provide new insights into Ad biology.
| |
INTRODUCTION |
Replication-deficient adenoviruses
(Ad) (20) efficiently transfer and express candidate
therapeutic genes into a variety of dividing and postmitotic cell types
(6, 27, 51, 57). For these reasons such viruses constitute
effective vectors for direct in vivo gene therapy (5, 16, 19,
24). However, several drawbacks, such as toxicity, host
inflammatory response (14), or transient in vivo transgene
expression (18, 41), impair the full success of Ad vectors
in human gene therapy protocols. Multiple factors are involved, among
which some viral proteins whose functions are often not fully understood.
We focused our attention on the study of the product of gene IX (pIX)
from Ad serotypes 2 and 5 (Ad2 and Ad5) (3, 8). Protein
pIX is a small polypeptide of 140 residues (14.3 kDa) that is
incorporated into the mature viral capsid. It is associated with hexon
proteins to form group-of-nine hexons (GON) that make up the central
region of each facet of the icosahedron (8, 10). Precise
determination of the stoichiometry of this assembly has revealed that
there are 12 molecules of pIX, organized as four trimers per GON, and
therefore 240 molecules per virion (56, 59, 60). The
protein acts as a capsid cement and thereby enhances the thermal
stability of the virions (17, 23). It is essential for
packaging 100% and more of the full-length Ad DNA (25). By themselves, these properties of pIX appear to be important enough to
be taken into consideration during the design of Ad vectors.
Additional observations strongly suggest that pIX is more than a capsid
protein and may serve additional functions during the infectious cycle
(44): (i) gene IX is the only structural protein coding
gene which is uncoupled from the Ad major late promoter (MLP); (ii) its
expression pattern follows a different time course and begins at
intermediate times postinfection (p.i.), much earlier than that of all
the other structural proteins; (iii) finally, pIX accumulates in the
infected cell nuclei with a speckled distribution. In agreement with
this nuclear localization, we have previously shown that pIX is a
transcriptional activator of several viral and cellular TATA-containing
promoters, among which are the Ad E1a, E4, and MLP promoters
(44). We therefore hypothesized that pIX could be involved
in the transactivation of Ad genome expression.
To precisely delineate the functional domains of pIX responsible for
the structural and transcriptional properties, we performed an
extensive mutational analysis of the pIX coding sequence. We show that
the highly conserved N-terminal part of the protein is essential for
the structural properties of the capsid, whereas the C-terminal leucine
repeat (putative coiled-coil domain) is critical for the
transactivating function. Accumulation of pIX results in the formation
of specific nuclear structures (the clear amorphous [c.a.]
inclusions), the function of which is presently unknown. Our results
suggest that formation of these structures involves self-assembly of
pIX through its coiled-coil domain.
| |
MATERIALS AND METHODS |
Cells and viruses.
Monolayer human A549 (54)
cells were grown in Dulbecco medium supplemented with 10% fetal calf
serum (FCS). 293 cells (28) were grown in Dulbecco
modified Eagle medium with 2% FCS. A549 cells (at 80% confluence)
were infected with wild-type (wt) Ad2 or Ad5 at a multiplicity of
infection (MOI) of 50 PFU per cell. Mutant viral genomes were
constructed as infectious plasmids by homologous recombination in
Escherichia coli, as described elsewhere (12).
All vectors contain, in addition to alterations of gene IX (see below),
a deletion in E1 (between nucleotides 459 and 3331) and in E3 (between
nucleotides 28592 and 30470) (Ad E1° E3°) (41).
Nucleotide numbering throughout this study conforms to that of
Chroboczek et al. (15). Mutant viruses were amplified in
293 cells. Viral growth, purification, titration, and storage were
previously described (27, 38, 41).
Recombinant eukaryotic expression vectors.
The sequence
encoding wt pIX was derived from the Ad5 genome by PCR amplification as
previously described (44) and inserted into three types of
expression vectors: (i) the pAT4 vector (gift from M. Vigneron), in a
site located 3' to the sequences encoding the F domain of the human
estrogen receptor (hER) (4), generating a wt pIX fusion
protein tagged at its N terminus (F/IX); (ii) the pXJ41 vector
(61), which yields an untagged protein; (iii) the pXX
vector, a pSG5-derived vector (29), in which the sequence encoding wt pIX was inserted in a site located 5' to the sequences encoding the F domain of the hER, generating a pIX fusion protein tagged at its C terminus (IX/F). In these vectors, the expression of
the wt or mutated pIX sequences was directed by the cytomegalovirus enhancer and herpes simplex virus type 1 (HSV-1) thymidine kinase gene
promoter (pAT4 and pXJ41), or the simian virus 40 promoter (pSG5-derivative pXX).
The Ad E1a promoter sequence (positions +100 to +560, with numbering
according to the study of Chroboczek et al.) was subcloned in front of
the chloramphenicol acetyltransferase (CAT) reporter gene of the
promoterless pBLCAT6 vector, as previously described (44).
Point mutations and deletions in the pIX coding sequence (as indicated
in Fig.
2A) were generated by following the protocol
of the QuickChange
Site-Directed Mutagenesis system (Stratagene).
All plasmids were
verified by
sequencing.
Transfections, cell extracts, and Western blotting.
A549
cells were transfected by calcium phosphate coprecipitation
(13). For CAT assays, the cells were harvested 36 h
after transfection, extracts were prepared, and aliquots, normalized by
protein concentration, were assayed for CAT activity as described earlier (7). CAT activities were determined from at least
three independent experiments and quantitated with a Bioimaging
analyser (Fuji Photo Film Co.).
For immunoprecipitations, the cells were harvested 36 h after
transfection by three cycles of freeze-thaw in buffer A (50
mM
Tris-HCl, pH 7.9; 20% glycerol; 1 mM dithiothreitol; 0.1% NP-40)
containing 0.4 M KCl. The expression of recombinant proteins was
verified by Western blotting. After an additional clearing step
on
protein G-Sepharose to adsorb nonspecific binding proteins,
cell
extracts were incubated for 2 h with 1 µg of the anti-F
antibody,
after which 30 µl of protein G-Sepharose beads were added
and
incubation was continued for an additional 2 h. The beads were
then
washed three times at room temperature with buffer A containing
200 mM
KCl and 0.5% NP-40 (mild-salt conditions). The resin was
then
dissociated by boiling for 5 to 10 min in sodium dodecyl
sulfate (SDS)
sample buffer. The bound proteins were detected
on Western blots with
specific antibodies using the ECL System
(Amersham), as previously
described (
7). Anti-pIX rabbit polyclonal
antibodies were
raised against purified recombinant glutathione
S-transferase-IX fusion protein (anti-pIX)
(
44). Monoclonal
antibodies against the F domain of the
hER (Mab3A6) have been
described (
44). Monoclonal
antibodies against the Ad5 penton
base were provided by Transgene
(Strasbourg).
Electron microscopy.
A549 cells near confluence were
infected at an MOI of 5 to 10 PFU of Ad5 per cell for 30 min.
Monolayers were then rinsed with phosphate-buffered saline (PBS), fresh
medium was added, and the cells were reincubated for 16 and 30 h
before fixation. A549 cells were transfected with the vector generating
the untagged wt pIX and cultured for 36 h. The cells were fixed with
4% formaldehyde (Merck) in 0.1 M Sörensen's phosphate buffer
(pH 7.2) at 4 to 8°C for 1 h. During the fixation step, the
cells were scraped from their plastic substrate and centrifuged. The
resulting pellets were dehydrated in increasing concentrations of
methanol and embedded in Lowicryl K4M (Polysciences Europe GmbH).
Polymerization was performed at 
30°C for 5 days under
long-wavelength UV light (Philips fluorescence tubes, TL 6W) and
subsequently at room temperature for 1 day. Ultrathin sections were
collected on Formvar-carbon-coated copper grids (mesh 200).
For identifying structures containing the pIX viral protein, grids
bearing Lowicryl sections were floated for 2 min over drops
of Aurion
BSA-C (purchased from Biovalley) (0.01% in PBS) in order
to prevent
background, with prior incubation (30 min at room temperature)
in the
presence of anti-pIX polyclonal antibody diluted 1/50 in
PBS. After a
rapid washing in PBS, grids were incubated at room
temperature for 30 min in the presence of goat anti-rabbit immunoglobulin
G (IgG)
conjugated to gold particles (10-nm diameter; British
Biocell
International, Ltd., Cardiff, United Kingdom) and stained
with uranyl
acetate prior to observation with a Philips 400 transmission
electron
microscope at 80 kV and 6,000- to 22,000-fold magnification.
To make
sure that secondary antibody did not bind nonspecifically
to biological
material, we verified that no labeling occurred
when primary antibody
was
omitted.
Immunofluorescence.
Immunofluorescence staining experiments
were carried out as previously described (44). A549 cells
were fixed with formaldehyde (2% [vol/vol] in PBS) and permeabilized
with 0.1% Triton X-100 in PBS. The primary antibodies were diluted in
PBS containing 0.1% Triton X-100. The anti-pIX rabbit polyclonal
antibody was used as described elsewhere (44) and the
Mab3A6 anti-F antibody was used at a 1/5,000 dilution in PBS containing
0.1% Triton X-100. After incubation for 1 h, the coverslips were
washed several times in PBS-0.1% Triton X-100 and then incubated with
goat Texas red-conjugated anti-rabbit IgG and/or donkey fluorescein
isothiocyanate (FITC)-labeled anti-mouse IgG (Sigma) at concentrations
recommended by the suppliers. Nuclei were counterstained with Hoechst
33258. After the staining, the coverslips were mounted and analyzed
using a confocal laser scanning microscope (Leica). Image enhancement
software was used to balance signal strength, and eightfold scanning
was used to separate signal from noise.
| |
RESULTS |
Peptide sequence and functional domains of pIX.
Multiple
sequence alignments between pIX proteins from human and animal Ad
serotypes (Fig. 1) revealed a high degree
of identity (95%) over the entire length of pIX from serotypes
belonging to the same subgenus. Although the extent of homology between
serotypes from different species was lower, two conserved domains could be identified when human and animal serotypes were compared: upon referring to the coordinates of pIX residues (Aa) from the human Ad2
serotype, these domains can be located at the N-terminal (Aa8-39) and
C-terminal (Aa100-121) ends of pIX, respectively. An additional, alanine-rich domain, specific to the human serotypes, could be delineated (Aa60-69).
View larger version (81K):
[in this window]
[in a new window]
|
FIG. 1.
Conserved sequence elements in pIX. Amino acid sequence
alignments (CLUSTAL X) (58) of pIX from several human (top
8 sequences) and animal (bovine [b], porcine [p], and canine [c])
Ad serotypes, as indicated on the left, were performed. Accession
numbers are as follows: Ad2 (p03282), Ad5 (p03285), Ad3 (J01962), Ad7
(03283); Ad9 (q9yl97), Ad12 (03284), Ad40 (p48312), Ad41 (p32539), Ad2b
(q65377), Ad3p (q9w9x3), Ad1c (q65944), and Ad2c (p14268). Dots
correspond to gaps inserted by the program to optimize alignments.
Conserved sequence elements are boxed. The symbols "*" and
"^" (bottom) denote identical or related amino acid residues,
respectively. Numbers in parentheses (top) refer to coordinates of
amino acids in Ad2 and Ad5 pIX, relative to the starting methionine.
|
|
No particular structural motif could be identified within the
N-terminal domain. The alanine-rich stretch, which is unlikely
to adopt
any particular structure, may serve as a flexible link
between the two
halves of the pIX molecule. In contrast, the C-terminal
domain clearly
revealed features of a leucine-repeat (or coiled-coil
domain), as
suggested by the helical wheel representation (Fig.
2B): 10 nonpolar amino acid residues
(leucine and valine), spaced
every three and four residues, align at
positions a and d on one
side of the wheel; these residues presumably
provide a hydrophobic
interface to interact with similar residues,
symmetrically positioned
(a' and d') on a second monomer (Fig.
2B),
thereby potentially
adopting a coiled-coil conformation (
36,
37,
39,
40).
Moreover, in the case of Ad2 and Ad5, ionized residues
of opposite
charge, located on either side of the helical wheel at
positions
e and g, may further stabilize protein assembly by
symmetrically
interacting with corresponding residues at positions e'
and g'
(
35).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 2.
Schematic representation of specific subdomains of pIX.
(A) The conserved pIX sequence domains, including the central
human-specific polyalanine stretch, are represented as boxes, with
relevant peptidic elements and coordinates from the Ad2 or Ad5 wt
sequence. Point mutations or small deletions are indicated above, while
larger deletions mentioned in the text are depicted below. (B) The
predicted helical-wheel representation of the C-terminal leucine repeat
of Ad2 or Ad5 pIX, from residues 100 (first L at position a) to 134 (S
at position g) is shown next to a symmetrically positioned wheel of the
same region. The potential hydrophobic interactions (a-d' and d-a')
between the two helices are suggested by the alignments of the residues
at positions a and d with positions d' and a', respectively, in a
putative coiled-coil structure. The charged residues ("+" or
""), which presumably stabilize these interactions, are
indicated. Residues (with corresponding coordinates) that are altered
by site-directed mutagenesis are indicated.
|
|
To determine the functional significance of these domains, the effects
of a series of deletions or point mutations altering
the conserved
sequence elements were examined (Fig.
2A). Within
the N-terminal half
of pIX, amino acid stretches that are conserved
between all (Aa13-15,
Aa22-23, Aa26-28, and Aa31-39) or only human
Ad serotypes (Aa63-70)
were deleted (
). The C-terminal leucine
repeat was disrupted by
changing, either separately or simultaneously,
leucine-114 and
valine-117 to proline and aspartate, respectively,
to generate mutant
L114P, V117D, or L114P-V117D (L-V). Interruptions
of the apolar series
at positions a and d by these residues were
indeed expected to disturb
the correct alignment (proline) or
hydrophobic bonding (aspartate)
(
36,
37,
39,
40). Two
mutated forms of pIX were also
constructed in which the net charge
at position e was inverted by
exchanging Aa106 or Aa113 with a
lysine residue (mutants Q106K and
E113K, respectively), thereby
triggering electrostatic repulsion
between protein monomers (
26,
35).
The N-terminal part of pIX critically contributes to its
incorporation into the capsid.
Earlier immunoelectron microscopy
studies with purified Ad virions and pIX-specific antisera
(2) revealed that only the C-terminal portion of pIX was
accessible to the antibodies. The authors of that study concluded that
the C-terminal part of the protein was exposed at the surface of the
virions, whereas its N-terminal domain was hidden inside the viral
capsid. To further define the pIX elements involved in capsid assembly,
we introduced a set of mutations into the E1-deleted viral genome by
homologous recombination to generate viruses expressing the pIX
variants during viral production. The mutations were designed to alter sequence elements that are most strictly conserved within both human
and animal serotypes (see above). Because all recombinant viruses are
E1 defective, they were produced on the 293 cells which constitutively
express the E1a and E1b Ad5 genes (28). Each pIX mutant
virus was grown on 293 cells, viral particles (Fig.
3, v) were purified, and equal quantities
were submitted to SDS-PAGE, in parallel with equal quantities of the
corresponding infected crude extracts (Fig. 3, e). As a control, viral
particles and extracts were assayed by Western blotting for the
presence of a viral protein (penton base) that was expressed to equal
levels by all variants, irrespective of pIX alteration. The presence of
pIX was examined by Western blot analysis, using anti-pIX antibodies. As an additional control, we verified that the wt pIX protein was
present both in infected-cell extracts and in purified Ad E1° IXwt virions (Fig. 3, lanes 1 and 2), whereas it was absent from
both fractions, in the case of a virus (Ad E1° IX°) lacking gene IX
(Fig. 3, lanes 3 and 4).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 3.
Integrity of the conserved N-terminal domain of Ad2 pIX
is required for incorporation into the capsid. CsCl-purified Ad5 E1°
virions (2 × 1010 particles of each recombinant
virus) expressing either wt pIX (Ad E1° IXwt), no pIX (Ad E1°
IX°), or specific pIX variants (as indicated on the top) were
disrupted by boiling in SDS sample buffer, fractionated by SDS-10%
PAGE, and analyzed by immunoblotting using monoclonal anti-penton (top)
and polyclonal anti-pIX (bottom) antibodies (even "v" lanes).
Extracts were prepared (44) from 293 cells that had been
infected by the same viruses (MOI of 20 PFU per cell) and collected at
36 h pi. Aliquots were analyzed by immunoblotting (odd "e"
lanes) next to the corresponding virions. Signals corresponding to
penton and pIX are indicated.
|
|
Mutations altering pIX within its C-terminal part (L114P, V117D, and
L-V) did not prevent incorporation of the mutant protein
into the
capsid (Fig.
3, lanes 12, 14, and 16), indicating that
the integrity of
the leucine repeat is not required for this function.
Moreover, capsid
stability (assessed as in references
17 and
22) appeared
not to be altered (data not shown). In contrast,
deletions within the
conserved N-terminal domain of pIX, such
as in mutants
22-23 and
26-28, completely abolished incorporation
of pIX into the capsid
(Fig.
3, lanes 8 and 10, respectively),
despite nearly normal levels of
pIX synthesis (Fig.
3, lanes 7
and 9, respectively). Mutation
(
13-15) did not impair virion
insertion of pIX (Fig.
3, lane 6), but
the resulting capsid was
less stable, as indicated by thermolability
measurements (assessed
as in references
17 and
22) (data
not
shown).
Together, these results define the N-terminal region spanning Aa22-28
as crucial for the correct and stable recruitment of
pIX into the viral
capsid, whereas the C-terminal region is not
involved at
all.
The integrity of the C-terminal leucine repeat and central alanine
stretch of pIX are essential for its transcriptional activity.
We
previously showed that pIX exhibits transcriptional properties
(44). Recombinant pIX efficiently stimulated, in a
dose-dependent manner, the activity of several viral and cellular
TATA-containing promoters. To precisely delineate the transactivating
domain of pIX, we examined the effect of the complete set of pIX
mutations on E1a promoter activation (Fig.
4). To this end, vectors expressing wt or
mutated pIX sequences as proteins fused at their N termini (F/IX) or C
termini (IX/F) to the F epitope tag were transfected together with a
CAT reporter gene driven by the E1a promoter. After we verified that
equal levels of pIX were expressed, as revealed by immunoblotting with
antibodies against the F epitope (data not shown), the CAT activities
were measured. Under these conditions, relative CAT activities will
reflect the intrinsic transcriptional activating capacity of each
recombinant protein (44).
View larger version (48K):
[in this window]
[in a new window]
|
FIG. 4.
The leucine repeat and central hinge region are crucial
for Ad2 pIX transcriptional activity. (A) Structure of the chimeric
pE1a-CAT reporter plasmid in which the promoter region of the Ad5 E1a
transcription unit (44) was fused to the CAT gene. (B)
A549 cells were transfected with 1 µg of the pE1a-CAT reporter
plasmid, either alone (column 1) or together with plasmids expressing
the wt or mutated pIX as F-tagged fusion proteins: F/IX derivatives
(0.1 µg; column 2 and columns 3 to 14, respectively) or IX/F
derivatives (0.5 µg; columns 15 and 16, respectively). Cells were
collected 36 h later, and extracts were prepared. Relative CAT
activities (means from three independent experiments) are represented
with corresponding standard deviations.
|
|
In agreement with our earlier structural analysis (
44),
truncated versions of pIX lacking half of the leucine repeat
(F/IX:
111-140)
or most of the N-terminal half (F/IX:
11-74) of the
protein, lost
their transactivating property (compare in Fig.
4B,
columns 2
and 14 or 7,
respectively)
Point mutations within the sequence encoding the leucine repeat in the
C-terminal end of pIX severely reduced reporter stimulation:
single
(L114P and V117D) or double (L-V) alterations had effects
similar to
complete deletion (
111-140) of the C-terminal part
of the protein
(Fig.
4B, compare column 2 with columns 11 to 14).
Point mutations
Q106K and E113K also reduced transactivation (Fig.
4B, compare column 2 and columns 9 to 10), stressing the contribution
of electrostatic
interactions in functional assembly of pIX monomers.
The
transactivation function of pIX also depends on the integrity
of the
central domain, since deletion of the corresponding polyalanine
stretch
(
63-70) led to a strong reduction of reporter stimulation
(Fig.
4B,
compare columns 2 and 8). In contrast, deletions within
the N-terminal
part of pIX had no detectable effect on its intrinsic
stimulatory
activity, since very similar levels of transactivation
were obtained
with F/IX:
13-15, F/IX:
22-23, F/IX:
26-28, and
F/IX:
31-39
pIX variants and the wt protein (Fig.
4B, compare
column 2 and columns
3 to 6). Very similar results were obtained
with vectors expressing
C-terminally instead of N-terminally tagged
pIX derivatives (Fig.
4B,
compare columns 15 and 16 with columns
2 and 12, respectively; also
data not
shown).
Together, our results suggest that the pIX transactivating function is
dependent on the integrity of the C-terminal leucine
repeat, as well as
on the central alanine-rich element. Interestingly,
the N-terminal
region, critically involved in the capsid integration
of pIX, is not
involved in this
function.
The integrity of the C-terminal leucine repeat and central alanine
stretch of pIX are essential for its self-interaction.
The
presence of a leucine repeat type of structure at the C-terminal end of
pIX suggests that the protein may dimerize (or multimerize) by
interacting through this element (36, 37). To test this
possibility, a vector expressing the nontagged wt pIX was cotransfected
into A549 cells with vectors expressing F epitope-tagged wt or mutant
pIX proteins (F/IX). As revealed by Western blot analysis of cell
extracts with monoclonal anti-F or polyclonal anti-pIX antibodies, the
transfected vectors were expressed to very similar levels (data not shown).
When these extracts were immunoprecipitated with the monoclonal anti-F
antibody under mild-salt conditions and subjected to
SDS-polyacrylamide
gel electrophoresis (PAGE), a single band of
nontagged wt pIX protein,
with the expected size, was revealed
with the anti-pIX antibody, as
indicated in Fig.
5, in addition
to the
F-tagged protein variants. As expected, the band corresponding
to the
nontagged pIX was not revealed by the anti-F antibody which
was applied
first on the blot (see legend to Fig.
5), ruling out
the possibility
that it may correspond to a proteolytic degradation
product of the
F-tagged pIX. As an additional control, we verified
that, when
expressed in the absence of F-tagged pIX, the nontagged
pIX was not
detected in the anti-F immunoprecipitate (not shown).
The results
indicate, therefore, that a detectable fraction of
the nontagged wt pIX
was coprecipitated together with the F epitope-tagged
wt pIX (F/IXwt;
Fig.
5, lane 1). The relative weakness of the
band corresponding to
nontagged pIX, compared to that of the F-tagged
protein, could be due
in part to the fact that self-association
of the F-tagged proteins may
compete for heteromeric interactions.
That this coprecipitation
involved the leucine repeat domain was
demonstrated by our observation
that single point mutations (E113K,
Q106K, and V117D) or a double point
mutation (L-V) disrupting
this structure reduced or abolished the
carrying effect (Fig.
5, compare lane 1 with lanes 5 to 8). Similarly,
a mutant lacking
the C-terminal mid-part of the leucine repeat
(
111-140) did not
coprecipitate wt pIX (data not shown). As
expected, mutations
within either N-terminal domain (
22-23 and
26-28 [Fig.
5, compare
lane 1 with lanes 2 and 3] or
13-15 and
31-39 [not shown]) had
no effect on the interaction. In contrast,
the central alanine-rich
element (
63-70) of pIX was essential for
coprecipitation (Fig.
5, compare lanes 1 and 4) and is therefore also
involved in the
oligomerization process. Furthermore, our observation
that the
same mutations affect both transactivation and interaction
properties
of pIX suggests that the two activities may be linked.
View larger version (42K):
[in this window]
[in a new window]
|
FIG. 5.
Self-interaction of pIX. A549 cells were transfected
with vectors expressing the wt pIX, together with vectors expressing
N-terminally F-tagged wt or mutated pIX proteins, as indicated. Cell
extracts were prepared, and aliquots were immunoprecipitated (IP) with
monoclonal antibodies directed against the F epitope. The
immunoprecipitates were subjected to Western blot analysis (WB), with
probing first with the anti-F monoclonal antibody. After exposure, the
same blot was washed and reprobed with polyclonal antibodies against
pIX. The position of bands corresponding to the untagged and F-tagged
wt or mutated pIX are indicated. The bands marked by the asterisk
likely correspond to proteolytic breakdown products of the F-tagged
derivatives. The position of the IgG heavy subunit [IgG(H)] is
indicated to show that equal amounts of antibody were used in the
immunoprecipitation reaction. Numbers on the right correspond to the
positions of molecular size markers (in kilodaltons).
|
|
Protein pIX accumulates in virus-induced clear amorphous
inclusions.
We have previously shown (44), by
immunofluorescence staining with pIX-specific antibodies, that pIX was
predominantly associated with infected cell nuclei, in accordance with
the transcriptional properties of the protein. In addition, we observed
that the nuclear staining of pIX was dynamic, showing a speckled
distribution at later times of infection (unpublished observation). To
study more precisely the intranuclear distribution of pIX protein,
Ad5-infected A549 cells were examined by immunoelectron microscopy at
different times p.i. from 16 to 30 h p.i., in order to observe the
accumulation of pIX as a function of the successive steps of nuclear
alteration during infection (46-49).
No significant labeling was observed with the anti-pIX antibody before
16 h p.i. (not shown). At this time, the infected cells
were mainly at
the intermediate stage of nuclear alteration (
49).
Among
several virus-induced structures (including sites of viral
DNA
replication or transcription and sites of viral genome or
single-stranded viral DNA accumulation) (
49), pIX was
detected
in small, irregularly shaped or spherical c.a. inclusions
(Fig.
6A).
View larger version (114K):
[in this window]
[in a new window]
|
FIG. 6.
Protein pIX actively induces specific nuclear c.a.
inclusions. (A) Ad2-infected A549 cells at the intermediate stage of
nuclear transformation (14 to 16 h pi) (49) were processed
for immunogold labeling with anti-pIX polyclonal antibody on Lowicryl
sections of formaldehyde-fixed cells. Gold particles are scattered over
the fibrillogranular network (fg), one component of the viral region,
and accumulate over an enclosed small irregularly shaped clear
amorphous inclusion (c.a. inclusion; star). The accumulation site of
viral single-stranded DNA (a), the other compartment of the viral
region, is entirely devoid of pIX protein. c, cytoplasm. Bar, 0.5 µm.
(B) Late stage of Ad-mediated nuclear transformation (24 to 30 h
p.i.) (49) are characterized by the presence of progeny
viruses. The roughly spherical c.a. inclusion (star) is intensely and
homogeneously labeled. It is located in the electron-translucent region
(e) which separates the perinuclear layer of host chromatin (ch) from
the large, centrally located viral region (vr). Some viruses (v), both
scattered in the electron-translucent region and clustered within the
viral region are labelled. c, cytoplasm. Bar, 0.5 µm. (C)
Overexpression of recombinant pIX protein induces the accumulation of
the protein within newly formed c.a. inclusions: A549 cells were
transfected with the vector expressing the untagged wt pIX. Gold
particles accumulate over the entire surface of an ovoid c.a. inclusion
present in the nucleoplasm. It clearly appears that the labelled
inclusion (star) is similar to those observed in panel B following Ad
infection. c, cytoplasm; ch, perinuclear layer of condensed chromatin;
nu, nucleolus. Bar, 0.5 µm.
|
|
At a later stage (24 to 30 h p.i.), when a central viral
compartment and a perinuclear electron-translucent area with protein
crystals and isolated viruses were apparent (
47,
49), pIX
remained concentrated within the c.a. inclusions, which became
more
frequent and spherical (Fig.
6B). Sometimes two or three
of these
inclusions were juxtaposed (unpublished data). In addition,
pIX was
observed over the crystalline arrays of viruses and the
isolated
viruses particles (Fig.
6B).
Hence, since pIX is efficiently neosynthesized and belongs the late
phase of infection, the protein is predominantly associated
with c.a.
inclusions that are dynamic in their shape and location
in the nucleus.
They grow in size as the infection progresses,
as the pIX protein
accumulates into the nucleus. Irrespective
of their shape, size, and
location within the nucleus, they were
always intensely and
homogeneously labeled with the anti-pIX
antibody.
Overexpression of pIX induces by itself the formation of c.a.
inclusions.
Our ultrastructural data suggest that pIX is the main
component of the virus-induced c.a. inclusions. To determine whether pIX was directly responsible for their occurrence, we attempted to
localize the protein in cells transfected with a wt pIX expression vector, i.e., in the absence of any other viral protein. The morphology of pIX-expressing cells was similar to that of nontransfected cells
except for the additional presence in the nucleoplasm of c.a.
inclusions (Fig. 6C) identical to those observed in lytically infected
cells (Fig. 6B). Depending on the amount of expressed pIX (i.e., as a
function of time posttransfection), c.a. inclusions were variable in
size and frequency but always showed the same amorphous aspect.
Immunogold detection of pIX protein resulted in an intense labeling of
each c.a. inclusion and in a slight labeling of the surrounding
nucleoplasm and cytoplasm. Therefore, in the absence of other viral
proteins, pIX is able to induce the formation of c.a. inclusions
similar to those induced by Ad infection.
Formation of c.a. inclusions requires the integrity of the pIX
leucine repeat.
Specific immunofluorescence staining of cells
infected with wt Ad5 (unpublished data) or transfected with the wt
pIX-expressing vector revealed a speckled distribution of the protein
in the nucleus (Fig. 7A), most likely
corresponding to the accumulation of pIX within the c.a. inclusions
observed by electron microscopy. To identify peptidic domains of pIX
which may be responsible for the formation of c.a. inclusions, we
examined the effect of our set of mutations on the nuclear distribution
of pIX in transfected cells. Mutations affecting either the N-terminal
(13-15, 22-23, 26-28, and 31-39) or central (63-70)
domains of pIX did not affect the formation and nuclear location of the
c.a. inclusions, since the corresponding mutant proteins all yielded
the same speckled distribution as had the wt pIX upon transfection
(data not shown). In contrast, alteration of the leucine repeat of pIX
by modification of the net charge of specific residues (mutations E113K
or Q106K) drastically changed the intracellular distribution of the
corresponding pIX variants which were confined to the cytoplasm, as
revealed by immunofluorescence staining (Fig. 7B and data not shown).
Furthermore, although the overall level of mutant expression was
similar to that of the wt protein (as revealed by Western blotting
[data not shown]), these variants accumulated with a microspeckled
pattern.
View larger version (110K):
[in this window]
[in a new window]
|
FIG. 7.
Formation of the nuclear inclusions is dependent on the
integrity of the pIX leucine repeat. A549 cells were transfected with
plasmids encoding the untagged and/or F-tagged wt or mutated pIX, as
indicated. At 36 h posttransfection, cells were processed for
immunofluorescence staining with polyclonal anti-pIX antibodies and
goat Texas red-conjugated anti-rabbit IgG (A to C and E and F), and/or
with mouse anti-F antibody and donkey FITC-labeled anti-mouse IgG (D to
F). Nuclei were counterstained with Hoechst 33258. Panels E and F are
the merged confocal images obtained with each fluorochrome separately.
Bar, 10 µm.
|
|
Similarly, both point mutations (L114P, V117D, and L-V) and deletions
(
111-140) affecting the integrity of the leucine repeat
abolished
formation of the c.a. inclusions and resulted in a diffuse
distribution
of the altered pIX throughout the nucleus and cytoplasm
(Fig.
7C and
data not shown). Fusion of the F epitope tag at the
C-terminal end of
wt pIX (IX/F) also prevented the nuclear accumulation
of the protein
and induced the same diffuse pattern, despite the
integrity of the
leucine repeat (Fig.
7D). This effect, most likely
related to the
steric hindrance imposed by the tag, suggests that
free access to the
leucine repeat was essential for nuclear retention
of pIX and c.a.
inclusion formation. Interestingly, when cells
were cotransfected with
vectors expressing the untagged (wt pIX)
and C-terminally F-tagged
(IX/F) proteins, both types of proteins
accumulated within the same
inclusion bodies, as revealed by the
merged immunofluorescence staining
(Fig.
7, compare panels D and
E). It appears, therefore, that the
unfocused IX/F protein distribution
was redirected by the coexpressed
wt pIX into its corresponding
nuclear accumulation sites. In contrast,
when the F-tagged partner
of the cotransfection carried an alteration
within the leucine
repeat (IX/F:L-V), this recruitment was nearly
abolished, as shown
by the persistent diffuse distribution pattern of
the F-tagged
mutant (Fig.
7F).
Together, our results clearly indicate that the leucine repeat of pIX
is chiefly involved in the formation and nuclear retention
of the c.a.
inclusions. Furthermore, our data strongly suggest
that the assembly of
the pIX-specific inclusion bodies is an active
process driven by pIX
itself, most likely through self-assembly.
| |
DISCUSSION |
Viruses, as obligatory cell parasites, usually evolved toward the
highest possible degree of simplification of their structure and
components to reach at minimal expense the most efficient rates of
proliferation. Ad comply with this rule, not only in exploiting the
coding capacity of their genome by using alternative reading frames but
also in producing proteins with multiple biological activities. The
product of the Ad gene IX is an example of such multifunctional
proteins: pIX (140 residues) is a structural component of the viral
capsid, acts as a transcriptional activator, and accumulates in
infected cell nuclei as specific structures (c.a. inclusions), the
function of which remains to be established. In the present study, we
have performed extensive site-directed mutagenesis to define the
corresponding functional domains of the protein.
Structural involvement of pIX in viral capsid assembly.
The
pIX protein has previously been described as a capsid cement between
the viral hexons (8, 10), thereby optimizing DNA packaging
capacity and thermal stability of Ad virions (17, 23).
Based on immunochemical approaches, it had been suggested that the
N-terminal portion of pIX sticks inside the capsid, while its
C-terminal part points outward (2). Our present results indicate that residues 22 to 28 are essential for pIX incorporation into the capsid and for virion thermostability. The importance of these
residues is further supported by their complete conservation among
human and animal Ad serotypes. Additional residues within the
N-terminal region of pIX likely contribute to this function, as
suggested by the effect of the 13-15 deletion, which did not impair
pIX integration into the capsid but affected virion stability.
Interestingly, our results rule out any contribution of the putative
coiled-coil element of pIX to these capsid properties.
This clearly
indicates that the corresponding capsid interactions
occur through
elements other than the coiled-coil domain and suggests
that the
N-terminal element defined above fulfills this function.
Since
coiled-coil elements have been shown to be responsible for
the
trimerization or oligomerization of other proteins (
36,
37,
39,
40), like the well-documented yeast GCN4 (
30,
31)
or human immunodeficiency virus type 1 gp41 (
53), it is
likely that the coiled-coil element of pIX might also be involved
in
the trimerization of this protein in the GON complexes, as
in fact
suggested by our immunoprecipitation experiments. This
raises the
question of whether the pIX molecules are still organized
as trimers in
the leucine repeat mutants or whether the N-terminal
domain of pIX, in
close contact with the hexon molecules, is primarily
responsible for
the integration of pIX trimers into the virion
capsid. Clearly,
additional experiments, including coimmunoprecipitation
assays and
three-dimensional structure analyses, will be required
to solve these
questions. Our conclusions (i) that only the N-terminal
part of pIX is
implicated in its integration into the virion capsid,
(ii) that its
C-terminal end points outward, and (iii) that this
extremity could be
modified without altering the overall structural
properties of the
molecule open up the potential to hook ligands
of interest onto this
C-terminal end in order to modify the cellular
tropism of the
virus.
Transcriptional activity of pIX.
We have previously reported
that recombinant pIX exhibits properties of a transcriptional activator
when assayed in transfection experiments or in a reconstituted in vitro
transcription system (44). Here we show that the integrity
of the leucine repeat element of pIX is mandatory for this
transactivating function, while the highly conserved residues within
the N-terminal half of the molecule are not associated to this
activity. Larger deletions in the N-terminal region (11-74) or just
removing the alanine-rich element (63-70) also impaired pIX
transcriptional activity, despite preserving the coiled-coil domain. As
suggested by predictive structure analyses (data not shown), the
polyalanine stretch may serve a hinge function in the pIX molecule. It
is therefore possible that mutations targeting this element disrupt the
global structure of pIX and thereby affect its essential functions.
Additional evidence (M. Rosa-Calatrava et al., unpublished data)
revealed that, soon after cell entry and virion decapsidation,
the
released capsid pIX accumulates in the cell nucleus, as soon
as 45 min
p.i. until 6 h p.i. These pIX molecules may then activate
the
highly responsive E1a promoter, thus behaving as a ready-to-use
transactivator, much like the VP16 factor in the case of HSV-1
infection (
1). Furthermore, it is likely that the newly
synthesized
pIX, which starts accumulating at intermediate times p.i.,
also
contributes to the activation of the late phase of Ad infection
by
stimulating MLP activity. Thus, it will be of interest to decipher
the
molecular mechanism of pIX-mediated
transactivation.
As expected from the absence of basic residues flanking its leucine
repeat (to make up a bona fide basic-leucine-zipper) (
36,
37) and from the lack of any other DNA-recognizing motif, pIX
has no DNA-binding activity (data not shown). In contrast, as
revealed
by our coimmunoprecipitation experiments, the leucine
repeat is
involved in the interaction of pIX with itself, allowing
its
homodimerization or oligomerization via a coiled-coil structure.
The
fact that the same mutations affect both pIX self-interaction
and
transactivation properties suggests that the two activities
are
directly correlated. Alternatively, or in addition, pIX might
interact
through this leucine repeat element with components of
the
transcription apparatus: preliminary results suggest indeed
that pIX
contacts specific RNA polymerase II subunits and general
transcription
factors (unpublished data), thus mimicking other
viral transactivators
such as Ad E1a (
34), HSV-1 pX (
50),
or VP16
(
32).
Interestingly, one feature that is shared by all pIX-responsive
promoters assayed so far, whether from a viral or a cellular
origin, is
the presence of a canonical TATA box (
44). It will
be of
interest to identify the mechanism of this specific promoter
targeting.
It is tempting to speculate that such a promoter preference
for pIX is
of some advantage for Ad propagation since all viral
promoters, with
the exception of the E2 promoter, contain a TATA
box. This hypothesis
is further supported by the fact that only
episomal genes (i.e., either
in plasmidic or viral form) but not
chromosome-integrated reporters
have been found to be responsive
to pIX (unpublished
observation).
Nuclear accumulation structures of pIX.
Despite the absence of
any detectable homology with consensus nuclear localization signals
(9) in its peptide sequence, pIX concentrates within the
cell nucleus (44). We show here that the leucine repeat of
pIX is essential for this nuclear accumulation, as well as for the
formation of the c.a. inclusions. Together, these observations indicate
that pIX, by virtue of its low molecular weight, freely diffuses from
the cytoplasm to the nucleus, where it is retained, most likely through
interactions involving the leucine repeat and specific nuclear
components. Preliminary biochemical evidence suggests that pIX actually
associates with fractions of the nuclear matrix (unpublished data). It
is likely, therefore, that the targeting of the nuclear matrix by pIX
constitutes the initial step in the assembly of the c.a. inclusions,
providing a nucleation point for pIX oligomerization. Since these
nuclear structures are built up in the absence of any other viral
protein except pIX, we conclude that they reflect an intrinsic property of pIX.
The leucine repeat plays a central role in both pIX transcriptional
activity and the ability to form c.a. inclusions. However,
the
following lines of evidence clearly indicate that these functions
are
two independent properties of pIX: (i) during the late phase
of
infection, c.a. inclusions were always found to be excluded
from the
viral transcription sites; (ii) neither RNA polymerase
II nor primary
transcripts could be detected within the c.a. inclusions
from
Ad-infected cells or cells transfected with wt pIX-expressing
vectors
(unpublished data); (iii) a mutant lacking part of its
alanine-rich
element (
63-70) lost its capacity to transactivate
the E1a promoter
(Fig.
4) and to self-associate under our immunoprecipitation
conditions
(Fig.
5) but retained its ability to accumulate into
nuclear c.a.
inclusions (data not shown); and (iv) conversely,
fusion of the F
epitope to the C-terminal end of pIX (IX/F), completely
abolished the
ability of the protein to form c.a. inclusions in
the nucleus (Fig.
7D)
but did not affect its transcriptional properties
(Fig.
4, lane 15),
unless the structural integrity of the leucine
repeat was altered (Fig.
4, lane 16). The most simple interpretation
to account for the apparent
unrelatedness of these pIX functions
is that pIX might exert its
transcriptional properties only at
low concentrations (i.e., at initial
times of infection and just
after the onset of pIX synthesis), while it
starts forming nuclear
inclusions at higher concentrations by
accumulating on nuclear
matrix structures, as observed later in
infection or after transfection.
Our finding that addition of the F tag
at the C-terminal end of
pIX differentially affects pIX transcriptional
activity and c.a.
inclusion formation may therefore merely reflect the
preferential
impairment of contacts implicated in the assembly of c.a.
inclusions
compared to those required for promoter
transactivation.
The physiological role of these c.a. inclusions remains puzzling. An
interesting indication might be provided by the earlier
observation
(
47) that the host cell PML protein was confined,
late in
Ad infection, inside virus-induced nuclear structures,
now identified
as the pIX-induced c.a. inclusions (unpublished
data). Thus, like the
Ad early E4orf3 product which has previously
been shown to relocalize
PML into viral "fibrous-like" structures
(
11,
21), pIX
might also contribute to the viral process of
alteration of the nuclear
PML oncogenic domains (
33,
45,
52,
55), but during the
late phase of
infection.
In conclusion, it appears that pIX plays multiple functions during
infection. Interestingly, it shares some of these functions
with the
product of the other Ad intermediate gene, pIVa2: both
proteins are
transcriptional activators (
42,
44), take part
in the
virus-induced alterations of the host cell by accumulating
as specific
nuclear inclusions (
43), and are present in the
mature
virion particles (
8,
43,
44,
59,
62). No doubt
these
multifaceted viral entities have yet additional secrets
to reveal and
thus clearly deserve attention when designing Ad-based
vectors for gene
therapy
protocols.
| |
ACKNOWLEDGMENTS |
We thank M. Courtney, M. Methali, and M. Lusky for help and
stimulating discussions; A. Bahr for sequence alignment; N. Messaddeq for crucial technical assistance; the IGBMC cell culture staff for
providing cells; and the chemistry staff for preparing oligonucleotides and sequencing DNA.
This work was supported by Transgene S.A., the Convention
Industrielle pour la Formation par la Recherche CIFRE, and by funds from the French Ministry of Science, the Institut National de la
Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the University Louis Pasteur, the Alsace Region, the Fondation pour la Recherche Médicale, and the
Association pour la Recherche sur le Cancer.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: IGBMC, BP 163, 67404 Illkirch Cedex, France. Phone: (33) 388-65-34-46. Fax: (33)
388-65-32-01. E-mail: kedinger{at}esbs.u-strasbg.fr.
| |
REFERENCES |
| 1.
|
Ace, C. I.,
T. A. McKee,
J. M. Ryan,
J. M. Cameron, and C. M. Preston.
1989.
Construction and characterization of a herpes simplex virus type 1 mutant unable to transinduce immediate-early gene expression.
J. Virol.
63:2260-2269[Abstract/Free Full Text].
|
| 2.
|
Akalu, A.,
H. Liebermann,
U. Bauer,
H. Granzow, and W. Seidel.
1999.
The subgenus-specific C-terminal region of protein IX is located on the surface of the adenovirus capsid.
J. Virol.
73:6182-6187[Abstract/Free Full Text].
|
| 3.
|
Alestrom, P.,
G. Akusjarvi,
M. Perricaudet,
M. B. Mathews,
D. F. Klessig, and U. Pettersson.
1980.
The gene for polypeptide IX of adenovirus type 2 and its unspliced messenger RNA.
Cell
19:671-681[CrossRef][Medline].
|
| 4.
|
Ali, S.,
Y. Lutz,
J. P. Bellocq,
M. P. Chenard-Neu,
N. Rouyer, and D. Metzger.
1993.
Production and characterization of monoclonal antibodies recognising defined regions of the human oestrogen receptor.
Hybridoma
12:391-405[Medline].
|
| 5.
|
Bellon, G.,
L. Michel-Calemard,
D. Thouvenot,
V. Jagneaux,
F. Poitevin,
C. Malcus,
N. Accart,
M. P. Layani,
M. Aymard,
H. Bernon,
J. Bienvenu,
M. Courtney,
G. Doring,
B. Gilly,
R. Gilly,
D. Lamy,
H. Levrey,
Y. Morel,
C. Paulin,
F. Perraud,
L. Rodillon,
C. Sene,
S. So,
F. Touraine-Moulin,
A. Pavirani, et al.
1997.
Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial.
Hum. Gene. Ther.
8:15-25[Medline].
|
| 6.
|
Benihoud, K.,
P. Yeh, and M. Perricaudet.
1999.
Adenovirus vectors for gene delivery.
Curr. Opin. Biotechnol.
10:440-447[CrossRef][Medline].
|
| 7.
|
Bocco, J. L.,
B. Reimund,
B. Chatton, and C. Kedinger.
1993.
Rb may act as a transcriptional co-activator in undifferentiated F9 cells.
Oncogene
8:2977-2986[Medline]. (Erratum, 9:999, 1994.)
|
| 8.
|
Boulanger, P.,
P. Lemay,
G. E. Blair, and W. C. Russell.
1979.
Characterization of adenovirus protein IX.
J. Gen. Virol.
44:783-800[Abstract/Free Full Text].
|
| 9.
|
Boulikas, T.
1994.
Putative nuclear localization signals (NLS) in protein transcription factors.
J. Cell. Biochem.
55:32-58[CrossRef][Medline].
|
| 10.
|
Burnett, R. M.,
M. G. Grutter, and J. L. White.
1985.
The structure of the adenovirus capsid. I. An envelope model of hexon at 6 Å resolution.
J. Mol. Biol.
185:105-123[CrossRef][Medline].
|
| 11.
|
Carvalho, T.,
J. S. Seeler,
K. Ohman,
P. Jordan,
U. Petterson,
G. Akusjarvi,
M. Carmo-Fonseca, and A. Dejean.
1995.
Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies.
J. Cell Biol.
131:45-56[Abstract/Free Full Text].
|
| 12.
|
Chartier, C.,
E. Degryse,
M. Gantzer,
A. Dieterle,
A. Pavirani, and M. Mehtali.
1996.
Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli J.
Virol.
70:4805-4810.
|
| 13.
|
Chen, C., and H. Okayama.
1987.
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol. Cell. Biol.
7:2745-2752[Abstract/Free Full Text].
|
| 14.
|
Christ, M.,
B. Louis,
F. Stoeckel,
A. Dieterle,
L. Grave,
D. Dreyer,
J. Kintz,
D. Ali Hadji,
M. Lusky, and M. Mehtali.
2000.
Modulation of the inflammatory properties and hepatotoxicity of recombinant adenovirus vectors by the viral E4 gene products.
Hum. Gene Ther.
11:415-427[CrossRef][Medline].
|
| 15.
|
Chroboczek, J.,
F. Bieber, and B. Jacrot.
1992.
The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2.
Virology
186:280-285[CrossRef][Medline].
|
| 16.
|
Clayman, G. L.,
A. K. el-Naggar,
S. M. Lippman,
Y. C. Henderson,
M. Frederick,
J. A. Merritt,
L. A. Zumstein,
T. M. Timmons,
T. J. Liu,
L. Ginsberg,
J. A. Roth,
W. K. Hong,
P. Bruso, and H. Goepfert.
1998.
Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma.
J. Clin. Oncol.
16:2221-2232[Abstract].
|
| 17.
|
Colby, W. W., and T. Shenk.
1981.
Adenovirus type 5 virions can be assembled in vivo in the absence of detectable polypeptide IX.
J. Virol.
39:977-980[Abstract/Free Full Text].
|
| 18.
|
Connelly, S.,
T. A. Smith,
G. Dhir,
J. M. Gardner,
M. G. Mehaffey,
K. S. Zaret,
A. Mclelland, and M. Kaleko.
1995.
In vivo gene delivery and expression of physiological levels of functional human factor VIII in mice.
Hum. Gene Ther
6:185-193[Medline].
|
| 19.
|
Crystal, R. G.,
E. Hirschowitz,
M. Lieberman,
J. Daly,
E. Kazam,
C. Henschke,
D. Yankelevitz,
N. Kemeny,
R. Silverstein,
A. Ohwada,
T. Russi,
A. Mastrangeli,
A. Sanders,
J. Cooke, and B. G. Harvey.
1997.
Phase I study of direct administration of a replication deficient adenovirus vector containing the E. coli cytosine deaminase gene to metastatic colon carcinoma of the liver in association with the oral administration of the pro-drug 5-fluorocytosine.
Hum. Gene Ther.
8:985-1001[Medline].
|
| 20.
|
Davis, A. R.,
N. A. Wivel,
J. L. Palladino,
L. Tao, and J. M. Wilson.
2000.
Construction of adenoviral vectors.
Methods Mol. Biol.
135:515-523[Medline].
|
| 21.
|
Doucas, V.,
A. M. Ishov,
A. Romo,
H. Juguilon,
M. D. Weitzman,
R. M. Evans, and G. G. Maul.
1996.
Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.
Genes Dev.
10:196-207[Abstract/Free Full Text].
|
| 22.
|
Fallaux, F. J.,
A. Bout,
I. van der Velde,
D. J. van den Wollenberg,
K. M. Hehir,
J. Keegan,
C. Auger,
S. J. Cramer,
H. van Ormondt,
A. J. van der Eb,
D. Valerio, and R. C. Hoeben.
1998.
New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses.
Hum. Gene Ther.
9:1909-1917[Medline].
|
| 23.
|
Furcinitti, P. S.,
J. van Oostrum, and R. M. Burnett.
1989.
Adenovirus polypeptide IX revealed as capsid cement by difference images from electron microscopy and crystallography.
EMBO J.
8:3563-3570[Medline].
|
| 24.
|
Gahery-Segard, H.,
V. Molinier-Frenkel,
C. Le Boulaire,
P. Saulnier,
P. Opolon,
R. Lengagne,
E. Gautier,
A. Le Cesne,
L. Zitvogel,
A. Venet,
C. Schatz,
M. Courtney,
T. Le Chevalier,
T. Tursz,
J. G. Guillet, and F. Farace.
1997.
Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.
J. Clin. Investig.
100:2218-2226[Medline].
|
| 25.
|
Ghosh-Choudhury, G.,
Y. Haj-Ahmad, and F. L. Graham.
1987.
Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes.
EMBO J.
6:1733-1739[Medline].
|
| 26.
|
Gonzalez, L., Jr.,
D. N. Woolfson, and T. Alber.
1996.
Buried polar residues and structural specificity in the GCN4 leucine zipper.
Nat. Struct. Biol.
3:1011-1018[CrossRef][Medline].
|
| 27.
|
Graham, F. L., and L. Prevec.
1992.
Adenovirus-based expression vectors and recombinant vaccines.
Biotechnology
20:363-390[Medline].
|
| 28.
|
Graham, F. L.,
J. Smiley,
W. C. Russell, and R. Nairn.
1977.
Characteristics of a human cell line transformed by DNA from human adenovirus type 5.
J. Gen. Virol.
36:59-74[Abstract/Free Full Text].
|
| 29.
|
Green, S., and P. Chambon.
1988.
Nuclear receptors enhance our understanding of transcription regulation.
Trends Genet.
4:309-314[CrossRef][Medline].
|
| 30.
|
Harbury, P. B.,
P. S. Kim, and T. Alber.
1994.
Crystal structure of an isoleucine-zipper trimer.
Nature
371:80-83[CrossRef][Medline].
|
| 31.
|
Harbury, P. B.,
T. Zhang,
P. S. Kim, and T. Alber.
1993.
A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants.
Science
262:1401-1407[Abstract/Free Full Text].
|
| 32.
|
Hayashi, F.,
R. Ishima,
D. Liu,
K. I. Tong,
S. Kim,
D. Reinberg,
S. Bagby, and M. Ikura.
1998.
Human general transcription factor TFIIB: conformational variability and interaction with VP16 activation domain.
Biochemistry
37:7941-51[CrossRef][Medline].
|
| 33.
|
Hodges, M.,
C. Tissot,
K. Howe,
D. Grimwade, and P. S. Freemont.
1998.
Structure, organization, and dynamics of promyelocytic leukemia protein nuclear bodies.
Am. J. Hum. Genet.
63:297-304[CrossRef][Medline].
|
| 34.
|
Horikoshi, N.,
K. Maguire,
A. Kralli,
E. Maldonado,
D. Reinberg, and R. Weinmann.
1991.
Direct interaction between adenovirus E1A protein and the TATA box binding transcription factor IID.
Proc. Natl. Acad. Sci. USA
88:5124-5128[Abstract/Free Full Text].
|
| 35.
|
Hu, J. C.,
N. E. Newell,
B. Tidor, and R. T. Sauer.
1993.
Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis.
Protein Sci.
2:1072-1084[Medline].
|
| 36.
|
Hurst, H. C.
1995.
Transcription factors. 1:bZIP proteins.
Protein Profile
2:101-168[Medline].
|
| 37.
|
Hurst, H. C.
1994.
Transcription factors. 1:bZIP
Protein Profile
1:123-168[Medline].
|
| 38.
|
Legrand, V.,
D. Spehner,
Y. Schlesinger,
N. Settelen,
A. Pavirani, and M. Mehtali.
1999.
Fiberless recombinant adenoviruses: virus maturation and infectivity in the absence of fiber.
J. Virol.
73:907-919[Abstract/Free Full Text].
|
| 39.
|
Lupas, A.
1996.
Coiled coils: new structures and new functions.
Trends Biochem. Sci.
21:375-382[CrossRef][Medline].
|
| 40.
|
Lupas, A.
1996.
Prediction and analysis of coiled-coil structures.
Methods Enzymol.
266:513-525[Medline].
|
| 41.
|
Lusky, M.,
L. Grave,
A. Dieterle,
D. Dreyer,
M. Christ,
C. Ziller,
P. Furstenberger,
J. Kintz,
D. A. Hadji,
A. Pavirani, and M. Mehtali.
1999.
Regulation of adenovirus-mediated transgene expression by the viral E4 gene products: requirement for E4 ORF3.
J. Virol.
73:8308-8319[Abstract/Free Full Text].
|
| 42.
|
Lutz, P., and C. Kedinger.
1996.
Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter.
J. Virol.
70:1396-1405[Abstract].
|
| 43.
|
Lutz, P.,
F. Puvion-Dutilleul,
Y. Lutz, and C. Kedinger.
1996.
Nucleoplasmic and nucleolar distribution of the adenovirus IVa2 gene product.
J. Virol.
70:3449-3460[Abstract].
|
| 44.
|
Lutz, P.,
M. Rosa-Calatrava, and C. Kedinger.
1997.
The product of the adenovirus intermediate gene IX is a transcriptional activator.
J. Virol.
71:5102-5109[Abstract].
|
| 45.
|
Maul, G. G.,
D. Negorev,
P. Bell, and A. M. Ishov.
2000.
Review: properties and assembly mechanisms of ND10, PML bodies, or PODs.
J. Struct. Biol.
129:278-287[CrossRef][Medline].
|
| 46.
|
Puvion-Dutilleul, F.,
J. P. Bachellerie,
N. Visa, and E. Puvion.
1994.
Rearrangements of intranuclear structures involved in RNA processing in response to adenovirus infection.
J. Cell Sci.
107:1457-1468[Abstract].
|
| 47.
|
Puvion-Dutilleul, F.,
M. K. Chelbi-Alix,
M. Koken,
F. Quignon,
E. Puvion, and H. de The.
1995.
Adenovirus infection induces rearrangements in the intranuclear distribution of the nuclear body-associated PML protein.
Exp. Cell Res.
218:9-16[CrossRef][Medline].
|
| 48.
|
Puvion-Dutilleul, F., and E. Puvion.
1990.
Replicating single-stranded adenovirus type 5 DNA molecules accumulate within well-delimited intranuclear areas of lytically infected HeLa cells.
Eur. J. Cell Biol.
52:379-388[Medline].
|
| 49.
|
Puvion-Dutilleul, F.,
R. Roussev, and E. Puvion.
1992.
Distribution of viral RNA molecules during the adenovirus type 5 infectious cycle in HeLa Cells.
J. Struct. Biol.
108:209-220[CrossRef][Medline].
|
| 50.
|
Qadri, I.,
H. F. Maguire, and A. Siddiqui.
1995.
Hepatitis B virus transactivator protein X interacts with the TATA-binding protein.
Proc. Natl. Acad. Sci. USA.
92:1003-1007[Abstract/Free Full Text].
|
| 51.
|
Russell, W. C.
2000.
Update on adenovirus and its vectors.
J. Gen. Virol.
81:2573-2604[Free Full Text].
|
| 52.
|
Seeler, J. S., and A. Dejean.
1999.
The PML nuclear bodies: actors or extras?
Curr. Opin. Genet. Dev.
9:362-367[CrossRef][Medline].
|
| 53.
|
Shu, W.,
H. Ji, and M. Lu.
1999.
Trimerization specificity in HIV-1 gp41: analysis with a GCN4 leucine zipper model.
Biochemistry
38:5378-5385[CrossRef][Medline].
|
| 54.
|
Smith, B. T.
1977.
Cell line A549: a model system for the study of alveolar type II cell function.
Am. Rev. Respir. Dis.
115:285-293[Medline].
|
| 55.
|
Sternsdorf, T.,
T. Grotzinger,
K. Jensen, and H. Will.
1997.
Nuclear dots: actors on many stages.
Immunobiology
198:307-331[Medline].
|
| 56.
|
Stewart, P. L.,
R. M. Burnett,
M. Cyrklaff, and S. D. Fuller.
1991.
Image reconstruction reveals the complex molecular organization of adenovirus.
Cell
67:145-154[CrossRef][Medline].
|
| 57.
|
Trapnell, B. C., and M. Gorziglia.
1994.
Gene therapy using adenoviral vectors.
Curr. Opin. Biotechnol.
5:617-625[CrossRef][Medline].
|
| 58.
|
Thompson, J. D.,
T. J. Gibson,
F. Plewniak,
F. Jeanmougin, and D. G. Higgins.
1997.
The ClustalX windows interface: flexible strategies for multiple sequence alignment aided byquality analysis tools.
Nucleic Acids Res.
24:4876-4882.
|
| 59.
|
van Oostrum, J., and R. M. Burnett.
1985.
Molecular composition of the adenovirus type 2 virion.
J. Virol.
56:439-448[Abstract/Free Full Text].
|
| 60.
|
van Oostrum, J.,
P. R. Smith,
M. Mohraz, and R. M. Burnett.
1987.
The structure of the adenovirus capsid. III. Hexon packing determined from electron micrographs of capsid fragments.
J. Mol. Biol.
198:73-89[CrossRef][Medline].
|
| 61.
|
Xiao, J. H.,
I. Davidson,
H. Matthes,
J. M. Garnier, and P. Chambon.
1991.
Cloning, expression, and transcriptional properties of the human enhancer factor TEF-1.
Cell
65:551-568[CrossRef][Medline].
|
| 62.
|
Zhang, W., and M. J. Imperiale.
2000.
Interaction of the adenovirus IVa2 protein with viral packaging sequences.
J. Virol.
74:2687-2693[Abstract/Free Full Text].
|
Journal of Virology, August 2001, p. 7131-7141, Vol. 75, No. 15
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7131-7141.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Fabry, C. M. S., Rosa-Calatrava, M., Moriscot, C., Ruigrok, R. W. H., Boulanger, P., Schoehn, G.
(2009). The C-Terminal Domains of Adenovirus Serotype 5 Protein IX Assemble into an Antiparallel Structure on the Facets of the Capsid. J. Virol.
83: 1135-1139
[Abstract]
[Full Text]
-
Funston, G. M., Kallioinen, S. E., de Felipe, P., Ryan, M. D., Iggo, R. D.
(2008). Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping. J. Gen. Virol.
89: 389-396
[Abstract]
[Full Text]
-
Saban, S. D., Silvestry, M., Nemerow, G. R., Stewart, P. L.
(2006). Visualization of {alpha}-Helices in a 6-Angstrom Resolution Cryoelectron Microscopy Structure of Adenovirus Allows Refinement of Capsid Protein Assignments. J. Virol.
80: 12049-12059
[Abstract]
[Full Text]
-
Marsh, M. P., Campos, S. K., Baker, M. L., Chen, C. Y., Chiu, W., Barry, M. A.
(2006). Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX. J. Virol.
80: 11881-11886
[Abstract]
[Full Text]
-
Havenga, M., Vogels, R., Zuijdgeest, D., Radosevic, K., Mueller, S., Sieuwerts, M., Weichold, F., Damen, I., Kaspers, J., Lemckert, A., van Meerendonk, M., van der Vlugt, R., Holterman, L., Hone, D., Skeiky, Y., Mintardjo, R., Gillissen, G., Barouch, D., Sadoff, J., Goudsmit, J.
(2006). Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells.. J. Gen. Virol.
87: 2135-2143
[Abstract]
[Full Text]
-
Krause, A., Joh, J. H., Hackett, N. R., Roelvink, P. W., Bruder, J. T., Wickham, T. J., Kovesdi, I., Crystal, R. G., Worgall, S.
(2006). Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.. J. Virol.
80: 5523-5530
[Abstract]
[Full Text]
-
Le, L. P., Le, H. N., Dmitriev, I. P., Davydova, J. G., Gavrikova, T., Yamamoto, S., Curiel, D. T., Yamamoto, M.
(2006). Dynamic Monitoring of Oncolytic Adenovirus In Vivo by Genetic Capsid Labeling. JNCI J Natl Cancer Inst
98: 203-214
[Abstract]
[Full Text]
-
Bauer, U., Flunker, G., Bruss, K., Kallwellis, K., Liebermann, H., Luettich, T., Motz, M., Seidel, W.
(2005). Detection of Antibodies against Adenovirus Protein IX, Fiber, and Hexon in Human Sera by Immunoblot Assay. J. Clin. Microbiol.
43: 4426-4433
[Abstract]
[Full Text]
-
Vellinga, J., Van der Heijdt, S., Hoeben, R. C.
(2005). The adenovirus capsid: major progress in minor proteins. J. Gen. Virol.
86: 1581-1588
[Abstract]
[Full Text]
-
Gilcrease, E. B., Winn-Stapley, D. A., Hewitt, F. C., Joss, L., Casjens, S. R.
(2005). Nucleotide Sequence of the Head Assembly Gene Cluster of Bacteriophage L and Decoration Protein Characterization. J. Bacteriol.
187: 2050-2057
[Abstract]
[Full Text]
-
Vellinga, J., van den Wollenberg, D. J. M., van der Heijdt, S., Rabelink, M. J. W. E., Hoeben, R. C.
(2005). The Coiled-Coil Domain of the Adenovirus Type 5 Protein IX Is Dispensable for Capsid Incorporation and Thermostability. J. Virol.
79: 3206-3210
[Abstract]
[Full Text]
-
Sargent, K. L., Meulenbroek, R. A., Parks, R. J.
(2004). Activation of Adenoviral Gene Expression by Protein IX Is Not Required for Efficient Virus Replication. J. Virol.
78: 5032-5037
[Abstract]
[Full Text]
-
Vellinga, J., Rabelink, M. J. W. E., Cramer, S. J., van den Wollenberg, D. J. M., Van der Meulen, H., Leppard, K. N., Fallaux, F. J., Hoeben, R. C.
(2004). Spacers Increase the Accessibility of Peptide Ligands Linked to the Carboxyl Terminus of Adenovirus Minor Capsid Protein IX. J. Virol.
78: 3470-3479
[Abstract]
[Full Text]
-
Bertolotti-Ciarlet, A., Crawford, S. E., Hutson, A. M., Estes, M. K.
(2003). The 3' End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein. J. Virol.
77: 11603-11615
[Abstract]
[Full Text]
-
Mei, Y.-F., Skog, J., Lindman, K., Wadell, G.
(2003). Comparative analysis of the genome organization of human adenovirus 11, a member of the human adenovirus species B, and the commonly used human adenovirus 5 vector, a member of species C. J. Gen. Virol.
84: 2061-2071
[Abstract]
[Full Text]
-
Dmitriev, I. P., Kashentseva, E. A., Curiel, D. T.
(2002). Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX. J. Virol.
76: 6893-6899
[Abstract]
[Full Text]
Top
Abstract
Introduction
