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Cell-binding domain of adenovirus serotype 2 fiber

American Society for Microbiology
Journal of Virology
Authors:
N Louis
N Louis
N Louis
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Pascal Fender at French National Centre for Scientific Research
Pascal Fender
A Barge
A Barge
A Barge
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Abstract and Figures

The adenovirus fiber appears as a long, thin projection terminated by a knob (head). The fiber consists of a trimeric protein whose head domain is thought to interact with cell receptors. The head part (amino acids 388 to 582) of adenovirus type 2 fiber was produced in a baculovirus expression system. The purified protein was shown to cross-link into trimers. It was very resistant to proteolytic attack and seemed to attain a high degree of compactness. The head domain efficiently inhibited attachment of adenovirus to receptors on the surface of HeLa cells, thereby confirming the hypothesis that the head domain interacts with viral receptors.
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JOURNAL
OF
VIROLOGY,
June
1994,
p.
4104-4106
Vol.
68,
No.
6
0022-538X/94/$04.00
+
)
Copyright
©)
1994,
American
Society
for
Microbiology
Cell-Binding
Domain
of
Adenovirus
Serotype
2
Fiber
NATHALIE
LOUIS,'
PASCAL
FENDER,'
ANNIE
BARGE,2
PAUL
KITTS,3t
AND
JADWIGA
CHROBOCZEKI*
Institut
de
Biologie
Structurale,
38027
Grenoble,'
and
European
Molecular
Biology
Laboratory,
Grenoble
Outstation,
38042
Grenoble,2
France,
and
National
Environment
Research
Council,
Institute
of
Virology
and
Environmentdl
Microbiology,
Oxford,
United
Kingdom3
Received
29
December
1993/Accepted
23
March
1994
The
adenovirus
fiber
appears
as
a
long,
thin
projection
terminated
by
a
knob
(head).
The
fiber
consists
of
a
trimeric
protein
whose
head
domain
is
thought
to
interact
with
cell
receptors.
The
head
part
(amino
acids
388
to
582)
of
adenovirus
type
2
fiber
was
produced
in
a
baculovirus
expression
system.
The
purified
protein
was
shown
to
cross-link
into
trimers.
It
was
very
resistant
to
proteolytic
attack
and
seemed
to
attain
a
high
degree
of
compactness.
The
head
domain
efficiently
inhibited
attachment
of
adenovirus
to
receptors
on
the
surface
of
HeLa
cells,
thereby
confirming
the
hypothesis
that
the
head
domain
interacts
with
viral
receptors.
One
of
the
major
constituents
of
the
adenovirus
outer
capsid
is
an
elongated
structure,
the
fiber,
protruding
from
each
of
the
twelve
fivefold
vertices
of
the
icosahedral
virion
(for
a
review,
see
reference
10).
These
fibers
play
a
crucial
role
in
adenovirus
infection
by
attaching
the
virus
to
specific
receptors
on
the
cell
surface
(2,
13,
18).
The
adenovirus
type
2
(Ad2)
fiber
appears
as
a
long,
thin
projection
terminated
by
a
knob
(head)
(15,
16)
and
is
a
trimer
of
three
identical
subunits
(17).
Ad2
fiber
polypeptide
(582
amino
acids)
can
be
divided
into
three
regions
(4):
a
short
amino-terminal
tail
region,
a
shaft
consisting
of
repeating
units
each
of
approximately
15
amino
acids
whose
relative
hydro-
phobicities
rather
than
strict
identities
tend
to
be
conserved,
and
a
carboxy-terminal
part
of
about
180
residues.
The
polarity
of
the
polypeptide
chain
in
the
fiber
structure
has
been
determined;
the
fiber
is
attached
to
the
virion
through
its
N
terminus
located
in
the
tail
region,
and
the
distal
head
contains
the
C
terminus
(3).
The
globular
head
of
the
fiber
is
thought
to
interact
with
the
cellular
adenovirus
receptor,
but
this
was
never
confirmed
by
experimental
data.
This
paper
describes
the
structural
and
functional
properties
of
the
re-
combinant
head
domain
of
Ad2
fiber.
Cloning,
expression,
and
purification.
Total
DNA
was
ex-
tracted
from
Ad2
virions
(12)
and
digested
with
TaqI.
Frag-
ment
TaqI-B
(nucleotides
29860
to
33055,
numbering
after
Roberts
et
al.
[14]),
which
includes
the
fiber
gene
(nucleotides
31054
to
32883),
was
cloned
into
the
unique
AccI
site
of
pUC19,
resulting
in
the
plasmid
pTaq.
A
fragment
coding
for
the
head
of
the
fiber
protein
(nucleotides
32215
to
32883)
was
obtained
by
PCR,
with
priming
oligonucleotides
containing
BamHI
restriction
sites
and
with
pTaq
as
a
template.
Ampli-
fied
DNA
was
digested
with
BamHI
and
cloned
into
the
BamHI-cleaved
and
dephosphorylated
vector
pAcCL29
(8).
The
recombinant
plasmid
containing
the
insert
in
the
correct
orientation
relative
to
the
polyhedrin
promoter
was
cotrans-
fected
with
linearized
baculovirus
DNA
in
the
presence
of
lipofectin
(6),
into
Sf9
cells.
Expression
of
the
head
protein
in
Sf9
cells
infected
with
recombinant
virus
was
demonstrated
by
Western
immunoblot
analysis
with
rabbit
serum
prepared
against
a
peptide
consisting
of
the
last
16
C-terminal
amino
acids
of
fiber
polypeptide.
Recombinant
baculovirus
isolates
*
Corresponding
author.
Mailing
address:
CNRS,
Institut
de
Biolo-
gie
Structurale,
41
Avenue
des
Martyrs,
38027
Grenoble,
France.
t
Present
address:
Clontech
Laboratories,
Palo
Alto,
Calif.
were
subjected
to
three
rounds
of
plaque
purification.
Three
days
after
infection
with
recombinant
baculovirus,
cells
were
collected,
incubated
in
10
mM
Tris
buffer
(pH
8)
containing
protease
inhibitors
for
20
min
at
4°C,
and
broken
by
homog-
enization.
The
supernatant
obtained
after
lysis
of
expressing
cells
was
applied
to
a
column
of
Q-Sepharose
Fast
Flow
equilibrated
with
20
mM
Tris
buffer
(pH
8)
containing
pro-
tease
inhibitors.
The
recombinant
protein
was
eluted
with
the
same
buffer
containing
0.05
M
NaCl.
The
head
protein
ob-
tained
had
a
purity
of
better
than
95%.
Biochemical
and
electron
microscopic
analysis.
The
C-
terminal
fragment
starts
at
Ala-388
and
ends
at
Glu-582.
Its
monomer
has
an
apparent
molecular
weight
of
about
21,000.
Upon
cross-linking
with
glutaraldehyde,
monomers,
dimers,
and
trimers
of
head
protein
could
be
observed
on
a
denaturing
Kd
80
49,5
_
_
32,5
18,5
1
05
0,1
OlmM]
FIG.
1.
Cross-linking
studies
of
the
recombinant
head
domain.
A
freshly
prepared
solution
of
glutaraldehyde
was
added
to
each
portion
(10
p.g)
of
protein
to
the
required
final
concentration.
After
30
min
at
room
temperature,
samples
were
boiled
for
5
min
in
Laemmli
(7)
sample
buffer,
subjected
to
(0.1%
sodium
dodecyl
sulfate-15%
poly-
acrylamide
gel
electrophoresis,
and
analyzed
by
Western
blot
with
a
serum
prepared
against
the
last
16
C-terminal
amino
acids
of
fiber
polypeptide.
4104
VOL.
68,
1994
1
23
45
6
A
60
_
U
FIG.
2.
Chymotrypsin
digestion
of
the
head
domain.
Portions
(5
50
--
,ug)
of
the
head
domain
were
treated
with
chymotrypsin
in
50
mM
Tris
buffer
(pH
8)
containing
5
mM
EDTA-10
mM
CaCl2
at
room
40
,
temperature
at
different
enzyme/protein
(e/p)
ratios.
Samples
were
/
,
analyzed
by
sodium
dodecyl
sulfate-15%
polyacrylamide
gel
electro-
X
30
A
phoresis
and
stained
with
Coomassie
brilliant
blue.
Lane
1,
untreated
J.
sample;
lane
2,
molecular
weight
markers;
lane
3,
30-min
treatment,
2
A
e/p
=
1:100;
lane
4,
30-min
treatment,
e/p
=
1:10;
lanes
5
and
6,
10-
4C
and
30-min
treatments,
respectively,
e/p
=
1:1.
The
molecular
weight
markers
were
soybean
trypsin
inhibitor,
Mr
=
21,500,
and
lysozyme,
Mr
10
=
14,400
(Bio-Rad).
0
20
40
60
80
100
gel
(Fig.
1).
The
amount
of
trimer
increased
with
increasing
Tume
(min)
concentration
of
the
cross-linking
agent.
B
Native
fiber
can
be
cleaved
with
chymotrypsin
after
Tyr-17
(in
the
tail
domain)
and
then
at
Met-448
(in
the
head
domain),
at
500
rather
high
enzyme/protein
ratios
(3).
The
head
protein
expressed
in
baculovirus
could
be
partially
cleaved
with
chymotrypsin,
giving
v
A
rise
to
two
fragments
with
approximate
Mrs
of
15,000
and
6,000,
400
but
only
when
the
ratio
of
enzyme
to
protein
was
1:1
(Fig.
2,
lanes
5
and
6).
These
results
suggest
that,
as
for
native
protein,
the
head
300
domain
can
be
cleaved
at
Met-448.
E
Native
Ad2
fiber
contains
0-linked
GlcNac
(1,
5,
9),
and
it
seems
that
the
site
of
glycosylation
is
in
the
shaft
(11).
200
Accordingly,
mass
spectroscopy
performed
on
the
sample
of
head
protein
expressed
in
the
baculovirus
system
showed
that
100
this
protein
does
not
contain
any
carbohydrate
residue.
-
X
*
In
the
electron
microscopic
images
of
the
purified
head
0
0
20
40
60
80
100
-
r--.-Z
~
Time
(min)
A
virus
a
head
protein
+
virus
FIG.
4.
Interaction
of
the
head
domain
with
HeLa
cells.
(A)
Binding
of
head
domain
to
HeLa
cells.
Cells
were
washed
in
phos-
phate-buffered
saline
(PBS)
and
resuspended
in
PBS
containing
3%
bovine
serum
albumin
at
a
concentration
of
5
x
107
cells/ml.
[35S]me-
1X
.4
.
.......
thionine-labeled
recombinant
protein
(1
,ug;
specific
activity,
8,500
-l
FX;
/-.>
cpm/,ug)
was
incubated
at
37
and
4°C
with
1
ml
of
cells.
At
the
times
indicated,
100-iLl
aliquots
were
withdrawn,
the
cells
were
collected
by
centrifugation
and
washed
twice
in
PBS,
and
the
radioactivity
was
1'
_
.>
+*~s¢
measured
in
a
scintillation
counter.
Nonspecific
binding
was
measured
-_
Q_-
j
i
%
}by
addition
of
a
100-fold
excess
of
unlabeled
protein.
(B)
Inhibition
of
-,
t
Ad2
binding
by
the
recombinant
head
protein.
Two
PBS-washed
cell
samples
(1
ml
each)
were
prepared.
To
one
sample,
1.2
,ug
of
recombinant
protein
was
added.
After
15
min
of
incubation
at
37°C,
"4C-labeled
Ad2
was
added
(2.5
x
1012
virions;
specific
activity,
1
cpm/6.25
x
i07
virions)
to
both
samples,
and
the
incubation
at
37°C
was
continued.
Samples
of
100
,ul
were
withdrawn
at
different
times
and
diluted
in
900
[il
of
PBS,
the
cells
were
recovered
by
centrifuga-
.-
i
11
1
e
^tion,
and
their
radioactivity
was
determined
in
the
scintillation
counter.
protein
expressed
in
the
baculovirus
system,
we
observed
FIG.
3.
Electron
microscopy
of
the
Ad2
fiber
head
expressed
in
the
p
baculovirus
system.
The
protein
was
negatively
stained
with
1%
globular
particles
that
often
appeared
triangular.
Some
of
sodium
silicotungstate.
Low-dose
electron
microscopy
and
measure-
them
are
shown
in
Fig.
3
by
arrowheads.
The
mean
(±
ments
were
as
described
elsewhere
(15).
Arrowheads
in
the
main
standard
distribution)
dimension
of
the
side
of
these
triangles
image
indicate
triangular
shapes.
The
particles
in
the
left
panel
show
is
58
+
4
A
(5.8
±
0.4
nm)
(n
=
105),
which
is
similar
to
the
triangles
made
up
of
three
subunits.
The
bar
represents
20
nm.
side
view
of
the
heads
of
the
native
Ad2
fibers,
56
+
4
A
(5.6
NOTES
4105
4106
NOTES
±
0.4
nm)
(15).
Sometimes,
the
triangles
were
resolved
into
three
subunits,
which
clearly
indicates
the
trimeric
nature
of
this
recombinant
head
domain.
Some
of
them
are
presented
in
the
left
panel
of
Fig.
3.
In
conclusion,
proteolytic
analysis,
cross-linking,
and
elec-
tron
microscopy
of
the
C-terminal
fragment
expressed
in
the
baculovirus
system
show
that
it
is
a
compact,
trimeric
protein
with
morphology
similar
to
that
of
the
head
domain
in
the
native
Ad2
fiber.
Functional
analysis
of
baculovirus-expressed
head
domain.
Radioactive
recombinant
protein
was
allowed
to
interact
with
HeLa
cells
alone
or
in
the
presence
of
excess
nonlabeled
protein.
The
results
obtained
after
subtraction
of
nonspecifi-
cally
bound
proteins
(approximately
35%)
showed
that
each
HeLa
cell
fixed
about
I05
molecules
of
head
domain
at
37°C
(Fig.
4A).
This
result
agrees
well
with
the
amount
of
native
Ad2
fiber
which
can
be
attached
to
cells
(13).
In
order
to
determine
if
the
recombinant
protein
recognizes
Ad2
cellular
receptors,
we
used
a
competition
binding
assay
between
Ad2
and
the
recombinant
head
domain.
HeLa
cells
could
bind
about
4,500
virus
particles
in
the
absence
of
the
competitor.
When
the
cells
were
incubated
with
the
recombinant
protein
15
min
prior
to
the
addition
of
virus,
binding
of
virus
was
inhibited
by
over
70%
(Fig.
4B).
This
value
is
close
to
the
90%
inhibition
of
Ad2
attachment
observed
for
native
fiber
protein
(13,
18).
In
conclusion,
the
recombinant
head
domain
produced
in
the
baculovirus
system
is
trimeric,
structurally
similar
to
the
appro-
priate
part
of
native
protein,
and
is
competent
in
binding
to
adenovirus
receptors.
When
the
fiber
polarity
was
resolved,
revealing
that
the
N-terminal
part
is
embedded
in
the
penton
base
in
the
virion
structure
(3),
it
was
hypothesized
that
the
fiber
head
interacts
with
the
cell
during
infection.
This
work
supplies
an
experimental
confirmation
that
this
is
indeed
so.
The
biochemical,
morphological,
and
functional
analysis
of
the
Ad2
fiber
head
domain
allows
us
to
define
it
as
a
cell-binding
domain.
The
technical
assistance
of
Evelyne
Gout
is
warmly
acknowledged.
We
are
indebted
to
Anne-Marie
Di
Guilmi
for
help
in
the
initial
stage
of
this
work,
to
Eric
Forest
for
the
mass
spectrography
analysis,
and
to
M.-L.
Caillet-Boudin
for
a
gift
of
anti-C-terminus
serum.
We
are
grateful
to
B.
Jacrot
for
discussions.
The
financial
support
of
INSERM
(grant
910106)
is
acknowledged.
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... Some FAdV types (FAdV-1, 4, 10) have two fibres per penton base, while the rest possess only one (KAJAN et al., 2019). The knob and distal head domains of the fibre harbour the receptor-binding sites and thus play a critical role in virus attachment to the host cell (LOUIS et al., 1994;HENRY et al., 1994). ...
Comparative immune efficacy of recombinant penton base and fibre proteins against fowl adenovirus - 2/11 infection in chickens
Article
  • Mar 2024
Fowl adenovirus (FAdV) has 12 serotypes. However, recently hybrid types have also been reported. FAdV2/11 has emerged as the most frequently isolated FAdV type from Inclusion Body Hepatitis (IBH) and Hepatitis– Hydropericardium Syndrome (HHS) cases in chickens, causing severe economic impact worldwide. In an attempt to develop a subunit vaccine against FAdV-2/11 infection, viral capsid proteins, penton base and fibre, were expressed in E. coli and their immune potential was evaluated in domestic chicken. Purified recombinant proteins were administered on day 14 to specific pathogen-free (SPF) chickens followed by a challenge with the virulent virus on day 35 of life. The fibre induced the best immune response against the homologous challenge, with 80% protection. A moderate protective effect, resulting in 66% protection, was observed for penton base + fibre followed by the penton base vaccinated group manifesting 56% protection. Faecal excretion of the virus in immunized birds following the challenge was also studied. The study concludes that recombinant fibre protein may be a potential candidate for a subunit vaccine against FAdV-2/11 infection in chickens.
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... The Hexon gene is used for detection by PCR extraction. Penton is another structural protein that consists of an icosahedral capsid and is responsible for attaching to the host cell, called a knob (28), as well as virulence (29). Virus morphology was detected by electron microscopy from liver extract (30). ...
A Comprehensive Review on Angara Disease in Poultry
Article
Full-text available
  • Sep 2023
Hepatitis-hydropericardium syndrome (HHS) or Angara disease is an immunosuppression and contagious disease that often occurs in broiler poultry farms with a high mortality rate and sudden death. Furthermore, weight loss and sudden death have a significant and apparent economic impact on poultry farms, especially in fast-growing countries. Fowl adenovirus (FAdV) is a causative agent of HHS found in various animals, such as chicken. It causes rapid death and outbreak in young broiler farms, with an accumulation of jelly-like and straw-colored liquid in the heart pericardium, swollen and pale liver, heart muscles petechial and ecchymosis hemorrhages, swollen and pale kidneys, and atrophy thymus seen in gross pathology. Age, host, farm management, biosecurity plan, co-infectious, and level of immunity are risk factors for HHS. Angara is mainly found in Asian countries in Central and South America. The disease is transmitted vertically, like egg-transmitted, and horizontally by oral-fecal route. Rapid diagnostics and active monitoring systems in poultry farms can prevent and control Angara disease in farms. Vaccination and farm-specific biosecurity strategies reduce the initiation of outbreaks in farms. Treatment is based on activating humoral immunity against the virus, similar to vaccination.
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... Oncolytic adenoviruses (OAds) are well-studied due to their powerful capacity in oncolysis and immune response stimulation, and oncorine was the first approved OAd in China (22). OAd infects tumor cells through the interaction of adenoviral fiber knob with receptors on the cell surface (23,24). HAdV-5 was the most com monly used serotype for the design of conventional OAds and binds primarily to the coxsackievirus and adenovirus receptor (CAR) (25,26). ...
Toward the understanding of DSG2 and CD46 interaction with HAdV-11 fiber, a super-complex analysis
Article
Full-text available
Among human species B adenovirus, HAdV-11, has the particular feature of exploiting two primary receptors, desmoglein 2 (DSG2) and CD46, to mediate cell attachment. This serotype represents a particularly promising class of oncolytic viral vaccines. For instance, Enadenotucirev, a species B HAdV-11/HAdV-3 chimeric adenoviral vector, demonstrated preclinical tumor-selective cytotoxicity. Understanding the mechanism of HAdV-11 entry is a major challenge for the development of powerful vectors. Interactions between HAdV-11 and CD46 are well-characterized and clearly depend on the fiber protein, but the mechanism whereby HAdV-11 engages DSG2 has been still unresolved. In this study, we described a new cryo-EM structure of the HAdV-11 fiber in complex with DSG2. Kinetic interaction studies demonstrated that the binding stability of the HAdV-11 in complex with DSG2 was significantly lower than with CD46. Furthermore, competition assays with pseudotyped viruses suggested that the interaction of the HAdV-11 fiber with DSG2 was not required for infection but that DSG2 could serve as a receptor in the absence of CD46. Unexpectedly, we succeeded to isolate a HAdV-11 fiber/DSG2/CD46 “super complex” showing that these two receptors could simultaneously bind to the same trimeric HAdV-11K. Our work deciphers the use of two independent receptors by HAdV-11 from a biochemical and structural point of view, which can have an impact for future vectors design. IMPORTANCE The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there’s still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.
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... A concentration of polymorphisms was noted in the region of the short fiber which maps to the C-distal knob. Considering that the knob is the responsible domain for binding (29), and that particularly the FAdV-1 short fiber is critical for host-cell specific attachment (30), it may be hypothesized that this specific region of the short fiber is under increased selective pressure. ...
Reduced Performance Due to Adenoviral Gizzard Erosion in 16-Day-Old Commercial Broiler Chickens in Iran, Confirmed Experimentally
Article
Full-text available
  • Feb 2021
Adenoviral gizzard erosion (AGE) in broilers is an emerging infectious disease with negative impact on flock productivity. Despite of known primary etiological role of fowl adenovirus serotype 1 (FAdV-1) in AGE, there are a limited number of field reports worldwide, possibly because the disease is less noticeable and clinically difficult to assess. The present study documents an outbreak of AGE in 16-day-old broiler chickens on a farm in the north of Iran and the reproduction of the disease in an experimental setting. In the field, a sudden onset of mortality was noticed in affected broilers resulting in 6% total mortality and decreased weight gain leading to approximately 1-week delay to reach the target slaughter weight. Necropsy findings in dead broilers revealed black colored content in crop, proventriculus and gizzard together with severe gizzard erosions characterized by multiple black-brown areas of variable size in the koilin layer and mucosal inflammation. Microscopic examination revealed necrotizing ventriculitis marked with severe dissociation of koilin layer and degeneration of glandular epithelium with infiltration of mononuclear inflammatory cells. FAdV-1 was isolated from affected gizzards. Phylogenetic analysis of the hexon loop-1 (L1) sequence of the isolated virus showed 100% identity with pathogenic FAdV-1 strains previously reported from broiler chickens with AGE. Subsequently, an in vivo study infecting day-old commercial layer chickens with the field isolate demonstrated characteristic lesions and histopathological changes of AGE together with decreased weight gain in the infected birds. For the first time, the progress of a natural outbreak of AGE in Iran is described and experimental reproduction of the disease is demonstrated. The findings highlight the economic impact of the disease for regional poultry production due to mortality and impaired weight gain of the affected broilers.
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... For example, the shaft measures 9 nm for HAdV3 and 36 nm for l'HAdV40. The knob corresponds to the globular C-terminal of the fiber and interacts with receptors, allowing virus attachment to the cells [15]. Therefore, the fiber's knob dictates the viral tropism. ...
The Adenovirus Dodecahedron: Beyond the Platonic Story
Article
Full-text available
  • Jul 2020
Many geometric forms are found in nature, some of them adhering to mathematical laws or amazing aesthetic rules. One of the best-known examples in microbiology is the icosahedral shape of certain viruses with 20 triangular facets and 12 edges. What is less known, however, is that a complementary object displaying 12 faces and 20 edges called a ‘dodecahedron’ can be produced in huge amounts during certain adenovirus replication cycles. The decahedron was first described more than 50 years ago in the human adenovirus (HAdV3) viral cycle. Later on, the expression of this recombinant scaffold, combined with improvements in cryo-electron microscopy, made it possible to decipher the structural determinants underlying their architecture. Recently, this particle, which mimics viral entry, was used to fish the long elusive adenovirus receptor, desmoglein-2, which serves as a cellular docking for some adenovirus serotypes. This breakthrough enabled the understanding of the physiological role played by the dodecahedral particles, showing that icosahedral and dodecahedral particles live more than a simple platonic story. All these points are developed in this review, and the potential use of the dodecahedron in therapeutic development is discussed.
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... The hexon proteins have responsibilities to elicit the humoral responses in virus neutralizing and serotyping. The fiber proteins have knob head domains containing the receptor-binding sites which help the virus attach to the host cell membranes (Henry et al., 1994;Louis et al., 1994). Initially, liver homogenates collected from broiler chickens infected with FAdV-4 were used widely in some countries as inactivated vaccine materials for prevention of HHS (Admad et al., 1990). ...
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Targeted Molecular Therapy for Ovarian Cancer Patients
Chapter
  • Oct 2022
Over 230,000 women are diagnosed with ovarian cancer each year worldwide, and this disease is responsible for an estimated 140,000 deaths per year worldwide. In the USA, in 2022 about 19,800 new cases of ovarian cancer will be diagnosed and 12,810 women will die of the disease. There are only a limited number of chemotherapeutic agents with reasonable effectiveness against epithelial ovarian cancer. Most patients develop recurrent disease, which commonly acquires chemoresistance. The standard therapy involves a combination of optimal cytoreductive surgery with chemotherapy that consists of a platinum agent and a taxane compound. The response rate of the standard regimen for ovarian cancer exceeds 80%, but about 70% of patients with advanced ovarian cancer experience recurrence within 5 years, and they die because their cancer disease becomes resistant to platinum and taxane. Most of these patients are subsequently treated with other agents, such as liposomal doxorubicin, gemcitabine, topotecan, or etoposide. The overall response rates to these other drugs are 10–25%. The identification of the genes and their protein products in ovarian cancer that contribute to the malignant phenotype has increased our knowledge of human tumorigenesis. There are several cell surface receptors, signaling pathways, and nuclear proteins, which are possible targets for molecular therapeutic approach. Targeting specific cellular mechanisms associated with ovarian tumorigenesis and progression may serve to improve oncologic outcome and limit toxicity in patients with ovarian cancer. Targeted molecular therapy could improve oncologic outcome for patients with advanced and recurrent ovarian cancer. The ideal molecular target ought to be differentially expressed by the tumor, have a potential chemosensitive molecular site, and be necessary for the viability of the cancer cell. In this chapter, antiangiogenic therapy, classical transgene delivery therapy, DNA vector therapy, immunotherapy, epithelial growth factor receptor (EGFR) therapy, and poly (adenosine diphosphate ribose) polymerase (PARP) inhibition therapy are discussed.
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Adenovirus receptors on antigen‐presenting cells of the skin
Article
  • Jul 2022
Skin, the largest human organ, is part of the first line of physical and immunological defense against many pathogens. Understanding how skin antigen‐presenting cells (APCs) respond to viruses or virus‐based vaccines is crucial to develop antiviral pharmaceutics, and efficient and safe vaccines. Here, we discuss the way resident and recruited skin APCs engage adenoviruses and the impact on innate immune responses. The skin is part of the first line of physical and immunological defense against many pathogens. Understanding how antigen‐presenting cells present in the skin respond to viruses or virus‐based vaccines is critical to the development of effective and safe antiviral pharmaceuticals and vaccines. Here we discuss how resident and recruited skin antigen‐presenting cells engage directly, or indirectly, human adenoviruses, and the impact on innate immune responses.
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Mystery Eye: Human Adenovirus and the Enigma of Epidemic Keratoconjunctivitis
Article
  • Dec 2019
  • PROG RETIN EYE RES
Known to occur in widespread outbreaks, epidemic keratoconjunctivitis (EKC) is a severe ocular surface infection with a strong historical association with human adenovirus (HAdV). While the conjunctival manifestations can vary from mild follicular conjunctivitis to hyper-acute, exudative conjunctivitis with formation of conjunctival membranes, EKC is distinct as the only form of adenovirus conjunctivitis in which the cornea is also involved, likely due to specific corneal epithelial tropism of its causative viral agents. The initial development of a punctate or geographic epithelial keratitis may herald the later formation of stromal keratitis, and manifest as subepithelial infiltrates which often persist or recur for months to years after the acute infection has resolved. The chronic keratitis in EKC is associated with foreign body sensation, photophobia, glare, and reduced vision. However, over a century since the first clinical descriptions of EKC, and over 60 years since the first causative agent, human adenovirus type 8, was identified, our understanding of this disorder remains limited. This is underscored by a current lack of effective diagnostic tools and treatments. In part, stasis in our knowledge base has been encouraged by the continued acceptance, and indeed propagation of, inaccurate paradigms pertaining to disease etiology and pathogenesis, particularly with regard to mechanisms of innate and adaptive immunity within the cornea. Owing to its often persistent and medically refractory visual sequelae, reconsideration of key aspects of EKC disease biology is warranted to identify new treatment targets to curb its worldwide socioeconomic burden.
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Relative accessibility of N-acetylglucosamine in trimers of the adenovirus types 2 and 5 fiber proteins
Article
Full-text available
  • Dec 1990
Fiber is an adenovirus capsid protein responsible for virus attachment to the cell surface and contains O-linked N-acetylglucosamine (GlcNAc). Results of both amino acid analysis and Dionex chromatography indicated that 3 to 4 and 1.7 to 2.5 mol of GlcNAc are attached per mol of affinity-purified adenovirus type 2 (Ad2) and Ad5 fibers, respectively. Fiber shares an epitope with nuclear pore proteins containing O-linked GlcNAc, as shown by reactivity to monoclonal antibody RL2 directed against these pore proteins. GlcNAc on fiber was found to serve as an acceptor for the transfer of galactose from UDP-galactose by 4 beta-galactosyl-transferase in Ad2 and Ad5 but not in Ad7; quantitation by labeling with UDP-[U-14C]galactose in this reaction gave a 100-fold-lower estimate of the GlcNAc content of fiber, suggesting that these monosaccharides are buried within fiber trimers and are not accessible to the transferase. Affinity chromatography on lectin-bound Sepharose beads showed that Ad2 and Ad5 fibers bound weakly to wheat germ agglutinin and did not bind to ricin or concanavalin A; weak binding to wheat germ agglutinin suggests either that GlcNAc is not easily accessible or that there are not sufficient GlcNAcs for efficient binding. These data suggest that O-linked GlcNAc might be important for Ad2 and Ad5 fiber assembly or stabilization.
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Linearization of Baculovirus DNA Enhances the Recovery of Recombinant Virus Expression Vectors
Article
Full-text available
  • Nov 1990
Engineered derivatives of Autographs californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera fruglperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently In use can thus be facilitated by linearization of the viral DNA at the appropriate location.
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The Architecture of Adenoviruses
Chapter
  • Jan 1984
Viruses are “organized associations of macromolecules,” and this definition predetermines our way of dealing with the problems of virion architecture. This review, therefore, is not a pure descriptive morphology, but aims at defining and describing the basic structural elements and complexes that constitute the adenovirion, their localization in the virion, and their mutual interactions. Since several reviews on the structural proteins of adenoviruses or virus morphology have been published over the past five years, the papers quoted herein have been selected on the basis of direct applicability to the aspect under discussion; in many cases, the reader is directed to the appropriate review for other references.
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O‐Linked GlcNAc in serotype‐2 adenovirus fibre
Article
  • Sep 1989
  • Eur J Biochem
Serotype-2 adenovirus fibre is shown to possess an O-linked GlcNAc residue and to have affinity for wheat germ agglutinin. The cytoplasmic and nuclear fibres are both glycosylated. Glycosylation seems to take place in the cytoplasm since most of the [14C]GlcN-labelled fibre is found in this compartment, little label being associated with the microsomes. Glycosylation of the fibre was not affected by inhibitors of N- and O-glycosylation. A variation in fibre glycosylation is observed among adenovirus. Among the serotypes tested, only serotype-5 adenovirus (another subgroup C virus) also incorporated [14C]GlcN into its fibre, but did not possess affinity for wheat-germ agglutinin. The GlcNAc is located in the N-terminal two-thirds of the fibre and more probably in the N-terminal one-third. The free or penton-base-associated fibres are similarly glycosylated. These results suggest that glycosylation is not involved in viral adsorption and in assembly with the capsid penton base. Thus, glycosylation might be a characteristic feature of subgroup C viruses.
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Deletion analysis of functional domains in baculovirus-expressed adenovirus type 2 fiber
Article
  • Dec 1991
Various forms of Ad2 fiber were expressed in insect cells using recombinant baculoviruses and phenotypically characterized with respect to the following properties: trimerization, binding to penton base, nuclear targeting, and glycosylation. The morphology and dimensions of full-length fiber produced by invertebrate cells were indistinguishable from those observed in extracts from lytically infected mammalian cells. The domain required for trimer formation was mapped to the C-terminus, between amino acids 541 and 582. The N-terminal domain, between amino acids 1 and 16, negatively influenced the trimerization efficiency. Fiber gene products reduced to the shaft portion of the fiber capsomer formed significant amounts of stable dimers. Recognition with penton base only occurred with trimeric forms of fiber and was apparently not affected by deletion of the first 60 amino acids from the N-terminus. Fiber deleted of the Met1-Gly60 sequence was found to localize within the nucleus at levels similar to those of full-length fiber. All recombinant fibers, including tail-and-know-deleted forms, were found to be glycosylated using three separate assays, (i) in vivo labeling with [3H]glucosamine, (ii) binding to WGA, and (iii) reaction with monoclonal antibody RL2 directed against O-GlcNAc-containing glycopeptide. This implied that Ad2 fiber is a substrate for GlcNAc O-seryl transferase in insect cell cytoplasm and that at least one major glycosylation site is located in the shaft domain, between Met61 and Asn410.
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Human adenovirus-host cell interactions: Comparative study with members of subgroups B and C
Article
  • Sep 1990
Host cell interactions of human adenovirus serotypes belonging to subgroups B (adenovirus type 3 [Ad3] and Ad7) and C (Ad2 and Ad5) were comparatively analyzed at three levels: (i) binding of virus particles with host cell receptors; (ii) cointernalization of macromolecules with adenovirions; and (iii) adenovirus-induced cytoskeletal alterations. The association constants with human cell receptors were found to be similar for Ad2 and Ad3 (8 x 10(9) to 9 x 10(9) M-1), and the number of receptor sites per cell ranged from 5,000 (Ad2) to 7,000 (Ad3). Affinity blottings, competition experiments, and immunofluorescence stainings suggested that the receptor sites for adenovirus were distinct for members of subgroups B and C. Adenovirions increased the permeability of cells to macromolecules. We showed that this global effect could be divided into two distinct events: (i) cointernalization of macromolecules and virions into endocytotic vesicles, a phenomenon that occurred in a serotype-independent way, and (ii) release of macromolecules into the cytoplasm upon adenovirus-induced lysis of endosomal membranes. The latter process was found to be type specific and to require unaltered and infectious virus particles of serotype 2 or 5. Perinuclear condensation of the vimentin filament network was observed at early stages of infection with Ad2 or Ad5 but not with Ad3, Ad7, and noninfectious particles of Ad2 or Ad5, obtained by heat inactivation of wild-type virions or with the H2 ts1 mutant. This phenomenon appeared to be a cytological marker for cytoplasmic transit of infectious virions within adenovirus-infected cells. It could be experimentally dissociated from vimentin proteolysis, which was found to be serotype dependent, occurring only with members of subgroup C, regardless of the infectivity of the input virus.
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Structure of adenovirus fibre. II. Morphology of single fibres.
Article
  • Nov 1990
Adenovirus type 2 fibres in crystals appear to be significantly longer than found previously (accompanying paper). We therefore examined isolated fibre by electron microscopy and measured a length of 370 A, consistent with the length found in the crystals. The specific N-terminal structure of the fibre caused a heterogeneity in the length that may at least partially explain the values of 280 to 310 A published previously. Green et al. described a 15 amino acid repeat in the primary structure of the shaft of the fibre thought to be associated with the specific three-dimensional folding of the shaft. We compared the adenovirus type 2 (with 22 repeats) and type 3 (with 6 repeats) fibre lengths and derived a contribution of 13.2 A to the length of the shaft per 15 amino acid repeat. Specific morphological features of the fibre are discussed in relation to its amino acid sequence.
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Baculovirus expression vectors with single strand capability
Article
  • Apr 1989
Full textFull text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (102K), or click on a page image below to browse page by page. 2366
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O-linked GlcNAc in serotype-2 adenovirus fibre
Article
  • Oct 1989
Serotype-2 adenovirus fibre is shown to possess an O-linked GlcNAc residue and to have affinity for wheat germ agglutinin. The cytoplasmic and nuclear fibres are both glycosylated. Glycosylation seems to take place in the cytoplasm since most of the [14C]GlcN-labelled fibre is found in this compartment, little label being associated with the microsomes. Glycosylation of the fibre was not affected by inhibitors of N- and O-glycosylation. A variation in fibre glycosylation is observed among adenovirus. Among the serotypes tested, only serotype-5 adenovirus (another subgroup C virus) also incorporated [14C]GlcN into its fibre, but did not possess affinity for wheat-germ agglutinin. The GlcNAc is located in the N-terminal two-thirds of the fibre and more probably in the N-terminal one-third. The free or penton-base-associated fibres are similarly glycosylated. These results suggest that glycosylation is not involved in viral adsorption and in assembly with the capsid penton base. Thus, glycosylation might be a characteristic feature of subgroup C viruses.
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Crystallization, enzymatic cleavage, and the polarity of the adenovirus type 2 fiber
Article
  • Dec 1987
Crystals of the fiber protein of adenovirus type 2 have been grown. Analysis of these crystals (type I crystals) showed that they were composed of fiber polypeptide with a lower apparent molecular weight (60 kDa) than that of the soluble or virion-incorporated fiber (62 kDa). N-terminal sequencing revealed that the fiber polypeptide chain of 60 kDa was cleaved at tyrosine17 from the N-end. The C-terminus remained intact. Assays with protease inhibitors suggested that the spontaneous cleavage of the fiber occurring upon its crystallization was due to a cellular, calcium-dependent, chymotrypsin-like protease co-purifying with the fiber and activated during hydroxyapatite chromatography. Crystallization of fiber purified in the presence of chymostatin provided crystals of a different structure under the electron microscope (crystals of type II), composed of 62-kDa fiber polypeptide units. The 62-kDa fiber from the type II crystals, as well as the 62-kDa fiber isolated from infected cell extracts, were able to associate with the penton base in vitro to form a penton capsomer. The 60-kDa fiber has lost this capacity. The accessibility of the N- and C-termini of the fiber inside the penton structure was probed by anti-peptide sera after limited proteolysis. The results are consistent with a polarity of the fiber in which its N-terminus is oriented toward the penton base, the C-terminal domain corresponding to the distal knob.
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