Reduced Infectivity of Adenovirus Type 5 Particles and Degradation
of Entering Viral Genomes Associated with Incomplete Processing of
the Preterminal Protein
Sayuri E. Kato, Jasdave S. Chahal, and S. J. Flint
Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Princeton, New Jersey
mantling of virus particles prior to the transport of viral genomes
to and into the infected cell nucleus. The nonenveloped, icosahe-
rus receptor, Car, in the case of species C adenoviruses such as
serotype 5 (Ad5) (4, 70, 89). Interactions of RGD sequences pres-
ent in loops that project from the surface of each subunit of the
pentameric penton base with ?v integrins on the cell surface (14,
endocytosis (18, 50, 75, 80, 101; reviewed in reference 1). Subse-
quent escape from early endosomes into the cytoplasm is coordi-
nated with, and dependent on, initial uncoating reactions that
remove capsid proteins.
It is well established that uncoating occurs in several discrete
stages (80), the first being dissociation of fibers at the cell surface
(9, 30, 57, 62). Within the endosome, additional structural pro-
teins are released, including peripentonal hexons and minor cap-
sid proteins IIIa, VIII, and (importantly) protein VI (30, 79; re-
viewed in reference 80). The latter protein was implicated in
of partially uncoated Ad5 particles to disrupt membranes in vitro
(102). Antibodies or specific substitutions in protein VI that im-
pair membrane lysis activity in vitro reduce the transduction of
viral genomes into cells (56, 59, 60), indicating that this protein
mediates the lysis of endosomal membranes in infected cells. The
genome-containing, partially dismantled particles that enter the
cytosol, which retain the majority of the hexons (30) and some
protein VI (105), are transported on microtubules, with net
uccessful initiation of the human adenovirus infectious cycle
depends on a complex set of interactions among viral and
movement toward the microtubule organizing center (MTOC)
and nucleus (8, 49, 54, 88). Such transport requires the microtu-
49, 54, 88). Neutralizing monoclonal antibodies (MAbs) that rec-
ognize hexons have been reported to impair the intracellular
transport of partially disassembled particles and block their accu-
mulation at the MTOC (78), suggesting that a hexon-dynein in-
teraction is required for transport to the nucleus in infected cells.
this process: substitutions in a PDxY motif present in protein VI
that prevents the ubiquitinylation of this viral protein by Nedd4
the nucleus and the association of intracellular particles with mi-
crotubules but had no effect on endosomal escape (105).
pore complexes (29), but whether partially uncoated particles
must first traffic to the MTOC, where they have been observed to
accumulate (2, 16, 49), is not clear (reviewed in reference 38). At
nuclear pore complexes, the particles bind to the nucleoporin
Nup214, and histone H1 becomes associated with hexons (90).
Examination of the fate of proteins present in these partially dis-
assembled particles using conformation-specific anti-hexon anti-
bodies, anti-protein VII antibodies, or radioisotopically or fluo-
rescently labeled proteins has established that major core protein
Received 28 August 2012 Accepted 24 September 2012
Published ahead of print 3 October 2012
Address correspondence to S. J. Flint, email@example.com.
S.E.K. and J.S.C. contributed equally to this work.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.orgJournal of Virologyp. 13554–13565December 2012 Volume 86 Number 24
the mechanism by which viral genomes packaged by protein VII
particles to release capsid fragments and nucleoporins (85). This
action of kinesin also increases the permeability of the nuclear
of viral DNA-protein VII nucleoproteins into the nucleus via im-
portin family receptors (39).
thesized as larger precursors (preIIIa, etc.) from which viral par-
ture particles initially assembled also contain the precursor of the
the protein primer for initiation of DNA synthesis (36, 52). Pro-
cessing of precursor proteins is essential to form mature, infec-
coding sequence for the viral cysteine protease that prevents en-
capsidation of this viral enzyme (67, 97) results in the accumula-
tion at nonpermissive temperatures of noninfectious particles
containing uncleaved precursor proteins (reviewed in reference
mal kinetics, but in contrast to the wild type, they fail to escape
from these vesicles and are transported to late endosomes and
lysosomes (21, 29, 40). This intracellular fate can be attributed to
measured by the ability of the cointernalized protein synthesis
inhibitor ?-sarcin to penetrate the cytoplasm (102). Immature
Ad2tsl particles are also more stable at low pHs and increasing
temperatures than wild-type virions are (64, 102).
actions among structural proteins (64, 76). One unique feature of
the noninfectious particles is an additional “molecular stitch” be-
tween the groups-of-nine hexons (72) and the ring of peripen-
tonal hexons surrounding each vertex. It is thought that precur-
sor-specific segments of proteins IIIa and VIII contribute to this
structure and that its removal upon precursor cleavage would be
required to facilitate the release of vertex capsomers (reviewed in
reference 72). Additional protein was also observed in noninfec-
tious Ad2ts1 particles inside the cavities of each hexon, which
open on the inner surface side of the capsid, and has been attrib-
uted to preVI. As interaction with hexons blocks the membrane
lysis activity of proteins VI and preVI in vitro (76), this more
extensive hexon-preVI interaction seems likely to impair the re-
lease of protein VII from Ad2 ts1 particles and hence account for
their defect in endosomal escape. A third major difference is the
more ordered, compact core structure (64, 76), which may be, at
least in part, the result of more extensive interactions of preVII
than of VII with DNA within virus particles (13).
Although these structural studies have provided plausible ex-
and their lack of infectivity, the relative contributions of the indi-
vidual precursor-specific segments of the structural proteins, or
preTP, are not known. Indeed, apart from Ad2ts1, relatively few
include the protein VI substitutions that inhibit membrane lysis
activity described above (59, 60) and deletion of the protein V
coding sequence (92). In addition, particles that lack the fiber or
that carry fibers with substitutions in the Car binding surface of
the knob or shorter or longer shafts exhibit reduced Car-depen-
dent entry (41, 47, 55, 74, 75, 96). Here we report the serendipi-
tous discovery of a previously unrecognized low-infectivity phe-
notype, degradation of the great majority of viral genomes soon
after entry, and its association with a mutation in the preTP cod-
MATERIALS AND METHODS
Cells and viruses. 293 cells and human foreskin fibroblasts (HFFs) were
grown as monolayer cultures in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 5% and 10% fetal calf serum and bovine
growth serum, respectively. Primary human small airway epithelial cells
(SAECs) and bronchial/tracheal epithelial cells (NHBECs) were obtained
from BioWhittaker, Inc., and cultured using predefined medium and
growth conditions according to the manufacturer’s recommendations.
Wild-type Ad5 and the E1B 55-kDa-null mutant Hr6 (34), were propa-
were determined by assaying plaque formation on these same cells as
described previously (104).
Analysis of accumulation of viral DNA. Proliferating or quiescent
and harvested after increasing periods of infection. DNA was purified
from cells or isolated nuclei as described previously (24) or by using the
DNeasy tissue kit (Qiagen) according to the manufacturer’s protocol.
sequence detection system and a TaqMan probe (Applied Biosystems) of
an amplicon within the ML transcription units, 90 bp long (nucleotides
CTT CGC GGT TCC AGT ACT C-3=; ML Rev, 5=-CAG GCC GTC ACC
CAG TTC TAC-3=; ML probe, VIC-ATC GGA AAC CCG TCG GCC
TCC-TAMRA. Reaction mixtures contained TaqMan Universal PCR
master mix with AmpErase (Applied Biosystems), 2 ?l sample DNA (di-
luted as necessary), 300 nM each primer, and 200 nM TaqMan probe. In
human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter
as an internal control and Sybr green detection, as described previously
(11). Relative DNA concentrations were determined by the standard-
curve method, and all measurements were performed in triplicate.
Illumina sequencing of viral genomes. Ad5 and Hr6 particles were
purified from 293 infected-cell lysates containing ?5 ? 1010PFU by se-
quential centrifugation in discontinuous CsCl gradients and centrifuga-
tion to equilibrium in continuous CsCl gradients (27). The purified par-
pH 8.0, prior to the addition of 1 volume of 0.01 M Tris HCl, pH 8.0,
were extracted with (1:1) phenol-CHCl3and ethanol precipitated. The
isolated DNA was resuspended in 100 ?l 0.5 M Tris-HCl, pH 8.0, and
concentrations were determined from the absorbance at 260 nm mea-
DNA extracted from wild-type Ad5 and Hr6 was performed at Princeton
University’s Lewis-Sigler Institute Microarray Facility with an Illumina
Genome Analyzer II using SCS 2.3 software. One microgram of purified
sequences to distinguish wild-type Ad5 and Hr6 libraries, exactly accord-
ing to the manufacturer’s protocols. DNA from each library was se-
quenced over 51 cycles in a single flow cell.
Analysis of sequencing data. The Illumina output was analyzed by
using tools available at the Princeton Galaxy bioinformatics local work-
An Unusual Reduced-Infectivity Phenotype of Ad5
December 2012 Volume 86 Number 24jvi.asm.org 13555
file format, and 0.1% of the sequence reads acquired were selected arbi-
trarily and used to construct the full-length genome sequences of wild-
genome reference sequence AY339865 (86) by using Burrows-Wheeler
Aligner (51). Variations in the resulting BAM alignments were then de-
tected using FreeBayes (http://bioinformatics.bc.edu/marthlab/Free
Bayes). Alignments and polymorphisms were visualized using the Inte-
the final aligned wild-type sequence with ?30-fold sequence coverage,
except within ?150 bp of the genome ends, where sequence coverage
dropped to no lower than 16-fold. Thesequencesoftheseterminalregions
were confirmed by conventional sequencing and matched those of align-
ments. Once assembled, the wild-type Ad5 genome sequence was used to
align the Hr6 Illumina reads as described above. This alignment was again
Immunoblotting. HFFs, SAECs, or NHBECs at approximately 75 to
the periods of infection indicated and washed with phosphate-buffered
saline (PBS), and extracts were prepared as described previously (11).
Extracts were sonicated in 30-s bursts on ice until sample viscosity de-
at 4°C for 5 min. Proteins were detected by SDS-polyacrylamide gel elec-
against the E1A proteins and the E2 DNA-binding protein (DBP), M73
visualized with a horseradish peroxidase-labeled anti-?-actin MAb
To examine TPs covalently bound to viral DNA, virus particles were
purified from Ad5- and Hr6-infected cells as described above. Purified
particles were disrupted by incubation at 60°C for 10 min (64), and viral
DNA was digested with 1,250 U/ml Benzonase nuclease (Sigma) for 30
min at 37°C. TPs were detected by immunoblotting with anti-preTP
MAbs (98) kindly provided by R. Hay. Protein V, detected by immuno-
blotting with MAb F58#1 (53), served as an internal control.
Immunofluorescence. To examine viral replication centers, HFFs
grown on coverslips to approximately 90% confluence were mock in-
fected or infected with Ad5 or Hr6 for various periods and processed for
immunofluorescence as described previously (25). The viral E2 DBP was
with Cy5 (Jackson ImmunoResearch Laboratories Inc.). To visualize the
viral genome soon after entry, HFFs on coverslips were incubated, with
rocking, with Ad5, Hr6, or DMEM only (mock infection) at 4°C for 30
min. After removal of the inoculum, cells were washed twice with cold
PBS prior to the addition of DMEM containing 5% (vol/vol) bovine
growth serum prewarmed to 37°C. Cells were processed for immunoflu-
orescence as described previously (25), except that they were fixed and
purified rabbit anti-protein VII polyclonal antibody (42), which was
kindly provided by D. Engel, and Alexa fluor 488-conjugated goat anti-
rabbit IgG (Invitrogen). Nuclei were stained with 4=,6-diamidino-2-phe-
described previously (25). Late endosomes and microtubules were de-
tected by using a mouse anti-Rab7 MAb (Rab7-117; Sigma-Aldrich) with
a Cy5 anti-mouse IgG secondary antibody (Jackson Immuno Research
Laboratories Inc.) and a rat anti-?-tubulin antibody (Abcam) with an
Viral DNA accumulation is impaired in normal human epithe-
lial cells and fibroblasts in the absence of the E1B 55-kDa pro-
tein. In the Hr6 genome, deletion of bp 2347 alters the coding
sequence of the E1B 55-kDa protein but not those of the related
lower-molecular-mass proteins made from alternatively spliced
mRNAs (103). The consequent shift in the reading frame of the
E1B 55-kDa protein coding sequence introduces a termination
codon a short distance downstream of the deletion, but no trun-
cated E1B protein can be detected in Hr6-infected cells by using
various antibodies that recognize N-terminal epitopes (46, 103).
DNA synthesis was reported to occur normally in quiescent hu-
man SAECs infected with a second E1B-null dl1520 mutant,
ONYX-015 (63). It is well established that replication of E1B-null
mutants in established human cell lines is dependent on the host
differences in cell type might account for the reported differences
in mutant phenotypes. We therefore examined viral genome ac-
Hr6. We also included NHBECs in these experiments: as sub-
group C adenoviruses, such as Ad5, are associated with upper
either SAECs, which are derived from the lower respiratory tract,
or HFFs. Viral DNA concentrations were measured at various
times after infection by using real-time PCR amplification of a
sequence within the major late transcription unit, as described in
Materials and Methods.
parallel and cells were infected with each virus at 30 PFU/cell.
Nevertheless, we consistently observed that Hr6-infected cells
as illustrated in Table 1 for proliferating cells. Very similar results
were obtained when quiescent cells were infected (data not
after the adsorption of Ad5 and Hr6 to the three types of host cell
varied to a small degree. Such differences are presumably the re-
sult of variations in the efficiency of entry. Nevertheless, Hr6-
essary to form a plaque, that is, that these mutant virus particles
are less infectious than wild-type virions. To test this interpreta-
TABLE 1 Comparison of viral DNA concentrations entering Ad5- and
Hr6-infected cells and in virus particles
Entering infected cellsb
Hr6/Ad5 ratioHr6 Ad5
12.1 ? 0.09
7.96 ? 0.24
7.25 ? 1.16
0.28 ? 0.01
0.38 ? 0.01
0.20 ? 0.02
Present in 1,000 PFU
aViral DNA concentrations are in arbitrary units.
bViral DNA 2 h after infection of proliferating cells.
Kato et al.
jvi.asm.orgJournal of Virology
results of this analysis of the Ad5 and Hr6 preparations used for
the experiments summarized in the top part of Table 1 are shown
in the bottom part of Table 1 (experiment 1), and very similar
data demonstrate that an infectious unit of Hr6 contains signifi-
cantly more viral DNA than does an infectious unit of Ad5, in
other words, that most DNA-containing Hr6 particles are not in-
To permit the comparison of viral DNA accumulation in cells
than an order of magnitude, all DNA concentrations were ex-
pressed relative to the input value measured 2 h after infection.
The accumulation of viral DNA was some 10-fold less efficient in
Hr6-infected, proliferating HFFs than in cells infected with Ad5
(Fig. 1A), consistent with the results of our previous experiments
in which viral DNA concentrations were examined by hybridiza-
infection proceeds more rapidly in these epithelial cells than in
HFFs. This temporal difference may account for the higher viral
DNA concentrations attained by 30 h after Ad5 infection of the
two types of epithelial cells than in HFFs (Fig. 1B and C). Never-
theless, the Hr6 mutant also exhibited apparent defects in viral
DNA accumulation in proliferating SAECs or NHBECs (Fig. 1).
lication proteins. Adenoviral DNA synthesis requires the three
viral replication proteins encoded within the E2 transcription
stranded DBP (see references 5, 36, and 52). An obvious explana-
tion for the defects in viral DNA accumulation observed in Hr6-
infected cells is, therefore, that viral early gene expression and
synthesis of these replication proteins are impaired. To assess this
were compared in proliferating normal human cells infected with
Ad5 or Hr6. Total cell extracts were prepared 24 and 30 h after
infection, and the viral proteins examined by using immunoblot-
ting. No significant differences in the accumulation of the imme-
diate early E1A proteins (data not shown) or of the E2 DBP were
observed in Ad5- and Hr6-infected HFFs, NHBECs, or SAECs
(Fig. 2A). As the E2 transcription unit encodes the viral DNA
result implies that the reduced accumulation of Hr6 genomes is
not the result of failure to produce viral replication proteins.
We also examined the formation of viral replication centers
containing the E2 DBP by immunofluorescence. In adenovirus-
infected cell nuclei, the DBP forms two morphologically distinct
structures, small dot-like foci and larger globular or ring-like
structures (87, 95). The small foci appear early in infection, and
their formation is independent of viral DNA synthesis. In con-
trast, the ring-like structures that are associated with newly syn-
thesized viral DNA (61, 66, 95) do not appear when viral DNA
synthesis is blocked by drugs or mutations (87, 95). Both types of
DBP-containing structures were observed in proliferating HFFs
(data not shown), but the number of DBP-containing structures
was not substantially, or noticeably, higher in Hr6-infected cells
than in Ad5-infected cells (Fig. 2B). When infected cells were
quantified in terms of the presence of the different types of repli-
cation centers, fewer Hr6-infected than Ad5-infected HFFs and
NHBECs were found to contain the larger, ring-like structures
the reduced accumulation of viral DNA in the mutant-infected
cells (Fig. 1).
The majority of viral genomes are degraded in Hr6-infected
cells. Although Hr6-infected normal cells contained higher con-
centrations of input viral DNA than did cells infected with Ad5
(Table 1), neither the formation of a larger number of replication
was observed (Fig. 2). This apparent discrepancy suggested that
template for viral gene expression and DNA synthesis within in-
fected cell nuclei. To investigate this possibility, the concentra-
tions of intranuclear viral DNA were measured during both the
early and late phases of infection in HFFs, which are robust and
simple to culture. Proliferating HFFs were infected with Ad5 or
Hr6 at 30 PFU/cell, and DNA purified from isolated nuclei after
increasing periods of infection. Viral DNA concentrations were
tified in parallel to provide an internal control, as described in
Materials and Methods. In two independent experiments, Hr6-
Ad5 or Hr6, and DNA was isolated from cells harvested 2, 18, 24, and 30 h after infection. Viral DNA was quantified by real-time PCR as described in Materials
and Methods. Relative concentrations of viral DNA were calculated as the increase in concentration at each time point over the value measured at 2 h after
infection. Results represent the average of two independent experiments, and error bars represent the standard deviation.
An Unusual Reduced-Infectivity Phenotype of Ad5
December 2012 Volume 86 Number 24jvi.asm.org 13557
ciated viral DNA at 2 h postinfection (p.i.) than did Ad5-infected
cell nuclei, somewhat less than when DNA was purified from
whole cells (Table 1, top). The relative concentrations of viral
DNA were lower in Hr6-infected cell nuclei throughout the early
temporal analysis established that viral DNA concentrations de-
clined sharply in Hr6-infected cells between 2 and 12 h p.i., de-
reduced in concentration by only 40% during the same period
(Fig. 3B). As the ML amplicon used to detect viral DNA by quan-
titative PCR was only 90 bp in length, we conclude that the ma-
jority of Hr6 genomes, in contrast to wild-type genomes, are de-
graded very extensively within a few hours of entry into HFFs.
Such a fate accounts for the similar numbers of viral replication
centers formed in Ad5- and Hr6-infected cells (Fig. 2B).
cells, it seemed possible that the extensive loss of mutant viral
high concentration of entering viral DNA molecules. To address
this possibility, we compared the concentrations of viral DNA
cell Ad5 but with Hr6 under conditions designed on the basis of
measurements like those shown in Table 1 to yield an equal num-
ber of entering genomes. The results of a typical experiment, in
which the ratio of the concentrations of entering Hr6 and Ad5
DNA was 1.8, is shown in Fig. 3D. Despite the presence of similar
concentration from 6 to 18 h after infection, whereas only a small
decrease was observed in Ad5-infected cells (Fig. 3D).
As the minimal number of genomes competent to serve as the
templates for replication cannot be determined accurately from
infected with Ad5 or Hr6 or mock infected and harvested after the periods indicated. Total cell lysates were examined by immunoblotting as described in
Materials and Methods, with anti-E2 DBP MAb B6 and an anti-?-actin antibody. (B) Proliferating HFFs were infected with Ad5 or Hr6 or mock infected and
5-labeled anti-mouse IgG and is shown false colored in green. Nuclei were stained with DAPI (blue). Expanded, ring-like replication centers and dot-like
structures are indicated by the orange and white arrows, respectively. These two types of replication centers were quantified in Ad5- and Hr6-infected HFFs (C)
and NHBECs (D). Shortly after the onset of viral DNA synthesis, between 100 and 200 cells were analyzed for those in which the DBP was present only in small,
dot-like structures (Small foci) or present in both these foci and large, ring-like structures (Large rings).
from cells harvested at the times indicated, and viral DNA concentrations were determined by quantitative PCR as described in Materials and Methods. The
the average of two independent experiments. Error bars show standard deviations. (B) The 2- to 18-h data from the experiments shown in panel B are replotted
and error bars show the standard deviations.
Kato et al.
jvi.asm.orgJournal of Virology
data like those shown in Fig. 3, it was not possible to make an
appropriate comparison of increases in viral DNA concentration
in Ad5- and Hr6-infected cells. To circumvent this problem and
replication in normal human cells, we exploited a mutant virus
containing the Hr6 frameshift mutation (deletion of bp 2347 in
the Ad5 genome) in an E1-containing derivative (43) of AdEasy
(37). Analysis of this E1B 55-kDa-null mutant, AdEasyE1?2347
(43), has established that the timely synthesis of the E1B 55-kDa
proteins is required for efficient viral DNA synthesis in normal
human cells (11). However, greater numbers of entering viral ge-
nomes were not observed in cells infected with AdEasyE1?2347,
compared to its wild-type parent (11), indicating that deletion of
the poor infectivity of Hr6 virus particles. We therefore conclude
that the Hr6 genome must contain at least one additional muta-
tion responsible for this phenotype.
Hr6 and Ad5 DNAs were subjected to high-throughput sequenc-
ing. This host range mutant was isolated by virtue of its impaired
cells (28), following the exposure of Ad5 to nitrous acid (34). Hr6
and the Ad5 strain from which it was derived were obtained orig-
inally from J. Williams, Carnegie Mellon University, and have
been maintained by preparation of master stocks from which all
working stocks are amplified. Consequently, the preparations of
Ad5 and Hr6 used in these experiments were derived by only a
limited number of low-multiplicity passages from the stocks re-
Viral DNA was isolated from purified Ad5 and Hr6 particles
and used to prepare Illumina genomic libraries as described in
Materials and Methods. More than 370,000 reads from each virus
of our stock of Ad5 was mapped by alignment of reads to the
human adenovirus C serotype 5 complete genome reference se-
quence AY339865.1 (86). A total of 98.51% of the reads were suc-
cessfully mapped to this reference sequence, with the lowest cov-
erage at the most terminal ?150-bp regions at the ends of the
genome (Fig. 4). In these regions, coverage was no less than 16-
fold, and their sequences determined by deep sequencing were
confirmed by conventional sequencing. This analysis identified
two deletions that distinguished our wild type from the reference
Ad5 genome (Fig. 4), a deletion of one A-T base pair at position
14073, from a poly(A-T) stretch of 13 bp in the reference and a
deletion of one T-A base pair at position 34338 of the reference
coding sequences changes. The T-A deletion at bp 34338 was
found to be present in the Hr6 genome, described below. How-
ever, the A-T deletion at bp 14073 was not; at this locus, the Hr6
genome appears to be identical to the allele present in the refer-
ence sequence with accession no. AY339865.1 (15), containing a
poly(A-T) stretch of 13 bp. The Hr6 genome was mapped as de-
scribed above to the wild-type sequence depicted in Fig. 4, with
98.56% of the reads successfully aligned with our wild-type refer-
ence. Nine mutations unique to Hr6 were identified upon align-
ment with the wild type (Fig. 4; Table 2). Of these, four transition
sequences (Table 2). The deletion of bp 2347 and the G-to-T
transversion at bp 2947 correspond exactly to the mutations pre-
viously identified in the E1B 55-kDa protein coding sequence of
Hr6 by conventional sequencing (103). The remaining mutations
introduce amino acid substitutions into virion proteins. The C-
to-A transversion at bp 9655 results in replacement of Gly315 in
preTP with Val, while a transition at bp 32252 introduces Val in
place of Ala406 in fiber protein IV (Table 2).
The A406V substitution in protein IV lies close to the Car-
binding surface of the fiber knob, which has been identified by
mutational analysis and structural studies (6, 45, 70). However,
this residue is not conserved among the fibers of adenovirus sero-
types that bind to Car (70, 93). Indeed, residue 406 (or its equiv-
alent) is Asp in the fiber knob of the very closely related species C
serotype Ad2 and several species A and B serotypes (93). Consis-
Ad5 fiber knob carrying an A406K substitution was reported to
compete as efficiently as the wild type for binding to Car on Chi-
that impair the fiber knob-Car interaction decrease the efficiency
of genome transduction (41, 47), whereas Hr6-infected cells con-
after infection than do those infected with Ad5 (Table 1; see also
Fig. 6). As these observations argue strongly that the fiber muta-
tion cannot account for the poor infectivity of Hr6, subsequent
studies focused on the consequences of the preTP substitution.
Proteolytic processing of preTP is impaired by the G315V
substitution. The 671-amino-acid E2 preTP serves as the protein
FIG 4 Differences between the sequences of the Ad5 and Hr6 genomes. The horizontal line at the top represents the Ad5 genome in kilobase pairs. The fold
represent silent mutations detected in the Ad5 genome compared to the reference sequence or in Hr6 compared to the Ad5 sequence (see the text). The Hr6
mutations shown in blue and red indicate the previously described E1B 55-kDa coding sequence mutations (103) and newly discovered mutations in the preTP
and fiber genes that introduce substitutions, respectively.
An Unusual Reduced-Infectivity Phenotype of Ad5
December 2012 Volume 86 Number 24 jvi.asm.org 13559
primer for viral DNA synthesis when it becomes covalently at-
52). Subsequently, this precursor is processed by the viral L3 pro-
tease to the mature TP (77, 98). Initial cleavage by the protease at
two closely spaced sites (Fig. 5A) generates an ?62-kDa interme-
diate, termed intermediate TP (iTP) (77, 98). This reaction can
take place prior to the encapsidation of viral genomes during the
assembly of virus particles, in contrast to the production of ma-
ture TP (99), which comprises the C-terminal 322 residues of the
precursor (Fig. 5A). As the sequence of TP is not altered by the
this G315V substitution on preTP processing was investigated.
described in Materials and Methods, and equal concentrations of
for various forms of TP (98) (Fig. 5A) or for core protein V. We
tially or fully processed forms of preTP in Ad5 and Hr6 particles.
However, this antibody reacted strongly with a pair of proteins mi-
grating close to the 50-kDa molecular mass marker, as well as with
three more slowly migrating species (data not shown). As it was not
possible to identify the TP or its precursor unambiguously with this
antibody, we exploited precursor-specific MAb 53E (Fig. 5A) to in-
vestigate preTP processing. A significantly greater concentration of
the iTP processing intermediate was observed in Hr6 than in Ad5
particles (Fig. 5B). Quantification of the iTP signals shown, as de-
scribed Materials and Methods, using protein V as an internal con-
mutant virus particles. In a second experiment using Ad5 and Hr6
purified after the infection of cells with independent virus stocks, a
7.8-fold higher concentration of iTP was observed in Hr6 particles.
No corresponding differences in the concentration of unprocessed
preTP were observed upon longer exposure of MAb 5E3 immuno-
blots (Fig. 5B). These data indicate that the G315V substitution im-
pairs the final viral protease cleavage that liberates TP but not the
Higher concentrations of viral DNA at 2 h p.i. were observed in
Hr6-infected cells than in Ad5-infected cells when DNA was pu-
rified from unfractionated cells or from isolated nuclei. However,
as noted previously, this difference was less pronounced when
DNA was prepared from isolated nuclei. Furthermore, the mild
extraction of cells with nonionic detergent used to isolate nuclei
the cytoplasm (109) but does not remove cytoskeletal compo-
by immunofluorescence. To promote the synchronous entry of
4°C prior to removal of the inoculum and incubation at 37°C, as
described in Materials and Methods. Viral genomes were visual-
using polyclonal antibodies against viral core protein VII (42),
which remains associated with viral genomes that enter the nu-
cleus throughout the early phase of infection (12, 29, 35, 42, 107).
Discrete foci or dots of protein VII were readily detected 2 h after
infection with Ad5 in both the nucleus and the cytoplasm (Fig.
7 h p.i., the number of protein VII foci detected was somewhat
TABLE 2 Mutations identified in the Hr6 genome compared to the Ad5 genome
Site (bp) MutationGenomic location
E1B 55-kDa CDSa
E1B 55-kDa CDS
IIIa CDS termination codon
E3 gp19k CDS
E3 14.7k CDS
E4 Orf4 CDS
Frameshift and truncationb
Substitution at aac310, TGT (C)¡TTT (F)b
Substitution at aa 315, GGC (G)¡GTC (V)
Silent, TAC (Y)¡Tat (Y)
Silent, AGC (S)¡AGT (S)
Silent, AAG (K)¡AAA (K)
Substitution at aa 406, GCT (A)¡GTT (V)
Silent, AGG (R)¡AGA (R)
aCDS, coding sequence.
bAs previously reported (103).
caa, amino acid.
FIG 5 Comparison of TP precursors in Ad5 and Hr6 virus particles. (A) The
preTP is depicted to scale by the rectangle, with the precursor-specific and
mature TP sequences shown in white and gray, respectively. The vertical ar-
rows at the top indicate the sites at which preTP is cleaved by the viral L3
protease, and the arrow at the bottom shows the position of the G315V sub-
stitution in Hr6 preTP (77, 98). The epitopes recognized by the 5E3 (residues
184 to 200) and 11FH (residues 608 to 671) anti-preTP MAbs (99) are indi-
cated by the bars below the protein. (B) Equal concentrations of proteins
recovered from Ad5 and Hr6 particles, purified from equal number of infec-
tious units as described in Materials and Methods, were examined by immu-
noblotting with the MAbs against protein V and preTP indicated at the top.
5E3 (long) indicates longer exposure of the blot shown to the left. The posi-
tions of molecular mass markers (sizes are in kilodaltons) are indicated at the
left, and those of iTP, TP, and protein V are at the right.
Kato et al.
jvi.asm.orgJournal of Virology
lower and the majority were localized in nuclei (Fig. 6c). A strik-
ingly larger number of protein VII-associated viral genomes were
p.i. (Fig. 6d and e). This result of direct observation of Hr6 ge-
nomes is in excellent agreement with the rapid initial decrease in
the viral DNA concentration measured by quantitative PCR in
Hr6-infected cells (Fig. 3B). At 2 h after Hr6 infection, most pro-
tein VII foci were present in the cytoplasm (Fig. 6d). This popu-
lation decreased substantially by 7 h p.i. (Fig. 6, compare panels d
and e), indicating that most of the entering Hr6 genomes are de-
graded prior to or soon after entry into the nucleus.
Transport of noninfectious Hr6 genomes to the lysosome via
late endosomes, the intracellular destination of noninfectious
Ad2ts1 particles that cannot escape early endosomes (see intro-
therefore investigated whether Hr6 genomes were diverted to late
endosomes. Cells were infected synchronously for 0.5 or 2.0 h or
mock infected, and viral genomes were visualized as described in
the previous paragraph, while late endosomes were detected by
Microtubules were also examined, by using a rat anti-? tubulin
antibody, as described in Materials and Methods. Rab7-staining
late endosomes and microtubules were clearly discernible in both
uninfected and infected cells (Fig. 7). However, protein VII
FIG 6 Visualization of viral genomes during the initial period of Ad5 and Hr6 infection. HFFs were infected with 100 PFU/cell Ad5 or Hr6 for the periods
indicated as described in Materials and Methods or mock infected (M), and viral genomes were visualized by immunofluorescence using an anti-protein VII
polyclonal antibody (42). Nuclei were stained with DAPI (blue). Z stack projections of representative fields are shown.
stained with mouse anti-Rab and rat anti-?-tubulin-antibodies as described in Materials and Methods.
An Unusual Reduced-Infectivity Phenotype of Ad5
December 2012 Volume 86 Number 24 jvi.asm.org 13561
puncta representing viral genomes were not observed to be com-
0.5 or 2.0 h after infection (Fig. 7b to e). Furthermore, both Ad5
and Hr6 genomes were seen to congregate around juxtanuclear
microtubules by 2 h after infection. Indeed, the only difference
between Ad5- and Hr6-infected cells was the presence of protein
panels b and c), as was also evident 2 h after infection in the
experiments shown in Fig. 6.
Initial attempts to compare viral DNA synthesis when the E1B
cells revealed a previously unreported phenotype of the mutant
than of its parent Ad5 (Table 1). Such poor infectivity cannot be
attributed to defects in the initial reactions in the infectious cycle,
endocytosis (see introduction); much higher concentrations of
viral DNA were also detected in Hr6-infected cells than in Ad5-
infected cells 2 h after infection (Table 1). Rather, the majority of
intracellular Hr6 DNA molecules were degraded as the infectious
cycle progressed (Fig. 3B). This phenomenon was also observed
when the quantities of Hr6 and Ad5 DNA that initially entered
infected cell nuclei were closely similar (Fig. 3C). As the destruc-
tion of mutant DNA cannot be ascribed to induction of host re-
sponses by the much higher concentration of viral DNA in Hr6-
infected than in Ad5-infected cells following infection at equal
ceptible to extensive intracellular degradation.
This unusual phenotype is not the result of the failure to accu-
of the Hr6 E1B frameshift mutation into the phenotypically wild-
tion of either entry of greater quantities of mutant than wild-type
viral DNA into infected cells or increased degradation of mutant
genomes (11). The Hr6 genome must contain at least one addi-
cation of high-throughput sequencing to Ad5 DNA of our labo-
ratory strain, which was derived from that which served as the
mutations. The Ad5 DNA sequence exhibited two differences
from the reference strain (accession no. AY33986.1) (86) (Fig. 4).
a TA base pair near the 3= end of the genome (Fig. 4) occurred
within long runs of identical base pairs, consistent with slippage
errors by the viral DNA polymerase (20). The Hr6 genome was
found to contain seven mutations not described previously, five
silent and two that introduce amino acid substitutions (Table 2).
Like the insertion and deletion in Ad5 DNA discussed above, the
GC deletion responsible for the host range phenotype of Hr6 (bp
2347) may be the results of a slippage error during viral DNA
synthesis, as it lies in the sequence TTGT. The five transitions
nation by nitrous acid, the mutagen used during the derivation of
Hr6 (34). The presence of two transversions (bp 2947 and 9655)
was, however, surprising in view of the overall stability of the Ad5
genome: such mutations are not induced upon the exposure of
DNA to nitrous acid and must therefore have arisen spontane-
and the initial entry of higher concentrations of Hr6 than of Ad5
DNA (Table 1) provide strong evidence that the mutation that
results in an A406V substitution in fiber protein IV (Table 2)
cannot be responsible for the low infectivity of Hr6 particles. The
entry of Hr6 genomes into the cytosol upon escape from endo-
somes (Fig. 7), a late reaction that depends on the exposure of
protein VI upon partial disassembly triggered by the initial loss of
fibers bound to Car (see introduction), provides additional sup-
port for this conclusion. Furthermore, analysis of the forms of TP
present in viral particles indicated that processing of preTP from
to mature TP (Fig. 5A) is impaired in Hr6 particles (Fig. 5B).
Quantification using protein V as an internal control indicated
that, relative to Ad5, Hr6 particles contained, on average, an 8.9-
fold ? 1.1-fold higher concentration of iTP. This value is in rea-
tious unit of Hr6 (Table 1), particularly if an incompletely
to render that genome noninfectious. However, the G315V sub-
stitution in Hr6 preTP does not alter the TP sequence or the viral
protease cleavage site that produces mature TP but rather lies
some 34 amino acids nearer the N terminus (Fig. 5A).
Previous studies have established that the sites at which preTP
is cleaved by the viral protease are accessible in in vitro reactions
(98). However, the rate of production of mature TP was much
lower than that of iTP formation (100). Furthermore, in infected
cells, preTP that is not covalently linked to viral DNA can be
processed only to iTP (100). These observations indicate that a
conformational change is required upon the packaging of preTP
(or iTP) covalently linked to the viral genome to confer access of
the protease to the cleavage site (MTGG-V) that forms the N ter-
minus of mature TP. The substitution in Hr6 preTP introduces a
bulky Val residue in place of Gly in a sequence that comprises
three Gly residues in the wild type. This substitution would be
it is perhaps noteworthy that analysis of the domain organization
of preTP with MAbs indicated the importance of conformation
viral origin of replication and to the viral DNA polymerase (99).
The results of quantification of intracellular viral DNA mole-
cules (Fig. 3) and their visualization (Fig. 6) established unequiv-
ocally that the majority of Hr6 genomes are degraded within a
relatively short period after infection. Nevertheless, mutant ge-
nomes were not observed to associate with late endosomes, the
destination of noninfectious Ad2ts1 particles (see introduction),
indicating that escape from early endosomes into the cytosol was
icantly larger number of viral genomes, concentrated in the cyto-
difference in the localization of such mutant versus Ad5 genomes
could be discerned (Fig. 7). Furthermore, attempts to visualize
cessful. Consequently, the data currently available cannot estab-
lish whether noninfectious Hr6 DNA molecules are degraded
Kato et al.
jvi.asm.orgJournal of Virology
prior to nuclear entry or soon after transport into that organelle.
Tracking of the movement and intracellular destination of indi-
vidual viral genomes in real time in living cells is required to dis-
tinguish these possibilities. Nevertheless, if degradation were in-
tranuclear, the low rate of loss of entering Hr6 genomes (Fig. 3B)
predicts that, during the initial period of infection, a significantly
the cytoplasm, and in Hr6-infected than in Ad5-infected cell nu-
clei. Neither of these patterns was observed (Fig. 6), suggesting
that degradation of Hr6 genomes within the cytoplasm is more
likely. This scenario implies that incomplete processing of cova-
lently attached preTP impedes the nuclear entry of genomes, ren-
dering them susceptible to attack by cytoplasmic DNases. Such
enzymes include the abundant 3= ¡ 5= exonuclease Trex1, which
is responsible for the cytosolic degradation of DNA products of
HIV-1 reverse transcription (108), as well as cytoplasmic DNA
molecules that can activate innate immune responses (83, 110).
Our conclusion that the fate of entering Ad5 genomes is gov-
erned by processing of the covalently attached TP is consistent
with previous observations. For example, MAbs that react with
only preTP or iTP detected the protein(s) only in discrete nuclear
foci thought to be viral replication centers, whereas antibodies
that bind to other regions revealed TP throughout the nucleus
(100). Furthermore, mutations in precursor-specific segments of
genomes with the operationally defined structure termed the nu-
clear matrix and transcription of viral intermediate-early and
early genes (73), although whether preTP processing was im-
paired was not determined.
We have reported previously that the E1B 55-kDa protein re-
presses the expression of genes associated with immune defenses,
particularly innate and antiviral responses (58). This conclusion,
which was based on comparison of cellular gene expression in
considerable period following Hr6 infection (Fig. 3 and 6) could
well be detected by the cytoplasmic sensors of foreign DNA, such
immune response genes (3, 94). However, the properties of addi-
tional mutants carrying alterations in the E1B 55-kDa protein
as a repressor of expression of these genes. The null mutant
2347) does not exhibit the reduced-infectivity phenotype of Hr6,
and higher concentrations of entering viral genome are not ob-
its wild-type parent (11). Nevertheless, the expression of several
Hr6-infected cells was also substantially higher in cells infected
ent (11). Furthermore, this same response is induced by muta-
tions that result in the substitution of specific residues in the E1B
55-kDa protein (J. S. Chahal, C. Gallagher, and S. J. Flint, unpub-
We thank Daniel Engel and Ronald Hay for generous gifts of antibodies
against adenoviral protein VII and TP, respectively, Moriah Szpara for
advice and instruction on Illumina library construction, Lance Parsons
with preparation of the manuscripts.
This work was supported by grants from the National Institute of
(RO1A11058172 and R56A11091785), to S.J.F.
1. Bai M, Harfe B, Freimuth P. 1993. Mutations that alter an Arg-Gly-Asp
cell-rounding activity and delay virus reproduction in flat cells. J. Virol.
2. Bailey CJ, Crystal RG, Leopold PL. 2003. Association of adenovirus
with the microtubule organizing center. J. Virol. 77:13275–13287.
3. Barber GN. 2011. Cytoplasmic DNA innate immune pathways. Immu-
nol. Rev. 243:99–108.
4. Bergelson JM, et al. 1997. Isolation of a common receptor for Coxsackie
B viruses and adenoviruses 2 and 5. Science 275:1320–1323.
5. Berk AJ. 2007. Adenoviridae: the viruses and their replication, p 2355–
2394. In Knipe DM, Howley PM (ed), Fields virology, 5 ed, vol 2. Lip-
pincott Williams & Wilkins, Philadelphia, PA.
6. Bewley MC, Springer K, Zhang YB, Freimuth P, Flanagan JM. 1999.
cellular receptor, CAR. Science 286:1579–1583.
7. Blankenberg D, et al. 2010. Galaxy: a web-based genome analysis
tool for experimentalists. Curr. Protoc. Mol. Biol. Chapter 19:Unit
8. Bremner KH, et al. 2009. Adenovirus transport via direct interaction of
cytoplasmic dynein with the viral capsid hexon subunit. Cell Host Mi-
9. Burckhardt CJ, et al. 2011. Drifting motions of the adenovirus receptor
CAR and immobile integrins initiate virus uncoating and membrane
lytic protein exposure. Cell Host Microbe 10:105–117.
10. Cervera MM, Dreyfuss G, Penman S. 1981. Messenger RNA is trans-
lated when associated with the cytoskeletal framework in normal and
VSV-infected HeLa cells. Cell 23:113–120.
11. Chahal JS, Flint SJ. 2012. Timely synthesis of the adenovirus type 5 E1B
mal human cells. J. Virol. 86:3064–3072.
12. Chatterjee PK, Vayda ME, Flint SJ. 1986. Adenoviral protein VII pack-
ages intracellular viral DNA throughout the early phase of infection.
EMBO J. 5:1633–1644.
13. Chatterjee PK, Vayda ME, Flint SJ. 1986. Identification of proteins and
protein domains that contact DNA within adenovirus nucleoprotein
cores by ultraviolet light crosslinking of oligonucleotides32P-labeled in
vivo. J. Mol. Biol. 188:23–37.
14. Chiu CY, Mathias P, Nemerow GR, Stewart PL. 1999. Structure of
adenovirus complexed with its internalization receptor, alphavbeta5 in-
tegrin. J. Virol. 73:6759–6768.
15. Chroboczek J, Bieber F, Jacrot B. 1992. The sequence of the genome of
adenovirus type 5 and its comparison with the genome of adenovirus
type 2. Virology 186:280–285.
16. Dales S, Chardonnet Y. 1973. Early events in the interaction of adeno-
viruses with HeLa cells. during vectorial movement of the inoculum.
17. Edwards SJ, et al. 2002. Evidence that replication of the antitumor
suppressor genes. J. Virol. 76:12483–12490.
18. Einfeld DA, et al. 2001. Reducing the native tropism of adenovirus
vectors requires removal of both CAR and integrin interactions. J. Virol.
19. Engelke MF, Burckhardt CJ, Morf MK, Greber UF. 2011. The dynactin
complex enhances the speed of microtubule-dependent motions of ade-
novirus both towards and away from the nucleus. Viruses 3:233–253.
20. Field J, Gronostajski RM, Hurwitz J. 1984. Properties of the adenovirus
DNA polymerase. J. Biol. Chem. 259:9487–9495.
An Unusual Reduced-Infectivity Phenotype of Ad5
December 2012 Volume 86 Number 24jvi.asm.org 13563
21. Gastaldelli M, et al. 2008. Infectious adenovirus type 2 transport
through early but not late endosomes. Traffic 9:2265–2278.
22. Giardine B, et al. 2005. Galaxy: a platform for interactive large-scale
genome analysis. Genome Res. 15:1451–1455.
23. Goecks J, Nekrutenko A, Taylor J. 2010. Galaxy: a comprehensive
approach for supporting accessible, reproducible, and transparent com-
putational research in the life sciences. Genome Biol. 11:R86.
24. Gonzalez R, Huang W, Finnen R, Bragg C, Flint SJ. 2006. Adenovirus
E1B 55-kilodalton protein is required for both regulation of mRNA ex-
port and efficient entry into the late phase of infection in normal human
fibroblasts. J. Virol. 80:964–974.
25. Gonzalez RA, Flint SJ. 2002. Effects of mutations in the adenoviral E1B
26. Goodrum FD, Ornelles DA. 1998. p53 status does not determine out-
come of E1B 55-kilodalton mutant adenovirus lytic infection. J. Virol.
27. Graham FL, Prevec L. 1991. Manipulation of adenovirus vectors. Meth-
ods Mol. Biol. 7:109–128.
28. Graham FL, Smiley J, Russell WC, Nairn R. 1977. Characteristics of a
human cell line transformed by DNA from human adenovirus type 5. J.
Gen. Virol. 36:59–72.
29. Greber UF, et al. 1997. The role of the nuclear pore complex in adeno-
virus DNA entry. EMBO J. 16:5998–6007.
30. Greber UF, Willetts M, Webster P, Helenius A. 1993. Stepwise disman-
tling of adenovirus 2 during entry into cells. Cell 75:477–486.
31. Grosshans BL, Ortiz D, Novick P. 2006. Rabs and their effectors:
achieving specificity in membrane traffic. Proc. Natl. Acad. Sci. U. S. A.
32. Harada JN, Berk AJ. 1999. p53-Independent and -dependent require-
ments for E1B-55K in adenovirus type 5 replication. J. Virol. 73:5333–
33. Harlow E, Franza B, Jr, Schley C. 1985. Monoclonal antibodies specific
for adenovirus early region 1A proteins: extensive heterogeneity in early
region 1A products. J. Virol. 55:533–546.
34. Harrison TJ, Graham F, Williams JF. 1977. Host range mutants of
adenovirus type 5 defective for growth on HeLa cells. Virology 77:319–
35. Haruki H, Gyurcsik B, Okuwaki M, Nagata K. 2003. Ternary complex
formation between DNA-adenovirus core protein VII and TAF-Ibeta/
SET, an acidic molecular chaperone. FEBS Lett. 555:521–527.
36. Hay RT, Freeman A, Leith I, Monaghan A, Webster A. 1995. Molecular
interactions during adenovirus DNA replication. Curr. Top. Microbiol.
Immunol. 199(Pt 2):31–48.
37. He TC, et al. 1998. A simplified system for generating recombinant
adenoviruses. Proc. Natl. Acad. Sci. U. S. A. 95:2509–2514.
38. Henaff D, Salinas S, Kremer EJ. 2011. An adenovirus traffic update:
39. Hindley CE, Lawrence FJ, Matthews DA. 2007. A role for transportin in
40. Imelli N, Ruzsics Z, Puntener D, Gastaldelli M, Greber UF. 2009.
Genetic reconstitution of the human adenovirus type 2 temperature-
sensitive 1 mutant defective in endosomal escape. Virol. J. 6:174.
41. Jakubczak JL, et al. 2001. Adenovirus type 5 viral particles pseudotyped
B-adenovirus receptor-bearing cells. J. Virol. 75:2972–2981.
42. Johnson JS, et al. 2004. Adenovirus protein VII condenses DNA, re-
presses transcription, and associates with transcriptional activator E1A.
J. Virol. 78:6459–6468.
43. Kato SE, Huang W, Flint SJ. 2011. Role of the RNA recognition motif of
44. Kelkar SA, Pfister KK, Crystal RG, Leopold PL. 2004. Cytoplasmic
45. Kirby I, et al. 2000. Identification of contact residues and definition of
the CAR-binding site of adenovirus type 5 fiber protein. J. Virol. 74:
46. Lassam NJ, Bayley ST, Graham FL. 1979. Tumor antigens of human
tive host range mutants. Cell 18:781–791.
47. Leissner P, et al. 2001. Influence of adenoviral fiber mutations on viral
encapsidation, infectivity and in vivo tropism. Gene Ther. 8:49–57.
48. Lenk R, Penman S. 1979. The cytoskeletal framework and poliovirus
metabolism. Cell 16:289 301.
49. Leopold PL, et al. 2000. Dynein- and microtubule-mediated transloca-
tion of adenovirus serotype 5 occurs after endosomal lysis. Hum. Gene
50. Li E, et al. 2001. Integrin alpha(v)beta1 is an adenovirus coreceptor. J.
51. Li H, Durbin R. 2009. Fast and accurate short read alignment with
Burrows-Wheeler transform. Bioinformatics 25:1754–1760.
52. Liu H, Naismith JH, Hay RT. 2003. Adenovirus DNA replication. Curr.
Top. Microbiol. Immunol. 272:131–164.
53. Lunt R, Vayda ME, Young M, Flint SJ. 1988. Isolation and character-
ization of monoclonal antibodies against the adenovirus core proteins.
simplex virus infections. J. Virol. 76:9962–9971.
55. Magnusson MK, Hong SS, Boulanger P, Lindholm L. 2001. Genetic
fiber. J. Virol. 75:7280–7289.
56. Maier O, Galan DL, Wodrich H, Wiethoff CM. 2010. An N-terminal
domain of adenovirus protein VI fragments membranes by inducing
positive membrane curvature. Virology 402:11–19.
57. Martin-Fernandez M, et al. 2004. Adenovirus type-5 entry and disas-
sembly followed in living cells by FRET, fluorescence anisotropy, and
FLIM. Biophys. J. 87:1316–1327.
58. Miller DL, Rickards B, Mashiba M, Huang W, Flint SJ. 2009. The
adenoviral E1B 55-kilodalton protein controls expression of immune
response genes but not p53-dependent transcription. J. Virol. 83:3591–
59. Moyer CL, Nemerow GR. 2012. Disulfide-bond formation by a single
cysteine mutation in adenovirus protein VI impairs capsid release and
membrane lysis. Virology 428:41–47.
60. Moyer CL, Wiethoff CM, Maier O, Smith JG, Nemerow GR. 2011.
Functional genetic and biophysical analyses of membrane disruption by
human adenovirus. J. Virol. 85:2631–2641.
61. Murti KG, Davis DS, Kitchingman GR. 1990. Localization of adenovi-
rus-encoded DNA replication proteins in the nucleus by immunogold
electron microscopy. J. Gen. Virol. 71(Pt 12):2847–2857.
62. Nakano MY, Greber UF. 2000. Quantitative microscopy of fluorescent
adenovirus entry. J. Struct. Biol. 129:57–68.
63. O’Shea C, et al. 2004. Late viral RNA export, rather than p53 inactiva-
tion, determines ONYX-015 tumor selectivity. Cancer Cell 6:611–623.
64. Pérez-Berná AJ, et al. 2009. Structure and uncoating of immature ade-
novirus. J. Mol. Biol. 392:547–557.
65. Puntener D, et al. 2011. Stepwise loss of fluorescent core protein V from
human adenovirus during entry into cells. J. Virol. 85:481–496.
66. Puvion-Dutilleul F, Puvion E, Icard-Liepkalns C, Macieira-Coelho A.
1984. Chromatin structure, DNA synthesis and transcription through
the lifespan of human embryonic lung fibroblasts. Exp. Cell Res. 151:
67. Rancourt C, Keyvani-Amineh H, Sircar S, Labrecque P, Weber JM.
1995. Proline 137 is critical for adenovirus protease encapsidation and
activation but not enzyme activity. Virology 209:167–173.
68. Reich NC, Sarnow P, Duprey E, Levine AJ. 1983. Monoclonal antibod-
binding protein. Virology 128:480–484.
69. Robinson JT, et al. 2011. Integrative genomics viewer. Nat. Biotechnol.
70. Roelvink PW, Mi Lee G, Einfeld DA, Kovesdi I, Wickham TJ. 1999.
Identification of a conserved receptor-binding site on the fiber proteins
of CAR-recognizing adenoviridae. Science 286:1568–1571.
71. Rothmann T, Hengstermann A, Whitaker NJ, Scheffner M, zur
Hausen H. 1998. Replication of ONYX-015, a potential anticancer ade-
novirus, is independent of p53 status in tumor cells. J. Virol. 72:9470–
72. San Martín C. 2012. Latest insights on adenovirus structure and assem-
bly. Viruses 4:847–877.
73. Schaack J, Ho WJ-W, Frimuth P, Shenk T. 1990. Adenovirus terminal
tion of adenovirus DNA. Genes Dev. 4:1197–1208.
74. Seki T, et al. 2002. Artificial extension of the adenovirus fiber shaft
Kato et al.
jvi.asm.orgJournal of Virology
inhibits infectivity in coxsackievirus and adenovirus receptor-positive
cell lines. J. Virol. 76:1100–1108.
75. Shayakhmetov DM, Lieber A. 2000. Dependence of adenovirus infec-
tivity on length of the fiber shaft domain. J. Virol. 74:10274–10286.
76. Silvestry M, et al. 2009. Cryo-electron microscopy structure of adeno-
virus type 2 temperature-sensitive mutant 1 reveals insight into the cell
entry defect. J. Virol. 83:7375–7383.
77. Smart JE, Stillman BW. 1982. Adenovirus terminal protein precursor.
Partial amino acid sequence and the site of covalent linkage to virus
DNA. J. Biol. Chem. 257:13499–13506.
78. Smith JG, Cassany A, Gerace L, Ralston R, Nemerow GR. 2008.
bule-dependent cytoplasmic transport. J. Virol. 82:6492–6500.
by human alpha-defensins. Cell Host Microbe 3:11–19.
80. Smith JG, Wiethoff CM, Stewart PL, Nemerow GR. 2010. Adenovirus.
Curr. Top. Microbiol. Immunol. 343:195–224.
81. Somsel Rodman J, Wandinger-Ness A. 2000. Rab GTPases coordinate
endocytosis. J. Cell Sci. 113(Pt. 2):183–192.
82. Stenmark H, Olkkonen VM. 2001. The Rab GTPase family. Genome
83. Stetson DB, Ko JS, Heidmann T, Medzhitov R. 2008. Trex1 prevents
cell-intrinsic initiation of autoimmunity. Cell 134:587–598.
84. Stewart PL, et al. 1997. Cryo-EM visualization of an exposed RGD
epitope on adenovirus that escapes antibody neutralization. EMBO J.
85. Strunze S, et al. 2011. Kinesin-1-mediated capsid disassembly and dis-
ruption of the nuclear pore complex promote virus infection. Cell Host
86. Sugarman BJ, Hutchins BM, McAllister DL, Lu F, Thomas KB. 2003.
The complete nucleic acid sequence of the adenovirus type 5 reference
material (ARM) genome. Bioprocessing J. 2:27–32.
87. Sugawara K, Gilead Z, Wold WSM, Green M. 1977. Immunofluores-
cence study of the adenovirus type 2 single-stranded DNA binding pro-
tein in infected and transformed cells. J. Virol. 22:527–539.
88. Suomalainen M, et al. 1999. Microtubule-dependent plus- and minus
end-directed motilities are competing processes for nuclear targeting of
adenovirus. J. Cell Biol. 144:657–672.
mouse cellular receptors for subgroup C adenoviruses and group B cox-
sackieviruses. Proc. Natl. Acad. Sci. U. S. A. 94:3352–3356.
90. Trotman LC, Mosberger N, Fornerod M, Stidwill RP, Greber UF.
2001. Import of adenovirus DNA involves the nuclear pore complex
receptor CAN/Nup214 and histone H1. Nat. Cell Biol. 3:1092–1100.
91. Turnell AS, Grand RJ, Gallimore PH. 1999. The replicative capacities of
large E1B-null group A and group C adenoviruses are independent of
host cell p53 status. J. Virol. 73:2074–2083.
92. Ugai H, Borovjagin AV, Le LP, Wang M, Curiel DT. 2007. Thermo-
human adenovirus type 5 is rescued by thermo-selectable mutations in
the core protein X precursor. J. Mol. Biol. 366:1142–1160.
93. van Raaij MJ, Louis N, Chroboczek J, Cusack S. 1999. Structure of the
human adenovirus serotype 2 fiber head domain at 1.5 A resolution.
94. Vilaysane A, Muruve DA. 2009. The innate immune response to DNA.
Semin. Immunol. 21:208–214.
95. Voelkerding K, Klessig DF. 1986. Identification of two nuclear sub-
classes of the adenovirus type 5-encoded DNA-binding protein. J. Virol.
96. Von Seggern DJ, Chiu CY, Fleck SK, Stewart PL, Nemerow GR. 1999.
A helper-independent adenovirus vector with E1, E3, and fiber deleted:
structure and infectivity of fiberless particles. J. Virol. 73:1601–1608.
97. Weber J. 1976. Genetic analysis of adenovirus type 2 III. Temperature
sensitivity of processing of viral proteins. J. Virol. 17:462–471.
98. Webster A, Leith IR, Hay RT. 1994. Activation of adenovirus-coded
protease and processing of preterminal protein. J. Virol. 68:7292–7300.
99. Webster A, Leith IR, Hay RT. 1997. Domain organization of the ade-
novirus preterminal protein. J. Virol. 71:539–547.
100. Webster A, Leith IR, Nicholson J, Hounsell J, Hay RT. 1997. Role of
preterminal protein processing in adenovirus replication. J. Virol. 71:
101. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. 1993. Integrins
not virus attachment. Cell 73:309–319.
102. Wiethoff CM, Wodrich H, Gerace L, Nemerow GR. 2005. Adenovirus
J. Virol. 79:1992–2000.
103. Williams J, et al. 1986. The adenovirus E1B 495R protein plays a role in
regulating the transport and stability of the viral late messages. Cancer
104. Williams JF. 1970. Enhancement of adenovirus plaque formation on
HeLa cells by magnesium chloride. J. Gen. Virol. 9:251–253.
105. Wodrich H, et al. 2010. A capsid-encoded PPxY-motif facilitates ade-
novirus entry. PLoS Pathog. 6:e1000808. doi:10.1371/journal.p-
106. Wold WSM, Horwitz MS. 2007. Adenoviruses, p 2395–2436. In Knipe
Wilkins, Philadelphia, PA.
107. Xue Y, Johnson JS, Ornelles DA, Lieberman J, Engel DA. 2005.
Adenovirus protein VII functions throughout early phase and interacts
with cellular proteins SET and pp32. J. Virol. 79:2474–2483.
108. Yan N, Regalado-Magdos AD, Stiggelbout B, Lee-Kirsch MA, Lieber-
man J. 2010. The cytosolic exonuclease TREX1 inhibits the innate im-
mune response to human immunodeficiency virus type 1. Nat. Immu-
109. Yang U-C, Huang W, Flint SJ. 1996. mRNA export correlates with
activation of transcription in human subgroup C adenovirus-infected
cells. J. Virol. 70:4071–4080.
110. Yang YG, Lindahl T, Barnes DE. 2007. Trex1 exonuclease degrades
ssDNA to prevent chronic checkpoint activation and autoimmune dis-
ease. Cell 131:873–886.
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