The Human Adenovirus Type 5 E1B 55 kDa Protein
Obstructs Inhibition of Viral Replication by Type I
Interferon in Normal Human Cells
Jasdave S. Chahal, Ji Qi¤, S. J. Flint*
Princeton University, Department of Molecular Biology, Lewis Thomas Laboratory, Princeton, New Jersey, United States of America
Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune
responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a
set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes
many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN) was
therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least
500-fold, compared to only 5-fold, for wild-type (WT) virus replication. To investigate the mechanistic basis of such
inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively,
in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes
detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated
cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects
could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize
components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to
prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a
potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the
previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible
genes, and hence to facilitate formation of viral replication centers and genome replication.
Citation: Chahal JS, Qi J, Flint SJ (2012) The Human Adenovirus Type 5 E1B 55 kDa Protein Obstructs Inhibition of Viral Replication by Type I Interferon in Normal
Human Cells. PLoS Pathog 8(8): e1002853. doi:10.1371/journal.ppat.1002853
Editor: Michael Imperiale, University of Michigan, United States of America
Received May 18, 2012; Accepted June 26, 2012; Published August 9, 2012
Copyright: ? 2012 Chahal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by grants to S.J.F. from the National Institute for Allergy and Infectious Disease, National Institutes of Health, AI101058172 and
AI1091785 (www.niaid.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America
A major obstacle to the therapeutic application and efficacy of
adenoviral vectors is the induction of powerful innate and pro-
inflammatory immune responses following systemic delivery [1–4],
independently of viral gene expression [5–9]. The constellations of
chemokines produced in response to adenovirus vector infection
depend on the host cell type and its species of origin, as do the
mechanisms by which infection is detected by host cell pattern
recognition receptors to activate signal transduction pathways and
transcription of genes that encode these immunomodulators [2–4].
Nevertheless, production of several chemokines, including Rantes,
Mip1-a and IL-8, and such cytokines as interferon (IFN) a and b,
Tnf-a and IL-6 has been observed upon infection of a wide variety
of established and primary human and murine cells in culture and
in vivo [4,7–19]. Interferon a and b, designated hereafter IFN, bind
to the same heterodimeric receptor to establish a front line of anti-
viral defense via stimulation of transcription of numerous genes
[20–23]. The products of such interferon-stimulated genes (ISGs)
inhibit replication of a wide variety of viruses by multiple direct or
indirect mechanisms [22,24–27]. Proteins encoded by ISGs also
reinforce synthesis of IFN and other cytokines, promote processing
and presentation of antigens, and modulate the activity of
important effector cells of the immune system [22,25–29].
The replication of human adenovirus type 5 (Ad5), from which
nearly all vectors have been derived, is refractory to IFN in several
lines of established human cells [30–32], as a result of the actions
of several viral gene products that counter the effects of the
cytokine. The first to be identified, the small viral RNA, VA-RNA
I  binds to, and prevents activation of, the interferon-induced,
double-stranded RNA-activated protein kinase, which phosphor-
ylates elF2-a to inhibit translation during the late phase of
infection . More recently, it has been established that the viral
E4 Orf3 protein is required to prevent inhibition of viral DNA
synthesis in type I IFN-treated cells . This function of the E4
Orf3 protein correlates with reorganization of the promyelocytic
leukemia protein (Pml) from the discrete, nuclear Pml bodies
present in uninfected cells to track-like structure that also contain
the viral protein, and is abrogated by shRNA-mediated knock-
down of Pml or Daxx . In addition, the viral E1A proteins
suppress transcription of interferon-sensitive genes in infected cells
[35–39] and both block activation of the Jak-Stat signaling
PLoS Pathogens | www.plospathogens.org1 August 2012 | Volume 8 | Issue 8 | e1002853
pathway that induces transcription of ISGs and interact directly
with Stat 1 co-activators [37–43]. The contributions of these viral
products to modulation of innate immune responses in vivo have
not been investigated intensively. Nevertheless, both the 243R
E1A protein and the E3 gene, which encodes several proteins that
inhibit inflammatory responses and apoptosis induced by binding
of their ligands to the Tnfa and related receptors [44,45], have
been shown to decrease such responses to adenoviral vectors in
various murine organs or tissues [46–49]. Comparison of
induction of edema in mouse ears by vectors carrying different
combinations of E1A, E1B and E3 coding sequences also
implicated the E1B 19 kDa and 55 kDa proteins in inhibition of
inflammatory responses . The anti-inflammatory activity of
the E1B 19 kDa protein was proposed to be the result of the anti-
apoptotic activity of this viral Bcl-2 homologue [50,51].
The E1B 55 kDa protein makes an important contribution to
optimizing the host cell environment for efficient viral replication
[52,53] via formation of a virus-specific E3 ubiquitin ligase that
also contains the viral E4 Orf6 protein, Cul5 and several other
cellular proteins [54,55]. The activity of this enzyme targets the
cellular proteins p53, Mre11, Rad50, DNA ligase IV and integrin
a3 for proteasomal degradation [54–60]. The destruction of
Mre11 and Rad50 facilitates inhibition of the DNA double
stranded break repair response and helps circumvent inhibition of
viral DNA in infected cells [61–63], while degradation of DNA
ligase IV contributes to prevention of genome concatamerization
. Assembly of the virus-specific E3 ubiquitin ligase is also
necessary for induction of selective export from the nucleus of viral
late mRNAs [64,65].
One of the earliest functions ascribed to the E1B 55 kDa protein
was repression of transcription of genes regulated by the tumor
suppressor p53 in in vitro and transient expression assays [66,67].
This activity correlates with the ability of the E1B protein to
cooperate with E1A proteins to transform rodent cells [68–71]. It
has long been supposed that inhibition of p53-dependent
transcription by the E1B 55 kDa protein in infected cells would
contribute to preventing induction of cell cycle arrest or apoptosis
upon stabilization and activation of p53 by the viral E1A proteins
(e.g. [52,72,73]). However, when p53 accumulates to high
concentrations in cells infected by Ad5 mutants that cannot direct
synthesis of this E1B protein (E1B 55 kDa null-mutants),
expression of p53-activated genes is not increased [74–76].
Indeed, as assessed by microarray hybridization, the reversal of
the p53 transcriptional program is as complete in normal human
cells infected by an E1B 55 kDa-null-mutant as in wild-type Ad5-
infected cells . However, in the absence of the E1B protein,
expression of some 340 genes highly enriched for those associated
with immune responses and anti-viral defense was increased
significantly . In particular, we observed that this set contained
(MDA5), IF1T2, MX2 and TAP1 . These observations
suggested that repression of expression of such genes by the E1B
55 kDa protein might protect Ad5-infected cells against anti-viral
measures induced by type I IFN. We now report the results of
experiments designed to test this hypothesis, which demonstrate
that the E1B 55 kDa protein represses expression of ISGs and
blocks type I IFN-induced inhibition of viral DNA synthesis and
replication in normal human cells.
The E1B 55 kDa protein blocks inhibition of Ad5
replication by type I interferon in normal human cells
In initial experiments to investigate whether repression of
expression of interferon-stimulated genes (ISGs) by the E1B
55 kDa protein protects Ad5-infected cells from the anti-viral
defenses induced by this cytokine, replication of Ad5 and the E1B
55 kDa null-mutant Hr6 were compared in HFFs treated with
exogenous IFN. Cells were maintained in the presence of 500
units/ml IFN, or of vehicle only control, for 24 hrs., prior to and
during infection with 3 p.f.u./cell Ad5 or Hr6. They were
harvested after increasing periods of infection, and viral yields
measured by plaque assay on complementing 293 cells as
described in Materials and Methods. This IFN treatment regimen
decreased the yields of Ad5 by less than 3 fold (Figure 1A), in
agreement with previous observations (see Introduction). In
contrast, replication of Hr6 was reduced to a much greater
degree, up to 500-fold.
The Hr6 mutant was isolated after nitrous acid mutagenesis of
Ad5 by virtue of more efficient replication in complementing 293
cells [77,78] than in non-complementing cells . The mutation
responsible for this phenotype was mapped to the E1B 55 kDa
protein coding sequence by marker rescue and sequencing .
We have observed recently that the Hr6 genomes contain at least
one additional mutation outside the E1B gene that substantially
reduces the infectivity of virus particles (S. Kato, J. C. and S.J. F.,
manuscript in preparation). As described above, adenoviral VA-
RNA I, E1A proteins and the E4 Orf3 protein have been reported
previously to protect Ad5 replication against the inhibitory effects
of IFN. It was therefore essential to determine whether the
increased sensitivity of Hr6 replication to inhibition by IFN was
the result of mutations in the E1B gene, or elsewhere in the
genome. To this end, we exploited a mutant carrying the Hr6 E1B
55 kDa frameshift mutation (deletion of base-pair 2347) 
introduced into the genome of a phenotypically wild-type, E1-
containing derivative of AdEasy . As reported elsewhere ,
no E1B 55 kDa protein can be detected in HFFs infected by this
mutant (AdEasyE1D2347), and, as expected in the absence of this
viral protein [80,83–86], expression of viral late genes was
impaired. HFFs maintained in the absence or presence of IFN
were infected with 30 p.f.u./cell AdEasyE1D2347 or its parent
AdEasyE1 , and yields determined after a single cycle of
The most frequently used therapeutic vectors for gene
transfer or cancer treatment are derived from human
adenovirus type 5 (Ad5). We have observed previously that
the E1B 55 kDa protein encoded by a gene routinely
deleted from these vectors represses expression of
numerous cellular genes regulated by interferon (IFN) a
and b, which are important components of the innate
immune response to viral infection. We therefore com-
pared synthesis of pre-mRNA from IFN-inducible genes,
viral yields and early reactions in the infectious cycle in
normal human cells exposed to exogenous IFN and
infected by wild-type or E1B 55 kDa null-mutant viruses.
We report that the E1B 55 kDa protein is a potent
repressor of expression of IFN-regulated genes, and
protects viral replication against anti-viral actions of IFN
by blocking inhibition of formation of viral replication
centers and genome replication. These observations
provide the first information about the function of the
transcription repression activity of E1B during the infec-
tious cycle. Importantly, they also suggest new design
considerations for adenoviral vectors that can circumvent
induction of innate immune responses, currently a major
Ad5 E1B 55 kDa Protein Blocks Action of Interferon
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replication at 2 days p. i. Consistent with the results described
above, replication of AdEasyE1 was inhibited by only a modest
degree (,4-fold) in IFN treated cells (Figure 1B). However, in the
presence of IFN, the yield of AdEasyE1D2347 was inhibited nearly
300-fold (Figure 1B), indicating that E1B 55 kDa protein prevents
IFN-induced inhibition of replication in HFFs.
As described in the Introduction, it has been reported previously
that the E1B 55 kDa protein can repress transcription in simplified
experimental systems. This property suggested that this protein is
likely to inhibit transcription of ISGs in infected cells, but the
microarray hybridization data collected previously  cannot
distinguish among the multiple mechanisms by which RNA
concentrations can be regulated. The concentrations of pre-
mRNAs of representative ISGs increased in expression in Hr6-
compared to wild type-infected HFFs  were therefore
examined in the presence and absence of the E1B protein. HFFs
that were not exposed to IFN were infected with 30 p.f.u./cell
AdEasyE1 or AdEasyE1D2347 for 30 hrs, and primary transcripts
detected by using reverse transcription with random priming,
followed by PCR with primers specific for three ISG pre-mRNAs,
that is, spanning exon-intron junctions (see Materials and
Methods). To provide an internal control, GAPDH mRNA was
examined in parallel. Pre-mRNAs transcribed from the IL6, IFIT2
and STAT1 genes were present in mock-infected cells, and
decreased significantly in concentration following infection with
AdEasyE1, whereas only minimal differences in GAPDH mRNA
were detected (Figure 2A). In contrast, synthesis of these ISG pre-
mRNAs was not repressed in AdEasyE1D2347-infected HFFs, but
rather these RNAs accumulated to higher concentrations than
observed in uninfected or wild type-infected cells (Figure 2A). For
example, quantification of signals as described in Materials and
Methods indicated that the concentration of IL6 pre-mRNA was
14-fold higher in mutant compared to wild-type-infected cells,
whereas that of GAPDH mRNA was only 1.2-fold greater. These
observations, which are consistent with our microarray hybrid-
ization data , indicate that the E1B 55 kDa protein is a potent
repressor of ISG pre-mRNA synthesis in infected cells.
Although HFFs are permissive for adenovirus replication in
tissue culture, we wished to confirm the sensitivity to IFN of E1B
55 kDa null-mutants in normal human bronchial/tracheal
epithelial cells (NHBECs), which better represent the host cell
type encountered by serotype C adenoviruses in their natural site
of infection, the upper respiratory tract . To investigate the
sensitivity of NHBECs to IFN, the concentrations of ISG pre-
mRNAs were compared before and after IFN-treatment. Because
phenotypes exhibited by E1B 55 kDa-null-mutants of Ad5 have
been reported to be cell-type dependent [83,88–91], we also
examined the effects of infection in the presence or absence of the
E1B 55 kDa protein on ISG expression. NHBECs treated with
IFN or control as described in Materials and Methods were
infected with 30 p.f.u./cell AdEasyE1 or AdEasyE1D2347 for
24 hrs, and the concentrations of pre-mRNAs and of GAPDH
mRNA examined by RT-PCR. As observed in HFFs (Figure 2A),
IL6 and STAT1 pre-mRNAs were detected in uninfected,
untreated NHBECs, and accumulated to reduced concentrations
in AdEasyE1-, but not in AdEasyE1D2347-, infected cells
(Figure 2B). The same pattern was observed for GBP1 mRNA.
In these cells, IFIT2 pre-mRNA could be detected only following
infection, and was present in significantly greater quantities in the
absence of the E1B 55 kDa protein (Figure 2B). With the
exception of IL6, the RNA products of these genes accumulated
to increased concentrations in mock-infected cells exposed to IFN,
indicating that NHBECs respond to this cytokine. Expression of all
the ISGs examined was repressed in IFN-treated cells when
infected by the wild-type virus, but not following AdEasyE1D2347
infection (Figure 2B).
To confirm that the sensitivity of E1B 55 kDa null-mutant virus
replication to IFN was not specific to HFFs, the replication of the
E1B 55 kDa-null-mutants Hr6 and AdEasyE1D2347 was com-
pared to that of the corresponding wild-type virus in IFN-treated
or untreated NHBECs. In these experiments, IFN had only a
minor effect on replication of Ad5 or AdEasyE1 (Figures 3A and
B). Treatment with 250 U/ml IFN reduced Hr6 and AdEa-
syE1D2347 titers by between two and three orders of magnitude
Figure 1. Effect of IFN treatment on wild type and E1B 55 kDa null-mutant virus replication in HFFs. (A) Viral yields were determined by
plaque assay on 293 cells after infection of IFN-treated and untreated HFFs with 3 p.f.u./cell Ad5 or Hr6 for the periods indicated, and are shown as
the ratios of these values. (B) Yields were determined 2 days after infection with 30 p.f.u./cell AdEasy or AdEasy E1D2347. The averages of two
independent experiments and the standard deviations calculated as described in Materials and Methods are shown in both panels.
Ad5 E1B 55 kDa Protein Blocks Action of Interferon
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PLoS Pathogens | www.plospathogens.org15 August 2012 | Volume 8 | Issue 8 | e1002853