JOURNAL OF VIROLOGY, Nov. 2006, p. 10565–10578
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 21
Herpes Simplex Virus Type 1 ICP27-Dependent Activation of NF-?B?
Danna Hargett,1† Stephen Rice,3and Steven L. Bachenheimer1,2*
Department of Microbiology and Immunology1and Lineberger Cancer Center,2University of North Carolina, Chapel Hill,
North Carolina 27599-7290, and Department of Microbiology, University of Minnesota Medical School,
420 Delaware Street SE, Mayo Mail Code 196, Minneapolis, Minnesota 554553
Received 31 May 2006/Accepted 11 August 2006
The ability of herpes simplex virus type 1 (HSV-1) to activate NF-?B has been well documented. Beginning
at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-?B-dependent
(p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation
requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss
of both I?B? and I?B?. In this study, we identified loss of I?B? as a marker of NF-?B activation, and infection
with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was
necessary for I?B? loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region
from amino acids 21 to 63 as being necessary for I?B? loss. Additional experiments with mutant viruses with
combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-?B activation may be
augmented by functional ICP4. We also analyzed two additional markers for NF-?B activation, phosphoryla-
tion of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection
and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both I?B? and
Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphory-
lation was not affected during inhibitor treatment. These results demonstrate that there are several layers of
regulation of NF-?B activation during HSV infection, highlighting the important role that NF-?B may play in
NF-?B is a cellular integrator of diverse signaling pathways
leading to programs of immune, inflammatory, and antiapop-
totic gene expression. These pathways are initiated through the
engagement of cell surface receptors by a variety of chemical
ligands, such as cytokines, phorbol esters, lipopolysaccharide,
and virion glycoproteins, or by stresses such as UV irradiation
or changes in osmolarity. NF-?B is normally sequestered in the
cytoplasm through interactions with its inhibitory binding part-
ner I?B. While a variety of NF-?B activation pathways have
been characterized, many ultimately converge on and activate
the I?B kinase (IKK) complex, resulting in phosphorylation of
I?B. Once I?B is phosphorylated, polyubiquitin-dependent
proteolysis of I?B occurs, releasing NF-?B and allowing its
translocation to the nucleus, where it participates in transcrip-
tion activation in conjunction with other transcription factors
Transcription of the herpes simplex virus (HSV) genome
during lytic infection is temporally regulated (see reference 62
for a review). Three immediate-early (IE) proteins have im-
portant roles in regulating the temporal pattern of gene ex-
pression. ICP4 is essential for E and L gene expression and
colocalizes with viral DNA (22, 23, 85), RNA polymerase II
holoenzyme, and general transcription factors (10, 79). ICP0
can trans-activate viral IE, E, and L promoters alone or in
combination with ICP4 and/or ICP27 in transient reporter
assays. Studies that evaluated its function in the absence of
ICP4 or ICP27 demonstrated a role for ICP0 in efficient tran-
scription of IE and E genes (68). ICP0 affects disruption of
ND10 that has relocalized to viral DNA (20–22, 29, 44) and
also inhibits the function of a gene silencing complex (28) in
order to promote efficient viral transcription.
ICP27, a second essential regulatory protein, affects both
transcriptional and posttranscriptional aspects of viral gene
expression, as well as the metabolism of host cell mRNA.
Specifically, ICP27 contributes to negative regulation of IE
gene expression (61, 64) while positively regulating expression
of essential E genes for DNA replication except the ICP8 gene
(86). ICP27 mutants also express reduced amounts of leaky-
late (?1) genes and fail to express true-late (?2) genes (35, 45,
64). ICP27 has additional functions dependent on protein-
protein interactions as well as protein-RNA interactions, in-
cluding export of mRNA (11, 38, 50, 57, 69, 80), relocalization
of casein kinase 2 (CK2) (39), interaction with splicing factors
(8, 31, 43, 72, 76), and stimulation of mRNA 3?-end processing
The ability of HSV type 1 (HSV-1) to activate NF-?B has
been well documented. Beginning at 3 to 5 h postinfection
(hpi), HSV-1 induces a robust and persistent nuclear translo-
cation of NF-?B and increased p50/p65 heterodimer-depen-
dent DNA binding activity, as measured by electrophoretic
mobility shift assay (EMSA) (1, 56, 63). Persistent NF-?B
activation requires virus binding and entry, as well as de novo
infected-cell protein synthesis, including expression of func-
tional viral IE proteins ICP27 and ICP4 (1, 56). Activation is
accompanied by increased IKK activity (1) and loss of both
I?B? and I?B? (56). Interference with NF-?B activation oc-
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, 837 MEJB, University of North Carolina,
Chapel Hill, NC 27599-7290. Phone: (919) 966-2445. Fax: (919) 962-
8103. E-mail: firstname.lastname@example.org.
† Present address: Lewis Thomas Lab, Department of Molecular
Biology, Princeton University, Washington Rd., Princeton, NJ 08544-
?Published ahead of print on 23 August 2006.
curs following overexpression of a dominant-negative version
of I?B? (DN-I?B) containing alanine substitutions for critical
serine residues 32 and 36. The resulting substantial reduction
in NF-?B EMSA activity correlates with a reduction in virus
yield (1, 56). The latter may be related to the reported role of
NF-?B in preventing HSV-1-induced apoptosis (26), though
this may be dependent on cell type or on the timing of expres-
sion of viral protein US11 (84). Finally, HSV-1 infection of
HEp-2 cells transfected with a luciferase reporter plasmid con-
taining three copies of the major histocompatibility complex
NF-?B site (3? NF-?B-luc) resulted in a 3.5-fold induction of
luciferase activity, which was abolished in the presence of DN-
The foregoing results argue that HSV may have evolved to
evade the observed persistent NF-?B activation or to utilize
this host response to promote efficient virus replication. With
regard to the latter, one way this may occur is through sup-
pression of apoptosis, as mentioned above. Additionally, acti-
vation may contribute directly to transcriptional regulation of
viral genes. Nucleotide sequences which bind purified NF-?B
were identified in intron 1 and exons 2 and 3 of ICP0, as well
as upstream of the late UL46 and UL47 genes (63). NF-?B is
bound to the ICP0 promoter during infection (2). These sites
also bind nuclear factors induced by HSV infection or phorbol
myristate acetate treatment. Several examples of HSV-induced
expression of cellular genes regulated by NF-?B have been
reported. In keratinocytes, association of NF-?B with the I?B
promoter is reduced, leading to an impairment in the recon-
stitution of I?B levels after NF-?B activation (2). In lymphoid
cells, such as peripheral blood mononuclear cells treated with
gamma interferon and infected by HSV-1, expression of inter-
leukin-6 (IL-6) increased between 2 and 3 hpi (54). While
VP16 and IE proteins ICP4, ICP0, and ICP27 were not re-
quired for IL-6 mRNA induction, repression of IL-6 induction
occurred following infection of a macrophage cell line express-
ing DN-I?B. The chemokine RANTES/CCL5 was induced in
macrophages and fibroblasts by both HSV-1 and HSV-2, de-
pendent on NF-?B and IRF-3 activation and on virus-induced
PKR and ICP0 (51, 52). Glucocorticoid receptor expression
has been reported to increase during HSV infection (18). Be-
cause the anti-inflammatory effects of glucocorticoids act in
part by repressing p65 (58), down-regulation of the NF-?B-
dependent proinflammatory response may also occur during
virus infection. In summary, both increased NF-?B DNA bind-
ing activity and NF-?B-dependent gene activation in HSV-1-
infected cells have been documented. The role of viral proteins
in activation and how NF-?B contributes to infection outcomes
have not been clearly defined in all cell types investigated.
The experiments presented here were designed to evaluate
the roles of different stages in virus replication for activating
NF-?B in CV-1 cells, a cell type often used in HSV infection
studies. We assayed for NF-?B nuclear translocation, I?B deg-
radation, and p65 phosphorylation as surrogate markers for
NF-?B activation. We determined that virus attachment and
introduction of the viral tegument into the cell, without sub-
sequent gene expression, were not sufficient for activating NF-
?B. We established that expression of, among the kinetic
classes of viral genes, IE genes was sufficient for the activation
of NF-?B. Of the IE gene products, ICP27 and ICP4 were
required for full NF-?B activation. The results presented here
suggest that ICP27 was necessary to activate NF-?B, while the
role of ICP4 may be to augment the ICP27-induced activation.
We further determined that a small region of the ICP27 amino
terminus was required for the activation of NF-?B. This was
the same region of ICP27 that we identified as being required
for the activation of Jun N-terminal protein kinase (JNK) and
p38 (32). The use of a pharmacological inhibitor of JNK sup-
pressed HSV-induced NF-?B activation, suggesting that ICP27
may activate NF-?B indirectly as a downstream target of JNK.
MATERIALS AND METHODS
Cells and viruses. CV-1 cells were originally obtained from Saul Silverstein
(Columbia University) and were grown in Dulbecco’s minimal essential medium
supplemented with 5% bovine calf serum, 100 U/ml penicillin, 1% streptomycin,
and 1% L-glutamine (all from Gibco). Cells were seeded into 100-mm dishes at
a density of 2 ? 106cells per plate. Both mock infection and infection with virus
were carried out in spent media for one hour at 37°C. The inoculum was then
replaced with virus-free spent medium. Spent media were produced by collecting
the seeding media from plates prior to infection and were used as inoculum
media and overlay media, in order to minimize the introduction of growth factors
and other signaling stimulants found in fresh media. We have observed that the
background NF-?B activation in mock-infected samples using spent media was
lower than that when fresh or low-serum media were used (data not shown). The
KOS 1.1 strain of HSV-1 was used in all experiments unless otherwise noted. The
mutant tsB7 (6, 37), the parental HFEM strain of HSV-1, and the KOS ICP27
deletion mutant d27-1 (61) were provided by David Knipe (Harvard University).
The ICP4 mutants vi13 and n12 (14, 16, 77) were provided by Neal DeLuca
(University of Pittsburgh). The IE multiple mutants d100 (ICP0?/ICP4?), d103
(ICP4?/ICP22?/ICP47?), d106 (ICP4?/ICP22?/ICP27?/ICP47?), d107 (ICP4?/
ICP27?), and d109 (ICP0?/ICP4?/ICP22?/ICP27?/ICP47?) were also provided
by Neal DeLuca (67). The ICP6 mutant ICP6? (25) was provided by Sandra
Weller (University of Connecticut). The ICP0 mutant 7134 (9) was provided by
Priscilla Schaffer (Harvard Medical School). The titer of 7134 used in these
experiments was determined on complementing O-28 cells. The ICP27 C-termi-
nal truncation mutants n59r and n504r (61), the in-frame deletion mutants dLeu
(41), dAc (41), d5-6 (49), d1-2 (60), d2-3, d6-7 (5), d3-4, and d4-5 (48) and the
point mutants m11 (59) and M50T (42) were all provided by Stephen Rice
(University of Minnesota). The temperature-sensitive ICP27 mutant vBSLG4
and revertant vBS3-3 (73, 80) were provided by Saul Silverstein (Columbia
University). A fuller description of these mutants is provided in the tables.
Inhibitor treatment. Viral gene expression was limited to the IE phase by the
treatment of cells with 0.5 ?g per ml of cycloheximide for 30 min prior to
infection. The same concentration of drug was present in the inoculum and
replacement medium. At three hours postinfection, monolayers were rinsed with
spent medium and then treated with actinomycin D at a concentration of 0.4 ?g
per ml in spent medium. To prevent expression of true-late (?2) viral genes, the
viral DNA replication inhibitor phosphonoacetic acid (PAA) (Sigma) was
present in the virus inoculum and replacement medium at a concentration of 400
?g per ml (34). When using pharmacological inhibitors of signaling, subconfluent
cultures were treated 30 min prior to infection with vehicle control dimethyl
sulfoxide (DMSO) (0.5 ?l/ml), the p38 inhibitor SB203580, the JNK inhibitor
SP100625, or the MEK inhibitor UO126, all from Calbiochem. Inhibitor was
maintained in the media throughout infection and was present in the overlay
Preparation of whole-cell lysates and immunoblotting. At the time of harvest,
cells were placed on ice to prevent artifactual induction of stress responses. The
medium was removed and the monolayers rinsed with ice-cold Dulbecco’s phos-
phate-buffered saline. Cells were scraped directly into 1 ? sodium dodecyl
sulfate (SDS) sample buffer (3.85 mM Tris base [pH 6.8], 9.1% ?-mercaptoeth-
anol, 1.82% SDS, 4.6% glycerol, 0.023% bromophenol blue [in 100% ethanol])
and denatured by boiling. Nuclear and cytoplasmic fractions were prepared as
previously described (56). Cell-equivalent amounts of lysate were separated by
12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were trans-
ferred to PolyScreen polyvinylidene difluoride membranes (PerkinElmer Life
Sciences) followed by blocking in TBST (150 mM NaCl, 20 mM Tris [pH 7.6],
0.05% Tween 20) with 5% dry milk. All probing and washing of membranes was
done with TBST. Rabbit polyclonal antibody for I?B? (C-21, catalog no. SC-371;
Santa Cruz Biotechnology) was used at a 1:1,000 dilution overnight at 4°C per the
manufacturer’s instructions. Rabbit polyclonal antibody for p65 (antibody A,
catalog no. SC-109) was used at a 1:1,000 dilution for 1 h at room temperature.
10566 HARGETT ET AL.J. VIROL.
Rabbit polyclonal antibody for poly(ADP-ribose) polymerase (PARP) (catalog
no. SC-7150) and goat polyclonal antibody for GRP-78 (catalog no. SC-1050)
were used at 1:500. Donkey anti-goat secondary antibody (horseradish peroxi-
dase conjugated; Novus Biologicals) was used at 1:3,500. Mouse monoclonal
antibody for ?-tubulin (B-5-1-2; Sigma) was used at a 1:20,000 dilution. Rabbit
polyclonal antibodies for p38 (9219), phospho-p38 (9211), JNK (9252), phospho-
JNK (9251), phospho-p65 serine 276 (3037), and phospho-p65 serine 536 (3031)
were purchased from Cell Signaling Technology and used at a 1:1,000 dilution
overnight at 4°C per the manufacturer’s instructions. Mouse monoclonal anti-
bodies for the viral proteins ICP4 (1101), ICP0 (1112), and ICP27 (1113 and
1119) were purchased from the Rumbaugh-Goodwin Institute for Cancer Re-
search (Plantation, FL) and used at 1:800, 1:800, 1:800, and 1:4,000 dilutions,
respectively. Rabbit polyclonal antibody for VP16 (Clontech) was used at a
1:5,000 dilution. Rabbit polyclonal antibody against ICP8 (3-83) was a generous
gift from David Knipe and used at a 1:20,000 dilution. Rabbit polyclonal antibody
against glycoprotein C (gC) (R47), used at a dilution of 1:5,000, was a generous
gift of Gary Cohen and Roselyn Eisenberg (University of Pennsylvania). Goat
anti-rabbit and anti-mouse secondary antibodies were purchased from Amer-
sham Biosciences. The secondary antibody was detected using the SuperSignal
West Pico chemiluminescent substrate agent (Pierce). Films were scanned and
images stored as 8-bit grayscale JPEG files. The density of each band was
determined using the Image J program (NIH). Relative density values were
corrected for average background by subtracting out the density of a blank
portion of the film. The corrected values were then used to calculate either
average percent change in independent experiments (sample value/mock value ?
standard deviation) (see Fig. 3) or the percentage of uninfected cell levels (see
Fig. 2, 4, and 6) by using Microsoft Excel. All membranes for I?B? Western blots
were stripped and reprobed for ?-tubulin. Levels of ?-tubulin were used to
normalize I?B? band intensities to protein concentrations in the lysate.
Loss of I?B? after HSV-1 infection correlates with nuclear
translocation of p65. Amici et al. reported that HSV-1 infec-
tion of HEp-2 cells resulted in activation of a 3? NF-?B-
luciferase reporter gene and that this activation was abolished
in the presence of DN-I?B (1). Similarly, we and others have
reported that HSV-1 infection of human cell lines C33-A (56)
and A549 (1, 56, 63) induced loss of I?B? and the appearance
of NF-?B-dependent EMSA activity. Furthermore, DN-I?B
abolished this activation, and loss of I?B? was accompanied by
an increase in the amount of nuclear p65 (1, 56, 63). As this
result was reported for murine cells, we first determined
whether this link also existed in nonhuman-primate cells (Fig. 1).
We prepared nuclear and cytoplasmic extracts from mock-
infected and HSV-1-infected CV-1 cells at 12 hpi and assayed
for the distributions of p65 and I?B? within cytoplasmic and
nuclear compartments. In mock-infected cells (lanes 1 and 2),
the great majority of p65 (?95%) was localized in the cyto-
plasm, while after wild-type (wt) HSV infection (lanes 3 and 4),
?57% of p65 was localized in the nucleus. Following wt HSV-1
infection, there was a correlation between loss of I?B? and
relocalization of a portion of p65 to the nucleus. No residual
I?B? was detectable in the wt-KOS-infected cytoplasmic ex-
tract. Detection of PARP was used to control for nuclear
contamination of the cytoplasmic fraction. A trace amount of
cytoplasmic contamination of the nuclear fraction was detected
following Western blotting for GRP-78, a mitochondrial pro-
tein (data not shown). These results confirm a correlation
between loss of I?B? and nuclear translocation of p65, a cor-
relation which supports the use of I?B? loss as a reliable
marker for NF-?B activation.
I?B? degradation depends on viral gene expression. Be-
cause we previously reported that HSV-1 infection triggers loss
of I?B? and I?B?, as well as persistent nuclear translocation of
NF-?B (56), we set out to determine if viral protein expression
was necessary for activation of the NF-?B pathway. Initially,
we carried out infections with tsB7, a mutant that expresses a
temperature-sensitive UL36 tegument protein involved in the
release of DNA from the nucleocapsid. Thus, at the nonper-
missive temperature (NPT), any host cell responses to virus
binding and entry should occur normally and allow us to de-
termine if any of these early events in the viral life cycle are
important in the activation of the NF-?B pathway. Replicate
cultures of CV-1 cells were infected at 33, 37, or 39°C with the
parental HFEM strain or mutant tsB7 at a multiplicity of in-
fection (MOI) of 5, and whole-cell extracts were prepared at 8
hpi. Results of Western blot analysis are shown in Fig. 2A,
while Fig. 2B shows the results of quantification of I?B follow-
ing wild-type or mutant infection at each temperature. Results
displayed in Fig. 2 are a data set representative of three inde-
pendent experiments. In each case, tsB7 infection at the NPT
was impaired in I?B? loss, though to different extents, com-
pared to the parental HFEM strain at the same temperature.
Therefore, the results of the three experiments were not aver-
aged. In the characterization of I?B?, only the lower band of
the doublet seen in this experiment was quantified. The upper
band was determined to be nonspecific due to our failure to
detect it in other Western blot assays (see Fig. 5 and 6). Mock-
infected samples displayed constant levels of I?B? at all three
temperatures (Fig. 2A, lanes 1 to 3). Wild-type infection in-
duced 70, 56, and 64% decreases in I?B? levels at 33, 37, and
39°C, respectively (lanes 4 to 6), relative to the levels in the
corresponding mock-infected cells. Infection with tsB7 resulted
in a 73% decrease in I?B? at the permissive temperature (PT)
of 33°C but no decrease at 37 and 39°C (Fig. 2A, lanes 7 to 9,
and B) relative to the mock-infected cells at the corresponding
temperature. To ensure that tsB7 inhibited the expression of
viral genes at elevated temperatures, we performed Western
blot analysis for the IE proteins ICP0 and ICP4 and deter-
mined the levels of ?-tubulin as a loading control. Viral pro-
teins were expressed in all wild-type infections and at 33°C
after tsB7 infection (Fig. 2A, lanes 4 to 7). Levels of viral
proteins were severely inhibited in tsB7 infection at 37°C and
were undetectable at 39°C (lanes 8 and 9). The residual level of
viral protein detected at 37°C was insufficient to induce I?B?
FIG. 1. Loss of I?B? after HSV-1 infection correlates with nuclear
translocation of p65. Cytoplasmic (C) and nuclear (N) extracts were
prepared as previously described (56) from mock-infected and infected
(MOI ? 5) CV-1 cells and analyzed by Western blotting for p65, I?B?,
VOL. 80, 2006 ICP27 ACTIVATION OF NF-?B 10567
degradation, as the level of I?B? detected was comparable to
the level found in mock-infected cells at 37°C. Band densities
were quantified as described in Materials and Methods and
normalized to levels of ?-tubulin in each lane.
A recent report demonstrate that UV-irradiated virus was
able to transiently activate the NF-?B pathway during entry but
was unable to support the sustained NF-?B activation we ob-
serve, which begins around 3 hpi (2). We confirmed these
observations by infecting cells with equivalent amounts of un-
irradiated or UV-irradiated virus and assaying for I?B? loss.
As expected, unirradiated virus induced efficient loss of I?B?
when assayed at 8 hpi, while UV-irradiated virus failed to
display levels of I?B? comparable to those seen in mock-
infected cells (data not shown). We conclude that the events
prior to viral genome entry into the nucleus are not sufficient
for the sustained induction of the NF-?B pathway.
Immediate-early gene expression is sufficient for I?B? deg-
radation. Previously we demonstrated degradation of I?B? as
early as 6 hpi (56). At that time, there was significant expres-
sion of the IE and E proteins, but there were lower levels of L
proteins. Therefore, we hypothesized that either IE or E pro-
teins controlled the degradation of I?B?. We took advantage
of drugs that selectively inhibit protein synthesis (cyclohexi-
mide), mRNA synthesis (actinomycin D), or viral DNA repli-
cation (PAA) to define the requirements for viral gene expres-
sion in NF-?B activation. Whole-cell lysates were prepared and
analyzed by Western blot for all three classes of viral genes
(Fig. 3A). No viral proteins were detected in mock-infected
cells (lane 1), but we detected ICP0, ICP8, and VP16 in lysates
from infected, untreated cells (lane 2). Following the reversal
of cycloheximide and the addition of actinomycin D, cells ac-
cumulated ICP0 but not ICP8 and VP16 (lane 3). Finally,
infection in the presence of PAA resulted in the accumula-
tion of both IE and E proteins but not VP16 (lane 4).
Probing for I?B? revealed its loss from all infected-cell
lysates regardless of drug treatment, suggesting that only
immediate-early gene expression was required for the acti-
vation of the NF-?B pathway.
To confirm this observation, we infected CV-1 cells with
either wt KOS or the ICP4 mutant vi13 (77). This mutant
contains a two-amino-acid (aa) substitution in the DNA bind-
ing domain of ICP4 with a resulting protein synthesis pheno-
type marked by overexpression of IE genes and absence of E or
L gene expression. We probed whole-cell extracts prepared at
8 hpi for accumulation of ICP4, ICP0, and ICP8. Figure 3B
illustrates a typical result: wt virus and vi13 both expressed
ICP4 and ICP0 (lanes 1 and 2) while only the wt virus ex-
pressed ICP8 (lane 3). The overaccumulation of ICP4 and
ICP0 in vi13-infected cells was consistent with the results of
other reports using this mutant (56, 77). The level of I?B? was
undetectable in the wt-KOS-infected lysate (lane 2) and was
reduced in the lysate from vi13-infected cells compared to that
from mock-infected cells (lanes 1 and 3). Panel C of Fig. 3
shows the quantification of band intensities relative to those of
mock-infected cells from three independent experiments,
while the Western blots from panels A and B are representa-
tive of one of the repeats. From the results presented in panels
A and B, we conclude that immediate-early gene expression is
required and sufficient for the I?B? degradation seen during
HSV infection. Of note, however, is that while IE proteins
were overexpressed during vi13 infection, the mutant was not
as efficient as wt virus in causing nuclear translocation of p65 or
loss of I?B? (Fig. 1 and 3B).
ICP27 is necessary for loss of I?B?. Having demonstrated
that IE protein synthesis was sufficient for loss of I?B? to be
observed, we conducted a time course infection with several
mutants defective in IE protein expression to determine which
was necessary for this effect. This experiment was conducted
twice with similar results. The panel of immediate-early mu-
tants included (i) n12, a nonsense mutant which expresses a
?41-kDa ICP4 protein of aa 1 to 251 (15); (ii) 7134, which
contains a lacZ substitution for the ICP0 open reading frame
(9); (iii) ICP6?, from which the ICP6 open reading frame is
deleted (25); and (iv) d27-1, from which the entire ICP27 gene
is deleted (61). The n12 mutant was reported to express IE
proteins and the E protein ICP6 only (14, 15). Whole-cell
lysates were prepared at 6, 8, and 12 hpi, separated by SDS-
PAGE, and analyzed by Western blotting for I?B?. Western
blots of I?B?, IE proteins ICP4, ICP27, and ICP0, and E
protein ICP8 in the 12-hpi lysates are shown in Fig. 4A, and the
quantification of IE protein accumulations from all three time
points is summarized in Table 1. Western blots of viral proteins
shown in panel A were from purposefully overexposed auto-
FIG. 2. I?B? degradation is dependent on viral gene expression.
(A) Replicate CV-1 cultures were mock infected or infected with
HSV-1 strain HFEM or mutant tsB7 (MOI ? 5) at the indicated
temperatures, and lysates were prepared at 8 hpi. Western blot analysis
of proteins was performed as described in Materials and Methods.
Lysates fractionated on a 12% polyacrylamide gel were sequentially
probed for I?B? and ?-tubulin, and lysates fractionated on a 6%
polyacrylamide gel were simultaneously probed for ICP0 and ICP4.
Two additional independent experiments yielded similar results. NS,
nonspecific. (B) Band intensities of I?B? from the Western blot in
panel A were quantified using Image J as described in Materials and
Methods, and the results presented are normalized to the values from
mock infection. Filled bars, mock infected; open bars, HFEM infected;
hatched bars, tsB7 infected.
10568HARGETT ET AL. J. VIROL.
radiograms to confirm the viral mutant phenotypes, while all
quantifications, using Image J, were performed with exposures
which were in the linear range. Figure 4B summarizes the
levels of I?B? throughout the 12-h time course of the wt and
mutant infections. Levels of I?B? in mock-infected cells re-
mained constant throughout the time course, while levels in wt
virus-infected cells gradually decreased over the 12 h of the
analysis. Following infection with the ICP4 mutant n12, the
level of I?B? remained similar to those in mock-infected
cells at 6 and 8 hpi and was reduced to 55% of the level in
mock-infected cells by 12 hpi (Fig. 4A, lane 3, and B).
Analysis of I?B? in 7134- and ICP6?-infected cells revealed
a delay in loss of protein relative to that of mock-infected
cells until 12 hpi, when 80% and 88% losses, respectively,
were detected (Fig. 4A, lanes 4 and 5, and B). In contrast,
over the entire time course of infection, d27-1-infected cells
retained I?B? levels near those of the mock-infected cells
(Fig. 4A, lane 6, and B). This result is consistent with our
previous finding that d27-1 was impaired in nuclear trans-
location of NF-?B in C33-A cells (56).
To confirm the status of viral protein synthesis under con-
ditions of wt or mutant virus infection, lysates prepared at 6, 8,
and 12 hpi were also analyzed for ICP4, ICP27, ICP0, and
ICP8. In Fig. 4A, the results of Western blotting for viral
proteins and values for accumulation at 12 hpi relative to wt
KOS are shown. As expected, each IE null mutant failed to
express the product of its cognate deleted gene but expressed
the remaining IE proteins. Relative levels of viral protein ac-
cumulation at 6-, 8-, and 12-hpi time points are presented in
Table 1. Infection with n12 resulted in a large relative overex-
pression of ICP0 at 6 h similar to what we observed with
another ICP4 mutant, vi13 (Fig. 3) (47). Levels of ICP27 in
cells infected with n12 differed little from those observed in
wt-infected cells at earlier times and were somewhat elevated
at 12 h. Under our conditions of 7134 infection (a MOI of 5
based on the titer obtained on complementing O-28 cells),
there were slightly reduced levels of ICP4, ICP27, and ICP8
accumulation relative to wt KOS, while infection with ICP6?
resulted in reduced relative levels of ICP4 and ICP27 at the 8-
and 12-h time points only. These results, combined with those
in Fig. 3A and B, indicate that ICP27, and to a lesser extent
ICP4, is necessary for maximum loss of I?B? during HSV
infection. The residual I?B? in ICP0 and ICP6 mutant-in-
fected cells can be explained at least in part by reduced ICP27
and ICP4 accumulation. This in turn is consistent with delayed
protein expression phenotypes of ICP0 mutants (12, 19, 83)
and the slower kinetics of ICP6? virus production on conflu-
ent, resting cell monolayers or under conditions of serum star-
vation (24, 25). Alternatively, it might reflect a role for these
viral proteins in modulating aspects of ICP27 and ICP4 intra-
cellular localization or posttranslational modification and,
The p65 subunit of NF-?B is phosphorylated on serines 276
and 536 during HSV infection. Activation of NF-?B is regu-
lated in part through phosphorylation of p65 by distinct kinase
activities. Ser276 can be phosphorylated by protein kinase A or
mitogen- and stress-activated protein kinase 1 (MSK-1) and is
important for interactions with CREB-binding protein (CBP)/
p300 during transcription activation (87, 89–91). Ser536 is
phosphorylated by IKK after tumor necrosis factor (65, 66, 78)
or lipopolysaccharide treatment (88), increasing its transcrip-
tional activity. To characterize p65 phosphorylation during in-
fection, we performed Western blot analyses using antibodies
specific for phosphorylated Ser276 and phosphorylated Ser536.
FIG. 3. Immediate-early gene expression is sufficient for loss of I?B?.
(A) Replicate cultures of CV-1 cells were mock infected or infected with
KOS at an MOI of 5 under the conditions indicated and described in
Materials and Methods. Lysates were prepared at 8 hpi, and the accumu-
lations of indicated proteins were determined following fractionation on
12% polyacrylamide gels. The lysates were sequentially probed for I?B?,
VP16, and ?-tubulin or for ICP0 and ICP8. HSV/Cx rev, infection of cells
in the presence of cycloheximide followed by removal of cycloheximide
and addition of actinomycin D (ActD) at 5 hpi; HSV?PAA, infection of
cells in the presence of PAA. (B) Replicate cultures of CV-1 cells were
mock infected or infected with wt KOS or the ICP4 mutant vi13 (MOI ?
5). Lysates were prepared at 8 hpi, fractionated on 12% polyacrylamide
gels, and sequentially probed for I?B? and ?-tubulin or for ICP4 plus
ICP0 and ICP8. NS, nonspecific. (C) Relative amounts of I?B? from
three independent experiments were determined using Image J and av-
eraged, as described in Materials and Methods.
VOL. 80, 2006ICP27 ACTIVATION OF NF-?B 10569
FIG. 4. ICP27 is necessary for loss of I?B? and phosphorylation of p65 after HSV infection. (A) Replicate cultures of CV-1 cells were mock
infected or infected with the indicated viruses as described in the text (MOI ? 5). Whole-cell lysates were prepared at 6, 8, and 12 hpi, and
accumulations of I?B? viral proteins and ?-tubulin were determined by Western blotting. Shown are the results for lysates prepared at 12 hpi.
Under each panel, the relative amount of protein compared to the mock-infected sample (I?B?) or the wt-KOS-infected sample (ICP4, ICP27,
ICP0, or ICP8) as determined by Image J analysis is indicated. (B) Graphical representation of the rate of loss of I?B? over the 12-h course of
infection with wt KOS or the indicated mutant viruses. (C) Graphical representation of the phosphorylation of p65 at Ser276 over the 12-h course
of infection with wt KOS or the indicated mutant viruses. (D) Graphical representation of the phosphorylation of p65 at Ser536 over the 12-h
course of infection with wt KOS or the indicated mutant viruses.
10570HARGETT ET AL.J. VIROL.
The protein lysates analyzed were from the wt and mutant time
course infections that we had analyzed for I?B? (Fig. 4A and
B). Following the use of phospho-specific antibodies, the mem-
branes were stripped and reprobed for total p65. The resulting
Western blots were quantified using Image J (Fig. 4C and D).
For each sample, a phosphorylated p65-to-total p65 ratio was
calculated and the data were expressed as the change in acti-
vation over that in mock-infected cells (n-fold) at the same
time point. Strikingly, wt infection induced increasing amounts
of Ser276 phosphorylation over the 12-h time course. ICP0 and
ICP6 mutants also induced increasing amounts of Ser276 phos-
phorylation. The slower kinetics of phosphorylation was con-
sistent with the slower degradation of I?B? also seen with
these mutants (compare panels B and D), which we attribute to
delayed replication kinetics of these viruses. Consistent with
the previous finding that ICP4 and ICP27 were necessary for
NF-?B activation, Ser276 phosphorylation in ICP4 and ICP27
mutant infected cells was consistently between 5 and 33% of wt
virus induction, suggesting that expression of both of these IE
proteins is necessary for p65 phosphorylation at Ser276. At 6
hpi, wt HSV induced an 8.4-fold activation of Ser536 phosphor-
ylation, but this was not observed with any of the IE mutants
(Fig. 4D). At later times postinfection, HSV infection resulted
in only a 2- to 2.3-fold induction. By 12 hpi, all of the IE
mutants induced levels of Ser536 phosphorylation that were
similar to that induced by wt virus.
In order to determine the role of ICP27 on the stability of
I?B? during infection, we investigated a panel of viral mutants
expressing various combinations of IE genes as described in
Table 2. Three independent sets of infections with d100, d103,
d106, d107, and d109 mutants were conducted, and the levels
of I?B? from a representative experiment at 8 hpi are shown in
Fig. 5A. Infection with wt virus (lane 2) resulted in a 95% loss
of I?B? compared to the outcome with mock infection. Infec-
tion with the d103 mutant, which expresses only ICP0 and
ICP27, resulted in a 33% reduction of I?B? (lane 6). We have
reported that this mutant was capable of activating both p38
and JNK (32). The d100 mutant expresses only ICP22, ICP27,
and ICP47 among the IE proteins but failed to reduce I?B?
levels at any MOI tested (lanes 3 to 5). Infection with this
mutant resulted in phosphorylation of p38 as early as 8 hpi,
though activation of JNK was not detected until 16 hpi (32).
Additionally, we observed that lysates from all infections with
mutants that failed to express ICP27 (d106, d107, and d109)
had I?B? levels similar to or higher than the level in lysates
TABLE 1. Relative levels of infected-cell polypeptides (ICPs) in wt- and mutant-virus-infected cellsa
Virus (product of
Relative level of ICP at indicated hpi
ICP4ICP0 ICP27 ICP8
68 1268 1268 1268 12
aValues represent levels of ICPs at each time postinfection relative to wt-virus-infected cells. Accumulations of proteins in whole-cell lysates prepared at 6, 8, and
12 hpi were determined by Western blotting and quantified as described in Materials and Methods. Dashes indicate no detectable protein.
TABLE 2. IE deletion mutants
? gene product(s)
? gene product(s)
d27-1ICP0, ICP4, ICP22,
ICP22, ICP27, ICP47
ICP4, ICP22, ICP47
ICP4, ICP22, ICP27,
ICP0, ICP4, ICP22,
ICP0, ICP22, ICP4767
FIG. 5. ICP27 is necessary for I?B? loss. (A) Replicate cultures of
CV-1 cells were mock infected or infected with wt KOS, d100, d103,
d106, d107, or d109 at the indicated MOI (2.5, 5, or 10). Whole-cell
lysates were prepared at 8 hpi, and Western blotting was performed to
detect I?B?; the membranes were then stripped and reprobed for
?-tubulin. Amounts of I?B? normalized to ?-tubulin are indicated
under the appropriate lanes (see Materials and Methods). (B) CV-1
cells were mock infected or infected (MOI ? 5) with wt KOS, d27-1,
or d100 and harvested at 16 hpi. Lane 5 represents a d100 lysate
prepared at 24 hpi. Western blot analyses for I?B?, ICP27, and ?-tu-
bulin were performed through subsequent stripping and reprobing of
the same membrane.
VOL. 80, 2006 ICP27 ACTIVATION OF NF-?B 10571
with mock infection (lanes 7 to 9). Since the d107 mutant,
which expresses ICP22 and ICP47 in addition to ICP0, re-
tained more I?B? after infection than the d106 mutant, which
expresses only ICP0, we conclude that ICP22 and ICP47 have
no significant role in the degradation of I?B? during HSV
Because d100 infection was capable of activating both JNK
and p38 only at late times during infection (32), we compared
the levels of I?B of mock-infected, wt-infected, d27-1-infected,
and d100-infected cells at 16 hpi, as well as d100-infected cells
at 24 hpi (Fig. 5B). I?B? was not detectable in the lysate of
wt-infected cells at 16 h but I?B? levels were reduced in the
d27-1 and d100 lysates compared to that in the mock-infected
sample. I?B? was not detectable in the d100-infected lysate at
24 hpi (Fig. 5B), though we could detect I?B? in d27-1-in-
fected lysates at 24 hpi in other experiments (data not shown).
To determine whether the I?B? degradation we observed in
d100 infections at 24 hpi was due to induction of apoptosis,
levels of caspase 3 and 7 cleavage were determined. We did not
observe caspase cleavage with wt virus or d100 at either 16 or
24 hpi (data not shown). Cleavage of caspase proteins was
detected in d27-1 infections, consistent with findings that in-
fection with an ICP27 deletion mutant was abortive and in-
duced apoptosis (3–5). Collectively, these results were consis-
tent with the model that ICP27 expression may be sufficient for
the degradation of I?B in the context of viral infection.
Late gene transactivation function of ICP27 is not required
for loss of I?B?. Because of the requirement for ICP27 in
I?B? loss, we began a more detailed analysis of this protein. To
determine the importance of the transactivation functions of
ICP27 and consequent expression of true-late (?2) viral genes
for I?B? loss, we compared the ICP27 mutant vBSLG4, which
at the NPT of 39°C is incapable of stimulating ?2 gene expres-
sion, with revertant vBS3-3 (70, 71, 80). The results presented
in Fig. 6A are representative of the results from three inde-
pendent experiments. Significant loss of I?B? relative to the
mock-infected controls was detected at both the PT and NPT
in both mutant vBSLG4- and revertant vBS3-3-infected cells
(compare lanes 1, 3, and 5 with lanes 2, 4, and 6). At the same
time, ICP27 accumulation was reduced at 33°C compared to
that at 39°C because of the slower kinetics of the infection at
the lower temperature. Figure 6B presents the quantification
of I?B? levels under the various conditions of infection. Rel-
ative to the I?B? levels in mock-infected cells, reductions of 40
to 60% in I?B? levels were seen at 33°C and reductions of 80
to 90% were observed at 39°C, correlating with the amounts of
ICP27 accumulated (Fig. 6A). We analyzed levels of VP16 and
gC, the latter a ?2 gene transactivation target of ICP27, in
order to confirm loss of ICP27 transactivation activity at the
NPT in vBSLG4-infected cells. At 12 hpi, we detected compa-
rable amounts of VP16 and gC in the lysates from vBS3-3-
infected cells at either temperature (lanes 3 and 4). While both
L proteins accumulated at the PT in the lysate from vBSLG4-
infected cells (lane 5), considerably less gC and a reduced
amount of VP16 accumulated at the NPT (lane 6).
We also monitored the status of I?B? in cells infected with
the ICP27 point mutant m11 (48), which is also impaired in
late gene expression. We have consistently observed that in-
fection with the m11 mutant results in less ICP27 accumulation
than infection with wt virus. Therefore, to increase the amount
of m11 ICP27, infections were conducted at an MOI of 20.
While the amount of I?B? in mock- and d27-1-infected cells
remained high (Fig. 6C, lanes 1 and 3), the amount of I?B?
was greatly reduced in wt-KOS- and m11-infected cell lysates
(lanes 2 and 4). Therefore, we conclude that the transactiva-
tion function of ICP27 and ?2 viral gene expression are not
necessary for the activation of the NF-?B pathway, as evi-
denced by the loss of I?B?.
Residues 21 to 63 of ICP27 are required for the loss of I?B?.
We analyzed a series of ICP27 truncation and in-frame dele-
tion mutants to identify which domain of ICP27 was required
for the degradation of I?B?. Table 3 lists the mutants, the
deleted or mutated regions, and their replication capacities in
Vero cells. CV-1 cells were mock infected or infected with wt
HSV-1 or the panel of ICP27 mutants at an MOI of 5. Trip-
licate whole-cell lysates were prepared at 8 hpi, separated by
SDS-PAGE, and analyzed by Western blotting for I?B? and
FIG. 6. The late gene activation function of ICP27 is not required
for loss of I?B?. (A) Replicate cultures of CV-1 cells were mock
infected or infected (MOI ? 5) with either HSV-1 vBS3-3 or vBSLG4
at the indicated temperatures (°C). Whole-cell lysates were prepared
at 8 hpi, and Western blotting was performed to detect loss of I?B? or
accumulation of the indicated viral proteins. NS, nonspecific. (B) From
the results in panel A, I?B? band intensities relative to values from
mock-infected cells at the indicated temperatures (°C) were deter-
mined. Solid bars, mock infected; open bars, vBS3-3 infected; hatched
bars, vBSLG4 infected. (C) Whole-cell lysates were prepared from
mock-infected and infected (MOI ? 20) CV-1 cells as indicated, and
Western blotting was performed to detect I?B? and ICP27.
10572 HARGETT ET AL. J. VIROL.
ICP27 (Fig. 7A to C, which were representative of the results).
Mock-infected cells contained high levels of I?B?, as did cells
infected with the null mutant d27-1, the truncation mutant
n59r (panel A, lanes 1, 3, and 4), and the in-frame deletion
mutant d1-2 (panel B, lane 4). Reduced amounts of I?B? were
observed in wt-virus-infected cells (lane 2 of all panels) and
also following infection with the truncation mutant n504r
(panel A, lane 5) and the in-frame deletion mutants d2-3
(panel B, lane 5), d4-5, d5-6, and d6-7 (panel C, lanes 3 to 5).
Intermediate levels of I?B? were observed with the in-frame
deletion mutants dLeu and d3-4 (panel B, lanes 3 and 6),
suggesting that they may play an auxiliary role in ICP27-in-
duced loss of I?B?. Because the monoclonal antibody H1119
used to probe for ICP27 is directed at an epitope missing from
the dLeu mutant, the presence of this protein was verified
using the monoclonal antibody H1113 (data not shown).
Recently, we reported that d1-2 (aa 12 to 63 deleted) was
incapable of activating JNK and p38, while dLeu (aa 6 to 19
deleted) retained the ability to activate these stress kinases
(41). Since d1-2 was as impaired as d27-1 for loss of I?B?, two
additional mutants within the domain deleted from d1-2 were
characterized. The dAc mutation deletes amino acids 21 to 63
of ICP27, and the expressed protein was primarily nuclear,
though cytoplasmic staining was detectable (41). The affected
domain of ICP27 was termed the export control sequence
because of its ability to restrict shuttling (81). The second
mutant tested was M50T, which effects an amino acid substi-
tution conferring leptomycin B resistance to HSV replication
(53). Leptomycin B is an antifungal antibiotic originally iso-
lated from Streptomyces sp. (30) and was reported to be a
potent inhibitor of nuclear export signal-dependent and Crm-1
dependent export of proteins into the cytoplasm (40). Several
studies have described mechanisms of ICP27-mediated RNA
export by a Crm1-dependent pathway (69, 81) and an Aly/
REF-dependent pathway (11, 38, 50, 57, 69, 80).
To test the ability of dAc and M50T to activate stress-
activated protein kinases and degrade I?B?, we compared
them to wt HSV and d27-1. Three independent infections were
performed, two with an MOI of 5 and one with an MOI of 10,
and cells were harvested at 8 hpi. Western blots from one of
the experiments with infection at an MOI of 5 are shown in
Fig. 8. The data presented were representative of the outcomes
observed in all three experiments. Infection with wt HSV (lane
2) resulted in activation of both p38 and JNK, and as we
FIG. 7. A domain of ICP27 encompassing aa residues 12 to 63 is
necessary for loss of I?B?. Replicate cultures of CV-1 cells were mock
infected or infected with the indicated viruses at an MOI of 5. Whole-
cell lysates were prepared at 8 hpi, fractionated on a 12% polyacryl-
amide gel, and transferred to a membrane for Western blot analysis as
described in Materials and Methods. Membranes were sequentially
probed for I?B? and ICP27. (A) Analysis of wt virus, the null mutant
d27-1, and N-terminal truncation mutants n59r and n504r. Results of
probing the n504r lysate for I?B? and ICP27 were from a separate part
of the same autoradiograph containing the results in lanes 1 to 4.
(B) Analysis of wt virus and in-frame deletion mutants dLeu, d1-2,
d2-3, and d3-4. Because the monoclonal antibody H1119 epitope is
missing in the dLeu mutant, the presence of ICP27 was verified using
the monoclonal antibody H1113 (data not shown). Results of probing
mock-infected, wt-infected, and dLeu-infected lysates for I?B? and
ICP27 were from separate parts of the same autoradiograph contain-
ing the results in lanes 4 to 6. (C) Analysis of wt virus and in-frame
deletion mutants d4-5, d5-6, and d6-7.
TABLE 3. ICP27 mutants
aDeleted or mutated amino acid residues.
bBased on plaquing ratios (Vero/V27) reported in references 4 and 30. D,
defective for growth on Vero; I, inefficient growth on Vero; C, competent for
growth on Vero.
cReplicates at 33.5°C.
VOL. 80, 2006 ICP27 ACTIVATION OF NF-?B10573
previously reported (32), d27-1 showed little to no activation of
either p38 or JNK (lane 3), equivalent to the background
activation seen in mock infection (lane 1). Infection with both
dAc and M50T resulted in little to no activation (lanes 4 and
5). These results suggested that amino acids 21 to 63 were
required for the ICP27-induced activation of the stress-acti-
vated protein kinase and NF-?B pathways (as detected by
I?B? loss) and that the methionine at position 50 was impor-
tant in mediating the induction.
JNK activation is upstream of I?B? loss and Ser276 p65
phosphorylation. To understand if either p38 or JNK activa-
tion was upstream of NF-?B activation, we monitored I?B
levels and p65 phosphorylation after infection in the presence
of pharmacological inhibitors of signaling. Replicate cultures
of CV-1 cells were treated with DMSO or inhibitors for p38
(SB203580), JNK (SP600125), and MEK1 (UO126) for 30 min
prior to infection. Cells were then mock infected or infected
with wt KOS at an MOI of 5 in the presence of inhibitor and
harvested at 8 hpi. Western blot analysis for I?B or phosphory-
lated-p65 Ser276 or Ser536 was performed (Table 4). The
membranes were then stripped and reprobed for ?-tubulin or
total p65. Normalized I?B? and phospho-p65 levels were cal-
culated as described in Materials and Methods. The results
from two independent experiments could not be averaged be-
cause of differences in scale, derived during background cor-
rection but showed similar trends in I?B? and phospho-p65
levels due to inhibitor treatment. Table 4 displays the results of
one representative experiment. When the levels of I?B? were
compared across the inhibitors used, consistent degradation of
I?B? was observed in cells with DMSO- and UO126-treated
infections, as well as at low concentrations of SB203580. The
use of higher concentrations of SB203580 and all concentra-
tions of SP600125 resulted in levels of I?B? similar to those
resulting from mock infection (values in italics). Higher levels
of p38 inhibitor may also affect JNK activity. Therefore, JNK
activity, but not p38, may be required for ICP27-induced I?B?
loss. While HSV infection resulted in levels of Ser276 phos-
phorylation 2- to 10.8-fold higher than those in mock-infected
DMSO-, SB203580-, and UO126-treated cells, exposure to in-
creasing concentrations of SP600125 resulted in increasingly
lower levels of phosphorylated Ser276 (values in italics).
Though these levels were not reduced to levels shown by mock-
infected cells, decreased phosphorylation in the presence of
the inhibitor indicated a role for JNK activity in Ser276 phos-
phorylation. On the other hand, neither SP600125 nor any of
the other inhibitors prevented the modest induction of phos-
pho-Ser536 seen during HSV infection. Inhibition of p38 ki-
nase activity by SB203580 resulted in increased phospho-
Ser536 levels during infection, suggesting that p38 activity may
negatively regulate the ability of the virus to phosphorylate p65
on Ser536. Taken together, these results support a model in
which the loss of I?B? and phosphorylation of p65 at Ser276
TABLE 4. Effects of pharmacological inhibitors of p38, JNK, and
extracellular signal-regulated kinase on I?B? accumulation and
NF-?B p65 phosphorylation after HSV infectiona
SB203580 2.5 ?M
aInfections were performed with wt HSV-1 KOS at an MOI of 5 and lysates
prepared at 8 hpi.
bVehicle or inhibitor present for 30 min prior to and throughout infection.
cConcentrations of DMSO are equivalent to concentrations used to achieve
indicated doses of inhibitors dissolved in DMSO.
dPercent residual I?B? relative to the amount in mock-infected cells treated
with the maximum volume of DMSO or at the maximum drug concentration.
eIncrease (n-fold) in phosphorylated-p65 Ser276 or Ser536 relative to mock-
infected cells treated with the maximum volume of DMSO or at the maximum
FIG. 8. Loss of I?B? correlates with JNK and p38 phosphorylation
during infection with ICP27 mutants affecting aa residues 21 to 63.
Replicate cultures of CV-1 cells were mock infected or infected with
the indicated viruses at an MOI of 5. Whole-cell lysates were prepared
at 8 hpi, fractionated on a 12% polyacrylamide gel, and transferred to
a membrane for Western blot analysis as described in Materials and
Methods. Replicate membranes were sequentially probed for I?B?,
ICP27, and ?-tubulin or for phosphorylated (p) and total JNK and p38.
10574HARGETT ET AL. J. VIROL.
are positively regulated by JNK activity, while phosphorylation
of p65 at Ser536 is negatively regulated by p38 kinase activity.
In this report, we established a correlation between NF-?B
activation, namely p65 nuclear translocation, and the loss of
I?B? after HSV infection. Using I?B? as a surrogate marker,
we characterized requirements for the HSV-induced activation
of NF-?B. Virus binding and entry into the cell were not
sufficient for I?B? degradation, based on infections with the
uncoating mutant tsB7 or with UV-irradiated virus. Using
strategies to limit viral gene expression, we determined that IE
gene expression was sufficient for degradation of I?B. We
confirmed the sufficiency of IE gene expression with the ICP4
mutant vi13, which expresses IE genes only. We previously
reported that extracts prepared from both ICP4 mutant vi13-
and ICP27 mutant d27-1-infected C33-A cells did not display
increased NF-?B DNA binding activity, as measured by EMSA
(56), while infection with wt HSV or an ICP8 mutant resulted
in large increases in NF-?B DNA binding activity in C33-A or
U2-OS cells. I?B degradation was not evaluated for either cell
type (56). We speculate that the I?B? degradation observed
here following vi13 infection in CV-1 cells is regulated sepa-
rately from NF-?B DNA binding activity. In this model, ICP4
may play a role in the efficient or sustained activation of NF-?B
during HSV infection through regulation of DNA binding ac-
tivity, while ICP27 activity is required for loss of I?B and
subsequent nuclear translocation of NF-?B (Fig. 3). The ef-
fects of ICP4 on NF-?B may be associated with the ICP27
effects, since the vi13 mutant was not as efficient as wt virus in
causing loss of I?B?. ICP4 and ICP27 are known to physically
interact (55), and if complexed during NF-?B activation, loss
of one protein from the complex could affect the complex
structure and therefore its activities. Additional experiments to
determine which viral proteins interact with which cellular
factors in this pathway are needed to better define the rela-
tionship between ICP4 and ICP27 during NF-?B activation.
We further investigated the individual contributions of ICP4
and ICP27 in a panel of immediate-early deletion mutants.
Over a 12-h time course, wt-HSV infection resulted in I?B
degradation. ICP4, ICP0, or ICP6 mutant infection also re-
sulted in degradation, although at a reduced rate, suggesting
that complete degradation of I?B would eventually be ob-
served in these mutants. On the other hand, ICP27 mutant-
infected cells retained 99% of the level of I?B in mock-infected
cells even out to 12 hpi. In addition, infection with the d100
mutant, which expresses only ICP27, ICP22, and ICP47, even-
tually resulted in decreased levels of I?B, demonstrating that
ICP4 was not absolutely required for the degradation of I?B?
during the activation of the NF-?B pathway. This result was
consistent with I?B degradation seen in vi13 mutant-infected
We also characterized the phosphorylation status of p65
during HSV infection. The phosphorylation of p65 on Ser276
leads to increased NF-?B transcriptional activation (87) and
may lead to more efficient recruitment of CBP (91). Constitu-
tive phosphorylation of Ser536 was detected in mock-infected
lysates (data not shown), with a higher level of phosphorylation
seen after 6 hpi, which dropped to twofold at 8 hpi. As the peak
of Ser536 phosphorylation occurred as degradation of I?B?
(56) and phosphorylation of Ser276 were beginning, phosphory-
lation at Ser536 may be an earlier and transient event in the
NF-?B activation cascade. Since Ser536 phosphorylation was
less sensitive to ICP4 and ICP27 deletion than was Ser276
phosphorylation, especially at 12 hpi, the mechanism of Ser536
phosphorylation may require other viral factors or events in
the viral life cycle. Ser536 is located in the transactivation
domain of p65 (75), can be phosphorylated in an I?B?-inde-
pendent manner (75) and, when phosphorylated, mediates in-
creased transcriptional activity (65, 66, 78). We are continuing
our analysis of posttranslational modifications to p65 in wt-
and mutant-infected cells.
A recent report presented findings indicating that ICP0,
when overexpressed from a plasmid, could directly bind and
ubiquitylate I?B in vitro (17). Additionally, polyubiquitin
chains were not formed when a RING finger point mutant for
bovine herpesvirus 1 ICP0 (13G/51A) was expressed, suggest-
ing that ICP0-dependent I?B degradation and subsequent
NF-?B activation was dependent on this domain. Results of
our immediate-early mutant time course led us to conclude
that ICP0 is not required for I?B? loss. We have tested a
similar HSV-1 ICP0 mutant (C116G/C156A) (12, 55) for acti-
vation of p38, shown here to be an upstream activator of
NF-?B, and found that this RING finger domain mutant was
not impaired for activation of p38 (data not shown). We are
currently studying the ability of this RING finger mutant to
degrade I?B. Our studies evaluated the impact of deleting
ICP0 while delivering the rest of the viral genome. The differ-
ence between these two studies may suggest redundant mech-
anisms for the degradation of I?B. Alternatively, it may suggest
that during infection, ICP0 may be localized to areas where it
does not interact with I?B, demonstrating that the ICP0-de-
pendent ubiquitylation of I?B may reflect altered localization
of ICP0 in the absence of other viral proteins.
Using a panel of ICP27 mutant viruses, we identified a N-
terminal region (amino acids 12 to 63) previously implicated in
nuclear-cytoplasmic shuttling (50), as required for the ICP27-
dependent degradation of I?B in CV-1 cells. Viruses express-
ing ICP27 with deletions from this region are deficient in
HSV-induced apoptosis suppression (5). Other regions of
ICP27 involved in apoptosis suppression were C-terminal of
residue 263 or point mutations involved in ICP27 transactiva-
tion activity or ICP27 nuclear-cytoplasmic shuttling (5) in hu-
man cells. Since ICP27 mutants do not induce significant apop-
tosis in cells of nonhuman primate origin, we chose to evaluate
ICP27 mutants for NF-?B activation in CV-1 cells. Our obser-
vations on the importance of the N-terminal region of ICP27 in
NF-?B activation are consistent with previous reports demon-
strating (i) a requirement for NF-?B activation for apoptosis
suppression in HSV infections (24), (ii) a requirement for IE
gene expression but not VP16 in the induction of apoptosis
during HSV infection (74), and (iii) correlations between
ICP27 (3) and E and leaky-late (5) gene expression and the
ability to suppress apoptosis during HSV infection.
As other regions of ICP27 have been identified as being
important for apoptosis suppression, functions of ICP27 in
addition to NF-?B activation may be required for complete
suppression. In one model, sustained NF-?B activation could
be mediated by ICP27-regulated E or leaky-late proteins, per-
VOL. 80, 2006 ICP27 ACTIVATION OF NF-?B 10575
haps by phosphorylation of one or both upstream activators
IKK and I?B during infection (1, 56). The need for E and L
genes for sustained NF-?B activity could also explain the po-
tential role for ICP4, as it is required for expression of all E
and L genes. In another model, ICP27 could serve as the nexus
between a cellular factor, e.g., CK2 (7, 39), and key apoptosis
In this report, we also demonstrated that I?B degradation
was inhibited in the presence of the JNK inhibitor
SP100625, suggesting that JNK is upstream of NF-?B acti-
vation. Though an IKK-independent activation of NF-?B
mediated by a p38-CK2-I?B phosphorylation pathway has
been described previously (36), NF-?B activation during
HSV infection was dependent on the IKKs (2, 27). There-
fore, JNK-induced I?B degradation would have to target
IKK/I?B or a further upstream activator. A possible mech-
anism for JNK-dependent I?B phosphorylation during HSV
infection could involve I?B kinase complex-associated protein
(IKAP). IKAP interacts with JNK through a C-terminal do-
main (33) and was reported to induce JNK activation after
stimulation of HeLa or 293 cells with tumor necrosis factor
alpha and UV irradiation (33). IKAP can be detected in a
complex that includes NIK, IKK?, IKK?, I?B, and NF-?B
(13). IKK may be required for IKAP-I?B complex formation,
explaining its role in HSV-induced NF-?B activation. JNK may
interact with other proteins, such as ?-transducin repeat con-
taining protein, to cause I?B phosphorylation (82). Further
characterization of HSV-induced I?B phosphorylation is re-
quired to test these models.
An additional JNK-mediated mechanism for regulating
NF-?B involves phosphorylation of p65. MSK-1 was reported
to phosphorylate p65 on Ser276 (87), the same serine residue
phosphorylated after lipopolysaccharide treatment (90, 91).
MSK-1 phosphorylation of p65 can be mediated via either p38
or extracellular signal-regulated kinase (87). While JNK has
not previously been demonstrated to activate MSK, interac-
tions with viral proteins may affect the ability of JNK to phos-
phorylate a substrate and, therefore, cannot be ruled out as a
mechanism at this time.
This work was supported by NIH grant AI43314.
We thank Jessica Prince for technical support and helpful discus-
sions of the data and Devon Gregory for technical advice. We thank
Neal DeLuca, David Knipe, Priscilla Schaffer, and Saul Silverstein for
providing some of the HSV mutants and David Knipe, Gary Cohen,
and Roz Eisenberg for some of the antibodies used in these studies.
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