Analysis of the cell cycle regulatory protein (E2F1) after infection of cultured cells with bovine herpesvirus 1 (BHV-1) or herpes simplex virus type 1 (HSV-1).

Abstract

The E2F family of cellular transcription factors controls cell cycle progression and cell death. During cell cycle progression, activated cyclin-dependent kinases phosphorylate the retinoblastoma (Rb) protein, causing the release and activation of E2F family members. Previous studies demonstrated that bovine herpes virus 1 (BHV-1) productive infection increases E2F1 protein levels, the bICP0 early promoter is activated more than 100 fold by E2F1 or E2F2, and silencing E2F1 reduced the efficiency of productive infection. In this study, the effect of herpes simplex virus type 1 (HSV-1) productive infection on E2F protein levels and regulation of E2F dependent transcription was compared to BHV-1 infection in the same permissive cell line, rabbit skin (RS) cells. Silencing E2F1 with a specific siRNA reduced HSV-1 productive infection approximately 10 fold in RS cells, and total E2F1 protein levels increased during productive infection. In contrast to RS cells infected with BHV-1, a fraction of total E2F1 protein was localized to the cytoplasm in HSV-1 infected RS cells. Furthermore, E2F1 did not efficiently trans-activate the HSV-1 ICP0 or ICP4 promoter. When RS cells were transfected with an E2F reporter construct or the cyclin D1 promoter and then infected with BHV-1, promoter activity increased after infection. In contrast, HSV-1 infection of RS cells had little effect on E2F dependent transcription and cyclin D1 promoter activity was reduced. In summary, these studies indicated that silencing E2F1 reduced the efficiency of HSV-1 and BHV-1 productive infection. However, only BHV-1 productive infection induced E2F dependent transcription.

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Virus Research 160 (2011) 66–73
Contents lists available at ScienceDirect
Virus Research
journal homepage: www.elsevier.com/locate/virusres
Analysis of the cell cycle regulatory protein (E2F1) after infection of cultured cells
with bovine herpesvirus 1 (BHV-1) or herpes simplex virus type 1 (HSV-1)
Aspen Workmanb,c, Clinton Jonesa,b,c,∗
aSchool of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Fair Street at East Campus Loop, Lincoln, NE 68583-0905, United States
bSchool of Biological Sciences, University of Nebraska-Lincoln, Fair Street at East Campus Loop, Lincoln, NE 68583-0905, United States
cNebraska Center for Virology, University of Nebraska-Lincoln, Fair Street at East Campus Loop, Lincoln, NE 68583-0905, United States
a r t i c l ei n f o
Article history:
Received 22 February 2011
Received in revised form 10 May 2011
Accepted 11 May 2011
Available online 23 May 2011
Keywords:
Herpes simplex virus type 1
Bovine herpesvirus 1
E2F dependent transcription
Productive infection
a b s t r a c t
The E2F family of cellular transcription factors controls cell cycle progression and cell death. During cell
cycle progression, activated cyclin-dependent kinases phosphorylate the retinoblastoma (Rb) protein,
causing the release and activation of E2F family members. Previous studies demonstrated that bovine
herpes virus 1 (BHV-1) productive infection increases E2F1 protein levels, the bICP0 early promoter is
activated more than 100 fold by E2F1 or E2F2, and silencing E2F1 reduced the efficiency of productive
infection. In this study, the effect of herpes simplex virus type 1 (HSV-1) productive infection on E2F
protein levels and regulation of E2F dependent transcription was compared to BHV-1 infection in the
same permissive cell line, rabbit skin (RS) cells. Silencing E2F1 with a specific siRNA reduced HSV-1
productive infection approximately 10 fold in RS cells, and total E2F1 protein levels increased during
productive infection. In contrast to RS cells infected with BHV-1, a fraction of total E2F1 protein was
localized to the cytoplasm in HSV-1 infected RS cells. Furthermore, E2F1 did not efficiently trans-activate
the HSV-1 ICP0 or ICP4 promoter. When RS cells were transfected with an E2F reporter construct or the
cyclinD1promoterandtheninfectedwithBHV-1,promoteractivityincreasedafterinfection.Incontrast,
HSV-1infectionofRScellshadlittleeffectonE2FdependenttranscriptionandcyclinD1promoteractivity
was reduced. In summary, these studies indicated that silencing E2F1 reduced the efficiency of HSV-
1 and BHV-1 productive infection. However, only BHV-1 productive infection induced E2F dependent
transcription.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
During productive infection of cultured cells, gene expression
of herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1
(BHV-1), both alpha-herpesvirinae subfamily members, is tempo-
rally regulated in three distinct phases: immediate early (IE), early
(E), or late (L), reviewed in Jones (1998, 2003). In contrast to small
DNA tumor viruses, alpha-herpesvirinae subfamily members do
not appear to promote entry into the S phase of the cell cycle. For
example, HSV-1 encodes several genes, ICP0, ICP27, ICP22/US1.5,
andUL13,whichinhibitcellcycleprogression(Advanietal.,2000b;
Advani et al., 2000a; Flemington, 2001; Hobbs and DeLuca, 1999;
Lomonte and Everett, 1999; Orlando et al., 2006; Song et al.,
2001). However, drugs that interfere with cyclin dependent kinase
activity, roscovitine and olomucine, reduce the efficiency of pro-
∗Corresponding author at: School of Veterinary Medicine and Biomedical Sci-
ences, University of Nebraska-Lincoln, Fair Street at East Campus Loop, Lincoln, NE
68583-0905, United States. Tel.: +1 402 472 1890; fax: +1 402 472 9690.
E-mail address: cjones@unlnotes.unl.edu (C. Jones).
ductive infection (Schang et al., 1998, 1999) suggesting that certain
cell cycle regulatory proteins facilitate HSV-1 replication. The E2F
family of transcription factors includes cell cycle regulatory pro-
teins that interact with Rb family members (Rb, p107, and p130)
(Harbour and Dean, 2000). Phosphorylation of Rb family members
by cyclin dependent kinase/cyclin complexes leads to E2F release,
andconsequentlycertainE2Ffamilymembers(E2F1,-2,or-3)acti-
vate transcription (Attwooll et al., 2004; Harbour and Dean, 2000;
Nevins et al., 1997; Weintraub et al., 1992). Consensus E2F binding
sites are present in the promoters of many genes that control cell
cycleprogression(DeGregorietal.,1995;Nevinsetal.,1997;Ohtani
et al., 1995; Schulze et al., 1995; Wells et al., 1997). Many DNA syn-
thetic genes are activated by E2F family members (Harbour and
Dean, 2000) suggesting transient induction of E2F dependent tran-
scriptionbyalpha-herpesvirinaesubfamilymembersmayenhance
productive infection in highly differentiated cells.
TheE2FfamilyoftranscriptionfactorsstimulatesBHV-1produc-
tive infection and reactivation from latency. For example, a recent
study demonstrated that a siRNA directed against E2F1 inhibits
BHV-1 productive infection, E2F1 protein levels and binding activ-
ity increase after infection, and E2F1 or E2F2 stimulates the bICP0
0168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.virusres.2011.05.009

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A. Workman, C. Jones / Virus Research 160 (2011) 66–73
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early promoter more than 100 fold (Workman and Jones, 2010).
Furthermore,over-expressionofE2F4stimulatesBHV-1productive
infection and E2F1 or E2F2 trans-activates IEtu1 (immediate early
transcription unit 1) promoter activity (Geiser and Jones, 2003).
During dexamethasone-induced reactivation from BHV-1 latency,
sensory neurons that express abundant levels of lytic cycle genes
also express cyclin E and cyclin A (Winkler et al., 2000). Although
these studies suggest that increased E2F protein levels and bind-
ing activity are important for productive infection, BHV-1 may also
increase E2F1 protein levels because productive infection leads to
p53 dependent apoptosis (Devireddy and Jones, 1999).
WithrespecttoHSV-1productiveinfection,“free”E2F(notasso-
ciated with Rb family members) increases following infection of a
human tumor cell line (C33-A) (Hilton et al., 1995). Re-localization
of E2F4 to the nucleus occurs in human tumor cell lines (C33-A
and U2-OS) following HSV-1 infection (Olgiate et al., 1999). Fur-
ther support for E2F4 playing a role in HSV-1 replication comes
from the findings that infection of mouse cells that lack p107−/−
and p130−/−, two Rb family members that interact with E2F4,
leads to reduced infectious virus (Ehmann et al., 2001). In primary
human fibroblasts or Hela cells, the subcellular distribution of E2F4
isalteredfollowingHSV-1infection,whichisassumedtoinactivate
E2F4 (Advani et al., 2000b). This same study also concluded that
HSV-1 infection leads to post-translational modification of E2F1
and E2F5, translocation of E2F family members from the nucleus to
the cytoplasm, and reduced E2F binding to consensus E2F binding
sites. Based on these observations, HSV-1 appears to inactivate E2F
dependent signaling.
In this study, we compared the effects of HSV-1 versus BHV-1
infection on E2F1 and E2F dependent transcription in the same cell
line. Like BHV-1, an E2F1-specific siRNA reduced HSV-1 productive
infection following infection of rabbit skin cells. Although BHV-1
and HSV-1 both increased E2F1 protein levels, a significant amount
of the E2F1 protein was localized in a cytoplasmic fraction follow-
ing HSV-1 infection. Conversely, E2F1 was localized to the nucleus
followingBHV-1infection.BHV-1,butnotHSV-1,infectionofrabbit
skin cells enhanced E2F dependent transcription.
2. Results
2.1. An E2F1 specific siRNA reduced the levels of HSV-1
productive infection
Silencing E2F1 decreases the levels of BHV-1 productive infec-
tion, in part, because E2F1 stimulates the bICP0 early (E) promoter
more than 100 fold in transient transfection assays (Workman and
Jones, 2010). Since BHV-1 and HSV-1 are both alpha-herpesvirinae
subfamily members, we hypothesized that both viruses require
similar transcription factors, including E2F1, for efficient produc-
tive infection. To test whether E2F1 influenced HSV-1 productive
infection, we examined the effect of an E2F1 specific siRNA on viral
growth in rabbit skin (RS) cells, which supports productive infec-
tion by BHV-1 and HSV-1. RS cells are not transformed by a known
oncogene, they can be readily transfected with siRNA and plasmids
(WorkmanandJones,2010),andthusarewellsuitedforthesestud-
ies.RelativetothecontrolsiRNA,co-transfectionoftheE2F1siRNA,
but not the control siRNA, with the HSV-1 genome reduced the
number of plaques more than 10-fold (Fig. 1A).
Previous studies demonstrated that the E2F1 specific siRNA
reduced E2F1 protein levels, but not E2F2 or ?-actin protein levels
(Workman and Jones, 2010). Additional studies were performed to
evaluate the effect of the E2F1 siRNA on the cell cycle in uninfected
cells. The E2F1 siRNA reduced the percent of cells in S phase less
than 2 fold at 24h after transfection (Fig. 1B). However, silencing of
E2F1 did not dramatically increase apoptosis or cell death. By 48h
Fig. 1. Suppression of E2F1 reduced the levels of productive infection. (Panel A) to
test whether E2F1-specific siRNA affected HSV-1 productive infection, RS cells were
transfected with 100nM E2F1 siRNA or 100nM control siRNA for 24h, followed
by transfection of HSV-1 genomic DNA as described in the methods. Forty-eight
hours after transfection of genomic DNA cells were collected, subjected to three
rounds of freeze/thaw cycles and plaque assays performed on RS cells. The data is
an average of three independent studies and the error bars are standard deviations.
(Panel B) to examine the effect of the siRNAs on cell cycle, RS cells were transfected
with 100nM E2F1 siRNA or 100nM control siRNA. At 24 or 48h after transfection,
cells were collected, fixed, stained and analyzed by flow cytometry as described in
the methods section. These studies are representative of at least three independent
studies.
after transfection, there was no difference in the percent of cells
in S phase regardless of the treatment. In summary, these stud-
ies demonstrated that silencing E2F1 protein levels reduced HSV-1
productive infection, which was similar to the effect of the same
siRNA on BHV-1 productive infection (Workman and Jones, 2010).
2.2. Analysis of E2F1 trans-activation of HSV-1 promoter
constructs
Previous studies revealed that E2F1 trans-activated the BHV-1
bICP0 early (E) promoter more than 100 fold in transient trans-
fection assays (Workman and Jones, 2010). Two E2F1 responsive
regions (ERR) were identified within the bICP0 early promoter, one
adjacent to the TATA box (ERR1) and one approximately 600bp
upstream of the TATA box (ERR2). When cloned upstream of a
minimal HSV-1 thymidine kinase promoter, ERR1 stimulated pro-
moter activity 6 fold whereas ERR2 stimulate promoter activity
more than 20 fold. Mobility shift assays provided further evidence
that E2F1 interacted directly with these sequences (Workman and
Jones, 2010). Therefore, we hypothesized that E2F1 may stimulate
promoters of important HSV-1 regulatory genes.
To test this hypothesis, neuro-2A cells were cotransfected with
an HSV-1 ICP0 or ICP4 promoter construct plus an E2F1 expression
plasmid, and CAT activity was measured. Neuro-2A cells were use
for this study because these cells are neuronal-like and it was of
interest to test whether E2F regulates gene expression in neurons.
Furthermore, E2F1 over-expression is not as toxic in neuro-2A cells
when compared to RS cells (data not shown). The HSV-1 ICP0 pro-
moterwasactivatedonly3foldbyE2F1(Fig.2A).TheICP4promoter
wasconsistentlylowerthanbasalactivitywhencotransfectedwith
E2F1 (Fig. 2A).

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A. Workman, C. Jones / Virus Research 160 (2011) 66–73
Fig. 2. Analysis of E2F1 transactivation of HSV-1 promoter constructs. (Panel A) to examine the effect of E2F1 on HSV-1 promoters, neuro-2A cells were cotransfected with
1?g of the designated promoter-CAT construct and 0.1?g of the E2F1 expression plasmid. At 48h post-transfection, cells were collected and CAT activity measured. The
data is an average of three independent studies and the error bars are standard deviations. (Panel B) to test whether silencing E2F1 protein levels reduced basal activity
of HSV-1 promoters, neuro-2A cells were cotransfected with 1?g of the designated promoter-CAT construct and 100nM E2F1 siRNA or a control siRNA (cont-si). At 48h
post-transfection, cells were collected and processed for CAT activity. (Panel C) neuro-2A cells were cotransfected with 1?g of the designated promoter-luciferase construct
and 0.1?g of the E2F1 expression plasmid. At 48h post-transfection, cells were collected and processed for luciferase expression. The data is an average of three independent
studies and the error bars are standard deviations.
We may have under-estimated the effects of E2F1 on ICP0 and
ICP4 promoter activity because E2F family members can induce
apoptosis (Attwooll et al., 2004; Chang et al., 2000; Harbour and
Dean, 2000) and the HSV-1 ICP0 as well as the ICP4 construct had
high basal activities. Consequently, we examined whether reduc-
ing E2F1 protein levels by the E2F1 specific siRNA had an effect on
ICP0andICP4basalpromoteractivity.Neuro-2Acellswerecotrans-
fected with the designated promoter-CAT construct and the E2F1
siRNA or a control siRNA (cont-si). At 48h after transfection, cells
were collected and CAT activity measured. The E2F1 specific siRNA
reduced ICP0 promoter activity approximately 60%: whereas the
control siRNA did not reduce ICP0 promoter activity (Fig. 2B). The
E2F1 siRNA reduced ICP4 promoter activity by approximately 40%.
In contrast, the control siRNA reduced ICP4 promoter activity less
than 20% (Fig. 2B).
Finally, we tested whether E2F1 had an effect on a panel of
HSV-1promoter-luciferaseconstructs.Ofthoseexamined,theearly
UL42 promoter (L42-Luc column) was activated approximately 2.5
fold (Fig. 2C). UL42 encodes a protein that stimulates the viral
DNA polymerase (Gottlieg and Challberg, 1994). Since E2F1 acti-
vates expression of genes that are necessary for DNA replication
(DeGregori et al., 1995), this result suggested that under certain
conditions E2F1 might promote expression of certain viral genes
that are necessary for DNA replication. The late VP16 and gC pro-
moters as well as the early ICP6 promoter were not activated by
E2F1. In summary, the HSV-1 promoters we examined were not
efficiently trans-activated by E2F1 in neuro-2A cells.
2.3. Analysis of E2F1 protein levels during productive infection
Previous studies demonstrated that E2F1 protein levels
increased during BHV-1 productive infection in bovine kidney cells
(Workman and Jones, 2010). Additional studies were performed to
directly compare the effect of BHV-1 or HSV-1 productive infec-
tion on E2F1 protein levels in RS cells. RS cells were infected with
BHV-1 or HSV-1 using a moi of 5 and whole cell lysate collected at
thedesignatedtimespost-infection.Westernblotanalysisrevealed
that BHV-1 increased E2F1 protein levels by 16h after infection
(Workman and Jones, 2010 and Fig. 3A). Higher levels of E2F1 were
detectedinRScellwithin8hafterinfectionwithHSV-1usingamoi
of 5 (Fig. 3B). When RS cells were infected with HSV-1 at a moi of
0.5(Fig.3C)or0.1(datanotshown),enhancedE2F1levelswerenot
readily detected until 16h after infection. In RS cells, HSV-1 grows
faster than BHV-1 at a moi of 0.5 or lower; however, the end point
titers of BHV-1 and HSV-1 are similar when RS cells are infected
with a moi of 0.5 or higher (data not shown). We suggest that dif-
ferences in the growth rate of the two viruses was one reason why
higher levels of E2F1 were detected in HSV-1 infected RS cells at
8h after infection when RS cells were infected with a moi of 5.
The E2F1 band detected after HSV-1 infection appeared to
migrate differently compared to E2F1 after BHV-1 infection or
in mock-infected cells (Fig. 4A). E2F can be phosphorylated and
phosphorylation is crucial for regulating transcriptional activation,
localization, and stability (Garcia-Alvarez et al., 2007; Real et al.,
2010; Weei-Chin et al., 2011). To test whether E2F1 was phospho-
rylated, whole cell extract from HSV-1 infected cells was treated
with calf intestinal alkaline phosphatase (CIAP) and then western
blot analysis performed using an anti-E2F1 antibody (Fig. 4B). CIAP
treatment resulted in a decrease in the electrophoretic mobility
of E2F1 in RS cells (Fig. 4B) suggesting the observed shift in E2F1
mobility after HSV-1 infection was the result of phosphorylation.
To visualize E2F1 in mock-infected cells, 200?g of protein in the
cell lysate was loaded versus 20?g protein derived from infected
samples.SimilarresultswereobservedwhencomparingBHV-1and
HSV-1 infection in the human osteosarcoma cell line (U2OS) that is
permissive for both viruses (data not shown).
Additional studies were performed to examine E2F1 protein
levels in primary human embryonic lung (HEL) fibroblasts after
infection with HSV-1. The rationale for this study was to compare
the results obtained in RS cells to a low passage human cell type.

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Fig. 3. Analysis of E2F1 protein levels during productive infection, The effect of BHV-1 or HSV-1 infection on E2F1 protein levels was measured by western blot analysis.
RS cells were infected with BHV-1 (moi=5; Panel A), HSV-1 at a (moi=5; Panel B), or HSV-1 at a moi of 0.5 (Panel C) for the designated times after infection (h). Cells were
collected,lysed,and100?gofproteinwasanalyzedbywesternblotusinganE2F1polyclonalantibodydiluted1:10,000.Asacontrol,theblotwasalsoprobedwithantiserum
against ?-actin. Lanes M4 or M24 are 4 or 24h respectively after mock infection.
Confluent cultures of HEL fibroblasts were infected with HSV-1 at a
moi of 1 and whole cell lysate collected at the designated times (h)
after infection. In HEL cells, HSV-1 infection increased the steady
state E2F1 protein levels at 16h after infection (Fig. 5A). CIAP treat-
ment resulted in a decrease in the electrophoretic mobility of E2F1
in HEL cells (Fig. 5B). In summary, both BHV-1 and HSV-1 increased
E2F1proteinlevels,andHSV-1infectionledtonovelchangesinthe
phosphorylation status of E2F1 in RS and HEL cells.
2.4. Analysis of E2F1 localization following infection
A previous study revealed that E2F1 remained localized to the
nucleus following infection of bovine kidney cells with BHV-1
(Workman and Jones, 2010). Conversely, E2F1 was reported to
localize to the cytoplasm following HSV-1 infection of HeLa cells
(Advani et al., 2000b). Confocal microscopy suggested that a sub-
set of E2F1 was localized to the cytoplasm after infection of RS
cells with HSV-1 (data not shown). To confirm these findings and
comparethemtoBHV-1productiveinfection,biochemicalfraction-
ation studies were conducted in RS cells. Two additional moi, 0.1
or 0.5pfu/cell, were used for the HSV-1 studies. A moi of 5pfu/cell
was used for BHV-1 because BHV-1 does not grow as quickly as
HSV-1 in RS cells (data not shown). Consistent with previous stud-
ies (Workman and Jones, 2010), E2F1 was detected in the nuclear
extract, but not the cytoplasm, of BHV-1 infected cells at 16 or 24h
after infection (Fig. 6A). At a lower moi, E2F1 was also detected
Fig. 4. Analysis of E2F1 protein levels in infected RS cells. (Panel A) Cell lysate prepared from RS cells infected with BHV-1 (moi=5) or HSV-1 (moi=5) for 24h were separated
on the same SDS-polyacrylamide gel to show mobility differences of E2F1. (Panel B) to determine whether phosphorylation was responsible for the altered mobility of E2F1,
20?g of protein derived from cell lysate prepared from RS cells infected with HSV-1 for 24h was treated (or not) with calf intestinal alkaline phosphatase (CIAP). As a control,
200?g of mock-infected RS cells was analyzed. More protein was loaded in the mock lane to visualize the E2F1-specific bands.

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A. Workman, C. Jones / Virus Research 160 (2011) 66–73
Fig.5. AnalysisofE2F1proteinlevelsinnormallowpassagehumanlungfibroblasts.
(Panel A) Low passage human embryonic lung fibroblasts (HEL) were infected with
HSV-1 at a moi of 1 for the designated times after infection (h). Cells were collected,
lysed, and 100?g of protein analyzed by western blot using an E2F1 polyclonal
antibody diluted 1:10,000. As a control, the blot was also probed with antiserum
directed against ?-actin. (Panel B) Twenty micrograms of protein from the sample
at 24h after infection of HEL cells were treated (or not) with calf intestinal alkaline
phosphatase (CIAP) and ran next to 200?g of cell lysate from mock-infected cells.
More protein was loaded in the mock-infected lane (lane HEL) to visualize E2F1-
specific bands.
in the nucleus of RS cells infected with BHV-1 (data not shown).
Alternatively, E2F1 was detected in the nuclear and cytoplasmic
extract following infection with HSV-1 for 16 or 24h regardless of
whether the moi was 0.1 or 0.5 (Fig. 6B). Eight hours after HSV-1
infection (moi of 0.1 and 0.5pfu/ml) E2F1 was primarily detected
in the nucleus of infected cells. As a control for nuclear proteins, we
examined histone H3 and found, as expected, that histone H3 was
detected only in the nucleus after infection of RS cells with HSV-1
or BHV-1 (Fig. 6A and B).
2.5. Analysis of E2F-dependent transcription following infection
The studies in Figs. 3–6 indicated that HSV-1 and BHV-1
increased E2F1 protein levels, but these studies did not reveal
whether E2F dependent transcription correlated with increased
E2F1 protein levels. Therefore, we examined the effect of BHV-1
or HSV-1 productive infection on E2F dependent transcription. RS
cells were initially transfected with a luciferase construct contain-
ing a minimal promoter with three consensus E2F binding sites
(3xE2F) or the Cyclin D promoter (CycD). Cells were then infected
with BHV-1 or HSV-1 at a moi of 0.1, 0.5, or 5 for 24h. BHV-1 infec-
tion increased 3xE2F promoter activity approximately 5 fold and
CycD promoter activity approximately 4 fold in RS cells (Fig. 7). In
contrast, HSV-1 had little or no effect on the 3xE2F promoter con-
struct,andCycDpromoteractivitywasconsistentlyreduced.These
results indicated that E2F dependent transcription was stimulated
following infection of RS cells with BHV-1, but not HSV-1.
3. Discussion
In this study, we compared the effect that BHV-1 had on the
cell cycle regulated transcription factor E2F1 versus HSV-1. For
these studies, we used a spontaneously immortalized rabbit skin
cell line that supports productive infection of both viruses. The
studies clearly demonstrated that a siRNA directed against E2F1
reduced the plaque forming efficiency of HSV-1 (Fig. 1) and BHV-1
Fig. 6. Localization of E2F1 after infection with BHV-1 or HSV-1. (Panel A) RS cells were infected with BHV-1 at a moi of 5 for 8, 16, or 24h. Cells were then harvested and cell
fractionation conducted as described in the methods section. Cytoplasmic and nuclear fractions (100?g) were analyzed by western blot using an E2F1 polyclonal antibody
diluted 1:10,000. As a control for proteins that are in the nucleus, the respective fractions were probed with an antibody directed against Histone H3 that was diluted 1:500.
(Panel B) RS cells were infected with HSV-1 at a moi of 0.1 or 0.5 for 8 or 16h. Cellular fractions were analyzed as described in panel A. Lane M was cell lysate derived from
mock-infected cells.

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Fig. 7. Activity of E2F-responsive promoters following BHV-1 or HSV-1 infection.
RS cells were transfected with 1?g of the designated promoter-luciferase construct
(a promoter containing 3 consensus E2F1 binding sites: 3xE2F or the cyclin D pro-
moter: CycD). Twenty-four hours after transfection, cells were infected with BHV-1
orHSV-1atamoiof0.1,0.5,or5.At24hafterinfection,celllysatewascollectedand
luciferase activity measured. The results are the average of 3 independent experi-
ments. The data is an average of three independent studies and the error bars are
standard deviations.
(WorkmanandJones,2010).ForHSV-1,thiswassomewhatsurpris-
ing because E2F dependent transcription was not activated after
infection. In this study and a previous study, we demonstrated that
BHV-1 infection induced E2F dependent transcription and bind-
ing to a probe containing E2F consensus binding sites (Workman
and Jones, 2010). We suggest that silencing E2F1 prior to infection
reduces the number of cells that are cycling, which consequently
reduces the efficiency of productive infection. This conclusion is
supported by other studies demonstrating that cyclin dependent
kinase inhibitors, roscovitine and olomucine, reduce HSV-1 virus
replication and transcription (Schang et al., 1998, 1999). Roscovi-
tine also efficiently reduces BHV-1 productive infection (data not
shown).
Following infection with HSV-1, E2F1 appeared to be differen-
tially phosphorylated and a fraction of the total E2F1 protein was
localizedinthecytoplasmicfraction.Incontrast,E2F1waslocalized
tothenucleusafterBHV-1infection,asjudgedbybiochemicalfrac-
tionation (Fig. 6) and confocal microscopy (Workman and Jones,
2010). Since HSV-1 inactivated E2F dependent transcription, we
suggestthatphosphorylationofE2F1promoteslocalizationofE2F1
tothecytoplasm,resultinginalossofE2Fdependenttranscription.
Inactivating E2F1 by HSV-1 may be important for efficient pro-
ductive infection because E2F1 induces apoptosis following DNA
damage (Nip et al., 1997). HSV-1 encodes two protein kinases, US3
and UL13 that are packaged in the virion and tegument respec-
tively (Chee et al., 1989; Frame et al., 1987; Purves et al., 1987;
Smith and Smith, 1989). Plasmids over-expressing the two viral
kinases were cotransfected with E2F1 and there was no obvious
changes in the mobility of E2F1 (data not shown) suggesting the
viral encoded protein kinases do not directly phosphorylate E2F1.
E2F1 is directly phosphorylated by several cellular protein kinases,
including glycogen synthase kinase-3?, ATM (ataxia telangiectasia
mutated), and mitogen activated protein kinases (MAPK) (Garcia-
Alvarezetal.,2007;Realetal.,2010;Weei-Chinetal.,2011).HSV-1
activates MAPK and ATM during productive infection (Boutell and
Everett,2004;Corcoranetal.,2006;Hargettetal.,2005)suggesting
E2F1 was phosphorylated by one of these cellular protein kinases.
It is not known whether BHV-1 infection induces MAPK or ATM
protein kinases during the course of productive infection.
TranscriptionallycompetentE2Ffamilymembersareassociated
with a family of DP proteins (Helin et al., 1993) suggesting BHV-
1 stimulates E2F dependent transcriptional activity by promoting
interactions between E2F1 and DP family member. Conversely,
HSV-1 may uncouple E2F1 dependent transcription by inactivat-
ing DP family members. A recent study identified a novel DP family
member(DP-4)inseveralhumancelltypesthatexhibitshighlevels
ofexpressionfollowingDNAdamage(Ingrametal.,2011).Interest-
ingly, DP-4 binds to E2F1 resulting in a complex that does not bind
DNA. Consequently, E2F1 does not induce apoptosis when associ-
ated with DP-4. There is no commercially available DP-4 antibody,
thus it is not possible to test whether DP-4 is induced by HSV-1
infection in RS cells, or whether BHV-1 induces DP-4. It will be of
interest to examine the effect that HSV-1 has on DP-4 in human
cells.
In summary, it was surprising to find that E2F dependent tran-
scription was activated by BHV-1, but not HSV-1. Activation of
E2F dependent transcription appears to be important for BHV-
1 productive infection because E2F1 and E2F2 strongly stimulate
the bICP0 E promoter and to a lesser extent the IE promoter that
controls expression of bICP0 and bICP4 (Geiser and Jones, 2003;
Workman and Jones, 2010). We suggest that BHV-1 encodes or
induces a protein that maintains E2F1 transcriptional activity dur-
ing productive infection. Conversely, HSV-1 may encode or induce
a factor that prevents activation of E2F dependent transcription
during productive infection.
4. Methods
4.1. Cells and viruses
Murine neuroblastoma 2A (neuro-2A), human embryonic lung
(HEL), and rabbit skin (RS) cells were grown in Earle’s modified
Eagle’s medium (EMEM) supplemented with 5% fetal calf serum
(FCS). Bovine kidney cells (CRIB) were grown in EMEM supple-
mented with 10% FCS. All media contained penicillin (10U/ml) and
streptomycin (100?g/ml).
The Cooper strain of BHV-1 (wt virus) was optained from the
National Veterinary Services Laboratory, Animal and Plant Health
Inspection Services, Ames, Iowa. Stock cultures of BHV-1 were pre-
pared in CRIB cells.
The HSV-1 McKrae strain was obtained from S. Wechsler (U
of California – Irvine). Stock cultures of the McKrae strain were
prepared in RS cells.
4.2. Plasmids and measurement of promoter activity in
transfected cells
Plasmids expressing E2F1 or E2F2, pCMV-E2F1 and pCMV-E2F2
respectively, were obtained from J.R. Nevins (Duke University,
Durham, USA). CAT constructs used in this study, HSV-1 ICP0-
CAT (pAB5) and ICP4-CAT, were previously described (Devireddy
and Jones, 2000). Luciferase promoter constructs used in this
study, pGL3basic, VP16-luc, gC-Luc, UL42-Luc, and ICP6-Luc, were
obtained from P. Schaffer and have been previously described
(Kushnir et al., 2010). The empty vector pcDNA3.1 was purchased
from Invitrogen.
Measurementofchloramphenicolacetyltransferase(CAT)activ-
ity in neuro-2A cells was performed as described previously
(Workmanetal.,2009,2011;WorkmanandJones,2010).Luciferase
activity in transfected neuro-2A cells was performed using the
PromegaLuciferaseAssaySystemasdescribedbythemanufacturer
(E4030; Promega).
For productive infection assays, RS cells were transfected with
an E2F-responsive promoter construct using Lipofectamine 2000
according to the manufacturer’s instructions. Twenty-four hours
later, cells were infected with BHV-1 or HSV-1 at a moi of 0.1, 0.5,
or 5 for 24h. Cells were then collected and processed as above for
luciferase activity. Data for CAT and luciferase activity were aver-

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A. Workman, C. Jones / Virus Research 160 (2011) 66–73
agedfromtheresultsofmultipletransfectionsperformedinatleast
three independent experiments.
4.3. SDS-polyacryamide gels and western blotting of E2F
expression
RS cells or HEL cells were infected with wt BHV-1 or HSV-1 at
the moi indicated in the respective figure legend and cell lysate
collected at various times (h) after infection. Whole cell lysate was
preparedasdescribedpreviously(Workmanetal.,2011;Workman
andJones,2010),andproteinconcentrationswerequantifiedbythe
Bradford assay. Standard 12% SDS-polyacrylamide gels were used
to analyze E2F1 protein levels as described previously (Workman
and Jones, 2010). The E2F1 antibody (sc-193X; Santa Cruz Biotech-
nology) was diluted 1:10,000 in blocking solution. An antibody
directedagainst?-actin(SantaCruzBiotechnology,SantaCruz,CA)
was used as a loading control.
4.4. Nuclear and cytoplasmic fractionation
RS cells were infected with BHV-1 or HSV-1 at a moi of 0.1, 0.5,
or 5 for 8, 16 or 24h. Cells were harvested by centrifugation at
1000rpm for 5min and washed twice in cold phosphate buffered
saline (PBS). Cells were then suspended in buffer A (50mM NaCl,
10mM HEPES pH8, 500mM sucrose, 1mM EDTA, 0.5% NP40), vor-
texedbriefly,andcentrifugedat5000×gfor2min.Thesupernatant
was collected and saved as the cytoplasmic fraction. The nuclear
pelletwaswashedtwicewithlowsaltbufferB(50mMNaCl,10mM
HEPES pH8, 25% glycerol, 0.1mM EDTA). The pellet was then sus-
pendedinhighsaltlysisbufferC(350mMNaCl,10mMHEPESpH8,
25%glycerol,0.1mMEDTA).Lysiswascarriedoutat4◦Cfor30min.
Supernatantwasclearedbycentrifugingat13,000rpmfor15minat
4◦C. Protein concentrations were quantified by the Bradford assay
and standard 8% SDS-polyacrylamide gels were used as described
above to analyze E2F1 protein levels. An antibody directed against
histone H3 (Abcam; ab1191) was diluted 1:500 and used a control
for fractionation studies.
4.5. Analysis of HSV-1 productive infection
RS cells grown in 60mM dishes were transfected with 1?g
of blank pcDNA3.1 vector, 100nM E2F1 siRNA (sc-35247; Santa
CruzBiotechnology)or100nMcontrolsiRNA(44-2926;Invitrogen)
using Lipofectamine 2000 according to the manufacture’s specifi-
cations. At 24h after transfection, cells were transfected with 2?g
oftheHSV-1viralgenome.Forty-eighthoursaftertransfectioncells
werecollectedandsubjectedtothreeroundsoffreeze/thawcycles.
Celldebriswaspelletedbycentrifugation,andthesupernatantwas
analyzed by plaque assay on RS cells using 10 fold dilutions.
4.6. Cell cycle analysis by flow cytometry using the Telford
method
RS cells grown in 60mM dishes were transfected with 100nM
E2F1siRNAoracontrolsiRNAusingLipofectamine-2000according
to the manufacture’s specifications. At 24h or 48h after transfec-
tion, an aliquot of 1×106RS cells were pelleted, then suspended in
1mLofice-cold70%ethanol,androtatedat4◦for30min.Cellswere
then pelleted and suspended in Telford reagent (100mM EDTA,
0.1% TritonX-100, 75mM Propidium Iodide, and 2.5U/mL RNAse
A in PBS pH 7.4) and rotated at 4◦for 2h. Cells were then analyzed
using a Becton Dickson FACS caliber flow cytometer. The data was
analyzed using Cell Quest.
4.7. Alkaline phosphatase (CIAP) treatment
Whole cell lysate was collected as described above. Twenty
micrograms of infected cell lysate was treated with 10U of alka-
line phosphatase (04 898 133 001; Roche) and incubated at
37◦for 30min. Reactions were stopped by the addition of SDS-
polyacrylamide gel loading buffer. Samples were boiled for 5min
and assayed by immunoblotting.
Acknowledgements
This research was supported by grants from the USDA and
Agriculture and Food Research Initiative Competitive Grants Pro-
gram (08-00891 and 09-01653). A grant to the Nebraska Center for
Virology (1P20RR15635) also supported certain aspects of these
studies. Aspen Workman was partially supported by a fellowship
from a Ruth L. Kirschstein National Research Service Award 1 T32
AIO60547 (National Institute of Allergy and Infectious Diseases).
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