Deacetylation of the herpes simplex virus type 1 latency-associated transcript (LAT) enhancer and a decrease in LAT abundance precede an increase in ICP0 transcriptional permissiveness at early times postexplant.

Abstract

Only the latency-associated transcript (LAT) of the herpes simplex virus type 1 (HSV-1) genome is transcribed during latency, while the lytic genes are suppressed, possibly by LAT antisense mechanisms and/or chromatin modifications. In the present study, latently infected dorsal root ganglia were explanted to assess both relative levels of LAT and histone H3 (K9, K14) acetylation of the LAT locus and ICP0 promoter at early times postexplant. We observed that a decrease in both LAT enhancer histone H3 (K9, K14) acetylation and LAT RNA abundance occurs prior to an increase in acetylation, or transcriptional permissiveness, at the ICP0 promoter.

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JOURNAL OF VIROLOGY, Feb. 2006, p. 2063–2068
0022-538X/06/$08.00?0
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 4
doi:10.1128/JVI.80.4.2063–2068.2006
Deacetylation of the Herpes Simplex Virus Type 1 Latency-Associated
Transcript (LAT) Enhancer and a Decrease in LAT Abundance
Precede an Increase in ICP0 Transcriptional Permissiveness
at Early Times Postexplant
Antonio L. Amelio,† Nicole V. Giordani,† Nicole J. Kubat, Jerome E. O’Neil, and David C. Bloom*
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32610-0266
Received 6 September 2005/Accepted 22 November 2005
Only the latency-associated transcript (LAT) of the herpes simplex virus type 1 (HSV-1) genome is tran-
scribed during latency, while the lytic genes are suppressed, possibly by LAT antisense mechanisms and/or
chromatin modifications. In the present study, latently infected dorsal root ganglia were explanted to assess
both relative levels of LAT and histone H3 (K9, K14) acetylation of the LAT locus and ICP0 promoter at early
times postexplant. We observed that a decrease in both LAT enhancer histone H3 (K9, K14) acetylation and
LAT RNA abundance occurs prior to an increase in acetylation, or transcriptional permissiveness, at the ICP0
promoter.
Herpes simplex virus type 1 (HSV-1) is characterized by its
ability to establish latency as an episome in neurons (12).
During this time, transcriptional activity is virtually nonexist-
ent, with the exception of the latency-associated transcript
(LAT), an 8.3- to 8.5-kb noncoding RNA that can be spliced to
yield a 2.0-kb stable intron (5, 13, 16). One proposed function
of the LAT is the suppression of nearby lytic phase transcripts
ICP0, ?34.5, and ICP4 through antisense mechanisms, thereby
promoting the establishment and maintenance of latency (3).
Studies using LAT promoter and/or 5? exon mutants demon-
strate impaired establishment of latency and leaky expression
of lytic phase transcripts (3). In addition, a recent study has
demonstrated that the lytic gene regions of LAT mutants are
associated with less of the repressive histone H3 K9 dimethyl,
suggesting that the LAT plays a direct role in promoting a
transcriptionally nonpermissive environment for lytic genes
during latency (18). If the LAT is indeed responsible for sup-
pressing lytic phase transcripts during latency, one might ex-
pect reactivation to directly regulate LAT levels.
In addition to putative LAT-mediated suppression of lytic
transcripts during latency, mounting evidence suggests that
latent gene expression is also regulated at the chromatin level.
The latent viral genome is known to associate with nucleo-
somes (4). Investigation of chromatin modification, in partic-
ular, the acetylation of histone H3 lysine residues 9 and 14 (K9
and K14, respectively), demonstrates that during latency, the
lytic regions of the virus exist in a hypoacetylated, or transcrip-
tionally nonpermissive, state, while the LAT promoter and 5?
exon/enhancer remain hyperacetylated, or transcriptionally
permissive (9) (Fig. 1). However, LAT transcription is not a
prerequisite, nor is it necessary to maintain the hyperacety-
lated, or structurally relaxed, chromatin state (8), suggesting
that the enhancer within the LAT region is an important cis-
acting DNA element.
Reactivation of the latent viral genome has been linked to a
reactivation critical region (rcr) that encompasses the LAT
core promoter through the LAT 5? exon/enhancer, since re-
combinants lacking this region display greatly reduced reacti-
vation phenotypes (2, 6, 7, 10). However, this still does not
address whether the regulatory elements in the rcr act at the
RNA or DNA/chromatin level. An initial study using Northern
blot analysis had detected a decrease in LAT at 24 and 36 h
postexplant of latently infected murine trigeminal ganglia (14),
suggesting that LAT expression may be incompatible with re-
activation. Therefore, we sought to determine whether changes
in LAT transcription and/or histone acetylation occur at early
times during reactivation.
A model to study early molecular events of reactivation
involves explant of latently infected murine dorsal root ganglia
(DRG) into supplemented medium, a process that results in
reactivation of latent virus (15). In the present study, explanted
DRG were placed in medium and incubated for specific time
intervals, followed by RNA isolation or chromatin cross-link-
ing.
To determine the effect of explant on LAT levels, RNA was
isolated from female murine DRG and reverse transcribed
using random decamers, and the resulting cDNA was analyzed
by real-time (TaqMan) PCR. Relative quantities of the LAT
were normalized to either adenine phosphoribosyl transferase
(APRT), a cytoplasmic cellular transcript, or to Xist, a nuclear
noncoding RNA. As shown in Fig. 2, LAT abundance may
transiently increase initially (1.5- to 2-fold), before decreasing
between 2 and 3 h postexplant (hpe). Regardless of the cellular
control used for normalization, the overall pattern of expres-
sion appears the same, suggesting that the decreases observed
for the LAT following explant were not due to a general
decrease in either total cellular or nuclear RNAs.
The observed effect of explant on LAT RNA levels suggests
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, Box 100266, University of Florida College
of Medicine, Gainesville, FL 32610-0266. Phone: (352) 392-8520. Fax:
(352) 392-3133. E-mail: dbloom@ufl.edu.
† A.L.A. and N.V.G. contributed equally to this work.
2063

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that early events in explant-induced reactivation may alter
transcription of the LAT. Previously, we reported that the LAT
region is maintained in a hyperacetylated state during latency,
independently of LAT transcription; therefore, we sought to
determine whether there was a change in histone H3 (K9, K14)
acetylation following explant. Chromatin immunoprecipitation
(ChIP) analysis of the LAT promoter following explant dem-
onstrated a dramatic reduction in acetylation as early as 1 hpe
(Fig. 3A). In order to be certain that this effect was not due to
global changes in histone acetylation caused by explant-in-
duced stress, subsequent experiments (Fig. 3B and C) were
normalized to APRT, a constitutively expressed cellular gene.
In the third experiment (Fig. 3C), the decrease in acetylation at
the 1-hpe time point was not as dramatic, and in this experi-
ment the acetylation actually increased between 1 and 2 hpe.
Overall, the LAT promoter displayed a variable decrease in
histone H3 acetylation occurring within the first hour of ex-
plant.
Because it was previously reported that the LAT enhancer is
capable of increasing LAT transcription (1) and because acet-
ylation of the enhancer was shown to be independent of abun-
dant LAT transcription (8), it seemed plausible that the ob-
served decrease in LAT abundance might correlate with
changes in the acetylation of histones associated with the LAT
enhancer following explant-induced reactivation. Real-time
PCR analyses of the LAT 5? exon for the same three ChIP
experiments that were analyzed for the LAT promoter dis-
played marked decreases of at least fivefold in acetylation
occurring as early as 0.5 hpe (Table 1; Fig. 3A to C), with the
decrease by 1 hpe being statistically significant among the three
independent ChIP experiments (P ? 0.02). It should be noted
that in experiment 3, following a dramatic decrease in acety-
lation of the 5? exon at 0.5 hpe, there is an increase in acety-
lation. This increase parallels the increase in acetylation ob-
served at the LAT promoter in this same experiment and
possibly reflects a more rapid return to the “latent” acetylation
state in this set of ganglia. Overall, the dramatic and rapid
decreases in the acetylation of histone H3 associated with the
5? exon and LAT promoter suggest that a rapid change in
transcriptional permissiveness precedes the decrease in LAT
RNA abundance.
To determine whether the observed changes in the LAT
abundance are linked to a change in the transcriptional per-
missiveness of the ICP0 promoter, we also analyzed its acety-
lation status using the same ChIP experiments. As shown in
Fig. 3A to C (far right column), there is a net increase in ICP0
promoter acetylation occurring as early as 2 hpe and increasing
by as much as threefold by 3 hpe. Despite this increase in
acetylated histone H3 associated with the ICP0 promoter, no
significant increase in ICP0 transcription could be detected by
FIG. 1. Diagram of the HSV-1 genome. Regions of the long and short repeat (RLand RS, respectively) that have been analyzed for H3
acetylation are indicated by gray or black bars. The LAT promoter and enhancer components are encompassed by the reactivation critical region
(rcr). These elements are maintained in a hyperacetylated state during latency, as indicated by the gray bar. The antisense immediate-early ICP0
and ICP4 promoters exist in a hypoacetylated state during latency, as indicated by the black bars. UL, unique long region; US, unique short region.
FIG. 2. LAT RNA levels decrease at early times postexplant. RNA
was isolated and pooled from DRG (4/mouse) explanted from three
mice latently infected with 1 ? 105PFU/mouse of HSV-1 strain KOS
as described previously (8). cDNA was analyzed in triplicate by real-
time PCR using primers and a probe specific for the 5? exon of the
LAT (nucleotides 119326 to 119397) (8). Relative quantities of LAT
RNA were normalized to the PCR of the cellular genes Xist (8) or
APRT (forward, CTCAAGAAATCTAACCCCTGACTCA; reverse,
GCGGGACAGGCTGAGA; probe, CCCCACACACACCTC). The
results of two independent experiments are graphed as percent LAT 5?
exon RNA relative to the RNA level of the 0-hpe time point.
2064NOTES J. VIROL.

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FIG. 3. Explant causes a decrease in acetylation of the LAT enhancer, which precedes an increase in acetylation of the ICP0 promoter. ChIP analysis was performed as previously described
(8) using DRG (6/mouse) explanted from mice latently infected with 1 ? 105PFU/mouse of HSV-1 strain KOS. Samples were analyzed in triplicate by real-time PCR. (A) Results from
experiment 1 show the percentages of bound/input ratios relative to the 0-hpe time point for the postexplant times indicated. Two mice were used per time point. (B and C) Experiments 2 and 3, respectively, show the percentages of bound/input ratios normalized to APRT bound/input ratios and relative to the 0-hpe time point for the postexplant times indicated. Four mice wereused per time point. B/I, bound/input.
VOL. 80, 2006NOTES 2065

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4 hpe (data not shown). Taken together, these data show a
sequential process where changes in chromatin structure of the
LAT enhancer and decreased transcription of the LAT allow
for an increase in acetylation at the ICP0 promoter, perhaps
facilitating productive reactivation in at least some neurons.
In order to extend these analyses and determine whether an
increase in ICP0 transcription could be detected following
longer incubations of the explants, we performed three inde-
pendent ChIP and reverse transcription-PCR (RT-PCR) ex-
periments at 8 and 12 hpe. We chose 12 hpe as our latest time
point since it has been recently shown that infectious virus is
first detected in the majority of explants at 14 hpe (11). ChIP
analyses of the 8- and 12-hpe time points revealed that the
LAT and ICP0 promoters and the LAT 5? exon show compa-
rable levels of acetylation at 8 and 12 h (Fig. 4A). In compar-
ison to the levels of acetylation observed at 3 and 4 h, this
represents a slight decrease in transcriptional permissiveness
of the ICP0 promoter but is still higher than the baseline values
for time zero (latent expression). RT-PCR analyses of these
later time points clearly demonstrate that the levels of LAT
RNA remain low through the 12-h time point (Fig. 4B). These
results are consistent with, and extend, our observations at the
earlier time points (Fig. 2). Nonetheless, we still failed to
detect a significant increase in ICP0 transcription by 12 hpe. It
should be noted that the earliest detection of a net increase in
ICP0 transcripts in explanted ganglia was 96 h by Northern
blotting (14) or 24 h by RT-PCR (17). Since it has been shown
that the first round of reactivating virus produced by explant
TABLE 1. Calculation of changes in acetylation following explant-induced reactivation
DNA target
Expt
no.
Time
(hpe)
B/Ia
B/I relative to
APRT B/I
B/I relative to
B/I at 0 hpe
% B/I relative to
B/I at 0 hpe
Change (n-fold)
relative to 0 hpe
Difference (n-fold)
relative to 0 hpeb
LAT Promoter10
1
2
3
4
0
0.5
1
2
3
0
0.5
1
2
3
0
1
2
3
4
0
0.5
1
2
3
0
0.5
1
2
3
0
1
2
3
4
0
0.5
1
2
3
0
0.5
1
2
3
0.1430
0.0042
0.0039
0.0003
0.0068
2.4028
1.5483
0.0248
0.0096
0.2627
0.0098
0.0088
0.0109
0.0442
0.0368
0.1401
0.0213
0.0020
0.0000
0.0006
3.2863
2.7672
0.0951
0.0086
0.4751
0.0885
0.0073
0.0041
0.0956
0.1134
0.0029
0.0008
0.0006
0.0006
0.0100
0.0145
0.0262
0.0289
0.0529
0.0464
0.0053
0.0020
0.0060
0.0074
0.0299
NDc
ND
ND
ND
ND
295.7442
60.5022
1.4054
0.5462
12.7400
1.7692
1.9255
1.1141
5.2463
3.5309
ND
ND
ND
ND
ND
351.3441
17.0743
4.2162
0.5462
15.0993
14.9577
1.5404
7.7986
13.1157
9.9748
ND
ND
ND
ND
ND
1.7203
0.9733
1.3528
2.8914
2.1909
0.8496
0.3783
0.6691
0.9640
2.6436
1 100
2.9724
2.7223
0.2079
4.7537
100
20.4576
0.4752
0.1847
4.3078
100
108.8363
62.9720
296.5368
199.5778
100
15.2301
1.4622
0.0350
0.4541
100
4.8597
1.2000
0.1555
4.2976
100
10.2985
52.1381
87.6856
66.6869
100
25.7968
21.9441
19.9524
344.5042
100
56.5787
78.6380
168.0709
127.3556
100
44.5306
78.7506
113.4604
311.1441
10
0.0297
0.0272
0.0021
0.0475
1
0.2046
0.0048
0.0018
0.0431
1
1.0884
0.6297
2.9654
1.9958
1
0.1523
0.0146
0.0004
0.0045
1
0.0486
0.0120
0.0016
0.0430
1
0.1030
0.5214
0.8769
0.6669
1
0.2580
0.2194
0.1995
3.4450
1
0.5658
0.7864
1.6807
1.2736
1
0.4453
0.7875
1.1346
3.1114
33.6423
36.7332
480.9459
21.0362
1
4.8882
210.4340
541.4208
23.2138
1
0.9188
1.5880
0.3372
0.5011
1
6.5659
68.3894
2856.4697
220.2176
1
20.5774
83.3318
643.2079
23.2690
1
9.7102
1.9180
1.1404
1.4995
1
3.8765
4.5570
5.0119
0.2903
1
1.7674
1.2716
0.5950
0.7852
1
2.2456
1.2698
0.8814
0.3214
?32.6423
?35.7332
?479.9459
?20.0362
0
?3.8882
?209.4340
?540.4208
?22.2138
0
0.0812
?0.5880
0.6628
0.4989
0
?5.5659
?67.3894
?2855.4697
?219.2176
0
?19.5774
?82.3318
?642.2079
?22.2690
0
?8.7102
?0.9180
?0.1404
?0.4995
0
?2.8765
?3.5570
?4.0119
0.7097
0
?0.7674
?0.2716
0.4050
0.2148
0
?1.2456
?0.2698
0.1186
0.6786
2
3
LAT 5? exon 1
2
3
ICP0 promoter1
2
3
aB/I, bound input ratio; average bound quantity divided by the average input quantity.
bDifference ? 1 ? x, where x represents change (n-fold) relative to 0 hpe (value shown in preceding column).
cND, not determined.
2066NOTES J. VIROL.

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occurs by 14 h (11), it is likely that the inability to detect an
increase in ICP0 RNA is because ICP0 transcription occurs in
only the small subset of cells that ultimately go down the path
to productive reactivation. It is well documented that reacti-
vation occurs only in a small percentage of the total population
of latently infected neurons, so detection of this small increase
in ICP0 transcripts above the background of the entire latent
population would be difficult, if not impossible. It is also likely
that the ICP0 transcription detected at 24 h and later postex-
plant reflects secondary rounds of replication within the ex-
planted ganglia, possibly in nonneuronal support cells (11).
This study sought to determine the relationship between
regulation of the LAT and the LAT region acetylation status at
early reactivation times. The observations described here sup-
port the hypothesis that the LAT may act to suppress imme-
diate-early genes and that the chromatin status of the LAT
enhancer is linked to early reactivation events. Previous studies
using the LAT promoter and/or 5? exon mutants showed im-
paired establishment of latency and leaky expression of lytic
phase transcripts during latency. Our finding that a significant
and dramatic decrease in the LAT occurs at early times during
explant-induced reactivation supports the notion that the LAT
may be acting through antisense or other RNA-mediated
mechanisms to suppress nearby lytic phase transcripts. Fur-
thermore, the changes observed in the acetylation status of the
LAT enhancer indicate that the LAT enhancer is both sensi-
tive and responsive to reactivation signals. The increased level
of acetylation at the ICP0 promoter following deacetylation of
the LAT enhancer suggests that chromatin remodeling both at
the LAT locus and at the ICP0 promoter may be directly linked
during reactivation. It is therefore possible that the LAT en-
hancer functions to recruit a novel histone-modifying complex
that helps establish and maintain the active expression of the
LAT during latency. During reactivation, the complex may
quickly respond to restructure the chromatin within the LAT
region to facilitate nearby lytic phase gene expression. Work
aimed at identifying such a complex is currently under way.
This work was supported by grant AI48633 from the National Insti-
tutes of Health and in part by the Investigators in Pathogenesis Award
from the Burroughs Wellcome Fund (to D.C.B.). N.J.K. and N.V.G.
received support from NIH training grant AI07110.
The authors thank P. McAnany for excellent technical assistance
and J. Feller for helpful comments on the manuscript.
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FIG. 4. LAT RNA levels remain low and the ICP0 promoter re-
mains in a transcriptionally permissive state through 12 hpe. (A) Anal-
ysis of three independent ChIP experiments at 8 and 12 hpe. Three
mice per time point per ChIP were precipitated with anti-H3 K9, 14
acetyl and analyzed by TaqMan PCR with primers and probes for the
LAT promoter, 5? exon, and ICP0 promoter. The ICP0 promoter
remains hyperacetylated, though there is an apparent increase in the
relative level of LAT 5? exon acetylation by 12 hpe. (B) LAT and ICP0
RNA at 0, 8, and 12 hpe from three independent experiments of
explanted DRG was analyzed by TaqMan real-time PCR. Reverse
transcription using random decamer primers (Ambion) or a strand-
specific primer for the ICP0 transcript (LAT I-1, GACACGGATTG
GCTGGTGTAGTGGG; nucleotides 120797 to 120820) was per-
formed using Omniscript reverse transcriptase (QIAGEN) according
to the manufacturer’s instructions. The cDNA was then analyzed with
primers and probes for ICP0 (forward, GGCCGAGGGAGGTTTCC,
nucleotides 121385 to 121401; reverse, CCGCTTCCGCCTCCTC, nucle-
otides 121438 to 121453; probe, CTCCCAGGGCACCGAC, nucleo-
tides 121412 to 121427) or the LAT 5? exon and then normalized
relative to APRT and Xist cellular controls. A significant decrease in
LAT RNA occurs between 0 and 8 hpe (P ? 0.05) and remains at low
levels through 12 hpe. No significant change in the amount of ICP0
RNA was detected between 0 and 12 hpe.
VOL. 80, 2006NOTES 2067