During herpes simplex virus type 1 infection of rabbits, the ability to express the latency-associated transcript increases latent-phase transcription of lytic genes.
ABSTRACT Trigeminal ganglia (TG) from rabbits latently infected with either wild-type herpes simplex virus type 1 (HSV-1) or the latency-associated transcript (LAT) promoter deletion mutant 17DeltaPst were assessed for their viral chromatin profile and transcript abundance. The wild-type 17syn+ genomes were more enriched in the transcriptionally permissive mark dimethyl H3 K4 than were the 17DeltaPst genomes at the 5' exon and ICP0 and ICP27 promoters. Reverse transcription-PCR analysis revealed significantly more ICP4, tk, and glycoprotein C lytic transcripts in 17syn+ than in 17DeltaPst. These results suggest that, for efficient reactivation from latency in rabbits, the LAT is important for increased transcription of lytic genes during latency.
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ABSTRACT: Background: Herpes simplex virus, type 1 (HSV-1) commonly produces lytic mucosal lesions. It invariably initiates latent infection in sensory ganglia enabling persistent, lifelong infection. Acute HSV-1 encephalitis is rare and definitive evidence of latent infection in the brain is lacking. However, exposure untraceable to encephalitis has been repeatedly associated with impaired working memory and executive functions, particularly among schizophrenia patients. Methods: Patterns of HSV-1 infection and gene expression changes were examined in human induced pluripotent stem cell (iPSC)-derived neurons. Separately, differences in blood oxygenation level-dependent (BOLD) responses to working memory challenges using letter n-back tests were investigated using functional magnetic resonance imaging (fMRI) among schizophrenia cases/controls. Results: HSV-1 induced lytic changes in iPSC-derived glutamatergic neurons and neuroprogenitor cells. In neurons, HSV-1 also entered a quiescent state following coincubation with antiviral drugs, with distinctive changes in gene expression related to functions such as glutamatergic signaling. In the fMRI studies, main effects of schizophrenia (P = .001) and HSV-1 exposure (1-back, P = 1.76 × 10(-) (4); 2-back, P = 1.39 × 10(-) (5)) on BOLD responses were observed. We also noted increased BOLD responses in the frontoparietal, thalamus, and midbrain regions among HSV-1 exposed schizophrenia cases and controls, compared with unexposed persons. Conclusions: The lytic/quiescent cycles in iPSC-derived neurons indicate that persistent neuronal infection can occur, altering cellular function. The fMRI studies affirm the associations between nonencephalitic HSV-1 infection and functional brain changes linked with working memory impairment. The fMRI and iPSC studies together provide putative mechanisms for the cognitive impairments linked to HSV-1 exposure.Schizophrenia Bulletin 03/2014; · 8.61 Impact Factor
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ABSTRACT: After infecting peripheral sites, herpes simplex virus (HSV) invades the nervous system and initiates latent infection in sensory neurons. Establishment and maintenance of HSV latency require host survival, and entail repression of productive cycle ("lytic") viral gene expression. We find that a neuron-specific microRNA, miR-138, represses expression of ICP0, a viral transactivator of lytic gene expression. A mutant HSV-1 (M138) with disrupted miR-138 target sites in ICP0 mRNA exhibits enhanced expression of ICP0 and other lytic proteins in infected neuronal cells in culture. Following corneal inoculation, M138-infected mice have higher levels of ICP0 and lytic transcripts in trigeminal ganglia during establishment of latency, and exhibit increased mortality and encephalitis symptoms. After full establishment of latency, the fraction of trigeminal ganglia harboring detectable lytic transcripts is greater in M138-infected mice. Thus, miR-138 is a neuronal factor that represses HSV-1 lytic gene expression, promoting host survival and viral latency.Cell host & microbe 04/2014; 15(4):446-56. · 13.02 Impact Factor
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ABSTRACT: Herpes simplex viruses (HSV) are significant human pathogens that provide one of the best-described examples of viral latency and reactivation. HSV latency occurs in sensory neurons, being characterized by the absence of virus replication and only fragmentary evidence of protein production. In mouse models, HSV latency is especially stable but the detection of some lytic gene transcription and the ongoing presence of activated immune cells in latent ganglia have been used to suggest that this state is not entirely quiescent. Alternatively, these findings can be interpreted as signs of a low, but constant level of abortive reactivation punctuating otherwise silent latency. Using single cell analysis of transcription in mouse dorsal root ganglia, we reveal that HSV-1 latency is highly dynamic in the majority of neurons. Specifically, transcription from areas of the HSV genome associated with at least one viral lytic gene occurs in nearly two thirds of latently-infected neurons and more than half of these have RNA from more than one lytic gene locus. Further, bioinformatics analyses of host transcription showed that progressive appearance of these lytic transcripts correlated with alterations in expression of cellular genes. These data show for the first time that transcription consistent with lytic gene expression is a frequent event, taking place in the majority of HSV latently-infected neurons. Furthermore, this transcription is of biological significance in that it influences host gene expression. We suggest that the maintenance of HSV latency involves an active host response to frequent viral activity.PLoS Pathogens 07/2014; 10(7):e1004237. · 8.06 Impact Factor
JOURNAL OF VIROLOGY, June 2008, p. 6056–6060
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 12
During Herpes Simplex Virus Type 1 Infection of Rabbits, the Ability
To Express the Latency-Associated Transcript Increases
Latent-Phase Transcription of Lytic Genes?
Nicole V. Giordani,1Donna M. Neumann,2Dacia L. Kwiatkowski,1Partha S. Bhattacharjee,2
Peterjon K. McAnany,1James M. Hill,2and David C. Bloom1*
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida,1and
Department of Ophthalmology, Louisiana State University Health Sciences Center, New Orleans, Louisiana2
Received 14 December 2007/Accepted 27 March 2008
Trigeminal ganglia (TG) from rabbits latently infected with either wild-type herpes simplex virus type 1
(HSV-1) or the latency-associated transcript (LAT) promoter deletion mutant 17?Pst were assessed for their
viral chromatin profile and transcript abundance. The wild-type 17syn? genomes were more enriched in the
transcriptionally permissive mark dimethyl H3 K4 than were the 17?Pst genomes at the 5? exon and ICP0 and
ICP27 promoters. Reverse transcription-PCR analysis revealed significantly more ICP4, tk, and glycoprotein
C lytic transcripts in 17syn? than in 17?Pst. These results suggest that, for efficient reactivation from latency
in rabbits, the LAT is important for increased transcription of lytic genes during latency.
During herpes simplex virus type 1 (HSV-1) latency in sen-
sory neurons, there is an overall repression of transcription
from the viral genome, with the exception of the latency-asso-
ciated transcript (LAT) region. The latent genomes are main-
tained as nucleosome-associated episomes (4) that are not
repressed through DNA methylation (6, 11). Rather, histone
tail modifications appear to correspond with transcriptional
permissiveness. Specifically, during latency in the mouse, the
* 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: firstname.lastname@example.org.
?Published ahead of print on 9 April 2008.
FIG. 1. H3 K4 dimethylation status of rabbit TG latently infected with wild-type 17syn? (n ? 7 TG) (A) or core LAT promoter deletion virus 17?Pst
(n ? 6 TG) (B). Rabbits were infected by corneal scarification with 50,000 PFU of virus per eye. ChIP analyses were performed as previously described
(11) with TaqMan real-time PCR used for analysis of the LAT 5? exon/enhancer, ICP0 promoter, and ICP27 promoter. Bound/(bound ? unbound)
[B/(B?U)] values were normalized to B/(B?U) values of the cellular control, rabbit centromere. Mean lines denote the average value for each data set.
LAT promoter region is more enriched in acetylated histone
H3 K9 or K14, a marker of transcriptional permissiveness, than
are the lytic genes ICP27 and ICP0, and the LAT 5? exon/
enhancer region is more acetylated than the LAT promoter
(11). Further, Wang et al. demonstrated that for mice infected
with a wild-type virus, the HSV-1 genome’s lytic gene promot-
ers become associated with dimethyl histone H3 K9, a marker
of transcriptional repression, during establishment of latency,
while they become less associated with dimethyl H3 K4, a
transcriptionally permissive mark (14). In contrast, a LAT pro-
moter deletion virus has less-repressive histone modifications
associated with lytic genes during latency (14). These patterns
of histone modifications are consistent with a previous study
demonstrating that a LAT promoter deletion mutant is more
transcriptionally leaky during latency in mice, displaying in-
creased ICP4 and tk transcript accumulation compared to
those of wild-type HSV-1 (3). Taken together, these findings
suggest that in the mouse LAT plays a role in the repression of
lytic genes during latency.
While the mouse model is used extensively to study HSV-1
latency and has provided valuable insight into molecular events
surrounding the infection, the rabbit eye model is generally
considered a more relevant system to study reactivation and
the events leading up to reactivation because it more closely
parallels the biology of clinical HSV-1 reactivation in humans
(for a review see reference 13). In the rabbit eye model, latency
is established in the trigeminal ganglia (TG) following ocular
infection with HSV-1. Reactivation can be efficiently induced
by iontophoresis of adrenergic agents, such as epinephrine,
and the reactivating virus can then be detected in the tears (7,
9). The rabbit is also one of the only HSV-1 reactivation
models in which reactivation results in viral shedding and clin-
ical lesions that recur at the primary site of infection (1).
However, reactivation from latency in the rabbit eye model is
more LAT dependent than that in mouse models; specifically,
LAT promoter deletion mutants are severely reduced in reac-
tivation relative to the wild type (8, 12). Since the role of LAT
in facilitating efficient reactivation in the rabbit eye model is
not known, we sought to determine whether a LAT promoter
mutant exhibits alterations in its latent chromatin profile and
RNA transcript accumulation, characteristics that could give
new insight into the LAT’s mechanism of action.
To determine the transcriptional permissiveness of the LAT
region in latently infected rabbits, chromatin immunoprecipi-
tation (ChIP) was performed using anti-dimethyl H3 K4 (Mil-
lipore) on TG from rabbits latently infected with 17syn?, fol-
lowing the procedure described previously (11). Bound and
unbound fractions were analyzed in triplicate by TaqMan real-
time PCR. The relative quantity of the bound fraction was
normalized to that of the total of the bound plus unbound
fractions. During latency in the rabbit, the chromatin profile of
wild-type 17syn? indicated that the LAT region was more
transcriptionally permissive than lytic genes ICP0 and ICP27
(Fig. 1A; Table 1). This finding is consistent with the chromatin
profiles observed in latently infected mice (11). We also as-
sessed the viral chromatin profile of rabbits latently infected
with 17?Pst, a nonreactivating HSV-1 recombinant with a
202-bp deletion of the core LAT promoter (2, 5). A compar-
ison between the wild type and this LAT promoter deletion
virus showed that while the LAT region of 17?Pst was more
enriched in dimethyl H3 K4 than were the lytic genes examined
(Fig. 1B; Table 1; P ? 0.02 and P ? 0.03 for ICP0 and ICP4,
respectively), the level of enrichment was an average of 2.5-
fold less (P ? 0.004) than that observed for 17syn?. In addi-
tion, in 17?Pst the level of H3 K4 dimethylation was approx-
TABLE 1. Dimethyl H3 K4 enrichment values during latency
in the rabbit
and expt no.
LAT 5? exon
LAT 5? exon
aPreviously described in reference 10.
cRelative enrichment normalized to the B/(B?U) value of the cellular con-
trol, rabbit centromere.
VOL. 82, 2008 NOTES6057
imately twofold lower for ICP0 (P ? 0.1) and ICP27 (P ? 0.1)
than in 17syn?. These observations were in contrast to the
chromatin profile of a LAT-negative mutant previously de-
scribed for the mouse, in which the enhancer and ICP0 appear
slightly more transcriptionally permissive than in the wild type
Chen et al. previously reported that, during latency, a LAT
promoter deletion virus displayed greater leakiness of lytic
transcripts than did the wild type in mice infected via the
ocular route (3). Because our ChIP analysis of rabbit TG
revealed that the lytic genes of 17?Pst are less transcriptionally
permissive than those of 17syn?, we sought to determine the
relative levels of lytic gene transcription of the two viruses
during latency in the rabbit. RNA was isolated from latently
infected rabbit TG; cDNA for each TG was synthesized simul-
taneously in four separate reactions using random decamers
and then pooled to enable detection of low-abundance tran-
scripts. The resulting cDNA was analyzed by real-time PCR.
Relative quantities were first normalized to glyceraldehyde-3-
phosphate dehydrogenase (GAPDH), and the normalized val-
ues were further normalized to the relative viral genome quan-
tity. For the 17?Pst lytic genes, no transcripts were detectable
by TaqMan real-time PCR at the limits of sensitivity. There-
fore, values assigned in these cases represent the maximum
possible quantity. As shown in Fig. 2A and Table 2, while there
may have been some leaky expression of LAT in 17?Pst during
latency, it was almost 800-fold less (P ? 0.017) than that
observed for 17syn?. Assessment of lytic genes ICP4 (Fig. 2B),
thymidine kinase (tk) (Fig. 2C), and glycoprotein C (gC) (Fig.
2D), HSV-1 regions representative of the immediate-early,
early, and late gene classes, respectively, confirmed that these
RNAs are present at greater levels in rabbit TG latently in-
fected with 17syn? than in rabbit TG infected with 17?Pst.
Specifically, 17syn? displayed averages of at least 3-, 35-, and
154-fold more RNA for ICP4, tk, and gC, respectively, than did
17?Pst, strongly suggesting that the absence of LAT in 17?Pst
corresponds to a greater repression of lytic genes during la-
tency in the rabbit. The observed differences were not due to
variability in establishment, since the relative genome quanti-
ties were 0.0018 and 0.002 for 17syn? and 17?Pst, respectively
(P ? 0.84).
It had previously been shown that in mice latently infected
with a LAT promoter deletion mutant, the HSV-1 genomes
were less enriched in the repressive histone dimethyl H3 K9
than with the wild type (14). Therefore, in order to examine the
association of a repressive histone modification with the latent
HSV genome in the rabbit, a ChIP experiment was performed
on latently infected rabbit TG using anti-trimethyl H3 K9 (Mil-
lipore). The 17?Pst lytic genes tested (ICP4, tk, and gC)
showed levels of enrichment in this histone modification sim-
ilar to those of 17syn? (data not shown). This indicates that,
unlike during latency in the mouse, HSV-1 LAT-negative ge-
nomes in the rabbit do not become less enriched in repressive
histone marks relative to the wild type, therefore indicating
that the LAT does not seem to exert a repressive effect on the
chromatin state of latent genomes in rabbits.
The findings presented here for the transcriptional status of
a LAT-negative mutant during latency in the rabbit are the
opposite of what has been previously observed for the mouse,
where the LAT appears to play a role in repression of the
latent HSV-1 genome (3, 14). To the contrary, in the rabbit
LAT seems to exert a positive effect on facilitating transcrip-
tional permissiveness of the lytic genes, both at the level of H3
K4 dimethylation and at the level of viral transcripts detected
in latent ganglia. Further, when the average number of RNA
molecules per viral genome (values calculated through extrap-
olation of a DNA standard curve derived from TaqMan real-
time PCR) is compared with those determined by previous
analyses in the mouse, the overall abundance of lytic tran-
scripts detected is an order of magnitude less in the rabbit than
in the mouse (Table 3), suggesting that control of viral tran-
scription in the rabbit is more constrained than that in the
FIG. 2. 17syn? RNA is more abundant than that of 17?Pst for all
genes analyzed by reverse transcription-PCR during latency in the
rabbit TG. Relative quantities were normalized first to cellular control,
GAPDH, and then to the value of HSV-1 polymerase normalized to
GAPDH to account for any variations in establishment of latency.
Average values of each data set are denoted by the mean lines. n ? 5
TG per virus. (A) LAT is not abundantly transcribed in 17?Pst but is
abundant in 17syn? (P ? 0.017). (B) ICP4 levels are an average of
threefold higher in 17syn? than in 17?Pst (P ? 0.035). (C) Abundance
of tk RNA is approximately 35-fold higher in 17syn? than in 17?Pst
(P ? 0.011). (D) gC RNA is approximately 154-fold more abundant in
17syn? latent infection of rabbits than in 17?Pst (P ? 0.005).
6058NOTES J. VIROL.
In summary, our results suggest that in contrast to what
occurs in the mouse, in the rabbit LAT does not act as a
repressor of lytic genes but instead acts to keep the lytic ge-
nome in a more transcriptionally activated state. We believe
that in the rabbit eye model, the expression of LAT is impor-
tant in establishing or maintaining the lytic genes in a state that
is poised for reactivation.
This work was supported in part by NIH grants AI48633 (D.C.B.)
and EY006311 (J.M.H.) as well as an Investigator in the Pathogenesis
of Infectious Disease Award from the Burroughs Wellcome Fund
(D.C.B.) and a Research to Prevent Blindness, Senior Scientific Inves-
tigator Award (J.M.H.), and an LSUHSC Translational Research Ini-
tiative Grant (P.S.B.). D.M.N. was supported by an individual NRSA,
EY016316, and N.V.G. was supported by training grant AI07110 from
We thank J. Feller, L. Watson, and Z. Zeier for helpful comments
on the manuscript.
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TABLE 2. Relative RNA values during latency in the rabbit
and expt no.
LAT 5? exon
LAT 5? exon
aLAT 5? exon/enhancer previously described in reference 10, ICP4,
NC_001806, nucleotides 130278 to 130292 (forward), 130208 to 130224 (reverse),
130232 to 130246 (probe); tk, thymidine kinase, NC_001806, nucleotides 47638
to 47662 (forward), 47552 to 47573 (reverse), 47598 to 47611 (probe); gC,
NC_001806, nucleotides 96582 to 96598 (forward), 96622 to 96643 (reverse),
96601 to 96615 (probe).
bRelative quantities are normalized to those of the cellular control, rabbit
cRelative genome quantity, viral polymerase/GAPDH.
TABLE 3. Calculated average number of RNA molecules per
Avg no. of RNA molecules/viral genome
for model and strain:
LAT 5? exon
aData reported by Chen et al. (3).
bLAT?strain, KOS; LAT?strain, dlLAT1.8.
cLAT?strain, 17syn?; LAT?strain, 17?Pst.
dND, not determined.
VOL. 82, 2008 NOTES6059
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6060 NOTESJ. VIROL.