Mechanics
of
the
Herpes
Simplex
Virus
latency­reactivation
cycle
Kristen
Schroeder
Department
of
Veterinary
Microbiology
Western
College
of
Veterinary
Medicine
University
of
Saskatchewan
Canada
Prepared
as
part
of
the
requirements
for
Advanced
Virology,
VTMC
833
Table
of
contents
Abstract
Introduction
The
lytic
infection
cycle:
mechanism
and
regulation
Establishment
and
maintenance
of
viral
latency
in
the
neuron
Establishment
of
viral
latency
Figure
1:
Model
of
the
process
of
establishing
HSV
latency
Maintenance
of
the
latent
state
Viral
reactivation
in
response
to
stress
Figure
2:
Model
of
the
process
of
reactivation
from
latency
VP16:
A
viral
mediator
of
reactivation
from
latency?
Models
of
HSV
latency
Conclusions
Figure
3:
Summary
of
major
events
in
the
HSV
latency­reactivation
cycle
Sources
Abstract:
Herpes
simplex
virus
(HSV)
leads
a
double
life:
in
epithelial
cells
HSV
performs
lytic
replication,
however
in
sensory
neurons
the
virus
is
able
to
enter
latency
and
persist
indefinitely
in
this
state.
This
curious
double
life
has
been
the
topic
of
intense
scrutiny
for
decades,
and
recent
advances
have
illuminated
several
factors
that
may
explain
how
HSV
is
able
to
mediate
cell
type‐specific
infectious
cycles.
HSV
latency
is
marked
by
genetic
silence,
save
for
that
of
the
latency‐associated
transcript,
yet
upon
receiving
a
stress
stimulus
the
virus
is
able
to
reactivate
and
replicate
to
form
infectious
progeny.
This
specialized
infectious
cycle
is
the
product
of
a
complicated
interaction
between
HSV
and
the
neuronal
environment,
which
features
several
levels
of
regulation.
HSV
interacts
with
cellular
regulatory
machinery
to
effect
changes
in
its
own
transcriptional
permissiveness,
which
is
reflected
in
changes
to
the
secondary
chromatin
structure
associated
with
the
virus.
The
dynamics
of
the
interaction
between
HSV
and
the
neuron
are
discussed
in
the
context
of
establishment
and
reactivation
from
the
latent
state,
including
current
theory
on
how
the
viral
protein
VP16
may
mediate
exit
from
latency.
Introduction
Herpes
simplex
virus
(HSV)
is
a
large
double‐stranded
DNA
virus
that
spreads
through
the
human
population
in
a
manner
similar
to
other
alphaherpesviruses,
using
lytic
epithelial
infection
to
feed
the
establishment
of
a
latent
reservoir
in
sensory
neurons.
HSV
accomplishes
its
dual
life
cycle
through
means
of
multifunctional
viral
factors,
which
are
able
to
interact
with
host
cellular
regulatory
machinery.
While
innumerable
advances
have
been
made
in
the
study
of
HSV
in
lytic
infection,
the
mechanisms
by
which
HSV
latency
is
established
and
by
which
reactivation
occurs
remain
elusive.
It
has
been
established
that
the
HSV
latency‐reactivation
cycle
is
in
part
achieved
through
epigenetic
regulation
of
the
secondary
chromatin
structure
adopted
by
the
virus
upon
entry
into
the
nucleus.
Cell‐specific
transcriptional
activators
and
regulatory
mechanisms
also
have
a
role
in
the
interplay
between
neuron
and
virus,
however
the
molecular
machinery
responsible
for
effecting
this
regulation
remains
largely
mystified,
due
partly
to
the
difficulty
of
approximating
HSV
latency
experimentally.
As
HSV
has
adapted
to
the
neuronal
environment
and
can
persist
in
this
location
for
the
life
of
the
host,
determining
the
interactions
that
mediate
entrance
into
and
exit
from
latency
may
illuminate
specialized
ways
in
which
neurons
exert
transcriptional
control,
and
how
HSV
has
evolved
to
subvert
these
processes.
The
lytic
infection
cycle:
mechanism
and
regulation
HSV
is
able
to
replicate
in
a
wide
variety
of
cells
utilizing
a
lytic
program
of
gene
expression.
In
a
typical
epithelial
cell
lytic
infection
is
initiated
by
the
delivery
of
the
viral
genome
and
the
tegument
protein
VP16
to
the
nucleus,
wherein
VP16
is
able
to
induce
the
expression
of
the
five
immediate
early
(IE)
genes
(ICP0,
ICP4,
ICP22,
ICP27
and
ICP47)
through
its
activation
domain
(AD).
This
induction
occurs
via
the
formation
of
a
DNA‐binding
complex
that
includes
VP16
and
two
cellular
factors—host
cell
factor
C1
(HCF)
and
the
POU
domain
octamer
binding
protein
Oct‐1—and
binds
the
TAATGARAT
consensus
sequence
in
the
IE
promoters
to
activate
their
transcription
[1].
The
IE
genes
comprise
a
class
of
trans‐activating
proteins
necessary
for
induction
of
further
gene
expression
from
the
virus.
Through
induction
by
the
IE
proteins
the
early
(E)
class
of
genes
are
expressed
and
synthesize
proteins
involved
in
viral
DNA
replication.
Once
sufficient
levels
of
E
gene
products
have
accumulated
viral
replication
takes
place,
and
following
this
event
the
late
(L)
gene
class
is
induced.
The
late
class
is
comprised
of
genes
that
encode
structural
proteins
utilized
in
the
assembly
of
capsids
that
will
house
newly
synthesized
viral
genomes.
Late
genes
are
also
responsible
for
encoding
proteins—including
VP16—which
make
up
the
virion
tegument,
a
compartment
that
carries
viral
proteins
able
to
alter
the
environment
of
a
newly
infected
cell
and
initiate
subsequent
rounds
of
viral
replication.
Once
the
progeny
virions
have
been
assembled,
egress
occurs
and
they
are
released
to
continue
the
infectious
cycle.
In
addition
to
regulation
of
HSV
replication
by
viral
factors,
control
of
this
process
is
associated
with
epigenetic
regulation
by
cellular
factors;
the
incoming
HSV
genome
is
loaded
with
histones
and
assembles
into
unstable
nucleosomal
structures
similar
in
organization
to
cellular
chromatin
[2].
Histones
binding
the
HSV
genome
are
also
subject
to
covalent
modification
that
is
reflective
of
a
transcriptionally
active
state
[3].
The
viral
proteins
ICP0,
VP16,
and
ICP8
have
been
demonstrated
to
interact
with
chromatin
remodeling
machinery
that
can
effect
these
epigenetic
changes
[4‐6],
indicating
yet
a
further
level
of
regulation
by
the
virus.
HSV
mutants
unable
to
manufacture
the
IE
trans‐activators
required
for
proceeding
through
the
lytic
cycle
have
been
shown
to
become
progressively
restricted
by
the
accumulation
of
nucleosomes
[7],
therefore
it
is
likely
that
viral
influence
on
cellular
chromatin
dynamics
plays
a
role
in
2
successful
replication.
While
lytic
infection
results
in
the
production
of
progeny
virus,
active
blockade
of
this
well‐characterized
process
is
considered
to
be
the
basis
of
HSV
latency.
Establishment
and
maintenance
of
viral
latency
in
the
neuron
While
HSV
undergoes
a
lytic
infection
cycle
in
most
cell
types,
it
establishes
latent
infection
specifically
within
sensory
neurons.
During
lytic
infection
of
the
epithelium,
HSV
is
able
to
enter
the
axon
terminus
of
innervating
neurons
and
establish
a
latent
reservoir
within
nerve
cell
nuclei
in
the
trigeminal
ganglia
(TG).
This
latent
reservoir
can
persist
for
the
life
of
the
host,
and
the
transcriptional
silence
of
HSV
exclusively
within
the
neuron
suggests
active
repression
of
the
lytic
program
by
neuron‐specific
factors.
Due
in
part
to
the
lack
of
an
experimental
model
that
accurately
parallels
HSV
latency,
the
mechanisms
by
which
the
latent
state
is
achieved
remain
ill
defined.
As
no
viral
component
has
been
identified
as
an
active
effector
of
entrance
into
latency,
this
process
is
considered
to
be
a
complex
interaction
between
the
virus
and
the
neuronal
environment.
Entrance
into
latency
can
be
compartmentalized
into
two
distinctive
phases:
the
latent
state
is
first
established
in
the
neuron,
followed
by
an
indefinite
phase
of
maintenance
of
the
silenced
virus.
An
overview
of
the
main
points
presented
is
in
Figure
1.
Establishment
of
viral
latency
In
order
to
successfully
reach
the
neuronal
cell
body
and
gain
access
to
the
nucleus,
HSV
must
first
navigate
intracellular
traffic
and
travel
up
the
nerve
axon
through
association
with
cellular
motor
proteins
[8].
A
major
question
surrounding
this
phenomenon
is
if
a
lack
of
efficient
VP16
transport
from
the
virion
tegument
to
the
neuron
nucleus
contributes
to
the
establishment
of
latency.
A
recent
study
has
demonstrated
that
HSV
capsids
lacking
VP16
and
several
other
major
tegument
proteins
bind
cellular
transport
motors
more
efficiently,
which
is
consistent
with
studies
performed
in
related
alphaherpesviruses
indicating
that
homologous
VP16
dissociates
prior
to
axonal
transport
[9].
In
contrast,
a
separate
study
utilizing
GFP‐tagged
HSV
VP16
to
probe
the
mechanisms
of
neuronal
capsid
transport
has
shown
this
protein
to
travel
the
up
the
axon
as
for
other
capsid
proteins
[10].
While
a
lack
of
VP16
at
the
neuronal
nucleus
facilitating
viral
latency
is
an
attractive
hypothesis,
the
observation
that
HSV
is
able
to
undergo
local
replication
in
the
TG
alludes
to
the
fact
that
latency
is
not
simply
infection
of
a
non‐permissive
cell
[11].
It
is
also
possible
that
the
neuronal
location
and
abundance
of
the
cellular
factors
Oct‐1
and
HCF,
required
to
form
the
VP16‐induced
complex,
may
present
a
barrier
to
full
induction
of
lytic
transcription.
HCF
has
been
observed
to
be
tethered
to
the
Golgi
apparatus
by
the
cellular
basic
leucine
zipper
protein
Luman
in
latently
infected
sensory
neurons
[12,
13],
separated
from
the
nucleus
where
it
can
act
as
a
transcriptional
cofactor.
HCF
has
also
been
shown
to
be
involved
in
the
recruitment
of
the
repressor
element‐1
silencing
transcription
factor/neuronal
restrictive
silencing
factor
(REST/NRSF)
and
its
associated
coREST
and
histone
deacetylase
(HDAC)
complex
partners
to
the
HSV
genome
during
lytic
infection
[14].
The
REST/coREST/HDAC
repressor
complex
is
known
for
its
role
in
transcriptional
regulation
during
neurogenesis
and
silencing
of
neuronal
genes
in
non‐neuronal
cells
[15,
16],
and
has
recently
been
demonstrated
to
be
important
for
induction
of
the
HSV
lytic
genes
[17].
One
hypothesis
is
that
in
lytic
infection,
the
REST/coREST/HDAC
complex
is
recruited
to
the
IE
promoters
by
HCF
(complexed
with
VP16
and
Oct‐1)
and
functions
to
activate
IE
expression
via
demethylation,
however
in
latent
infection
this
sequence
of
events
does
not
occur.
The
contribution
of
the
REST/coREST/HDAC
complex
to
establishing
latency
remains
unresolved,
and
several
important
questions
remain,
such
whether
a
repressor
complex
with
the
function
of
suppressing
neuronal
genes
would
be
available
and
3
Figure
1:
Model
of
the
process
of
establishing
HSV
latency.
1)
HSV
enters
the
neuron
axon
and
dissociates
from
most
of
its
tegument
before
traveling
to
the
nucleus.
2)
The
neuronal
environment
does
not
support
induction
of
the
HSV
lytic
program
due
to
physiological
differences,
such
as
the
cytoplasmic
locale
of
HCF.
3)
Cellular
intrinsic
antiviral
mechanisms,
such
as
those
housed
in
the
ND10
nuclear
bodies,
repress
the
HSV
genome
4)
Repressive
chromatin
is
loaded
onto
the
HSV
genome.
5)
The
effective
result
is
a
blockade
of
transcription
of
the
IE
genes.
6)
The
LAT
is
transcribed
and
works
to
promote
the
maintenance
of
the
latent
state.
active
to
participate
in
genetic
regulation
of
differentiated
neurons.
Additional
difficulties
in
forming
the
VP16‐induced
complex
may
stem
from
a
reported
low
abundance
of
Oct‐1
in
neuronal
cells
[18].
While
it
has
been
supported
that
Oct‐1
is
required
to
induce
IE
gene
expression
and
that
other
POU‐domain
transcription
factors
more
prevalent
in
the
neuron
cannot
substitute
for
its
function
[19,
20],
a
correlation
between
neuronal
Oct‐1
levels
and
the
establishment
of
HSV
latency
has
not
been
defined.
In
this
manner,
the
silencing
of
HSV
during
entrance
into
latency
may
be
exerted
in
part
due
to
the
neuronal
environment.
The
entrance
of
HSV
into
latency
may
also
involve
the
influence
of
intrinsic
cellular
antiviral
mechanisms
that
engage
in
silencing
foreign
genetic
material
entering
the
nucleus
[21].
4
Following
nuclear
import,
the
HSV
genome
localizes
to
nuclear
domain
10
(ND10)
structures
(also
known
as
promyelocytic
leukemia
(PML)
nuclear
bodies)
[22],
which
in
lytic
infection
are
rapidly
disrupted
by
ICP0
[23].
In
cell
culture
models
of
HSV
latency,
infection
with
ICP0‐null
mutants
has
demonstrated
that
the
virus
stably
associates
with
ND10
structures
for
the
duration
of
quiescence.
The
presence
of
the
ND10
protein
constituents
PML,
Sp100,
Daxx
and
ATRX
has
been
shown
to
correlate
with
increased
viral
repression
in
this
scenario
[24‐26].
ND10
structures
are
involved
in
cellular
chromatin
dynamics,
and
Daxx
and
ATRX
are
known
to
be
specifically
involved
in
a
chromatin
remodeling
complex
able
to
alter
nucleosome
structure
[27,
28].
This
presents
a
potential
mechanism
for
HSV
transcriptional
silencing
in
the
absence
of
ICP0,
which
may
occur
if
viral
IE
translation
is
aborted.
It
remains
to
be
determined
if
this
repressive
system
is
relevant
at
the
site
of
HSV
latency
however,
as
it
is
not
clear
which
ND10
constituents
are
expressed
within
the
human
neuronal
context.
Certain
human
neuron
populations
have
been
demonstrated
to
accumulate
similar
PML‐staining
nuclear
bodies
in
response
to
trauma
and
replication
of
other
alphaherpesviruses
[29,
30],
though
it
has
also
been
noted
that
the
presence
of
ND10
structures
is
restricted
within
cells
of
neuronal
lineage
[31].
The
latter
finding
has
spurred
studies
into
the
subcellular
localization
of
ICP0
during
infection
of
neurons,
and
evidence
suggests
a
failure
of
this
protein
to
localize
to
the
neuron
nucleus
may
contribute
to
the
establishment
of
latency
through
ND10‐mediated
repression
[32].
Further,
the
human
neuronal‐
committed
NT2
cell
line
and
its
neuronally
differentiated
derivative
hNT
have
been
demonstrated
to
be
poor
hosts
for
HSV
replication,
and
this
reduced
permissiveness
has
been
suggested
to
involve
the
abnormal
ND10
structures
observed
in
these
cells
[33].
Therefore,
the
presence
of
intrinsic
antiviral
mechanisms
such
as
that
housed
in
ND10
structures
may
be
involved
in
establishing
HSV
latency.
At
the
level
of
the
HSV
genome,
epigenetic
regulation
may
contribute
to
the
establishment
of
latency
through
modification
of
viral
chromatin
structure.
The
establishment
of
latency
is
hypothesized
to
involve
several
levels
of
chromatin
regulation,
including
modification
of
histone
tails,
changes
in
nucleosome
organization,
and
association
of
proteins
involved
in
transcriptional
silencing.
The
nucleosome
structure
of
latent
HSV
exhibits
a
more
stable
organization
than
in
lytic
infection,
with
nucleosome
occupancy
appearing
more
similar
to
that
of
cellular
DNA
[7].
The
variant
histone
macro2A,
known
to
be
a
strong
repressor
of
cellular
transcription
[34],
has
also
been
shown
to
be
enriched
in
its
incorporation
into
latent
HSV
chromatin
structure
[35].
These
findings
are
consistent
with
a
decrease
in
the
accessibility
of
lytic
promoters
to
transcriptional
machinery,
reinforcing
transcriptional
silence
as
progression
into
latency
takes
place.
From
studies
performed
on
mouse
ganglia
harboring
latent
HSV,
it
has
been
demonstrated
that
the
histones
present
at
lytic
gene
loci
are
modified
to
reflect
a
transcriptionally
inactive
state
[35‐38].
Further,
it
has
been
shown
that
enrichment
with
histones
bearing
marks
associated
with
cellular
heterochromatin
is
a
gradual
process
enhanced
by
the
presence
of
a
functional
latency‐
associated
transcript
(LAT)
[37,
38]—a
phenomenon
discussed
in
greater
detail
below.
Taken
together,
these
findings
indicate
that
the
establishment
of
HSV
latency
features
a
corresponding
increase
in
features
that
demarcate
transcriptional
silence
in
a
cellular
context.
The
LAT
is
an
8.3kb
mRNA
that
is
processed
to
yield
a
2.0kb
stable
intron,
and
detection
of
this
transcript
has
long
been
described
as
the
hallmark
of
HSV
latency.
From
studies
performed
in
mice
transgenic
for
the
LAT
it
has
been
shown
that
accumulation
of
the
stable,
spliced
LAT
occurs
most
significantly
in
neural
tissues,
consistent
with
its
association
with
latency
[39,
40].
It
has
also
been
demonstrated
that
LAT‐defective
viral
mutants
are
able
to
establish
and
reactivate
from
latency,
although
less
efficiently,
indicating
that
the
function
of
the
LAT
locus
is
not
solely
5
responsible
for
this
process
[41,
42].
Several
potential
mechanisms
have
been
posited
for
how
the
LAT
contributes
in
establishing
latency,
with
much
work
describing
LAT‐derived
small
RNA
products
as
mediators
of
its
effect.
MicroRNAs
processed
from
the
LAT
locus
have
been
demonstrated
to
downregulate
the
expression
of
the
lytic
genes
ICP0
and
ICP4
via
post‐
transcriptional
modification
[43],
and
sRNAs
processed
from
the
stable
2.0kb
LAT
intron
have
also
been
shown
to
repress
ICP4
expression
[44].
In
this
manner,
the
LAT
may
act
to
help
extinguish
any
residual
lytic
gene
expression
and
facilitate
entrance
into
latency.
The
LAT
has
also
been
implicated
in
the
association
of
transcriptionally
repressive
histone
modifications
with
the
latent
HSV
genome
[37,
38].
This
association
between
a
non‐coding
RNA
species
and
the
accumulation
of
repressive
histone
marks
shares
similarities
with
cases
such
as
Xist‐mediated
transcriptional
silencing
of
the
X
chromosome
through
the
polycomb
repressor
complex
2
(PRC2)
[45].
The
polycomb
repressor
complex
1
(PRC1)
is
involved
in
maintaining
repressive
histone
modifications
added
by
PRC2,
and
has
been
demonstrated
to
bind
the
latent
HSV
genome
[35],
however
a
specific
targeting
mechanism
based
on
the
LAT
has
not
yet
been
defined.
The
existence
of
open
reading
frames
(ORF)
within
the
LAT
locus
has
fostered
the
hypothesis
that
a
functional
protein
may
be
translated
from
the
LAT,
and
may
contribute
to
its
effect.
For
several
reasons—such
as
the
LAT
exhibiting
nuclear
localization
and
higher
conservation
of
nucleotide
than
protein
sequence
between
strains—it
has
been
widely
held
that
the
LAT
does
not
function
via
a
protein
product.
However,
as
deregulation
of
LAT
ORF
has
yielded
functional
protein
products
[46],
mutation
of
the
LAT
ORF
start
codons
decreases
LAT
function
[47],
and
translated
LAT
ORF
have
been
detected
in
latently
infected
mice
[48],
the
possibility
of
a
LAT
protein
remains.
Regardless
of
the
mechanism
by
which
the
LAT
functions,
the
results
collectively
support
the
hypothesis
that
its
effect
is
to
promote
latency.
Maintenance
of
the
latent
state
Once
the
latent
state
has
been
established,
HSV
can
persist
in
the
neuron
indefinitely
until
a
stimulus
is
encountered
that
is
able
to
initiate
reactivation.
A
low
level
of
spontaneous
reactivation
has
been
demonstrated
to
occur
in
most
models
of
latency
[49‐51],
however
the
vast
majority
of
the
virus
maintains
its
latent
state.
Two
main
viral
factors
are
theorized
to
promote
the
maintenance
of
latency:
conformation
of
the
viral
genome,
and
the
effects
of
the
LAT.
In
quiescent
infection
of
both
neuronal
and
non‐neuronal
cells,
HSV
has
been
shown
to
most
stably
persist
as
a
circular
episome
[52,
53],
as
opposed
to
linear
or
other
conformations
potentially
more
susceptible
to
degradation.
Circularization
does
not
necessarily
reflect
an
active
viral
process
specific
to
favoring
latency
however,
as
a
circular
form
is
also
supported
as
being
the
initial
replication
template
during
lytic
infection
[54].
In
addition
to
viral
conformation
influencing
persistence
of
the
latent
state,
the
LAT
has
been
implicated
in
maintaining
latency.
The
LAT
is
the
only
actively
transcribed
viral
gene
during
latency,
and
is
associated
with
histones
marked
to
reflect
active
transcription
[38,
55].
The
importance
of
transcription
from
this
locus
is
reflected
by
the
binding
of
chromatin
insulators,
which
segregate
the
active
chromatin
structure
of
the
LAT
from
the
rest
of
the
transcriptionally
repressed
virus
[56].
Several
studies
have
indicated
a
role
for
the
LAT
in
preventing
the
neuron
from
undergoing
apoptosis
[57],
indirectly
influencing
maintenance
of
the
latent
HSV
reservoir.
Additional
support
for
this
role
has
been
shown
through
the
ability
to
functionally
substitute
the
LAT
with
unrelated
anti‐apoptosis
genes
[58,
59].
Deletion
analysis
of
the
LAT
locus
has
localized
the
anti‐apoptotic
effect
to
the
first
1.5kb
of
the
primary
transcript
[60],
and
there
is
evidence
to
support
a
role
for
two
small
RNA
products
from
this
region
in
enhancing
survival
of
infected
6
neuronal
cells
[44].
Again,
it
has
not
been
resolved
if
the
mechanism
of
the
LAT
effect
is
through
regulatory
RNA
species
or
if
a
protein
component
is
involved.
It
is
also
possible
that
the
LAT
may
keep
the
latent
HSV
genome
in
a
state
amenable
to
future
reactivation;
the
effect
of
heterochromatin
marks
becoming
increasingly
associated
with
the
virus
in
the
presence
of
the
LAT
transcript
is
not
observed
within
the
rabbit
model
[61],
which
is
regarded
as
a
closer
approximation
of
human
HSV
latency.
This
would
be
consistent
with
reports
that
reactivation
of
LAT‐deleted
mutant
HSV
in
the
rabbit
model
is
significantly
reduced
[62],
as
would
be
the
case
if
the
LAT
is
responsible
for
maintaining
a
viral
state
poised
for
reactivation.
While
further
insight
into
the
mechanism
behind
the
LAT
phenomenon
in
this
model
has
not
as
of
yet
been
provided,
it
is
possible
that
certain
functions
of
this
transcript
could
be
host‐specific.
The
LAT
has
primarily
been
supported
as
contributing
to
the
maintenance
of
latency
through
its
aforementioned
ability
to
dampen
lytic
gene
transcription
in
the
neuron,
and
by
promoting
survival
of
infected
neurons
through
its
anti‐apoptotic
effect.
Viral
reactivation
in
response
to
stress
HSV
infection
of
neurons
exists
as
a
reversible
state,
where
maintenance
of
latency
is
punctuated
by
episodes
of
viral
reactivation
which
lead
to
production
of
infectious
virus
at
the
epithelial
surface.
As
reactivation
of
HSV
in
the
TG
is
known
to
be
preceded
by
cellular
stress,
changes
in
the
neuronal
environment
due
to
the
stress
response
may
be
involved
in
creating
a
situation
permissive
for
viral
replication.
An
overview
of
some
of
the
main
features
of
reactivation
is
presented
in
Figure
2.
In
response
to
stress,
several
neuronal
events
take
place
that
may
facilitate
the
exit
of
HSV
from
the
latent
state.
Immediately
following
a
reactivation
stress
to
the
neuron,
HCF
translocates
from
its
position
at
the
Golgi
to
the
nucleus,
where
it
further
localizes
to
the
viral
IE
promoters
[63,
64].
A
possible
role
for
HCF
in
assisting
reactivation
from
latency
has
been
described
wherein
HCF
mediates
the
removal
of
repressive
histone
methylation
through
interaction
with
lysine‐specific
demethylase‐1
(LSD‐1),
a
participant
in
the
REST/coREST/HDAC
complex
whose
blockade
is
correlated
with
reduced
reactivation
ability
[14].
As
in
the
establishment
of
latency
the
role
of
Oct‐1
during
reactivation
is
not
well
defined,
although
some
evidence
supports
the
upregulation
of
Oct‐1
in
response
to
stressors
such
as
DNA
damage
and
chemotherapeutic
agents
[65],
indicating
a
potential
means
of
overcoming
low
neuronal
levels
of
this
cofactor.
Early
during
reactivation
a
sharp
decrease
in
the
amount
of
LAT
mRNA
is
observed
[66],
and
this
has
been
connected
to
regulation
of
the
LAT
promoter
by
the
inducible
cAMP
early
repressor
(ICER)
through
proximal
repressor
elements
[67].
This
observation
is
consistent
with
a
role
for
the
LAT
in
enforcing
maintenance
of
the
latent
state
by
repression
of
lytic
gene
expression.
In
addition,
reports
that
components
of
the
REST/coREST/HDAC
complex
are
upregulated
in
situations
of
neuronal
stress
indicates
this
complex
may
participate
in
induction
of
the
IE
genes
during
reactivation
[68].
Collectively,
these
observations
show
a
change
in
the
neuronal
environment
in
response
to
reactivation
stress
that
may
facilitate
the
initial
steps
in
HSV
exit
from
latency.
Global
changes
to
the
chromatin
structure
of
latent
HSV
also
occur
in
response
to
reactivation
stimuli,
including
changes
in
histone
modifications
and
nucleosome
organization
on
the
viral
genome.
It
has
been
reported
that
following
reactivation
stimuli,
histones
on
the
latent
HSV
genome
become
associated
with
activating
marks
[66,
69].
These
changes
are
transiently
observed
however,
and
there
is
difficulty
in
interpreting
the
relevance
of
these
results
due
to
the
use
of
HDAC
inhibitors,
which
may
increase
histone
acetylation
in
a
global
manner
that
does
not
bear
on
transcriptional
activation
[70].
In
a
comparison
between
an
efficiently
reactivating
strain
7
and
a
poorly
reactivating
strain
in
the
rabbit
model,
the
efficiently
reactivating
strain
demonstrated
an
increased
association
of
acetylated
histones
on
the
lytic
promoter
ICP4
that
correlated
with
an
increase
in
ICP4
mRNA
levels
when
a
non‐HDAC
reactivating
agent
was
utilized
[71].
The
results
of
this
study
suggest
that
epigenetic
regulation
does
play
a
role
in
reactivation
of
HSV,
however
the
exact
mechanisms
by
which
this
effect
is
mediated
remain
elusive.
Figure
2:
Model
of
the
process
of
reactivation
from
latency.
1)
The
neuronal
stress
response
genes
are
induced
following
the
reactivation
stimulus.
2)
Transcription
factors
accumulate
in
the
nucleus,
such
as
HCF,
which
is
translocated
from
the
cytoplasm.
3)
Transcription
from
the
LAT
is
shut
off.
4)
The
HSV
genome
is
loaded
with
transcriptionally
permissive
chromatin.
5)
Transcription
from
the
IE
genes
can
now
proceed.
6)
IE
gene
products,
such
as
ICP0,
reinforce
the
reactivation
stimuli
and
work
to
initiate
viral
replication.
The
viral
trans‐activating
protein
ICP0
has
been
the
subject
of
considerable
study
due
to
its
strong
association
with
events
that
occur
during
reactivation
from
latency.
From
the
intensive
8
study
of
the
role
of
this
protein
in
the
context
of
HSV
reactivation
it
has
become
clear
that
while
ICP0
is
involved
in
numerous
interactions
that
work
to
successfully
reactivate
the
virus,
it
does
not
appear
to
be
responsible
for
its
initiation
[51,
72‐74].
In
latent
or
quiescent
infection
by
HSV
mutants
defective
in
the
major
trans‐activators,
providing
ICP0
alone
in
trans
is
sufficient
to
overcome
host
repression
[75,
76],
however
it
has
been
demonstrated
that
mutant
viruses
lacking
functional
ICP0
are
also
able
to
successfully
rescue
the
defective
mutants
[72].
While
restriction
mechanisms
that
facilitate
reactivation
studies
in
cell
culture
systems
are
incompletely
understood
and
highly
variable
between
cell
types,
ICP0
has
been
demonstrated
to
target
repressive
cellular
mechanisms
during
reactivation
from
quiescence.
This
viral
protein
is
known
to
mediate
protein
degradation
through
ubiquitin
ligase
activity,
disrupt
ND10
nuclear
substructures,
and
dissociate
HDAC
machinery
[77]—all
of
which
may
contribute
to
bringing
latent
HSV
into
a
transcriptionally
active
state.
ICP0
has
also
been
associated
with
mediating
a
change
in
HSV
chromatin
structure
and
histone
modifications
during
reactivation
[4,
78,
79],
which
is
unsurprising
in
light
of
its
known
associations
with
chromatin
remodeling
complexes
during
lytic
infection.
That
ICP0
is
involved
in
these
processes
suggests
that
while
not
strictly
required
for
reactivation
to
occur,
presence
of
this
protein
greatly
favors
successful
reactivation.
Studies
targeted
at
elucidating
the
initial
genes
to
be
induced
following
reactivation
stimuli
have
found
several
HSV
promoters
to
be
upregulated
following
stress,
including
that
of
ICP0
[80].
It
has
been
theorized
that
the
HSV
genetic
program
during
reactivation
follows
a
different
pattern
from
that
seen
during
lytic
infection
[81,
82],
however
increased
sensitivity
in
detection
methods
has
indicated
that
HSV
reactivation
may
proceed
as
per
the
lytic
program
following
stress
stimuli
[80,
83].
In
sum,
the
exit
from
latency
is
seen
to
be
greatly
enhanced
by
the
function
of
the
IE
trans‐activators,
and
replication
of
HSV
may
proceed
in
a
manner
similar
to
lytic
infection
once
the
neuronal
environment
is
altered
to
favor
this
process.
VP16:
A
viral
mediator
of
reactivation
from
latency?
Despite
all
that
has
been
defined
so
far
in
the
HSV
latency‐reactivation
cycle,
the
mechanism
by
which
reactivation
is
initiated
has
not
yet
been
fully
elucidated.
The
most
recent
evidence
suggests
that
VP16
is
the
viral
mediator
of
reactivation
from
latency,
able
to
undergo
de
novo
synthesis
in
response
to
cellular
stress
effectors
and
induce
expression
of
the
IE
gene
class
in
a
manner
similar
to
lytic
infection.
It
is
important
to
also
consider,
however,
that
cellular
stress
response
effectors
may
stimulate
the
expression
of
another
viral
factor,
or
indirectly
stimulate
viral
gene
expression
through
stress‐induced
relief
of
cellular
repression
mechanisms
prior
to
VP16
synthesis.
Evidence
for
VP16
as
the
viral
linchpin
of
reactivation
is
discussed
in
the
following
section.
Because
of
the
obvious
suitability
of
VP16
for
the
role
of
reactivating
HSV
from
the
latent
state,
early
experiments
were
designed
to
determine
if
the
presence
of
this
protein
could
abort
entrance
into,
or
induce
exit
from
latency
[84].
A
recombinant
virus
was
constructed
carrying
a
second
VP16
gene
under
an
inducible
promoter,
and
mice
were
infected
and
induced
to
express
VP16
via
the
secondary
promoter.
As
significant
changes
to
establishment
and
reactivation
from
latency
were
not
observed,
it
was
concluded
that
VP16
expression
did
not
preclude
these
processes
and
was
therefore
not
involved.
The
results
of
this
study
must
be
interpreted
with
care,
as
some
methodological
questions
remain
unanswered—such
as
if
functional
VP16
was
translated
in
the
mouse
neuron
in
response
to
promoter
induction.
In
seeming
contrast,
the
results
of
contemporary
reactivation
studies
have
shown
that
VP16
provided
in
trans
is
able
to
mediate
reactivation
from
latency
[51,
76,
85],
and
recent
inquiry
has
sought
to
further
explore
9
the
potential
of
VP16
to
be
involved
in
initiating
the
reactivation
process.
Lending
support
to
the
hypothesis
that
de
novo
synthesis
of
VP16
can
mediate
reactivation
in
vivo
are
observations
that
HSV
mutants
deleted
for
the
VP16
AD
are
unable
to
exit
the
latent
state,
as
demonstrated
by
the
absence
of
viral
lytic
proteins
[85].
Further,
the
VP16
promoter
has
been
shown
to
be
activated
by
reactivation
stimuli
in
experiments
utilizing
a
reporter
gene
fused
to
the
VP16
promoter
at
a
secondary
site
on
the
HSV
genome
[85],
suggesting
the
block
to
reactivation
occurs
because
of
the
missing
AD.
These
results
prompt
a
closer
look
at
the
suitability
of
VP16
to
act
as
the
viral
mediator
of
reactivation.
VP16
is
transcribed
with
leaky
late
kinetics
during
lytic
infection
yet
has
several
properties
which
distinguish
it
from
the
prototypical
leaky
late
gene
VP5.
These
differences
may
be
responsible
for
the
reported
activity
of
VP16
in
the
earliest
phase
of
the
viral
response
to
stress
[85].
In
a
comparative
analysis
of
the
VP16
and
VP5
promoters,
several
differences
in
core
promoter
elements
were
described,
including
the
existence
of
an
E
box,
the
first
of
which
to
be
described
in
the
context
of
an
HSV
promoter
[86].
E
box
promoter
elements
are
able
to
interact
with
a
variety
of
transcription
factors,
and
have
been
documented
to
be
relevant
in
the
context
of
the
neuronal
transcriptional
response
to
stress
stimuli
[87].
As
the
promoter
of
VP5
has
been
shown
to
be
unable
to
functionally
substitute
for
the
VP16
promoter
[85],
it
is
possible
that
VP16‐specific
promoter
elements,
such
as
the
E
box
element,
are
able
to
mediate
its
early
expression
and
thus
initiate
exit
from
the
latent
state.
Recent
studies
on
the
stress
induction
of
HSV
promoters
has
indicated
that
the
VP16
promoter
is
modestly
induced
by
heat
shock
stress
in
the
absence
of
other
viral
factors
[80],
supporting
the
theory
that
VP16
may
interact
with
neuronal
stress
response
effectors.
The
role
of
viral
genes
responsive
to
neuronal
stress
that
are
also
known
to
regulate
VP16
in
the
lytic
context,
such
as
ICP27
[88,
89],
will
be
a
key
issue
in
investigating
the
ability
of
VP16
to
mediate
reactivation.
Central
to
the
issue
of
VP16‐mediated
reactivation
is
again
the
subcellular
transport
of
this
viral
protein.
The
findings
of
one
study
utilizing
UV‐irradiated
superinfecting
virus
as
a
reactivation
stimulus
demonstrated
a
lack
of
response
from
latent
HSV
in
mouse
trigeminal
ganglia
[90].
These
results
imply
that
VP16
from
the
tegument
is
insufficient
to
initiate
reactivation
and
may
indirectly
support
a
role
for
newly
synthesized
VP16
as
mediating
this
effect.
Several
unresolved
issues
remain
that
obscure
a
clear
understanding
of
the
role
of
VP16
beyond
that
of
IE
gene
trans‐activator,
and
bear
on
how
this
protein
interacts
with
the
neuronal
environment.
For
example,
the
VP16
protein
does
not
appear
to
have
a
classic
nuclear
localization
signal,
and
conflicting
data
exist
on
whether
cellular
HCF
is
able
to
act
as
a
chaperone
for
its
nuclear
import.
Through
use
of
a
transfected
plasmid
system
it
has
been
demonstrated
that
there
is
some
correlation
between
the
functional
binding
of
VP16
and
HCF
and
nuclear
import
of
both
proteins
[91],
however
as
siRNA
depletion
of
HCF
does
not
decrease
VP16
trans‐
activation
function
[92],
it
is
possible
that
VP16
reaches
the
nucleus
via
another
primary
method.
Because
HCF
is
cytoplasmic
prior
to
the
induction
of
neuronal
stress,
it
is
unclear
whether
VP16‐
HCF
interaction
represents
another
factor
in
the
balance
between
latency
and
reactivation.
In
addition
to
its
role
in
the
VP16‐induced
complex,
the
VP16
protein
has
also
been
demonstrated
to
interact
with
cellular
chromatin
regulation
machinery
in
lytic
infection.
It
has
been
shown
that
the
absence
of
the
VP16
AD
leads
to
increased
occupancy
of
histones
that
are
modified
to
reflect
a
transcriptionally
repressed
state
[5,
93,
94].
The
exact
mechanism
behind
this
phenomenon
has
not
as
of
yet
been
determined,
however
VP16
has
been
demonstrated
to
interact
with
histone
acetyltransferases
(HATs)
as
well
as
the
ATP‐dependent
chromatin
remodeling
complex
SWI/SNF
[95],
lending
support
to
the
notion
that
VP16
can
effect
epigenetic
10
change.
These
functions
may
play
a
role
in
increasing
transcriptional
activity
from
latent
HSV
in
the
context
of
reactivation
in
the
neuron.
In
sum,
described
functions
of
the
VP16
protein,
as
well
as
characteristics
of
the
VP16
promoter
lend
this
protein
well
to
a
role
in
HSV
reactivation.
Models
of
HSV
latency
Latency
and
reactivation
studies
are
routinely
performed
in
a
variety
of
model
systems,
however
demonstrated
difficulty
has
been
encountered
that
must
be
noted
to
be
able
to
interpret
findings
accurately.
In
the
small
animal
model
the
obvious
benefit
is
a
similarity
to
the
human
context
in
terms
of
infection
at
the
body
surface
feeding
establishment
of
a
latent
reservoir
in
the
TG.
However,
it
is
difficult
to
detect
genetic
changes
in
the
relatively
small
number
of
neurons
that
undergo
reactivation
during
natural
infection,
and
the
background
of
immune
and
surrounding
cells
must
be
also
be
accounted
for.
There
is
also
an
issue
of
host
restriction;
for
example,
it
is
known
that
mouse
Oct‐1
differs
from
human
Oct‐1
at
several
residues
important
for
interaction
with
VP16,
impeding
the
ability
of
some
HSV
strains
to
replicate
in
this
context
[96].
Use
of
ex
vivo
cultured
neurons
or
differentiated
neuronal
cell
lines
allows
for
isolation
and
control
of
the
occurrence
of
reactivation
and,
in
the
latter,
latency,
however
the
necessary
use
of
reagents
to
inhibit
lytic
replication
suggests
that
these
systems
approximate
natural
latency
less
well.
Notably,
a
model
using
isolated
porcine
neurons
in
a
two‐chambered
apparatus
that
permits
the
natural
HSV
infection
route
to
occur
has
been
recently
developed
[97],
and
avoids
several
of
the
aforementioned
issues.
Human
fibroblasts
have
been
utilized
as
a
simple
system
of
HSV
latency,
wherein
a
natural
restriction
of
HSV
lytic
replication
is
achieved
through
incubation
at
a
non‐permissive
temperature
[75].
This
system
provides
a
means
of
studying
HSV
repression
in
a
human
cell
context
that
may
mimic
several
aspects
of
neuronal
latency—such
as
the
existence
of
a
heterologous
population
of
quiescent
genomes
in
varying
states
of
silence
[98]—
however
it
is
not
clear
if
the
repression
mechanisms
active
in
these
cells
are
similar
to
those
present
in
primary
neurons.
Conclusions
Recent
evidence
has
demonstrated
a
role
for
VP16
in
reactivation
of
HSV
from
latent
infection
of
the
neuron,
a
fitting
finding
considering
the
role
of
this
protein
in
initiating
the
lytic
genetic
program.
While
the
same
intensive
study
of
VP16
has
not
yet
been
performed
as
it
has
for
other
HSV
factors
involved
in
the
latency‐reactivation
cycle—such
as
ICP0
and
the
LAT—this
factor
may
be
the
key
to
further
deciphering
the
complex
interactions
between
neuronal
environment
and
virus.
While
the
conversation
on
HSV
reactivation
has
occurred
over
the
better
part
of
the
past
century,
support
for
several
hypotheses
over
the
past
decade
has
helped
illuminate
the
molecular
mechanisms
by
which
the
process
of
latency
occurs.
Firstly,
epigenetic
regulation
through
modification
of
HSV
secondary
chromatin
structure
plays
an
important
role
in
the
regulation
of
HSV
at
all
points
of
its
infectious
cycle.
This
is
illustrated
especially
by
rapid
changes
to
chromatin
structure
following
reactivation
stimuli.
Secondly,
multiple
interactions
between
viral
factors
and
cellular
regulatory
machinery
belie
the
exquisite
control
HSV
exerts
over
the
cells
it
infects.
Finally,
HSV
has
evolved
to
exploit
the
specific
environment
of
the
neuron,
a
cell
with
a
distinctive
pool
of
transcription
factors
and
availability
of
regulatory
mechanisms,
to
establish
a
latent
reservoir
that
ultimately
fosters
the
spread
and
survival
of
this
virus
within
the
human
population.
11
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Figure
3:
Summary
of
major
events
in
the
HSV
latency‐reactivation
cycle.
For
details,
see
text.
12
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