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1
MBB 306 (2013 - 2014)
VIRUSES and INFECTIONS
of HUMANS
LECTURE: LATENT and PERSISTENT
VIRUS INFECTIONS
2
TIME (YEARS)
Chronic Infection (Hepatitis B and C)
Infectious virus
Virus shedding
Presence of
infectious virus
Varicella Zoster
Latent Infection (Herpesviruses eg Varicella-Zoster)
Non-infectious
Disease episode
TYPES of LONG-LASTING VIRAL
INFECTIONS i) Latent infections – the viral genome is present but infectious virus not produced, its
presence only detected by molecular methods such as PCR: e.g.
all herpesvirus infections.
ii) Chronic persistent
infections – infectious virus
constantly present and
detectable by
conventional laboratory
methods: e.g. hepatitis B
and C virus infections.
3
PERSISTENT/LATENT INFECTIONS of MAN
VIRUS GROUP CELL OR TISSUE
TYPE INVOLVED
STATE OF VIRAL
NUCLEIC ACID
Hepadnaviridae
(Hepatitis B virus)
Hepatocytes Partially integrated
Flavivirus
(Hepatitis C virus)
Hepatocytes; Cells of
lymphocytic origin
Not integrated; Replicates
in cytoplasm
Herpetoviridae:-
a)HSV (1 & 2)
b) VZV
c) EBV
d) Four other human
herpesviruses
Sensory neurones
Sensory neurones
B lymphocytes
Episomal
Episomal
Episomal
Papillomaviridae Keratinising epithelial cells Integrated and episomal
Retroviridae:-
a) HIV
b) HTLV-1
CD4+ lymphocytes
T lymphocytes
Episomal and integrated
Integrated
4
IMPLICATIONS of LATENT and
PERSISTENT VIRAL INFECTIONS
1. Epidemiological:– long-term asymptomatic virus
carriers as source of virus spread and new
infections.
2. Immunopathological: - immune response to long-
term infections resulting in disease.
3. Neoplasms:- long-term outcome of some persistent
infections.
4. Control - strategic use of vaccines and antiviral
drugs.
7
EM of a
HERPESVIRUS
PARTICLE
virion
membrane
(lipid envelope)
capsid
containing
viral dsDNA
tegument
SCHEMATIC
REPRESENTATION
of a HERPESVIRUS
PARTICLE
8
BIOLOGY of LATENT HSV INFECTIONS in HUMANS
• Following primary infection of epithelial cells in the oral cavity
(HSV-1) or the genital mucosa (HSV-2), local nerve endings are
invaded and virus is transported along axons to the local dorsal
root ganglion.
• Most often infected are trigeminal and superior cervical ganglia
(HSV-1) and sacral ganglia (HSV-2), and both viruses can establish
latent infection in any dorsal root ganglion. However, in about
50% of latently-infected individuals subsequent endogenous
recurrent infections do not occur.
• In the remaining individuals, a first reactivation occurs at around
4 months following the primary infection.
• Frequency of subsequent recurrences vary between rare (<1 per
year) to so common as to be virtually continuous.
9
HSV LATENCY and REACTIVATION
HSV Latency
in neurones
a) No attack by CTLs or antibodies as MHC antigens are normally down-regulated in
neurones.
b) The neuronal axon provides a direct pathway to the periphery and therefore to
susceptible epidermal cells and viral dissemination.
c) No need for viral genome to divide to maintain a fixed number of genome copies in the
cell, as neurones do not divide.
ADVANTAGES OF SURVIVAL IN NEURONES
10
HERPES SIMPLEX VIRUS (HSV)
LATENCY HSV-1 latency in sensory neurones is the default pathway
consequent on a failure to initiate HSV IE gene expression.
There are three stages:-
1. Establishment of Latency – virus latently established in sensory ganglia by
21-28 days post-infection. Lack of ICP0, VP16 and HCF repression
of HSV IE gene activity. LATs involved in establishment of latency.
2 Maintenance of Latency – presence of LATs (Latency Associated
Transcripts); form of chromatin; continued inhibition of ICP0; inhibition of
apoptosis no viral DNA replication; no detectable viral proteins.
3. Reactivation from the Latent State – At host level - ‘stress’ on neuronal cells
- through tissue trauma, UV-irradiation, chemotherapeutic agents, hormonal
changes. De novo synthesis of VP16; the transactivating function of VP16 is
necessary for virus exit from the latent state.
ICP0
11
Immediate Early promoter
VP16
HCF-1
HSV DNA
Histone H3K9 Oct-1
Histone H3K4
HATs
Histone
acetylation of
HSV DNA Transcription using host
cell RNA polymerase II
and viral IE proteins
HCF-1
Factors Contributing to Establishment of
HSV Latency in Neuronal Cells
Luman
NUCLEUS
CYTOPLASM
HSV DNA associated with methyl
groups (heterochromatin form)
NO TRANSCRIPTION of
any VIRAL PROTEINS
LATs
(miRNAs)
12
FACTORS in the ESTABLISHMENT of HSV-1
LATENCY in NEURONES
1. Reduction in VP16 activity and availability through :-
Loss during passage through the axon.
2. Lack of Oct-1:-
Oct-1 is not present in sensory neuronal nuclei and equivalent enzymes
in the nucleus (eg Oct-2) are far less efficient at binding VP16.
3. Reduction in HCF availability through:-
a) Lack of HCF (in cells HCF is found in the cell cytoplasm).
b) In neuronal cells HCF is sequestered in the cytoplasm by interaction
with Luman, a cell protein homologue of VP16.
4. Reduction in ICP0 availability:– despite expression of its mRNA, ICP0 does
not accumulate in the neuronal nucleus as it does in epithelial cells.
5. Function of LATs:-
a) Prevent apoptosis.
b) Give rise to miRNAs that inhibit ICP0 activity.
6. Role of Chromatin – histone posttranslational modifications involve
heterochromatin in association with HSV-1 genes during latency
completely preventing HSV gene transcription and expression.
13
LATENCY ASSOCIATED
TRANSCRIPTS (LATs) LATs are overlapping, uncapped, non-polyadenylated
antisense RNA transcripts. 1000s of LAT molecules are
present in neurones latently-infected with HSV. They are
transcribed from within the viral genomic long repeat
sequences.
LATS encode multiple, functional microRNAs (miRNAs),
non-coding RNAs of 21-24 nucleotides that regulate gene
expression based on sequence similarity to their target.
Phenotypes such as increased efficiency of latency
establishment, reactivation from latency and apoptosis are
associated with the LATs.
14
THE FUNCTION of LATENCY ASSOCIATED
TRANSCRIPTS (LATs) LATS down-regulate the genes required for lytic infection.
Two miRNAs encoded by HSV LATs are both antisense to, and
efficiently silence the HSV-ICP0 gene. Two other miRNAs silence
the ICP34.5 1gene of the virus. ICP34.5 is a regulator of HSV
DNA replication.
Presence and activity of ICP0 controls the balance between the lytic
and latent states.
HSV LATs also have extensive anti-apoptotic activity in HSV-
infected neurones.
HSV LATs facilitate the long-term stability of the latent cell
population within the infected host, ie maintain the latent state.
15
MAINTENANCE of HSV LATENT INFECTION
In HSV-infected human ganglia, there are thousands of latently-infected sensory
neurones. In any particular neurone, the HSV DNA copy number can vary
from <10 to >1000.
Five factors appear to contribute to the maintenance of HSV latency in human
sensory neurones over the life of the host.
1.Inhibition of HSV genome activity, especially the ICP0 and ICP4 genes, via
LATs and microRNAs.
2.Anti-apoptotic activity of LATs.
3.Presence of cellular repressor factors that can associate with the HSV DNA in
the absence of ICP0.
4.The repressive heterochromatin form of the HSV DNA through histone
methylation.
5.The non-cytolytic CD8+ T cell inhibition of neuronal HSV-1 viral replication.
16
HSV REACTIVATION from LATENCY - 1 1. Latent virus reactivates in stressed neurones, and the precise mechanism has
almost been worked out. In response to stress, several neuronal events take
place that facilitate exit of HSV from the latent state:-
2. In animal models, immediately following neuronal stress, HCF-1 can move
from the Golgi in the cytoplasm into the nucleus, and can recruit cellular
proteins such as LSD-1 to HSV IE promoters to reverse repressive histone
methylation.
3. Following stress, there is de novo expression of VP16. Recent studies in
animal models have indicated that the VP16 promoter is modestly induced
by heat shock stress, suggesting VP16 may interact with neuronal stress
response effectors.
4. There are reports of an up-regulation of Oct-1 during reactivation, although
this is disputed. Decrease in the amount of the LAT RNAs has been
observed.
5. Changes to the chromatin structure of the latent HSV genome occurs,
including histone modifications and nucleosome re-organisation.
17
HSV REACTIVATION from LATENCY - 2 1. ICP0 is not involved in the initiation of reactivation but is
required for progression to virus production following exit from
latency.
2. Reactivation involves neurones having a high copy number of
HSV DNA in their nucleus. The large reservoir of latently-
infected neurones allows repeated periodic reactivation to
occur.
3. In HSV infections, spontaneous reactivation leading to virus
shedding from the mucosa occurs at high frequency.
4. When the viral lytic cycle starts the neuronal cell dies. Neurone
loss is recuperated by natural re-routing of connections.
5. After reactivation, newly-synthesized viral DNA moves from
neurones to epithelial cells and the lytic cycle starts.
18
MOLECULAR EVENTS in LYTIC HSV
INFECTION
HCF-1 VP16
Oct-1
Cellular factors
promoting histone
acetylation, +
LSD-1 (lysine-
specific
demethylase-1)
TAATGARAT
HSV DNA
Acetylation of
histones
Chromatin
remodelling
Histone 3
HSV DNA
Histone modifiers
associated with
active
transcription
H3K9ac; H3K14ac
RNApol II
HSV IE Promoters
transcribed
19
MOLECULAR EVENTS in LATENT HSV
INFECTION
TAATGARAT
HSV DNA
Methylation of
histones
Deacetylation
of histones
Histone 3
HSV DNA
Histone modificers
associated with active
transcription
H3K9me; H3K27ac
RNApol II
HSV IE Promoters
transcribed
In absence of virus-
encoded transactivators,
methylated histones and
histone deacetylases
maintain viral
promoters in a repressed
state
20
MOLECULAR EVENTS in GENOME
DEREPRESSION of HSV LATENT INFECTION
HCF-1 VP16
Oct-1?
Cellular factors
promoting histone
acetylation, +
LSD-1 (lysine-
specific
demethylase-1)
TAATGARAT
HSV DNA
Acetylation of
histones
Chromatin
remodelling
Histone 3
HSV DNA
Histone modifiers
associated with
active
transcription
H3K9ac; H3K14ac
RNApol II
HSV IE Promoters
transcribed
21
LATENT HSV INFECTION in NEURONES
HSV-1 virions
Attachment to nerve cell termini
Entry into neurone axon
---------------------------------------------------------------------------------------------------------------------------
Retrograde axonal transport of virus
(with detachment of tegument and tegument proteins)
--------------------------------------------------------------------------------------------------------------------------
Entry of linear HSV genome into nucleus
Circularisation of viral genome
Low or absent viral factors
Low or absent host cell factors
Lack of IE transcripts
Establishment of latency
(LAT gene expression)
Presence of all necessary viral and
host proteins
Stimulation of IE transcripts
Further stimulation of viral
and/or cellular factors? Removal
or reduction of repressor factors?
Reactivation stimuli
(some form of ‘stress’ to the neurones)
-----------------------------------------------------------------------------------
PERIPHERY
AXON
NUCLEUS
EXTRACELLULAR
Activation of viral and
host factors
23
TIME (YEARS)
Chronic Infection (Hepatitis B and C)
Infectious virus
Virus shedding
Presence of
infectious virus
Varicella Zoster
Latent Infection (Herpesviruses eg Varicella-Zoster)
Non-infectious
Disease episode
TYPES of LONG-LASTING VIRAL
INFECTIONS i) Latent infections – the viral genome is present but infectious virus not produced, its
presence only detected by molecular methods such as PCR: e.g.
all herpesvirus infections.
ii) Chronic persistent
infections – infectious virus
constantly present and
detectable by
conventional laboratory
methods: e.g. hepatitis B
and C virus infections.
25
MECHANISMS CONTRIBUTING TO
HCV PERSISTENCE - 1
3’ 5’
E2 C E1 NS2 NS3 NS
4A NS
4B NS5B
NS
5A
E2 hypervariable region - extensive variation within HVR1, its
amino-terminal (hypervariable) region evades acquired immunity.
STRUCTURAL NON-STRUCTURAL
NS5A protein activity – induces down-regulation of the interferon-
stimulated genes through interleukin-8 (IL8).
NS5A and E2 proteins – bind cellular protein kinase R (PKR),
inhibiting its activity as a down-regulator of translation.
E2 protein – indirectly inhibits production of IL2 and interferon- γ
(IFN-γ) from peripheral blood cells.
26
MECHANISMS CONTRIBUTING TO HCV PERSISTENCE - 2
3’ 5’ E2 C E1 NS2 NS3 NS
4A NS
4B
NS
5A NS5B
NS3/4A protease activity - evasion of the main intracellular
defence mechanism of the host cell, IFN-α/β.
Core and NS3 protein activity in infected liver DCs – inhibits
their maturation and stimulatory functions,
including production of IL-12.
Core and NS3 protein activity in infected liver DCs – triggers
release of IL-10 and TNF-α and triggers apoptosis.
STRUCTURAL NON-STRUCTURAL
NS4A/NS4B protein suppresses Th-1 responses by stimulating
production of IL-10 from peripheral blood
monocytes.
27
HEPATITIS C VIRUS – HOST CELL INTERACTIONS and the
OUTCOME of INFECTION
Exposure to HCV
Acute infection
and triggering of
the host response;
RIG-1; TLR3
IFN production/ISG expression
Infection resolved
(15 – 25% of cases)
Reduction of ISG expression and function
Alteration of antigen presentation and immune
cell function
RIG-1 = retinoic acid inducible gene 1
TLR3 = Toll-like receptor 3
NS3/NS4A = HCV non-structural genes/proteins
IFN = interferon
ISG = interferon stimulated genes
Viral protein interference and
blocking of host cell responses
Evasion of IFN action and
persistent infection
75 – 85% cases
Signalling interference:
NS3/4A
Formation of viral quasispecies:
outgrowth/selection/diversification:
viral adaptation
VIRAL PERSISTENCE
28
HUMORAL IMMUNE RESPONSE to HCV ENVELOPE
PROTEINS: EVOLUTION of ANTIBODY ESCAPE MUTANTS
In a virus that circulates as a
quasispecies, antibodies against the
predominant strain exert selective
pressure.
A.
B.
C.
Minor variants will emerge and become
more prevalent. Different tissues
harbour different quasispecies.
Later, the immune system will
recognise and exert pressure on the
new dominant variant, and new
mutants will be selected.
D
Finally, toleration of the virus by the
host is established, and antibody is
superfluous and essentially non-
functional.