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MBB 306 (2013 - 2014)

VIRUSES and INFECTIONS

of HUMANS

LECTURE: LATENT and PERSISTENT

VIRUS INFECTIONS

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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.

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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

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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.

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1. LATENT INFECTION:

THE HERPES SIMPLEX VIRUSES

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OROFACIAL HERPES SIMPLEX VIRUS

LESIONS a) Primary infection b) Recurrent infection

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EM of a

HERPESVIRUS

PARTICLE

virion

membrane

(lipid envelope)

capsid

containing

viral dsDNA

tegument

SCHEMATIC

REPRESENTATION

of a HERPESVIRUS

PARTICLE

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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.

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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

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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

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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)

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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.

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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.

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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.

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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.

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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.

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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.

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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

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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

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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

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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

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PERSISTENT INFECTION:

THE HEPATITIS C VIRUS

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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.

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P ATIENT with ACUTE HEPATITIS

INFECTION

HCV GENOME and PROTEIN FUNCTION

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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.

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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.

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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

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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.

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END of LECTURE

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RETROGRADE = moving towards a goal, against the

direction of flow accompanied by a worsening or

deteriorating condition.

ANTEROGRADE = moving in the normal or usual

direction of flow.