MBB 306 - 2013-2014 VIRUSES and INFECTIONS of HUMANS

LECTURE: THE HUMAN HERPESVIRUSES

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CLASSIFICATION of the HUMAN HERPESVIRUSES

ALPHAHERPESVIRUSES: -- Epidermo-neurotropic viruses with a wide host range. Type 1 herpes simplex virus (HSV-1) Type 2 herpes simplex virus (HSV-2) Varicella-Zoster virus (VZV) HHV-1 HHV-2 HHV-3

BETAHERPESVIRUSES:-- Slow growing viruses with a narrow host range; grow in T-lymphocytes and/or leukocytes. Human cytomegalovirus (HCMV) Human herpesvirus 6 Human herpesvirus 7 HHV-5 HHV-6 HHV-7

GAMMAHERPESVIRUSES:-- These viruses have a predilection for B-lymphocytes.

Epstein-Barr virus (EBV) Kaposis sarcoma virus (KSHV)

HHV-4 HHV-8

[Herpesviruses infecting very many vertebrate animals, including other primates, frogs 2 and elephants have been identified].

DISEASES caused by the IMPORTANT HUMAN HERPESVIRUSES

VIRUS NUMBER HHV-1 HHV-2 COMMON NAME Herpes simplex virus type 1 (HSV-1) Herpes simplex virus type 2 (HSV-2) Varicella-Zoster virus (VZV) Epstein-Barr virus (EBV) DISEASE Oropharyngeal herpes; Encephalitis. Genital herpes; Encephalitis

HHV-3

Chickenpox and shingles

HHV-4

Glandular fever/Infectious mononucleosis; Burkitts lymphoma Cytomegalic inclusion disease (CID) in utero, in newborns and in the immunocompromised.

HHV-5

Human Cytomegalovirus (HCMV)

ELECTRON MICROGRAPH OF A HERPESVIRUS PARTICLE

Viral nucleocapsid containing the DNA and ICP0 protein Viral tegument containing VP16 and ICP4 proteins

Lipid envelope of the virus

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SCHEMATIC REPRESENTATION of a HERPESVIRUS PARTICLE

THE HSV GENOME

The linear 152kbp genome contains 3 origins of DNA replication and about 80 ORFs.

HSV DNA replication is by a rolling circle mechanism which takes place in discreet HSV replication compartments.
Replication generates long progeny HSV DNA concatamers which are cleaved and packaged into progeny viral particles.

CYTOPATHIC EFFECT PRODUCED by HERPES SIMPLEX VIRUS (HSV) in CELL CULTURE

MAJOR HSV-1 PROTEINS

THE TEGUMENT PROTEINS
Of the 22 proteins in the HSV tegument , four are structural, while four other proteins are important in altering the environment of the newly-infected cell. Major tegument proteins include:VP 16 (-TIF/transcription initiating factor-coded by gene UL48). ICP0 and ICP4 or immediate early proteins. ICP4:

THE or IMMEDIATE EARLY (IE) PROTEINS

- essential for transcription

ICP-27: - regulator of gene expression ICP-0: - promiscuous transactivator; degrades cellular repressors of HSV DNA transcription

ICP-22/ - deregulate the infected cell Us1.5: cycle ICP-47: - primarily a pathogenic protein

The vhs protein (virion host shut-off protein).

All the IE proteins are also defined by possessing VP16-response (TAATGARAT) elements within their promoters. 6 (ICP = Infectious cell polypeptide: Us = Unique short)

ENTRY of HSV-1 into HOST CELLS

gB fusogen; essential for cell entry gD - essential for cell fusion and penetration
gB Fusogen gH + gL Fusion Regulator gH + gL Receptors
HSV CAPSID + DNA

HSV Glycoproteins

gD Fusion Trigger
gD Receptors

gH+gL complex gB Receptors essential for fusion and cell entry

Host Cell

HSV PROTEINS regulating or operating against HOST CELL METABOLISM

Immediately after entering the cell the HSV-1 tegument protein, vhs, becomes free in the cell cytoplasm. The vhs protein:a). accelerates decay of host cell mRNA b). disrupts pre-existing host cell polysomes Replication of the HSV genome commences with transcription and expression of the HSV IE proteins. Activation and accumulation of these proteins drives transcription of the other viral genes and subverts host gene expression. In particular,

a). ICP0 an E3 ubiquitin ligase destabilising specific cellular proteins that would otherwise inhibit the HSV life cycle. Creates a cellular environment favourable for HSV DNA transcription
b). ICP 27 - inhibits the host cell splicing of pre-mRNA

All the IE proteins contribute to a redirection of cell metabolism from synthesis of host proteins to synthesis of viral proteins, and develop an active and robust 8 environment.

PROGRESSION of HSV and VIRAL DNA to CELL NUCLEUS

After penetration into the cell cytoplasm, the viral envelope is lost. VP16 (500-1000 molecules) and the other tegument proteins are freed into the cytoplasm. The capsid containing the viral DNA and remnants of the tegument is transported to the nuclear membrane along the microtubule network. The capsid docks at the nuclear pore and this facilites release of the viral DNA into the nucleus.

EM INTRACYTOPLASMIC HERPES SIMPLEX VIRIONS

CHROMATIN and HSV DNA REPLICATION

Chromatin is a complex of DNA and histones the nucleosome - through which DNA is structured and manoeuvered in the cell nucleus. Modifications of chromatin permit transcription processes. A nucleosome has a core of 8 histones, with DNA wrapped around the outside, and a single molecule of histone H1 to stabilise the complex. Two types of chromatin are recognised:Heterochromatin - highly condensed; DNA associated with it is inaccessible and therefore genetically inactive. Euchromatin - much less condensed allowing transcription of the associated DNA.

THE NUCLEOSOME

HOST CELL FACTOR (HCF-1), CHROMATIN and HSV LYTIC INFECTION

HCF is a co-activator unable to bind DNA alone. With activated histone acetyl transferases (HATs), HCF causes chromatin modifications that increase gene expression by binding to an activator having a DNA binding domain such as HSV VP16.

The chromatin modifications involving HCF are acetylation and methylation of the nucleosome histone core.
HCF/HAT activity opens the nucleosomes in the control regions of the promoters close to the enhancers, by relaxing the connections between the histones and associated DNA. High levels of acetylation make the chromatin more active, allowing transcription. With low or absent HCF, transcription is reduced as the promoters and enhancers cannot be accessed by transactivating factors. 12

VIRAL GENE CASCADE in LYTIC HSV INFECTION

The entire process of HSV replication takes 18-20h in fully permissive cell cultures.
Immediate Early () - genes -- transcribed and expressed from input viral DNA, induced by VP16 (-TIF) and responsible for synthesis of the 5 proteins (expressed 2-4h p.i.) -- Only transcribed after expression of the genes. (Maximal expression at 5-7h p.i.)

Early () and Late () - genes

Early () - genes
Late (-1 and -2) genes

-- Transcription and expression of the genes is not stimulated by viral DNA replication.
-- -1 genes are transcribed and expressed (at low levels) even in the absence of viral DNA replication, but are greatly stimulated when viral DNA is replicating. -- -2 genes are not transcribed or expressed in the absence of viral DNA replication.

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INITIATION of LYTIC HSV INFECTION in EPITHELIAL CELLS NUCLEUS

Histone (H3K9) acetylation groups form Euchromatin around the HSV DNA

HATs

Oct-1

Transcription of HSV DNA using host cell RNA polymerase II

HSV DNA
HATs HATs HATs (R HATs TAATGARAT = purine) sequences-Immediate early promoter

HCF VP16

VP16 HCF CYTOPLASM

MAJOR INTERACTIONS between HSV and the HOST CELL

HSV infection: elicits changes in cell cycle regulatory circuits causing cells to exit the cell cycle and arrest in G1, favourable for viral replication. interferes with host cell signal transduction pathways. Represses host cell apoptotic pathways, preventing cell death prior to virus release. Disrupts cytokine-mediated signalling networks.
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EXTERIOR ENVIRONMENT

Cell membrane

Cytoplasmic vesicle

RELEASE of HERPES SIMPLEX VIRUS Golgi FROM HOST CELL

Viral glycoproteins modified and matured in the Golgi Cytoplasmic vesicle

Association of nucleocapsid + tegument with membrane of vesicle bearing viral glycoproteins

Endoplasmic reticulum

CYTOPLASM
Precursor viral glycoproteins

Nucleocapsid + tegument

Nuclear membrane

NUCLEUS
Viral nucleocapsid

+
Empty viral capsid

Viral nucleic acid

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EMs showing BUDDING of IMMATURE HSV PARTICLE from NUCLEAR MEMBRANE

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REGULATION of VIRAL GENE EXPRESSION during LYTIC HSV-1 INFECTION

Viral entry to cell cytoplasm

Viral DNA transported to nucleus Immediate Early gene expression

VP16 transported to nucleus

Essential in vitro

ICP4

ICP27

ICP0

ICP22/Us1.5

ICP47 Non-essential
in vitro

X
Early gene expression Both ICP4 and ICP27 must be expressed for the lytic cycle to proceed
Viral DNA replication

Immune evasion

X
Late gene expression
Viral structural proteins

Assembly of virions and release

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DISEASES CAUSED by HSV-1 and HSV-2

Primary and recurrent oropharyngeal herpes (mainly HSV-1)

Primary and recurrent genital herpes (mainly HSV-2) Generalised and encephalitic neonatal herpes infection (mainly HSV-2) Herpetic keratoconjunctivitis and herpetic encephalitis (mainly HSV-1) Skin infections e.g. herpetic whitlow (mainly HSV-1)

Immunocompromised individuals are at increased risk of HSV infections, in terms of both frequency of infection and greater severity of illness.
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ACUTE, PRIMARY HERPETIC GINGIVISTOMATITIS

RECURRENT HERPETIC LESIONS COLD SORES

HERPETIC WHITLOW

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NATURAL HISTORY of HERPES SIMPLEX VIRUS INFECTIONS

SOURCE OF VIRUS SKIN LESIONS SALIVA (usually Type 1 virus) PASIVE IMMUNITY FROM MATERNAL ANTIBODY IN INFANCY SOURCE OF VIRUS GENITAL LESIONS GENITAL SECRETIONS (usually Type 2 virus)

PRIMARY INFECTION

NO IMMUNITY ASYMPTOMATIC SYMPTOMATIC IMMUNE RESPONSES

(90%-99%)

(1%-10%)

ESTABLISHMENT OF LATENT INFECTION

REACTIVATION OF LATENT VIRUS AND RECURRENT INFECTION

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VARICELLA-ZOSTER VIRUS (VZV)

VIRAL PROPERTIES
Size 120-250nm diameter; typical herpesvirus structure dsDNA linear in virion, circular in the infected nucleus. 72 open reading frames (ORFs)
Patient with chickenpox

Latent infection in neurones.

Genetically stable
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EPIDEMIOLOGY

VARICELLA-ZOSTER VIRUS (VZV)

Shingles in elderly patient

Highly infectious disease usually occurring in children. Virus spread from the upper respiratory tract prior to appearance of rash.

Virus present in skin lesions (as much as 1010 1011virions/ml). VZV causes severe infections in the immunocompromised.

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THE EPSTEIN-BARR VIRUS (EBV)

The most common disease caused by EBV is infectious mononucleosis (glandular fever).
About 70% of the worlds population are infected.

Often asymptomatic, but symptoms can include sore throat, swollen cervical lymph nodes and mild fever. May run a prolonged, episodic, course over 12 years. Associated with the cancer, Burkitts Lymphoma in some parts of the world.
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EM of the EPSTEINBARR VIRUS (EBV) PATIENT with INFECTIOUS MONONUCLEOSIS (GLANDULAR FEVER)

END of LECTURE
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INITIATION of LYTIC HSV INFECTION in EPITHELIAL CELLS NUCLEUS HCF

Histone (H3K9) acetylation forming Euchromatin

HATs

SETs

Histone methylation forming Heterochromatin

VP16

Oct-1 Oct-1
HSV DNA
Transcription of HSV DNA involving host cell RNA polymerase II

TAATGARAT sequences-Immediate early promoter

VP16 HCF HCF

VP16
CYTOPLASM

SETs

Histone methylation forming Heterochromatin

HCF

VP16

Oct-1

VP16 HCF