M229: Cell Biology and Pathogenesis

Molecular and cellular biology of herpesvirus pathogenesis

February 2002

Ren Sun

rsnu@mednet.ucla.edu

1. Introduction of herpesviruses2. Mechanism of herpesviral replication3. Herpesviral latency 4. Transformation by Epstein-Barr virus

Discussion of viral oncogene.

Take-home messages:

1. The purpose of viral life is to replicate itself. (its associated pathogenesis or human diseases are secondary effects)

2. Virus utilizes host machinery to replicate. (cellular and organal)

3. The ultimate form of parasitization is to co-exist with the host. (at both individual and population levels)

4. Studies of viruses have double meanings. (medically important and scientifically interesting)

5. Herpesvirus has two phases of the life cycle: latent infection and lytic infection.

herpein: to creep (Greek)

Herpesvirus particle

Electron cryomicroscopy and 3D reconstruction of HSV-1 B-capsidsZ.Hong Zhou et al. Science, 2000 April

Virion structure of herpesvirus

1. Herpesvirus particle is composed of an icosahedral capsid, containing a large linear double-stranded DNA genome.

2. Viral genome is a double-stranded DNA (150 kb to 250 kb), encoding about 100 genes. Only 40-60% of the genes are essential for viral replication in tissue culture.

3. The capsid is surrounded by a tegument and wrapped in a lipid envelope. The tegument, which consists of viral proteins, is unique to herpesviruses.

4. The particle is about 200 to 300 nm with 100 nm capsid.

Herpesviruses

• Large genome, complex gene expression and regulation

• Ubiquitous infections, primary infections usually in-apparent in childhood

• latent/persistent/recurrent infections

• Transition between lytic and latent replication

• Associated with acute and chronic/malignant diseases

Human herpesvirusesName Common name associated diseases subfamily size

HHV-1 Herpes Simplex Virus 1 (HVS-1) 150kbOral, ocular lesions; encephalitis

HHV-2 Herpes Simplex Virus 2 (HSV-2) 150kbGenital oral lesions; neonatal infections

HHV-3 Varicella Zoster virus (VZV) 130kbChickenpox, shingles

HHV-4 Epstein-Barr virus (EBV) 170kbInfectious Mononucleosis; tumors (BL, NPC, NHL)

HHV-5 Human Cytomegalovirus (HCMV) 230kbCongenital infection; systemic infection

HHV-6 Human herpesvirus 6 160kbExanthem subitum; systemic infection

HHV-7 Human herpesvirus 7 160kbExanthem subitum?

HHV-8 Kaposi’s Sarcoma associated herpesvirus 140kbKaposi’s Sarcoma, B cell lymphomas

Herpesvirus family

highly co-evolved with host widely disseminated froom human to flog, fish and snake

• Alphaherpesviruses

– Fast replication, cytolytic; latent in neurons (Herpes Simplex, Varicellar Zoster)

• Betaherpesviruses

– Intermediate replication, cytomegalic; latent in glands, kidneys (Cytomegalovirus, HHV-6, -7)

• Gammaherpesviruses

– Slow replication, latent in lymphocytes, lymphoproliferative, (Epstein-Barr virus, HHV-8)

At cellular level, Latency is the reversibly nonproductive infection of a cell by a replication-competent virus.

(1) They can successfully evade the host immune response.

(2) They enable their genome to persist in the latently infected cell, thereby in the host.

(3) They co-exist with the host cells and the host.

(4) The host becomes a active carrier for transmission.

All herpesviruses are capable of establishing latent infection.

Please note: The difference with abortive infection or infection with defective virus.

What is Latency? and why there is latency?

Mechanisms to establish latency

1. HSV infects non-dividing cells such as neurons.

No protein expression.

2. EBV infects dividing or mitotic cells such as B cells.

A set of viral protein(s) and origin of DNA replication initiation is required for replicating viral genome.

Function of partition is required to retain the viral genome in daughter cells during the latent infection.

3. Use RNAs to avoid antigenicity.

4. Down-regulation of presentation of viral antigen(s).

Life cycle of herpes simplex virus

“Fields Virology”

HSV-1 infection and lytic replication

HSV-1 latency and reactivation

“Fields Virology”

A Model of EBV Life Cycles in vivoPrimary infection of epithelium

Secondary infectionof epithelium

Resting B cellIII

III

Primary infectionPersistent infection

III

III III

IIIIII

I/IIIII

IIIIII

10 CTL response

20 CTL responseMemory CTLResting T cell

Kill Kill

Latency switch?

Lytic

Reactivation10 infection of B cell

Latency switch?

Viral entry of cell (simplex virus)

(1) Virus attachment to the host cell. envelope glycoproteins (gC and gB) binds heparin sulfate

(2) Binding of viral surface protein (gD) to one of the cellular receptors (herpesvirus entry mediators, Hve)

HveA (the tumor necrosis factor receptor family)HveB and C (nectins, Immunoglobulin superfamily)

(3) Fusion of the virus envelope with cellular membrane. gI and gE appear immediately on surface after entry

Multiple interactions during binding, penetration and uncoating.

(2) Virus attachment to the host cell.

EBV envelope glycoprotein gp350/220 binds to CD21(type 2 complement receptor, CR2) directly, in a fashion similar to that of C3d of complement. Activation of tyrosine kinase signal transduction, activate the B cell.gp42 (BZLF2) binds to HLA class II (MHC class II) as co-receptor.gp85/gp25/gp42 oomplex mediates membrane fusion.

HHV-7 binds to the CD4. CD4 is not by itself sufficient for entry. HHV-7 also can infect certain CD4-negative cells.

Each herpesvirus uses different viral envelope protein to bind different cellular receptor. Receptors for other herpesviruses are to be identified.

3) Penetration and uncoating of the virus particle.

For HSV-1, this occurs via a process of fusion of the viral envelope with the cell plasma membrane. HSV-1 glycoproteins gB, gD, gH and gL involve in the fusion.

Capsid binds to microtubules and is propelled by dynein (microtubule-dependant motor). Capsid moves to nuclear pore. Binding to nuclear pore complex causes conformationchange of the capsid and injection of genome into nucleus.

Functions of some virion proteins in the tegument:Host shut-off protein (vhs) Transcriptional activator (VP16) Protein kinasesViral mRNAs in virions of HCMV (Bresnahan et al Science 2000)

Herpesvirus genomes organization

Isomers

1

1

1

2

4

e.g.Channel catfish herpesvirus

e.g.Human herpesvirus-8

e.g.Epstein Barr virus

e.g.Pseudorabies virus

e.g.Herpes simplex virus

Circularization and replication of viral genome

For herpes simplex virus, the linear genome ends are held together by viral protein and immediately ligated by cellular enzymes (via recombination process). The genome remains nonnucleosomal during lytic replication (nucleosomal during latency).

For EBV, active DNA synthesis is required over 24 to 36 hours.

The DNA is replicated by viral DNA polymerase during lytic replication in a mechanism similar to rolling-circle model. Initiated at specific location(s) by viral protein(s).

Gammaherpesvirus (EBV and HHV-8) DNA is replicated by cellular DNA polymerase during latent infection in a mechanism similar to plasmid replication in E.coli. A viral protein Initiates the replication at a specific location and partition to daughter cells.

Viral DNA replication in the lytic infection

A. Replication origin.1. Origin of DNA replication during the lytic cycles,

initially learned from defective particles. 2. A/T rich palindromic sequences,

located near transcription initiation sites.3. At least two lytic origin in each wild type virus.

One is sufficient in vitro.

B. Mechanism of replication1. Theta form at the early phase.2. DNA recombination converted to rolling-circle model

with extensive branches.3. The final products are head-to-tail concatamers.

Cleavage and package of viral genome1. Lytic replication and terminal repeats are essential.2. pac 1 and pac 2 sequences, located at the each end of the genome,

are conserved in most herpesviruses.3. Each herpesvirus has a different terminal sequence arrangement.

Pac 1 Pac 2 Pac 1 Pac 2

Infection & circularization

Egress

Cleavage & package

Rolling circle replication

Lytic replication

Isomerization of HSV genome:

High efficiency and specificity, equal mole after one round of infection. Defect does not affect in vitro viral replication, but all wild isolates can isomerize.

UL US b b’ c c’

Replication and Isomerization

1:1

1:4

1:4

1:4

1:4

Lytic DNA replication and cleavage of gamma-herpesvirus genome

Vector Flag-Rta Rta

24 36 48 12 24 36 48 12 24 36 48I 12 hr p.t.

I : infected BHK cells

Multimer

Monomer

Rolling-Circle Replication

HindIII

Probe

Maturation of virion:

A. Capsid:1. Capsid is assembled in the nucleus. Empty capsid can self-

assemble in vivo and in vitro (from purified proteins).2. DNA is packed into pre-assembled capsids containing scaffolding

proteins, proteinase and other viral proteins.3. Head-full mechanism to measure the length of the DNA.4. Terminal repeat sequences (pac1 and pac 2) are required. Processing

massive and inter-connected, non-linear DNA. Capsid proteins binding to viral DNA, probably no histones. 70,000 spermidine and 40,000 spermine per capsid.

B. Envelope:1. Envelop at nuclear membrane, de-envelop in cytoplasm and re-envelop

at cell membrane.2. There are few cellular proteins and a lot viral proteins in envelope..

C. Tegument:1. 20 to 40 viral proteins (some are well organized & attached to capsid).2. Some RNAs, eg HCMV.

Cellular changes during lytic (productive) infection

1. Deposition of materials (tegument proteins) on nuclear membranes or into nucleus. Insertion of viral glycoproteins into cytoplasmic and plasma membranes.

2. Nucleolus enlarged, displaced toward the nuclear membrane and dis-aggregated later.

3. Fusion of infected cells with un-infected cells to form polykarycytes (polykarycytosis). Syncytium is common to many viruses.

4. Destabilization of all mRNAs by a tegument protein vhs (virion host shot-off),which removes preexisting host mRNAs. Vhs induce endoribonucleolytic cleavage. Host protein synthesis are shot-off at initial infection to ensure the expression of viral immediate-early genes.

Cellular changes during lytic (productive) infection

5. Viral immediate-early gene products stimulate cellular metabolism (eg. Inactivate p53 & Rb).

6. Host DNA and protein synthesis shot-off during viral DNA replication, to ensure the synthesis of viral DNA and structure genes.

7. Apoptosis is actively inhibited by viral functions in all stages of viral replication (encoding Bcl2, inactivating Rb & p53), but the cell is ultimately destructed.

8. A late protein 34.5 is required in neuron cells to prevent triggering of cellular stress responses that result in a premature total shut off of protein synthesis.

Gene expression Kinetics of HSV

0 20

Hours post-infection

Peak gene expression

2 4 5 7

Peak gene expression

15

Late (gene expression

DNA replication

10

Virion assembly

Immediate-early Early Late

After viral infection, p53 becomes activated and induces the apoptosis pathway. Most DNA viruses have evolved mechanisms to inhibit p53.

SV40 Large T-antigen binds to the p53 DNA-binding domain, preventing p53 from binding to its control elements in DNA.

Ad E1B 55K stimulates p53 DNA-binding, but it also contains a strong repression domain, so that it turns p53 from an activator into a repressor of genes regulated by p53.

HPV E6 protein binds to p53 and induce its degradation via ubiqutination and proteosome degradation.

EBV Zebra inactivates p53 by direct binding. EBV Rta binds to Rb.

HHV-8 LANA inhibits the trans-activation function of p53.(Friborg et al, Nature, 1999)

DNA viruses inactivate p53

M

G1

S

G2

G1 Cdk-Cyclins

E2F

Rb

Cki1

p53

Ad E1BSV 40 THPV E6EBV ZebraHHV-8 LANA

Cellular targets of DNA tumor virusesAd, SV40, HPV, EBV, HHV-8E1A, T, E7, Rta, Rta?

Apoptosis

EBV induces HHV-8 encodes

Bcl2Ad E1BEBV bcl2HHV-8 bcl2

Expression and Functions of Herpesviral Genes

genesImmediate-early

genesEarly

genesLate

Transcription activators,Suppressers of immune recognition

Non-structural regulatory proteins, Enzymes for viral DNA replication,Inhibitors of apoptosis

Major structural proteins, Regulatory factors in virion (VP16, vhs)

+

+ _

_

Latent genes

+

Suppressers of immune recognition,Activators of transcription & proliferation

?

DNA replication enzymes

• Defining characteristic of herpesviruses (reactivation from resting cells).

• Early () genes encode 2 groups proteins which increase DNA synthesis

– Increase DNA replication (viral DNA polymerase, single-stranded DNA binding protein, helicase/primase, etc)

– Increase nucleic acid metabolism (thymidine kinase, ribonucleotide reductase, dUTPase)

Thymidine

dTMP dTDP dTTP DNA

dUMP

dUTPdUDP dCTP

Ribonucleotide Reductase

UDP

UMP

Dihydrofolate

methylene THF THF

Dihydrofolate ReductaseThymidylate Synthase

Thymidine Kinase

dUTPase

ssDNA binding proteinDNA PolymeraseDHFRTSTKAlkaline ExonucleaseHelicase-primaseUracil DNA glucosidasedUTPaseRibonucleotide Reductase, smallRibonucleotide Reductase, large

Complement Binding ProteinIL-6vMIP1 (macrophage Inflammatory Protein)vMIP2vMIP3

Bcl-2Interferon Response Factor-1Cyclin DFLIPOX-2IL-8 receptor

AP-1 like transcriptional activatormyb like transcriptional activator

Human herpesvirus-8 encoded cellular homologues

In simplex virus, nearly 50 % of viral genes are not required for replication in fibroblast cells in culture.

Gene expression cascade during lytic replication

VP16

-genes -genes -genes+ +

+

+_

__

Virion$

+ _

The balance between latency and lytic replication

VP16, vhs

-genes -genes -genes+ +

+

+_

_+ _

_

Virion$latent-genes

Latency

Lytic replication

Latency of herpes simplex virus

1. Viral DNA exists as a circular double stranded DNA in the nucleus of neuron cells for life time until reactivation.

2. LATs (latency-associated transcripts, discovered by Jack Stevens at UCLA, are the only RNAs expressed during latency. 2 kb LATs are circular RNAs, resulted from splicing of a 8.3 kb primary transcript. Function is not clear.

3. LATs are non-coding nuclear RNAs. No viral gene proteins are expressed or required to establishing latency!!??

4. The molecular mechanism of HSV latency and reactivationis not clear.

Epstein-Barr virus (gammaherpesvirus)

• Lytic infection in epithelial cells of nasopharynx and salivary gland

• Latent in B lymphocytes

• Multiple copies of viral episome in nucleus are replicated synchronously with cellular chromosomal replication.

• Sets of latent gene products (proteins and RNAs) contribute to viral episome replication and cell proliferation.

Epstein-Barr virus and cancer• Nasopharyngeal carcinoma

– One of the most prevalent malignancies in East Asian

– Antibody responses to EBV antigens

– EBV genome always found in tumor cells

• Gastric carcinoma (NCNT)– 1000 % in NCNT 1

– 10 % in general gastric carcinoma

– EBV genome always found in tumor cells, high a-EBV IgA

• Hodgkin’s lymphoma– Widespread in W Europe and US

– EBV DNA in Reed-Sternberg cells (~50% cases)

• Burkitt’s lymphoma– Common in Africa, EBV always found in endemic BL tumor cells

– EBV genomes (parts) found in some sporadic BL

• Immunoblastic B-cell lymphomas– Common in the immunosuppressed patients

– EBV genome always found in tumor cells

Evidence for EBV is a tumor virus (First human tumor virus, 1964)

• Sero-epidemiology, elevated antibody titers: VCA (IgA), EA (IgG)

• Viral genome and expression in tumor cells• Monoclonal viral genome in tumor cells• Transform primary B cells in vitro• Cause tumor in new world monkey• Prevent and cure of immunoblastic

lymphoma by CTL specific forEBV antigens

Cleavage and package of viral genome1. Lytic replication origin and terminal repeats are essential.2. pac 1 and pac 2 sequences, located at the each end of the genome,

are conserved in most herpesviruses.3. Each herpesvirus has a different terminal sequence arrangement.

Pac 1 Pac 2 Pac 1 Pac 2

Infection & circularization

Egress

Cleavage & package

Rolling circle replication

Lytic replication

Lytic DNA replication, cleavage package and circularization of gamma-herpesvirus genome

Multimer

MonomerHindIII

Transformation & clonal tumor

Establishment of latency in primary B cells (in vitro):

Virology

1. Activation of B cells by binding of virions to the receptor CD21.

2. Circularization of genome requires (repair) DNA synthesis.

3. The copy number per cell increases in the first week to about 10 to 50 per cell without lytic replication, and remains stable thereafter. Viral genome is replicated, once and only once, by cellular DNA polymerase in early S phase. The only viral protein required for maintaining latent replication is EBNA1 (Epstein-Barr viral nuclear antigen 1).

4. The viral genome undergoes progressive methylation, except the OriP region, which contains multiple binding sites for EBNA1 and is the nuclear matrix attachment site and transcription regulation sequences for latent RNAs.

Transformation of primary B cells (in vitro)

1. Six viral nuclear antigens (EBNAs) are expressed. Many of them have transforming activities.

2. Three cytoplamic membrane protein (Latent membrane protein, LMPs) have transforming activities.

3. Two novel RNAs, Epstein-Barr virus encoded RNAs (EBER1, EBER2) are expressed to over one million copies per cell. Good diagnosis marker for EBV related diseases. Transforming activity?

4. The latency can be disrupted and switch to lytic replication. The frequency is dependent on the host and can be enhanced by TPA, sodium butyrate or -IgM antibody crosslinking. The switch is controlled by transcription factors Zebra and Rta. The lytic genes are expressed in a cascade.

Establishment of latency and transformation of primary B cells (in vitro, at least three steps)

Cell biology:

1. Activation of primary B cells by binding of the receptor.

2. Induce B cell gene expression and proliferation in the way similar that in responses to antigens, mitogens or IL-4 or -CD40. The cells are dependent of cytokines and high cell density. This is the results of the expression EBNA2 and LMP1 (type II latency).

3. The expression of other EBNAs and LMP2s leads to full transformation. EBV in these cells induces express of bcl-2, activates the signal transduction pathways of TRAFs, Notch, NFB and inactivates Rb.

Latent genes of Epstein Barr virus• 6 Epstein-Barr virus Nuclear Antigens (EBNA’s)

– EBNA 1 maintains viral episome via OriP.– EBNA 2, 3A, 3B, 3C and LP involve in proliferation via

Notch signaling pathway. (Hsieh, Science 1995, PNAS 1999)

• 3 latent membrane proteins (LMP’s)– LMP1 activates membrane signaling (TRAFs) activate

proliferation and block apoptosis.

– LMP2 A&B sequester B cell receptor-associated tyrosine kinase (-IgM reactivates EBV), inhibit reactivation from latency. Not directly contribute to transformation, but necessary.

• 2 small RNAs (EBERs): transformation?

TRAFs

TNFR

TRAFs

TNFRTNF

EBV infection

LMP1

NFkB

Growth

NFkB

Growth

TRAFs

Activation of TRAF signal transduction pathway by EBV LMP1

Notch signal transduction pathway

X

Notch

Notch IC

CBF/Su

SAPCIR

HDAC

Notch IC

CBF/Su

DeltaNotch

Protease cleavage

Notch IC

After ligand binding and cleavage, Notch IC with an activation domain, enters nucleus, replaces CIR and activates transcription.

Without ligand binding, CIR recruit SAP and HDAC which represses transcription.

Activation of Notch pathway by EBV

Notch

Notch IC

EBNA2 with an activation domain, enters nucleus, replaces CIR, release the suppression and activates transcription.

CBF/Su

EBNA2

Notch

Notch IC

CBF/Su

EBNA3

EBNA3A&C binds CBF, prevents DNA binding and releases the suppression.

Gene expression regulation during latency

EB

ER

s

Cp Wp Qp LMP1p LMP2p

EBNA2-6 EBNA1 LMP1 LMP2

Ori

P

TR

Circular genome

EBERs are always expressed.

Type I === use of Qp. Expression of EBNA1 only, no CTL recognition. EBNA1 is essential forgenomic Maintenance.

Type II === use of Qp and LMP2pExpression of EBNA1 and low levels of LMP2s, weak CTL recognition.

Type III=== use of Cp, Wp and LMP1&2p. Expression of all six EBNAs, and higher levels of LMPs. Presenting strong transforming activities and strong CTL epitopes.

Switch among different latent forms and lytic cycle

Latency I

Latency II Latency III

Lytic cycle

EBNA1EBERs

EBNA1EBERs

EBNA1, 2, 3A,3B,3C,LP EBERs

ZebraRta

LMP1,2sLMP1,2s

Virion$p53

Rb

A Model of EBV Life Cycles in vivoPrimary infection of epithelium

Secondary infectionof epithelium

Resting B cellIII

III

Primary infectionPersistent infection

III

III III

IIIIII

I/IIIII

IIIIII

10 CTL response

20 CTL responseMemory CTLResting T cell

Kill Kill

Latency switch?

Lytic

Reactivation10 infection of B cell

Latency switch?

Similarity of Gammaherpesvirus Genome Organization

20 40 60 80 100 120 140 160 Kb

EBV

HHV-8 804-11 17-50 * 72-7552-69

MHV-68 4-11 17-50 52-69 72-75*

*4-11 17-50 52-69 75

ORF72 cyclin DORF73 LANAORF74 IL-8R(GPCR)

ORF50 Rta

20 40 60 80 100 120 140 160 Kb

EBV

K1

IL-6,DHFR,K3,TS,MIP-II,K5,MIP-Ibcl-2 Zebra

vIRF,K10-11

K12,FLIP

OX-2

HHV-8

PAN

804-11 17-50 * 72-7552-69

BILF1BILF2 EBNA1

Zebra,BZLF2

EBNA3a-c

gp350/220BLLF2 BHRF1,EBNA2,EBNA4,vIL-10,LMP2

LMP1,2

BARF1BALF2

M7M5,K3,M6M1-4

tRNA 1-8 M8

M10a,b,c

bcl-2

M12-14

MHV-68 4-11 17-50 52-69 72-75*

*

*4-11 17-50 52-69 75

EBER1-2

Genomic difference among gammaherepesviruses

Latency Lytic replication

Immediate-early Early Late

Cyclin DvIL-6

vMIP-IvMIP-II

Bcl-2Bcl-2

sVCAPAN RNA

n-butyrate TPA

Latent

++Rta

DHFRTS

vIL-8 receptor

+

vMIP-IIIFLIP

LNA

TK

KSHV gene expression program

Latency Lytic replication

PAN RNA, vIL-6 &Other lytic genes

+Rta

Latentgenes

+PKC

NFBp65

+

+

+

Mechanism controlling the balance between latency and lytic replication of KSHV

The life cycle of gammaherpesvirus

Tegument

-genes -genes -genes

+

Virioncell death

$

Latency Lytic cycle

Transformation

Rta

Biological implication of complicated gene expression regulation of EBV

Multiple level of regulation:The switch between type I, II, and III latencies;The switch between latency and lytic replication.

Purposes:Evade the immune system, maximize production of

virions or proliferation of infected cells and minimize damageto the host.

Conclusion:The viral gene expression regulation plays a critical

role in determining the biology and pathogenesis of EBV Infection.

Take-home messages:

1. The purpose of viral life is to replicate itself. (its associated pathogenesis or human diseases are secondary effects)

2. Virus utilizes host machinery to replicate. (cellular and organal)

3. The ultimate form of parasitization is to co-exist with the host. (at both individual and population levels)

4. Studies of viruses have double meanings. (medically important and scientifically interesting)

5. Herpesvirus has two phases of the life cycle: latent infection and lytic infection.

herpein: to creep (Greek)