Molecular Car 2010 Lecture NOTES

8/8/2019 Molecular Car 2010 Lecture NOTES

http://slidepdf.com/reader/full/molecular-car-2010-lecture-notes 1/47

Viruses and Cancer: Herpesviruses

4/12/2010

Paul D. Ling, Ph.D.Department of Molecular Virologyand MicrobiologyBaylor College of MedicineMail Stop BCM-385

One Baylor PlazaHouston, Texas 77030

Ph: 713-798-8474Fax: 713-798-3586email: [email protected]://public.bcm.tmc.edu/molvir/Ling.html



Important facts and relevance

What percentage of malignancies world-wide are caused by or requireand infectious agent?a) 1-2% d) 25-35%b) 5-15% e) 35-50%c) 15-25%

Virus encoded oncoproteinsa) Do not existb) Were fundamental to unraveling the concept of tumor suppressors and their

function(s)c) Have no relevance to understanding cell biology

Some viruses have been proven to cause cancer in humansa) Trueb) False

1



Key areas of Interest

1. Definition: What is a herpesvirus? (e.g., what are its general structural andchemical features) How are herpesviruses classified? What are the differenttypes of herpesviruses and what distinguishes them from each other?

2. What Herpesviruses are associated with human cancer? Which viruses andwhat cancers?

3. Evidence for viral etiology of cancer: What evidence suggests that a virus plays arole in the etiology of a tumor?

4. What oncogenic pathways are usurped by Herpesviruses encoded proteins? Howdoes one assess the oncogenic activity of a protein?

2



1.Definition-architecture/morphology of virus particle

2. Herpesvirus genomes/genetic material and genome organization

3. Basic biological properties shared among herpesviruses-Basic differences

4. Classification of subfamilies

5. Human herpesviruses

General Introduction

3



4

Herpesvirus Particle

All herpesviruses have

identical morphology andcannot be distinguished byEM

(Linda Stannard, University of Cape Town, S.A.)



5

Herpesvirus cartoon

Glycoproteins

Membrane

Tegument

Capsid

DNA



6

A

B

C

D

E

F

LTR RTR

R1 R2 R3 R4

IR TRUL US

UL US

anb b¶ a¶n cc¶ a

Class Herpesvirus sequence/genome arrangements Isomers

1

1

1

2

4

1



7

Biological properties

Enzymology

Nuclear replication

Cytolitic

Latency**



NOMENCLATURE AND CLASSIFICAT ION

Alphaherpesvirinae

Betaherpesvirinae

Gammaherpesvirinae

S implexvirus (H SV-1 and H SV-2) and V aricellovirus ( VZV)

Cytomegalovirus (HCMV) and the proposed genera

Roseolovirus (HHV-6 and HHV-7)

Lymphocryptovirus (EBV) and Rhadinovirus (HHV-8)

Short reproductive cycle

Latency in sensory nerve ganglia

Restricted host range

Enlar gement of infected cells-cytomegaliaLatency in lymphoreticular cells

Very restricted host range

Replication in lymphoid and epithelial cells

Latency in lymphoid cells

8



9

Designation Common Name Subfamily Disease

HHV-1 Herpes simplex virus 1 Alpha Oral, ocular lesions (fever blisters); Encephalitis

HHV-2 Herpes simplex virus 2 Alpha Genital, anal lesions (fever blisters); Severe neonatalinfections

HHV-3 Varicella Zoster virus Alpha Chickenpox (primary infection); Shingles(reactivation)

HHV-4 Epstein-Barr virus Gamma Infectious mononucleosis(primary infection); TumorsTumors include B cell tumors (Burkitt¶s lymphoma,immunoblastic lymphomas of the immunosuppressed) plus

Nasopharyngeal carcinoma, some T cell tumors

HHV-5 Cytomegalovirus Beta Infectious mononucleosis; Severe congenital infection;Infections in allograft recipients (pneumonia)

HHV-6 Human herpesvirus 6 Beta Roseola in infants (primary infection); Infections in allograftrecipients (pneumonia, marrow failure); possible role in

multiple sclerosis suggested?

HHV-7 Human herpesvirus 7 Beta Some cases of roseola (primary infection); May reactivateHHV-6

HHV-8 KSHV, Kaposi's sarcoma associatedherpesvirus (aka Human herpesvirus 8)

Gamma Tumors. Tumors include Kaposi's sarcoma; some B celllymphomas



10

1. General Characteristics of EBV

2. Molecular biology of infection in vitro

3. Infection in vivo

4. Associated tumors-evidence for role in pathogenesis-characteristics of associated tumors

Topic areas



12

EBV-induced B cell immortalization

EBV

Resting B cell

Proliferating/immortalized cells orlymphoblastoid cell lines (LCLs)

In vitro model for lymphomagenic potential of the virus

What proteins are involved and how do they work?



Wp

LMP2A

LMP-1

EBNA3A,B,C EBNA1TRIR1

LMP2Ap

LMP-1p

LMP2BpCp

W1 W2W0

LMP2B

EBNA-LP, EBNA2 (W0/W1·)

Y1 Y2

EBNA2

EBNA-LP

EBV latent proteins

EBNA2 (W0/W1)

Theimagecannot bedisplayed. Your computer may nothaveenough memory toopen theimage,or theimagemay havebeen corrupted.Restartyour computer,and then open thefileagain.If thered x stillappears,you may havetodeletethe imageand then insertit again.

EBNA3s/EBNA1

Bam A RNAs

EBERs



15

EBNA2 functions

Transcriptional activating protein, required for

immortalization-viral latent genes (e.g., LMP-1, LMP-2, and

Cp/EBNAs)-cellular genes (e.g., c-myc, CD21, Hes-1): drive

cells from G0-G1?

EBNA2

EBNAs

LMP2s

LMP1c-mycHes-1

EBV genome

Theimage cannotbedisplayed. Your computer may nothaveenough memory toopen theimage,or theimagemay havebeen corrupted.Restartyour computer,and then open thefileagain.If thered x stillappears,you may havetodelete theimageand then insertit again.



16

EBNA2CR1 CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9

PPR

NLS

TAD

GTGGGAAA

CBF1

SKIP

EBNA2 target promoter

?SAD

SAD

TAD

NLS

Self-association domain

Transactivation domain

Nuclear localization signal

EBNA2 cellular cofactors

nur77 CBP

TFIIBTAF40

TFIIH

Theimagecannot bedisplayed. Your computer may nothaveenough memory toopen theimage,or theimagemay havebeen corrupted.Restartyour computer,and then open thefile again.If

thered x stillappears,you may havetodeletetheimage and then insertit again.



17

Notch Signaling

Delta/Jagged

CBF1Su(H)

Lag-1

S

S

Notch

Theimagecannot bedisplayed. Your computer may nothaveenough memory toopen theimage,

or theimagemay havebeen corrupted.Restartyour computer,and then open thefileagain.If thered x stillappears,you may havetodeletethe imageand then insertit again.

Proliferation, differentiation, programmed cell death



18

LMP-1 resembles CD40 (i.e., T NFR)



19

LMP-1 transformation (functional assays)

Growth in low serumColonies in soft agar

Tumors in nude mice



20

1. Oncoprotein in vivo and in vitro. Transforms Rat-1 cells, induces B cell tumors

in eMu/LMP-1 transgenic mice.

2. Interacts with TRAFs 1,2, and 3. Activates NFkB.

3. modulates the EGF receptor in epithelial cells.

4.Acts as a constitutively active CD40 receptor (Constitutively active CD40 can

transiently substitute for LMP-1 in EBV immortalization maintenance).

5. Induces adhesion molecules and activation markers (CD23,CD39, 40, 54 and58), haemopoietic growth factor receptors, induction of cyclin D2, anti-apoptotic by inducing bcl2 and A20.

6. Essential (but not sufficient) for B cell immortalization

7. Expressed in a large number of EBV-associated malignancies

LMP-1 functions



21

LMP2 resembles a BCR

AKT RAC

JNK p38

RAS

Erk1/2

Proliferation Anti-stress

GSK3B

Bcatenin

Anti-apoptosisOncogenesis



22

LMP2A

1. C-term domain contains 8 tyrosine residues, two of which form immunoreceptor tyrosine-based activation

motif (ITAM)

2. ITAM recruits and activates Src family protein tyrosine kinases (PTKs) and the Syk PTK; induces low level

of forward signaling mimicking BCR (B cell receptor). Signaling is constitutive.

3. Recent studies indicated LMP2A can transform some epithelial cells mediated in part by activation of the

phosphoinositide 3-kinase -Akt pathway



24

(RAG?)

BCR

Pi3kinase/AKT

Epithelial cell proliferation?

Cell cycle checkpointsAnti-apoptosis



26

Release:

Normal B cell biol ogy; the pa rallel with EBV



27

Immune system control of EBV



29

TABLE 2 Guidelines for relating a putative virus to a human cancerEpidemiologic

1. The geographic distribution of infection with the virus should be similar to that of the tumor

with which it is associated when adjusted for the age of infection and the presence of cofactorsknown to be important in tumor development.

2.The presence of the viral marker (high antibody titers or antigenemia) should be higher in casesthan in matched controls in the same geographic setting, as shown in case-control studies.

3.The viral marker should precede the tumor, and a significantly higher incidence of the tumorshould follow in persons with the marker than those without it.

4.Prevention of infection with the virus (vaccination) or control of the host·s response to it (such asdelaying the time of infection) should decrease the incidence of the tumor.

Virologic

1.The virus should be able to transform human cells in vitro into malignant ones.

2.The viral genome or DNA (or viral gene expression) should be demonstrated in tumor cells and notin normal cells.

3.The virus should be able to induce the tumor in a susceptible experimental animal andneutralization of the virus prior to injection should prevent development of the tumor.



30

(a)high rate of EBV presence in these tumors over othercontrol tumor samples (Southern blot, PCR, in situ

hybridization)

(b)selective expression of EBV gene products in tumor cells(in situ hybridization to EBV RNA, immunohistochemistry)

(c) viral monoclonality in tumor cells (detection of genometermini)

d) seroepidemiology.

In addition, EBV can immortalize primary B lymphocytes,encodes at least one oncoprotein that is expressed in severalEBV-related cancers, and EBV also causes lymphomas inmarmosets.

Evidence supporting EBV as a causative agent in some human cancers



31



32



33

EBV-Associated Hodgkin's Disease

Double labeling of malignant HodgkinDouble labeling of malignant Hodgkin--Reed Sternberg (HRS) cells showing coReed Sternberg (HRS) cells showing co--expressionexpression

of Epsteinof Epstein--Barr virus early RNAs (EBERs; brown/black) and latent membrane protein 1Barr virus early RNAs (EBERs; brown/black) and latent membrane protein 1

(LMP(LMP--1; red).1; red).

K J Flavell, et. al. J Clin Pathol: Mol Pathol 2000; 53:262K J Flavell, et. al. J Clin Pathol: Mol Pathol 2000; 53:262--269269



34

EBV-Associated Nasopharyngeal Carcinoma

Double labeling in situ hybridization and immunohistochemistry reveals expressionDouble labeling in situ hybridization and immunohistochemistry reveals expression

of the Epsteinof the Epstein--Barr virus (EBV) encoded RNAs (EBERs) (black grains) in virtuallyBarr virus (EBV) encoded RNAs (EBERs) (black grains) in virtually

all cytokeratin positive (red labeling) tumor cells of an undifferentiatedall cytokeratin positive (red labeling) tumor cells of an undifferentiated

nasopharyngeal carcinoma.nasopharyngeal carcinoma.

G Niedobitek J Clin Pathol: Mol Pathol 2000; 53:248G Niedobitek J Clin Pathol: Mol Pathol 2000; 53:248--254254



35

1. Immunoblastic lymphomas:

a)Lymphomas in congenitally susceptable patients: X-linked lymphoproliferative syndrome (XLP), acutelesions arising within a few weeks of primary infection. Likely to be defect in immune system.

b)posttransplant lymphomas: Increased risk of lymphoma in transplant recipients. Disease isinfluenced by EBV status of patient at the time of transplant. Seronegative patients who receive EBVfrom the donor organ or blood transplants have much more rapid onset of disease. Reduction ofimmunosuppressive therapy can result in clearance of tumors by the host immune system.-A small proportion of these tumors have undergone c-myc translocations. Other genetic alterationshave not been characterized.-Most tumors are refractive to traditional chemotherapy as well as antiherpes drugs.

c)Lymphomas in AIDS patients: about 50% of lymphomas in HIV infected patients are EBV positive.Many of these present in the CNS.

d)Correlation with animal models: Experimental infection of cotton-top tamarins with EBV orreconstitution of SCID mice with EBV positive lymphocytes results in rapid and efficient tumordevelopment. The tumors presentation (multifocal) and histology are very similar to those found inimmunocompromised individuals.

**All EBV associated tumors in this group of patients (and the animal models) look like EBVimmortalized cells established in vitro and display latency III phenotype. Under the umbrella ofimmunodeficiency, EBV is likely to be the direct cause of these tumors. Why? Reduction ofimmunosuppression or reconstitution of immunity (adoptive immunotherapy; see Cliona Rooney lecture)is able to control these tumors.

Most of these tumors are likely to have arisen from infection of bystander B cells (not naive) and areunable to differentiate. Without an intact immune system they are not eliminated.



36

PTLD in Lung



37

2. Burkitts lymphoma:

a)characterized by activation of the c-myc proto-oncogene by a chromosomal

translocation where myc is regulated by an immunoglobulin enhancer. Malaria is asignificant co-factor. The central dogma is that Malarial induced T cell suppressionallows an expansion of the pool of EBV infected B cells and increases the likelihood ofan ´accidentµ occurring, e.g., a chromosomal translocation. EBV gene products providesurvival functions in context of high c-myc activity.

b) Recent evidence from EBNA-1 transgenic mice indicate that EBNA-1 can behave as

an oncoprotein in vivo, perhaps by modulating recombination proteins RAG-1 and RAG-2.Role for Ebers has also been suggested but recently discounted. Possible role forBARTs?

c)BL patients have higher levels of anti-EBV antibodies than control groups

d)About 30% of Burkitt·s lymphoma have mutations in p53.

e)Burkitts lymphoma display a type I latency pattern. Why not other latent genes?

f)phenotype of B cells suggests it arose from memory or post-GC B cell



38



39

3. Nasopharyngeal carcinoma (NPC):

a) One of the most common cancers in Southeast Asia. Cancer of epithelial cells innasopharynx. Displays a type II latency pattern (Expresses LMP-1 and LMP2A). High

levels of anti-EBV antibodies over control groups have been observed.b) May also be environmental or genetic risk factors. Deletions in the short arm ofchromosome 3 and diet (salted fish, contain significant levels of nitrosamines) orexposure to carcinogens or physical irritants. Recent findings show that preinvasivelesions in the nasopharynx are EBV positive and that some of these patients went on todevelop cancer.

c) No mutations in p53 usually observed.

d)How does EBV get into epithelial cells? Why is there no lytic infection of EBV in NPC?

4. Hodgkins disease: Up to 50% of Hodgkins lymphoma are EBV positive. EBV is found inthe neoplastic Reed-Sternberg cell. Gene expression pattern of EBV (LMP-1/LMP2A)

and of B cell suggests EBV infection rescued GC B cell with a ´crippledµ Ig mutation

5. Other: EBV is also associated with nasal T cell lymphomas, gastric adenocarcinomas,and leiomyosarcomas (smooth muscle tumors) in pediatric AIDS or post transplantpatients.



40

Putative check points in the EBV life cycle that give rise to tumors



41



42

KAPOSI'S SARCOMA ASSOCIATED HERPESVIRUS(KSHV/HHV8)

Discovered using molecular biologic techniques

Eighth human herpesvirus

Large (~165 kb) dsDNA genome

Causes three diseases:

Kaposi's sarcoma (KS): vascular tumor consisting

Of spindle cells derived from endothelial lineage. Proliferation due to

direct viral infection or induced inflammatory cytokines?

Primary effusion lymphoma (PEL): lymphomas found

in visceral cavities. Often co-infection with EBV.

Multicentric Castleman's Disease (MCD): reactive

lymphadnopathy characterized by expanded germinal centers and

proliferation of endothelial vessels with involved lymph nodes.

Cultured in PEL cell lines, but difficult to transmit



44

KSHV LL L HOMOLOGS

CELL CYCLE CONT OL NTI- OPTOSIS

vCyclin vIL-6

v cl-2vFLIP

vI F

IMMUNE MODUL TORY PROLIFERATION

v-IRF vIRF

v-CBP vGPCR



45



46

Molecular Car 2010 Lecture NOTES

Documents

Transcript of Molecular Car 2010 Lecture NOTES