Epstein Barr virus and cancer Flashcards
What are the 3 classifications of the herpesvirus?
- alpha subfamily = herpes simplex 1 and 2 (HHV1/2) - associated w/ carcinoma of the resp. tract
- beta subfamily = cytomegalovirus (HHV5) associated with Kaposi’s sarcoma
- gamma family = EBV HH4 - associated with burkitts lymphoma and nasopharyngeal carcinoma
What are the characteristics of EBV?
- EBV is a member of the gamma herpesvirus family
- genome is 172kb linear double molecule which ciruculizes infected cells
- EBV infects the B cells and certain epithelial cells
- in culture EBV efficiently immortalises resting b cells
- EBV associated diseases. = infectious mononucleosis, burkitts lymphoma, Hodgkin lymphoma
When does EBV infect B cells?
- via the CD21 receptor found on B lymphocytes
- two regions of homology exist between the receptor and EBV, allowing binding and internalisation
- can only infect B lymphocytes at certain times during their growth and maturation
How does EBV enter the cells?
Epithelial cells - initial entry is via viral protein BMRF-1 that interacts with cellular beta-1 integrins
- followed by interaction of viral protein gH/gL with cellular integrins that triggers fusion of the viral envelope with the epithelial cell membrane
B lymphocytes - entrer via viral glycoprotein gp350, binds to cellular receptor CD21
- triggers fusion of the viral envelope with cell membrane, allowing entry
- human CD35 (CR1) is an additional attachment factor for gp350, and can provide a route for entry of EBV into CD21 - negative cells
- once EBV enters the cell, the viral capsid dissolved and the viral genome is transported to the cell nucleus
Describe lytic infection
- primary infection is usually asymptomatic in childhood
- virus is secreted in the saliva and human infection occurs through oral transmission - associated with infectious mononucleosis
- by age of 40 = nearly everyone is infected
- oropharyngeal infection = lytic infection where 80 viral genes may be expressed
Describe latent/persistant infection
- lytic infection is followed by infection of circulating b cells
- this leads to persistance of viral DNA in the nucleus, establishing a latent infection and a restrictive pattern of gene expression
How is lytic EBV treated?
- during lytic cycle replication is via viral DNA polymerase
- acyclovir is a guanosine analogue
- it is phosphorylated by viral kinases only expressed during the lytic cycle
- host cell kinases that convert it to acyclovir triphosphate, this then terminates DNA synthesis
Can latent EBV be treated with Acyclovir?
No
- does not generate viral kinases that activate the drug
insensitive to acyclovir
Where are the EBV particles found during lytic/primary infection?
- primary site of lytic replication in oropharynx, cells that evade T-cell response establish stable reservoir
Where are the EBV particles found during latent/persistant infection?
- persistant reservoir maybe recruited to germinal centres, re enter reservoir or differentiate into plasma cells and reactivate lytic cycle
How do we know what the latent genes are?
- in vitro = peripheral blood lymphocytes from EBV infected individuals are placed in culture, the few EBV infected B cells give rise to spontaneous out growth of EBV-transformed cell line known as lymphoblastoid cell lines (LCL)
- LCL can be generated by infecting resting b cells with EBV = valuable tool for research
What do LCLs express?
- 6 EBV nuclear antigens (EBNAs - 1, 2, 3A, 3B, 3C and -LP)
- 3 latent membrane proteins (LMPs 1, 2A, 2B)
- transcripts from the BamHI region (BART transcripts)
- 2 small, non-polyadenylated RNAs (EBER1 and EBER2)
What are the different types of latency?
- in epithelial cells only latency II
- Latency III usually found in naive B cells - enter germinal centre and proliferate, expanding pool of EBV cells
- Latency II = GC infected cells = then differentiate into memory B cells, some of these show Latency I gene expression
What is the function of EBNA-1
- expressed in all EBV-infected cells
- found in nucleus, role in maintenance of the genome by binding to the origin of replication
- not recognised by cytotoxic T cells
- Gly-Ala repeat sequences are thought to prevent proteosomal degradation of EBNA-1
- shown to inc. survival of infected B cells. Lowers p53 levels by binding to p53-regulatory protein, USP7 = ubiquitin specific protease that is key regulator of p53 and mdm2
- may provide target for further therapy
What is the function of EBNA-2?
- found in nucleus= transcriptional regulator and activator of several viral and cellular genes, such as LMP-1, LMP2 and CD23
- interacts with DNA-binding protein to activate CD23 and LMP proteins
- RBP-Jk is a downstream regulator of notch signalling pathway
- deletion of EBNA2 = inability to transform b lymphocytes
- phosphorylation of EBNA2 = suppresses activity
What is the function of EBNA3
- related to proteins EBNA - 3A, 3B and 3C - expressed only in latency III
- EBNA-2 are restricted to nucleus and function as transcriptional regulators
- 3A and 3C essential for b cell transformation in vitro - EBNA3B is dispensable
- 3C can upregulate CD21 and LMP1
- EBNA3 proteins associate with RBP-PK transcription factor
What are LMP1 and 2?
- LMP-1 = found in membrane, considered the man transforming protein of EBV, major effector of virus induced cellular changed = inhibits apoptosis by activating NFkappaB
- LMP-2- intrinsic membrane protein that prevents EBV infected cells from entry into the lytic cycle
What are EBER-1/2?
- Non polyadenylated RNAs that are essential for EBV transformation of B cells, can induce IL-10 which might suppress cytotoxic T cells
Describe endemic Burkitts lymphoma
- BL is a high grade lymphoma and needs urgent treatment. Affects children from age 2-16
- tumour doubles in mass every 24 h and is fatal
- treatment with cyclophosphamide (helps to induce apoptosis) = one dose can usually shrink tumour
What are the 3 types of BL?
- endemic - children and young adults in africa and new guinea
- sporadic -children and adults outside endemic area
- immunodeficiency-related - primarily associated with HIV infection
- most BL exhibit type I Latency
Pathogenesis of BL
- all BL cases carry one of three characteristic chromosomal translocations that place the MYC gene under regulation of the lg heavy chain
- many tumours also have mutation in the p53-ARF pathway.
- BL is associated with either malarial or HIV infections
- EBV may have an initiator role, providing pool of infected b-cells = risk of c-myc translocation
Describe nasopharyngeal carcinoma
- rare tumour arising from epithelium of nasopharynx
- common in certain regions of east asia and africa = dietary and genetic factors
- dietary risk factors - consumption of salt-cured fish high in nitrosamines and smoked food may play role
- other factors = smoking and alcohol consumption
What do NPC tumours look like?
- blood stained postnatal discharge, impaired hearing, tinnitus
- tumour presents in 3 forms :
- proliferative - growth causing nasal obstruction
- ulcerative - causing epistaxis
- infiltrative - which causes cranial nerve involvement
How are NPC tumours treated?
- external beam radiotherapy - primary mode of therapy
- chemotherapy can be delivered together with radiotherapy and can include : cisplatin, 5-fluorouracil
Evidence that LMP is involved in NPC
- LMP1 is key EBV-encoded protein required for b cell immortalisation
- In NPC - LMP1 behaves as oncogene
- in human epithelial cells it exerts a variety of growth-promoting effects and potentiated anchorage-independent growth - greatly enhances cell motility and invasion - upregulate anti-apoptotic EGFR
What is the structure of LMP1
- 66kDa integral membrane protein = short amino acid cytoplasmic n-terminus, 6 hydrophobic alpha helical transmembrane spanning regions and cytoplasmic c-terminal tail
- long c terminus which contains essential activating domain (CTAR)
What is the role of bcl2 and p53 in NPC?
- 90% of NPC tumours contain wt p53 genes
- in the 10% of tumours that have mut p53, LMP1 may provide growth advantage
- in 90% of NPC tumours LMP1 can modulate p53 activity
- LMP1 stimulated expression of mdm2 = promotes p53 turnover
- also repressed p53 mediated DNA repair
- LMP1 can also synergise with Bcl-2 to override the growth suppression induced by wtp53
What does EBER-1 bind to?
- PKR is induced by interferons
- 68kDa kinase, activity is enhanced by viral infection and cell stress
- active PKR phosphorylated the ser 51 residue on the alpha subunit of eIF2alpha = inhibition of protein synthesis
- activated by dsRNA
What is the structure of PKR?
- 2 double stranded binding motifs at n terminus = regulatory domain
- c terminus = kinase domain
- normally in cytoplasm PKR is found in inactive state
How is PKR activated?
- during viral infection, transcription leads to production of dsRNA
- these activate PKR as part of antiviral mechanisms
- dimerisation occurs = autophosphorylation
How does EBER-1 play a role in PKR activation?
- EBER-1 competes with PKR-activating dsRNA to prevent this inhibition of protein synthesis
- activity is inhibited
What is the function of EBER-1 in vivo?
- expression of EBER-1 in EBV negative cells protects against IFN-induced apoptosis
- EBER expression stimulated growth factors such as IL-9 and IL-10
