Abstract:

Epst ei n- Barr virus is one of t he most common human viruses, infecti ng

appr oxi mat el y 95 % of t he worl d’s popul ati on. EBV viral prot ei ns are abl e to mani pul ate

cell ul ar pat hways essential for mai nt ai ni ng homeostasis by disrupti ng nor mal prot ei n

interacti ons. The fiel d of syste ms bi ol ogy is i deal for st udyi ng t he effects of viruses on

hu man hosts because t he reducti onist approach t o understandi ng cell ular pathways and

disease pat hogenesis is challenged and disregarded. A yeast-t wo- hybri d met hod was

utilized t o screen 216 EBV pr ot ei ns agai nst 15, 483 human prot ei ns, producing 188

positi ve candi dat es. The discovery and anal ysis of EBV- hu man prot ei n i nteracti ons has

the potential to reveal how t he disrupti on of cell ular pat hways can result in the

devel opment of EBV- associ at ed diseases, incl udi ng cancer.

I. Introducti on:

Syste ms Bi ol ogy and Interacto mes

The fiel d of syste ms bi ology has e mer ged as a result of t he successful compl eti on

of t he Hu man Geno me proj ect, whi ch made entire geno me sequences for human and

nu mer ous model organisms easil y accessi ble. Before t he availability of entire genome

sequences researchers appr oached compl ex questions regardi ng cell ul ar pathways and

disease pat hways by focusi ng on i ndi vi dual components of mol ecul ar processes. Syst e ms

bi ol ogy challenges the reducti onist approach by proposi ng an i ntegrated appr oach t o

understandi ng bi ol ogi cal pr ocessesi

. Marc Vi dal expl ai ns, “t he idea of systems bi ol ogy

presupposes t hat no life for m can be i magi ned without compl ex syste ms for med by

interacti ng genes and macromol ecul es, or cells at a hi gher scal ei i

.”

The “syst e m” approach to understandi ng cell ular pat hways and disease

devel opment focuses on the mappi ng of compl et e int eracti on net wor ks, or int eract omes.

In t he fiel d of prot eomi cs, interact ome maps are graphi cal represent ati ons of indi vi dual

pr ot ei ns and t he prot ei n-pr ot ei n i nteracti ons that occur wit hi n a celli i i

. Once these

interacti ons are observed and vali dat ed, bi ol ogi cal processes can be better underst ood.

Mor e i mport antl y, compl et e i nteract ome maps may reveal how ext ernal factors, such as

viruses, have t he potential to mani pul ate cell ular processes.

The constructi on of i nteract ome maps requires t he availability of open-reading

fra mes ( ORFs), or t he prot ei n codi ng sequence of a gene from t he start codon t o t he st op

codon, excl udi ng t he 5’ and 3’ untranslated regi ons ( UTRs)i v

. Earl y large-scale proteomi c

anal ysis utilized cDNA pools, whi ch were prot ei n-encodi ng genes incl udi ng the 5’ and 3’

UTRs. In many i nstances, researchers reported hi gh i nstances of false positives as a result

of hybri d prot ei n expression i n t he wr ong readi ng fra me or from t he 5’ or 3’ UTRsv

.

ORFs, whi ch excl uded untransl ated regi ons, not onl y mi ni mi zed t he number of false

positi ves, but also be easily transferred i nt o numerous expressi on vect ors and used by a

variet y of prot ei n-i nt eraction screeni ng met hods. The effecti veness of open readi ng

fra mes has been best observed i n t he hi gh t hroughput yeast t wo- hybri d ( Y2H) screeni ng

met hod.

Y2 H Syste m

Si nce its devel opment i n 1989 by Fi el ds and Song, the Y2H syst e m has e merged

as a wi del y accept ed method for det er mi ni ng protei n i nteracti ons i n vivo. The syste m has

been successfull y used i n numer ous large-scal e st udi es, effecti vel y identifying prot ei n

interacti ons in humans and several model organisms. It is relati vel y inexpensi ve because

eli mi nates t he need for anti body producti on and protei n purificati on pri or to screeni ngv i

.

The Y2H screeni ng process is flexi bl e i n t hat it relies on readil y availabl e geno me

sequences, whi ch can be easil y transfor med i nt o a wi de variet y of expressi on vect ors

through recombi nati onal cl oni ng. Lastl y, the Y2H syste m is compati bl e with Gen Mat e ©,

Aquari us © and Genzy me© r obotic liqui d handli ng syste ms, maki ng t he screeni ng

pr ocess accurat e and relativel y rapi d.

The general Y2H procedure invol ves splitting a transcri ption fact or, commonl y

GAL 4, int o t wo domai ns: the DNA- bi ndi ng domai n ( DB) and t he acti vation domai n

( AD). The DNA- bi ndi ng do mai n acti vat es t he expressi on of an adj acent reporter gene by

bi ndi ng t o t he upstrea m acti vati ng sequence ( UAS), while t he acti vati on domai n is

invol ved i n asse mbli ng transcri ption fact ors needed for t he initiati on of transcri pti on. The

expressi on of t he reporter gene, often HI S3, is dependent on t he interacti on of AD and

DB, made possi bl e by fusi on prot ei ns. ORFs, which code for t he prot ei ns of interest, are

expressed as fusi on prot eins bound t o t he AD and DB do mai ns. When t he protei n

encoded by t he t wo different ORFs i nteract, transcri pti on is acti vat ed and the report er

gene is expressedv i i

.

Aut o-acti vat ors are prot eins, fused t o t he DB do mai n, whi ch do not require

recruit ment of AD t o the pr omot er sequence t o i nitiate transcri pti onv i i i

. Si mpl y, aut o

acti vat ors are abl e t o i nitiat e transcri pti on wit hout interacti ng wit h prot ei ns fused t o t he

acti vati on domai n. They are a common source for false positi ves because t hey appear t o

react wit h fusi on prot ei ns on AD, when no i nteraction is occurri ng. Aut o activat ors can

be sel ected for pri or t o starti ng t he screen and t hroughout t he phenot ypi ng pr ocess.

The Y2H screeni ng met hod for prot ei n i nteracti ons utilizes t wo hapl oi d yeast

strai ns, desi gnat ed as eit her AD or DB. ORFs, which are expressed as AD and DB fusi on

pr ot ei ns are transfor med int o the correspondi ng yeast strai n t hrough a gat eway

recombi nati on cl oni ng process. The t wo yeast strains are mat ed allowi ng for the

interacti on of AD and DB, as well as t he expression of t he reporter gene. The acti vati on

of t he report er gene, confir mi ng prot ei n-prot ei n i nteracti ons, can be det ect ed by a col or

change or growt h on selecti ve medi ai x

. Use of t he Y2 H syst e m has resulted in t he

constructi on of i nteract ome maps for numer ous model organis ms, as well as t he

identificati on of virus-host prot ei n i nteracti ons excl usi ve t o Epst ei n- Barr virus.

There are many advant ages t o utilizi ng S. cerevisiae duri ng t he Y2H screeni ng

pr ocess. The first, and possi bl y most i mport ant, is that many yeast genes are homol ogous

wi t h human genesx

. This offers an effecti ve way t o research and i dentify t he potential

cause of numer ous human diseases, wit hout usi ng human cells, thus avoi ding numer ous

et hi cal dile mmas. S. cerevisi ae has a full y sequenced geno me, i n additi on t o a fast and

si mpl e life cycl e, maki ng it a model organis m for the Y2H screeni ng process.

Gat e way Reco mbi nati on Cl oni ng

The Gat eway recombi nation cl oni ng syst e m, first devel oped by Invitrogen™

company, all ows for t he transfer of DNA fragments i nt o numer ous expression vect ors

wi t hout alteri ng t he open readi ng fra mes of t he prot ei ns of i nterest.x i

The devel opment of

the cl oni ng technol ogy has compl et el y revol uti onized t he Y2H screeni ng by all owi ng

DNA fragments t o be easily cl oned i nt o compati ble vect ors t hrough a t wo-step process.

Thi s process repl aces t he previ ousl y used enoduclease and li gase- based met hods, whi ch

were not onl y labori ous, but also affect ed by i nappr opriatel y positi oned restricti on

enzy me sites. The Gat eway © syst e m utilizes site-specific recombi nati on that i ncl udes

BP and LR reacti ons.

The gene of i nterest, the ORF i n many cases, contai ni ng t wo att B sites on either

si de, is transferred i nt o a GAL4- based donor vect ors, through a BP reacti on. The donor

vect ors cont ai n ccdB and CmR count er-sel ectabl e genes, all owi ng for negative sel ecti on

of unwant ed by-product pl as mi ds after recombi nation occurs.x i i

The t wo att B sites, att B1

and att B2, on t he ORF will interact wit h t he att B sites, att P1 and att P2, on the donor

vect or. An enzyme mi xt ure of BP Cl onase will generat e entry cl ones, flanked by aat L

sites, whi ch cont ai n DNA sequences from bot h att B and att P sites.x i i i

See Figure 1.

Fi gure 1: Overview of BP process perfor med t hrough Gat eway recombi national

cl oni ngx i v

.

The LR reacti on utilizes the LR cl onase enzyme t o transfor m entry cl ones int o

desti nati on vect ors. Every entry cl one will cont ai n aat L restricti on sites, which will

interact wit h t he aat R restricti on sites on t he destinati on vect ors, AD and DB, produci ng

an expressi on cl onex v

. See Fi gure 2.

Fi gure 2: Overview of LR process perfor med t hrough Gat eway recombi national

cl oni ngx v i

.

Epstei n- Barr vi rus

Epst ei n- Barr virus ( EBV) was first identified i n 1964 after virus-li ke particles

were observed i n cells fro m a Bur kitt’s l ympho ma bi opsy. Aft er its initial di scovery,

EBV beca me t he first known human virus directly i nvol ved i n t he devel opment of

mali gnant t umors. Ext ensi ve research has confir med t he role of EBV i n t he pat hogenesis

of Bur kitt’s l ympho ma, Hodgki n’s disease, non-Hodgki n’s l ympho ma, nasopharyngeal

carci noma and l ympho mas, and lei omyosarcomasx v i i

.

The Worl d Healt h Or ganization esti mat es t hat 95% of adults worl dwi de have

been infected wit h EBVx v i i i

. Upon initial infection, indi vi duals become lifel ong carriers

of t he virus. The maj ority of i nfect ed i ndi vi duals coexist wit h t he virus without seri ous

compli cati ons, but a s mal l popul ati on devel op mal ignanci es as a result.

EBV is trans mitted by saliva and pri maril y i nfects the stratified squa mous

epit heli um of t he oropharynx. Initial infecti on may be asympt omatic; however, t wo-t hirds

of i nfect ed i ndi vi duals manifest infecti ous mononucl eosisx i x

. Lat ent infecti on is thought

to occur when B-l ymphocyt es and i nfect ed oropharynx cells i nteract i n t he oropharyngeal

lymphoi d organs. The virus re mai ns i n me mor y B-cells i ndefi nitel y, all owi ng for t he

reacti vati on and devel opment of numer ous EBV- associ ated cancers wherever B-cells

circul atex x

.

EBV is one of si x known viruses t hat constit ute t he Herpesvirus fa mil y. All

herpesviruses possess t he sa me struct ural charact eristics, but are di vi ded i nto t hree

subfa milies (, , and ) based on geno mi c size, cont ent, and organi zati onx x i

. EBV is a

me mber of t he -herpesvirus subfa mil y and possesses a 184-kbp doubl e-stranded DNA

geno me t hat encodes for 89 genes, wit h onl y 28 bei ng EBV-specific and import ant i n

latent B-l ymphocyt e i nfecti on. Fort y-si x of t he 89 genes are “core” genes, found i n all

herpesvirus fa milies, which are essential for successful geno me replicati on, packagi ng,

and deli very i n all cells. Si x of t he re mai ni ng ei ghteen “noncore” genes found i n bot h the

- herpesvirus and -herpesvirus subfa milies, and the last t wel ve genes are specific t o t he

-herpesvirus subfa mil yx x i i

. Li ke all herpesviruses, EBV is charact erized by a toroi d-

shaped prot ei n core, a nucl eocapsi d wit h 162 capso meres, a prot ei n tegument, and an

out er envel ope wit h ext ernal gl ycopr ot ei n spi kesx x i i i

.

Tho mpson and Kurzrock reveal t hat EBV viral prot ei ns are abl e t o mani pulate

cell ul ar pat hways essential for mai nt ai ni ng homeostasis by i mitating several growt h

fact ors, transcription factors, and antiapopt otic factorsx x i v

. Once the virus has control over

essential cell ular pat hways t he potential for EBV- related disorders t o develop is vast.

Understandi ng t he inter mol ecul ar prot ei n i nteractions bet ween t he viral protei ns, as well

as i nteracti ons that occur bet ween EBV and a human host, may provi de alter nat e ways t o

prevent and treat EBV- associat ed diseases.

Si nce t he discovery of EBV and its potential to cause a wi de variet y of diseases,

ext ensi ve research has been focused on known EBV pr ot ei ns and t heir rol e in t he life

cycl e of t he virus. EBNA-1 is a phosphopr ot ei n required for t he replicati on and

mai nt enance of t he EBV geno me and is consistently found i n t umors i n i nfect ed

indi vi duals. The prot ei n bi nds t o DNA sequences on t he EBV geno me and uses t he host

enzy mes t o medi ate t he replication process. Because of its ability t o conti nuousl y

replicate t he EBV geno me, while acti vati ng t he expressi on of ot her EBV genes, EBNA- 1

is consi dered t o pl ay a central role i n mai nt ai ni ng lat ent i nfecti onx x v

.

EBNA- 2 is one of t he first prot ei ns det ect ed after EBV i nfecti on and is a known

transcri ptional acti vat or responsi bl e for t he expressi on of viral and cell ular genes. It is

essential for i mmort alization of human B l ymphocyt es and i ncreases t he expressi on of c-

myc, a gene known t o become an oncogene as a result of over expressi on. EBNA- 2

increases expressi on t hrough bi ndi ng wit h ot her transcri ption fact ors, not ably those

invol ved i n t he pat hogenesis of T-cell lympho max x v i

.

EBNA- LP, often referred to as EBNA- 5, is known to i nteract wit h EBNA shortl y

after EBV i nfecti on. The pr ot ei n is known t o activat e resti ng B l ymphocyt es, while

suppressi ng t he tumor repressor prot ei n, retinobl ast oma. EBNA- LP, t hrough i nteracti on

wi t h EBNA- 2, is also essential to t he i mmort alization of B l ymphocyt es and one of t he

first det ectable prot ei ns after EBV i nfecti on.x x v i i

EBNA- 3A, EBNA- 3B, and EBNA- 3C are all transcri pti onal regul at ors, differi ng

in specific functi on. EBNA- 3A and EBNA- 3C are essential for EBV i mmor talizati on

insi de a host cell. EBNA-3C is also known t o i nactivate retinobl ast oma and increase the

pr oducti on of LMP- 1, whi ch i nhi bits apopt osis of infect ed cells. The rol e of EBNA- 3B is

generall y unknown, however, is not essential for EBV i mmort alizati onx x v i i i

.

Thr oughout t he years t he EBV LMP- 1 prot ei n has been li nked t o cancer because

of its ability t o i nhi bit apopt osis of cancer cells by raisi ng levels of Bcl-2. The prot ei n is

abl e t o recruit an array of cellul ar genes by mi mi cki ng cell growt h si gnals, increasi ng t he

risk for cancer devel opment. LMP- 1 also up-regulates t he expressi on of B-cell adhesi on

mol ecul es, all owi ng for cell ular buil dup and t umor devel opment.x x i x

L MP- 2 prot ei ns, LMP- 2A AND LMP- 3B, are both essential for EBV l atency i n

B- cells. Recent st udi es have shown t he expressi on of LMP- 2A i n Hodgki n’s disease and

nasopharyngeal carci noma, suggesti ng an unknown rol e i n cancer devel op ment. A better

understandi ng of t he LMP- 2 prot ei ns may provi de insi ght i nt o t he virus’ ability t o

overtake cell ular pat hways, causi ng numer ous diseases.x x x

EBV- Encoded RNAs 1 and 2, known as EBERs 1 and 2, are noncodi ng RNAs

expressed is nearl y all EBV i nfect ed host cells and found i n all for ms of latency.

I mport antl y, bot h EBERs are known t o mai nt ai n the mali gnant phenot ype of Bur kitt’s

lympho ma cells, suggesting t heir role i n oncogenesis.x x x i

Previ ous Work

In 2007, t he first EBV- hu man i nteract ome map was construct ed by t he Cent er for

Cancer Syst e m Bi ol ogy (CCSB) at Dana- Farber Cancer Instit ute ( DFCI) i n collaborati on

wit h Mi chael Cal der wood from Bri gha m and Women’s Hospital. The primar y obj ecti ve

of t he screen was t o i dentify t he interacti ons of EBV pr ot ei ns wit h each ot her and wit h

hu man prot ei ns usi ng a stri ngent Y2H syst e mx x x i i

. The successful constructi on of

interact ome maps depi cting EBV- EBV and EBV- hu man prot ei ns may be hel pful i n

identifyi ng t he rol e of specific prot ei ns i n t he virus’ replicati on and reacti vati on

pr ocesses.

An EBV ORFeo me contai ni ng 80 full and 107 partial EBV ORFs, representi ng 85

of t he 89 known EBV prot ei ns, were transferred int o GAL4- DB and GAL4- AD vect ors

by gat eway recombi nati onal cl oni ng t o test EBV- EBV prot ei n interacti ons. The

successful fusi on of t he 187 EBV ORF t o the DB and AD GAL4 domai ns all owed for t he

testi ng of approxi mat el y 35, 000 EBV prot ei n interacti ons via t he Y2H syst em. The

resulti ng fusi ons were transfor med i nt o t wo different hapl oi d yeast strai ns, then mat ed

and anal yzed on selecti ve medi a. Pot ential interactors were det er mi ned t hrough

Pol ymerase Chai n Reaction ( PCR) a mplificati on and sequenci ng. The 43 EBV- EBV

pr otei n interacti ons, involvi ng 44 EBV protei ns, identified i n t he Cal der wood screen

were mer ged wit h published EBV i nt eracti ons to construct the current EBV- EBV

interact ome net wor k (Fi g. 1.)x x x i i i

Fi g 1. “EBV–EBV i nteract ome net wor k resulting from t he mer gi ng of int eracti ons identified in t he

Cal der wood et al. st udy wit h published i nt eracti ons. Previ ousl y i dentified published i nt eracti ons are shown

as purpl e li nes and i nt eracti ons identified i n t he Cal der wood et al. st udy are shown as red lines. Core

herpesviruses are shown as yellow circles and noncore proteins are shown as green circles. Hi gh

confi dence i nt eracti ons are shown as soli d li nes and l ow confi dence i nt eracti ons are shown as dashed lines.

The EBV- EBV i nteract ome consists of 52 protei ns invol ved in 60 i nt eracti onsx x x i v

”.

EBV- hu man prot ei n i nteracti ons were then screened usi ng si mil ar testi ng

techni ques. A compl et e hu man spl een cDNA li brary was fused t o t he Gal 4-AD do mai n

and 113 EBV ORFs, representi ng 85 EBV prot ei ns, were fused t o t he Gal 4- DB do mai n.

The resulti ng fusi ons were t hen transfor med i nt o two different yeast strai ns, all owi ng t he

screeni ng of 85 known EBV protei ns agai nst 100,000 t o 1, 000, 000 human pr ot ei ns. The

t wo yeast strai ns were mat ed and pot ential activators were anal yzed by PCR and

sequenci ng. The resulti ng EBV- hu man i nteract ome net wor k i ncl uded 173 different EBV-

hu man prot ei n i nteracti ons bet ween 40 different EBV prot ei ns and 112 human prot ei ns.

(Fi g. 2.)x x x v

Fi g 2. “The EBV- hu man i nt eract ome resulting from t he Calder wood et al. Y2H screen. Core herpesvirus

prot ei ns are shown as yell ow circl es and noncore protei ns are shown as green circles. Hu man protei ns are

shown as bl ue squares. The i nteracti ons are shown as soli d red lines. The EBV- hu man i nt eract ome

net wor k represents 40 EBV prot ei ns and 112 hu man prot ei ns connect ed by 173 i nt eracti onsx x x v i

.

In 2009, t he CCSB at DFCI perfor med a second EBV- hu man screen i n hopes of

expandi ng t he previ ousl y construct ed EBV- hu man interact ome net wor k. The maj or

difference bet ween t he pri mary EBV- hu man screen ( CCSB wit h Cal der wood) and t he

2009 screen ( CCSB wit hout Cal der wood) was t he use of t he human ORFeo me v. 3. 1

(hORFeo me v3. 1), instead of t he human spl een cDNA li braryx x x v i i

. Duri ng t he screen,

hORFeo me v3. 1 was the largest publicl y availabl e library of human open readi ng fra mes,

cont ai ni ng 12, 212 human ORFs, representi ng 10, 214 human genesx x x v i i i

.

The EBV ORFs, cont ai ning 85 full-lengt h genes and 127 fragments, were

expressed as fusi on prot eins t o t he Gal 4- DB do main. The hORFeo me v3. 1 library was

expressed as fusi on prot eins t o t he GAL 4- AD domai n, all owi ng t he screeni ng of 216

EBV prot ei ns agai nst appr oxi mat el y 12, 000 human prot ei ns i n a stri ngent Y2 H syst e m.

Pot ential acti vat ors were anal yzed usi ng PCR a mplificati on and sequenci ng and t he

resulti ng i nteracti ons were mer ged wit h the pri mary screen result. The CCSB screen

reveal ed 241 interactions bet ween 38 EBV protei ns and 124 human proteins. (Fi g. 3)x x x i x

Fi g 3. CCSB EBV- hu man i nt eract ome net work constructed in 2009.

Redundant i nteracti ons bet ween t he Cal der wood and CCSB EBV- hu man screen

were anal yzed and dis mi ssed i n order t o confir m 68 additi onal i nteracti ons from t he

secondary screen. The mergi ng of bot h screens identified 381 i nteracti ons bet ween 49

EBV prot ei ns and 219 human prot ei ns, resulti ng i n the constructi on and expansi on of an

updat ed EBV- hu man i nteract ome map. (Fi g. 4.)x l

Fi g. 4. EBV- hu man i nt eract ome net wor k constructed from the consoli dati on of the Cal der wood and CCSB

Y2 H syste ms. EBV prot ei ns are expressed as red circl es and the bl ue circles show t he int eracti ng hu man

prot ei n. The EBV i nteract ome net wor k represents 49 EBV protei ns and 219 hu man prot ei ns connect ed by

381 i nt eracti onsx l i

The EBV diseasome was compl et ed shortly after the 2009 CCSB EBV- human

screen by Nat ali Gul bahce, Han Yan, and Laszl o Barabasi. The diseasome interact ome

map was used t o depi ct the i nteracti ons EBV- hu man prot ei ns and t heir likelihood i n t he

pat hogenesis of vari ous EBV associ ated diseases. The diseases were charact erized as

eit her first or second degree based on results from the Onli ne Mendelian Inheritance i n

Ma n

® ( OMI M) dat abase, whi ch cont ai ns i nfor mat ion on all known genetic di seases and

12, 000 genes. First-degree diseases are t hose pot entiall y caused by t he EBV virus and

second- degree diseases are t hose t hat potentiall y devel op as nei ghbori ng human prot ei ns

are affect ed by viral intrusi on. See Fi g 5.

Fi g. 5. The EBV Di seaso me. The green squares represent the diseases, the yell ow circles represent human

prot ei ns, the red dia monds represent viral protei ns, and t he bl ue circles represent human genes.

Gul bahce utilized t he resulti ng EBV- hu man i nt eract ome maps from t he

Cal der wood and CCSB screens to hel p identify positi ve disease li nks. 49 EBV protei ns

were found t o i nteract wi th 257 human prot ei ns, de monstrating 436 EBV- hu man disease

links. The diseasome map is effecti ve in hi ghli ghting how EBV may be i mplicat ed in t he

devel opment of numer ous diseases, incl udi ng cancer. It provi des additi onal confir mati on

of i nteract ors i dentified in bot h EBV- hu man screens and numer ous medi cal journal s,

whil e summari zi ng t he devast ation t hat may result from EBV i nfecti on.

II. Mat eri als and Met hods:

Obj ecti ve:

The goal of t his st udy is to furt her expand t he EBV- hu man i nt eract ome networ k

by usi ng a stri ngent Y2H syste m. The Y2H approach will utilize human and EBV ORFs

expressed as fusi on prot eins i n t wo S. cerevisi ae strai ns t o i dentify positi ve interact ors.

An expanded EBV- hu man i nteract ome will be helpf ul i n i dentifyi ng t he role of

pr otei n interacti ons in t he virus’ ability t o replicate and persist. A better understandi ng of

the virus will be useful in treating and preventi ng EBV- associated disorders.

BP cl oni ng reacti on:

1. The BP reacti on was assembl ed based on t he foll owi ng chart.

5 μl 10 μl

PCR cl one 5 μl 5 μl

pDONR vect or 75 ng 150 ng

5x BP Buffer 1 μl 2 μl

TE pH 8. 0 To 4 μl To 8 μl

BP cl onase 1 μl 2 μl

Tabl e 1: Constructi ng t he BP Gat eway reacti onx l i i

.

1. The resulti ng entry cl ones were incubat ed at 25° overni ght.

2. 2 μl of Prot ei nase K sol ution was added t o each reacti on. The constructs were

incubat ed for 10 mi nut es at 37° C.

3. The constructs were transfor med i nt o compet ent cells and pl ated ont o soli d medi a

cont ai ni ng Specti nomycin.

LR cl oni ng reacti on

1. The LR reacti on was asse mbl ed based on t he followi ng chart.

5 μl 10 μl

Entry cl one (50-150 ng) 5 μl 5 μl

Desti nati on vect or 7 ng 150 ng

5X LR Buffer 1 μl 2 μl

TE pH 8. 0 To 4 μl To 8 μl

LR Cl onase 0. 5 μl 1 μl

Tabl e 2: Constructi ng t he Gat eway LR reacti onx l i i i

.

2. The resulti ng expressi on cl ones were incubat ed at 25° C overni ght.

3. The expressi on cl ones were transfor med i nt o compet ent cells and pl ated onto

soli d medi a cont ai ni ng Specti nomyci n.

EBV ORF Li brary:

The EBV constructs were generat ed i n Eric Johansson’s laborat ory and were

pr ovi ded t o Cal der wood and CCSB as EBV constructs harbored i n E. Coli. PCR anal ysis

was perfor med on t he sampl es t o confir m t he presence of t he EBV constructs, to

det er mi ne t he exact sizes of t he constructs, and t o isol ate si ngle EBV cl ones.

1. The PCR reacti on was perfor med based on t he foll owi ng chart:

Mat erials μl needed.

10X buffer 30

10 mM dNTPSs 6

Mg SO4 12

Pri mer 1 0. 3

Pri mer 2 0. 3

EBV t e mpl at e 1- 2

HI- Fi Taq pol ymerase 1. 2

Di stilled wat er 240

Tabl e 3: Constructi ng t he PCR reacti on t o confir m proper vect or presence in generat ed

EBV constructsx l i v

.

2. EBV i nserts cont ai ni ng the proper vect or presence were select ed from t he

ori gi nal plates. The i nserts were then organi zed i nto fragments, cont ai ni ng a full-

lengt h EBV ORF, and placed i nt o 96 round bott om Cost ar © cell cult ure plat es.

3. The EBV DNA fragments were first transfor med int o E. coli cells then purified

usi ng t he QI Aprep Spi n Mi ni prep Kit ©. Fragments were transfor med i nt o the

Y8930 yeast strai n and glycerol st ocks were made of t he resulti ng EBV li brary,

whi ch were frozen for future use.

The gl yercol st ocks, of t he EBV li brary, ori gi nall y made by Cal der wood and CCSB,

were t he starti ng poi nt for this st udy. The EBV ORFs were transfor med i nto t he DB

do mai n t hrough Gat eway recombi nati onal cl oni ng.

hORFeo me v. 5. 1

The l ong-ter m goal of t he Ma mmali an Gene Collecti on ( MGC) is to i dentify and

cl one all human cDNA clones, cont ai ni ng a functional ORF. The collecti on is readil y

availabl e t o t he research communit y and served as a starti ng poi nt for hORFeo me v5. 1

constructi on. Hu man ORFs were isol ated from MGC c DNA cl ones and were a mplified

usi ng PCR machi nery. The a mplified ORF were cloned and transfor med i nto E. coli

vect ors by a recombi national cl oni ng reacti on. PCR was used t o verify and sequence

present ORFs. Present ORFs were identified by BLAST anal ysis and hORFeo me v. 5. 1

was successfull y compl eted. Gl ycerol st ocks of t he li brary were made and frozen for

fut ure use. The hORFeome v5. 1 li brary is currently t he largest publicl y availabl e human

open readi ng fra me li brary, i ncl udi ng 15, 483 human ORFs, representi ng 12, 794 human

genesx l v

. The li brary i ncl udes all previ ous ORFeo me v1. 1 and v3. 1 products and t he

additi on of 3, 272 ne wl y identified ORFs from MGC t e mpl at es. The expansion of t he

hu man ORFeo me has i ncreased t he effecti veness and pot ential of Y2H screeni ng

met hods by provi di ng a larger library of potential interact ors. The hORFeome v. 51

library was transfor med int o t he AD do mai n t hrough Gat eway recombi national cl oni ng.

S. cerevisi ae strai ns

Two Y S. cerevisi ae strains, Y8800 and Y8930, were utilized t hroughout t his

st udy and served as t he expressi on vect ors. Bot h strai ns were generated by Xi aofeng Xi n

in Charles Boone’s laborat ory. S. cerevisi ae strai ns Y8800 ( MATa) and Y8930 ( MATα)

cont ai n three GAL4p i nduci bl e report er genes, proxi mal t o an upstrea m activati on

sequence, whi ch provi des four i ndi cat ors for positive interact ors. The HI S3 reporter gene

identifies i nteract ors by allowi ng growt h on medi a lacki ng histi di ne. The ADE2 and LacZ

reporter genes bot h utilize col ori metric det ecti on of Gal 4p acti vit y, whi ch varies

dependi ng on t he strength of expressi on. ADE2, in additi on t o provi di ng a not able col or

change, allows for growt h on medi a lacki ng adeninex l v i

. Alt hough LacZ is present in bot h

strai ns, it was not utilized i n t his st udy.

Y8800 and Y8930 were geneticall y engi neered t o cont ai n del eti ons of t he GAL4

and GAL80 genes, whi ch code for GAL4p and GAL80p, t wo regul at ory GAL-transcri ption

genes. The del etion of GAL80p, a repressor gene, prevents t he inhi biti on of GAL4

transcri ption t hroughout the phenot ypi ng process. Two auxotrophi c mar kers, leu2 and

trp1, were utilized t o select for yeast cells harbori ng bot h AD and DB domains. Use of

auxotrophi c mar kers all ows for selecti on of yeast cells t hat were successfully

transfor med. Bot h strai ns are cycl ohexi mi de resistant, ai di ng i n plas mi d shuffli ng and

identificati on of aut o activat orsx l v i i

.

In t his st udy, Y8800 was the AD yeast strai n and Y8930 was t hus the DB strai n.

The Y8800 strai n woul d thus harbor t he GAL4- AD do mai n, whi ch cont ai ned t he

hORFeo me v5. 1 li brary expressed as fusi on prot eins. The auxotrophi c marker for Y8800

was leu2 (-trp1), indi cating t hat trypt ophan coul d be used t o confir m t he successful

transfor mati on of t he AD plas mi d i nt o the yeast strai n. Y8930 was desi gnated for t he

GAL4- DB do mai n, whi ch cont ai ned t he EBV ORFeo me expressed as fusion prot ei ns.

The auxotrophi c mar ker for t he Y8930 strai n was trp1 (-leu2), all owi ng for confir mati on

of successful transfor mat ion of t he DB pl as mi d and EBV ORFs when grown i n leuci ne

liqui d medi a.

Yeast Strai n Har bors ORF Auxotrophi c

Mar ker

Gr ow on

Y8800 GAL4- AD

do mai n

Hu man Leu2 - Trp medi a

Y8930 GAL4- DB

do mai n

EBV Trp1 - Leu medi a

Y8800 +

Y8930

GAL4- DB

do mai n and

GAL4- AD

do mai n

Hu man and

EBV

N/ A -leu, -trp –his,

+1 mM 3-a mi no

triazol e (3AT)

Tabl e 4: Summar y of Y8800 and Y8930 strai ns, their cont ents, and t he selecti ve medi a

required t hrough t he Y2H screeni ng process.

Procedure:

1. The t he Gen mat e © robot ic liqui d handi ng syst e m was used t o i nocul ate 10μl of

AD cl ones, the Y8800 strai n was transfor med wit h the GAL4- AD do mai n and

hu man ORFs, int o 80μl of 1XSC- Tr p li qui d medi a. Inocul ation was performed i n

96 round bott om well Costar © cell cult ure pl ates. Synt hetic Co mpl et e (SC) drop

out medi a cont ai ns essential ami no aci ds and vitami ns needed for yeast growt h,

whil e still selecti ng for auxotrophi es. The cult ures were grown at 30° C for t wo

days.

2. Si milarl y, 10μl of DB clones, cont ai ni ng t he GAL4- DB do mai n wit h fused EBV

ORFS, were inocul ated int o 80μl of 1XSC-l eu li qui d medi a usi ng t he Genmat e ©

robot. The cult ures were gr own at 30º C for t wo days.

3. To test for t he presence of aut o acti vat ors pri or t o starti ng pri mar y and secondary

phenot ypi ng processes, 5μl of t he DB cl ones were pi petted ont o –l eu and –his

pl ates. Gr owt h was observed after 24 hours. If the DB cl ones survi ved on

-histi di ne pl ates, wit hout the presence of t he AD clones, they were aut o-activat ors

and re moved from t he screen. Additi onal steps were taken t hroughout t he screen

pr ocess t o i dentify aut o acti vat ors t hat were not detected pri or t o screeni ng.

4. The AD and DB cl ones that grew i n the 1XSC-trp and 1XSC-l eu and were not

dee med aut o activat ors were mat ed usi ng t he Genmat e © robot. The AD and DB

cl ones were spotted directly on t op of one anot her all owi ng for t he hapl oi d cells t o

beco me di pl oi d. Di pl oi d yeast cells t hen harbored bot h t he GAL4- AD domain and

GAL4- DB do mai n, cont aini ng bot h human and EBV ORFs, all owi ng for pot ential

pr ot ei n i nteracti ons to occur. 5μl of each yeast culture were spotted on YEPD

pl ates, whi ch provi de essential nutrients for yeast gr owt h. Si x controls were added

to t he bott om of t he YEPD pl at es, whi ch were then grown for 24 hours at 30º

The Si x Controls

As wit h any sci entific st udy, controls were used and monit ored t o det ect any

abnor mal growt h patterns or lack of growt h all toget her. Si x controls, specific t o Y8800

and Y8930, were utilized to ensure effecti ve screeni ng techni ques. Any deviations from

expected growt h patterns were consi dered before conti nui ng t he screen.

Control DB cl one AD cl one Expect ed Gr owt h

1 pPC97: An e mpt y

DB expressi on

vect or.

pPC86: An e mpt y

AD expressi on

vect or.

No growt h is

expected because

onl y e mpt y vect ors

are present.

2 Rb: Weak

Positi ve

E2F1: Weak Positi ve Little or no growt h

3 Fos HLH: Strong

Positi ve

Jun HLH: Strong

Positi ve

Fai nt growt h

4 GAL4 AD f ull prot ei n Ma xi mu m growt h

5 DP1: E2F- 1: Strong growt h

6 DP1 E2F- 1 wit h

cycl ohexi mi de

Equal t o control 5

Tabl e 5: The si x controls and t heir expected growt h patterns.x l v i i i

.

5. Aft er 24 hours of i ncubation, pri mar y phenot ypi ng began wit h replica pl ating.

Yeast col oni es were replicat ed ont o selecti ve media t o assess t heir ability t o grow

in an i nteracti on -dependent manner. Replica pl ating ont o sel ecti ve medi a

confir ms t hat t he yeast vect ors are transfor med wi th bot h AD and DB, and

pot ential interacti ons can occur. This process requires t he availability of replica

pl ati ng bl ocks and vel vet squares, bot h of whi ch must be sterile pri or t o use. The

replica bl ocks and vel vet squares were aut ocl aved to prevent t he probabl y of

outsi de cont a mi nati on.

6. Yeast cells were transferred ont o t he sterile vel vets by evenl y pressi ng t he bott om

of t he pl ate. The cells from t he vel vets were then transferred t o a new plate

cont ai ni ng sel ecti ve media t hrough t he sa me even pushi ng techni que. Yeast cells

were replica pl ate from the YEPD pl ate ont o

1. –His, +Ade, +3AT pl ates.

2. Cycl ohexi mi de pl ates.

Gr owt h on –His and +Ade i ndicat ed t he interacti on of AD and DB and t he

expressi on of bot h reporter genes. 1mM of 3-a mi no triazol e (3AT), whi ch inhi bits t he

HI S3 gene product, was used t o decrease the backgr ound expressi on associat ed wit h

HI 3x l i x

. The specific concentration of 3AT ensured that weak interactions are not lost, but

the probabilit y of false positi ves was eli mi nated. The cycl ohexi de pl ates were used t o

identify aut o acti vat ors.

7. Aft er 24 hours, the –Hi s, +Ade, +3AT and cycl ohexi mi de were cleaned. Cl eani ng

the plates required t he use of replica plating bl ocks, sterile vel vets, and a roller. A

sterile vel vet was pl aced on t he replica plating bl ock t hen t he plate was firml y

pl aced ont o t he vel vet. A roller was used t o carefully and evenl y roll across the

back of t he plate until the col oni es were no l onger vi si bl e. In some cases, t wo

vel vets were necessary. Cl eani ng ensured t hat t here were an equal and

comparabl e number of cells in each spot, by re movi ng background growt hl

. Plates

were grown at 30° C for 5 days.

8. Positi ve col oni es from t he - Hi s, +Ade, +3AT pl ates were pi cked usi ng a sterile

toot hpi ck i nt o 80μl of 2XSC- Leu- Tr p li qui d medi a. The t oot hpi cks were

aut ocl aved before use and discarded after pi cki ng each col ony. Candi dat es were

gr own at 30º C overni ght.

9. Gl ycerol st ocks of potential candi dat es from t he –Hi s, +Ade, +3AT pl ates were

made and st ored i n 40 % gl ycerol. They were frozen t o all ow for reuse during

fut ure st udi es.

10. The positi ve di pl oi d cells from t he li qui d medi a were spotted ont o –l eu, -trp

pl ates. Col oni es were grown for t wo days at 30º C. Thi s process began

phenot ypi ng t wo, where positi ve int eracti ons were t hen pi cked and sequenced

usi ng PCR anal ysis.

11. The –l eu, -trp plates were replica pl ated ont o the flowi ng pl ates, in t he gi ven

or der, after t he t wo days.

1. –Hi s, +3AT pl ates

2. +3AT, +cycl ohexi mi de pl ates

12. The pl ates were cleaned i mmedi at el y after compl eti ng t he replicati on process t o

re move any background gr owt h. The pl ates were gr own for fi ve days at 30º C.

13. The secondary pl ates were pl aced si de by si de for comparison. Each i ndi vidual

spot was compared bet ween t he –his, +3AT and +3AT, +cycl ohexi mi de plat es t o

det er mi ne t he presence of aut o acti vat ors before PCR anal ysis and sequencing.

Any growt h observed on the +3AT, +cycl ohexi mi de pl ates were noted on the -

Hi s, +3AT pl ates and deemed aut o acti vat ors. Coloni es t hat grew on t he +cycl o

pl ates were not pi cked because t hey were aut oactivat ors. Col oni es t hat showed

gr owt h on –Hi s, +3AT and not +3AT, +cycl oheximi de were candi dat es, whi ch

were t hus picked and analyzed.

14. A st erile t oot h was used to pi ck positi ve candi dat es from t he –His, +3AT plat es.

Candi dat es were then pi cked ont o –Leu, - Trp pl ates, whi ch were grown for 24

hours at 30º C.

15. Fi ve milliliters of Z-buffer, whi ch cont ai ned 1. 0 mi lli gra ms of zymol yase per 1. 0

mi lliliter of l ysis buffer, was made. The zymol yase enzyme l ysed t he yeast cells

and released t he DNA i nto sol uti on.

16. 15μl of Z-buffer was distri buted i nt o the each 96 round bott om well on the

Cost ar © cell cult ure plates. A s mall amount of each candi dat e was pi cked and

pl aced i nt o a round- bott om well. A ne w sterile t oothpick was used t o pi ck each

col ony.

17. The foll owi ng zymol yase/l ysis progra m was run, in t he gi ven order, in a t her mal

cycl er.

1) Incubati on- 37º C for 15 mi nut es.

2) Acti vat e zymol yase enzyme- 95º C for 5 mi nut es.

3) 10º C for t wo hours.

18. Aft er t he zymol yase/l ysis progra m was compl et ed and t he DNA was released

int o sol uti on, 100μl of distilled wat er was added to each Costar © well. The

Cost ar © plates were centrifuged for 5 mi nut es at 2000 rpm. This process dilut ed

the te mpl at e and was necessary for furt her PCR anal ysis.

19. Additi onal PCR anal ysis was run t o a mplify segments of t he AD and DB

segments. The foll owi ng pr ogra m was run i n t he given order.

1) 94º C for 2 mi nut es

2) 94º C for 30 seconds

3) 58º C for 30 seconds

5) 94º C for 30 seconds, 30 ti mes

6) 68º C for 5 mi nut es

7) 10º C for 5 mi nut es

20. The resulti ng AD and DB PCR pl ates were st ore at 4º C for fi ve days before t hey

were sent t o Agencourt Bioscience Cor porati on i n Beverl y, MA for sequencing.

III. Results

The EBV- hu man screen all owed for t he screeni ng of 216 EBV prot ei ns against

15, 483 human prot ei ns and was successfull y compl eted t hree ti mes. The t hree screens

yi el ded 188 pot ential candi dat es, whi ch were analyzed usi ng PCR machi nery then sent to

the Agencourt Bi osci ence Cor porati on for sequenci ng.

St udy Co mplicati ons

Nu mer ous compli cati ons arose throughout t he durati on of t his study. Many were a

result of human error, however syste matic errors also negati vel y affected t he EBV- hu man

screens.

In t otal, ei ght EBV- hu man screens were atte mpt ed, but onl y three were

successfull y compl et ed. The first t wo screens were disregarded after pri mary

phenot ypi ng was compl eted. Upon exa mi nati on, the pl ates showed fai nt growt h i n half of

the expect ed spots at best. It was det er mi ned t hat the replica pl ating or cleani ng processes

were not compl et ed correctl y. It is possi bl e t hat during t he replicati ng process, the

“ mot her” plates were not pressed fir ml y ont o the vel vets, thus causi ng t he insufficient

transfer of col oni es ont o the sel ecti ve medi a. It is also possi bl e t hat duri ng cl eani ng t he

roller was pushed t oo firml y ont o t he vel vets, for too l ong of a ti me peri od, causi ng t he

col oni es t o transfer sol ely ont o t he cleani ng vel vets.

The next t hree screens were also disregarded after pri mar y phenot ypi ng because

the sa me growt h pattern was observed on every plat e after t he t wo yeast strai ns were

mat ed. A different growt h pattern is expect ed on each pl ate t hroughout a screen because

each human gene can i nteract differentl y. Through collaborati on wit h ot her me mbers i n

the CCSB depart ment, it was concl uded t hat intermi xi ng of t he wells caused an aut o

acti vat or t o spill int o adj acent wells. Initiall y 120μl of 1XSC-trp and 1XSC- leu li qui d

medi a were used t o i noculate t he AD and DB cl ones, in step t wo of t he procedure.

Bef ore t he cl ones were spotted ont o t he –l eu and –his plates, the Cost ar © cell cult ure

pl ates needed t o be spun. It was later det er mi ned that t he wells shoul d hol d 80μl i nstead

of 120μl. Too much li quid medi a was used t he wells were inter mi xi ng when spun,

causi ng t he sa me growt h pattern to be present on every pl ate.

There were also numer ous instances when cont a mi nati on rui ned pl ates used

duri ng t he screens. Cont ami nati on was li kel y a result of i mpr oper techni ques and air

poll ut ants.

Co mpli cati ons conti nued to arise even after the successful compl eti on of t he t hree

screens. Three pl ates contai ni ng 188 candi dat es were placed i n t he col d room and st ored

at 4º C until PCR coul d be perfor med. Duri ng t his ti me t he candi dat es were di sposed of,

leavi ng one pl ate left for anal ysis.

I V. Discussi on

Pr ot ei ns are possi bl y the most i mport ant class of macr omol ecul es i n t he hu man

body, servi ng a variet y of functi ons. Each i ndi vi dual prot ei n has a specific role i n

ensuri ng t hat vital life processes are perfor med correctl y and safel y. Many pr ot ei ns do so

by i nteracti ng wit h ot her pr ot ei ns, catal yzi ng bi oche mi cal reacti ons, servi ng as anti bodi es

agai nst anti gens, acti ng as che mi cal messengers, or carryi ng i mport ant molecul es from

different sites wit hi n the body.

I mport ant questi ons are posed when ext ernal fact ors can disrupt t he nor mal

functi on of t hese indi vi dual prot ei ns and t he functions t hey serve. What happens when

pr ot ei ns st op i nteracti ng wi t h prot ei ns t hat hel p the m carry out i mportant life processes?

What is the effect when pr ot ei ns start interacti ng wi t h different prot ei ns? Ne w research

has shown t hat t he develop ment of numer ous human diseases is attri buted t o

malfuncti ons in nor mal protei n i nteracti on. Oft en ti mes, the host’s i mmune syste m is

compr omi sed, all owi ng for an ext ernal fact or t o hijack and disrupt i mport ant cell ular

pr ocesses.

Vi r uses, such as EBV, have t he ability t o ent er i nto a host undet ect ed t hrough

respirat ory air ways. They are essentiall y genes, protect ed by a prot ei n coat, whi ch require

a host cell to survi ve. Viruses use the host’s enzymes and ot her cellul ar machi nery t o

express t heir prot ei ns, synt hesize prot ei ns, and replicat e. The cells used for viral

replicati on rarel y survi ve, releasi ng numer ous ne wly for med viruses, whi ch attack

nei ghbori ng host cellsl i

. As the host cells are i nfected and l ysed, nor mal protei n

interacti on and cell ul ar functi on may be effected.

As nor mal cell ular processes are affected, host cells may not de monstrate nor mal

cell growt h patterns. Healthy functi oni ng cells shoul d show bot h growt h stimul at ory

si gnals and i nhi bit ory si gnals. Basi call y, cells will conti nue t o di vi de as l ong as t here is

enough room and t hey shoul d st op di vi di ng once they come i n cont act wit h one anot her.

Ho wever, virall y i nfect ed cells may not have excessi ve growt h sti mul at ory si gnals or t oo

few i nhi bit ory si gnals as a result of abnor mal protei n i nteracti ons, resulti ng in t umor

for mati onl i i

.

EBV is one of t he most co mmon human viruses, esti mat ed t o i nfect ed 95 % of t he

worl d’s popul ation. The virus has been i mplicated in numer ous diseases, incl udi ng

Bur kitt’s lympho ma, Hodgki n’s disease, non- Hodgki n’s l ympho ma, and nasopharyneal

carci noma. Unli ke most viruses, EBV is abl e t o mai nt ai n its viral genome wi t hout

da magi ng or mar ki ng host cells for destructi on. The virus’ ability t o establish latency has

pr oposed numer ous questions regardi ng t he effect on bot h viral and human pr ot ei ns and

the devel opment of cancer.

Ne w research has i dentified t he role of i ndi vi dual EBV prot ei ns, while suggesti ng

that prot ei ns t hat react wi th numer ous ot her prot eins may somehow be i nvolved i n

di sease devel opment. However, research focused on t he EBV virus still remai ns

inadequat e. Wit h such a large percent age of t he worl d’s popul ati on affect ed by t he virus,

mor e attenti on shoul d be focused on preventi ng and treating t he virus before t he disease

devel ops.

In t he 2007 EBV- hu man interact ome proj ect, CCSB and Cal der wood defi ned

EBV “hubs” as prot ei ns wi t h relati vel y short pat hways t o human prot ei ns or t hose wit h a

large number of prot ei n int eracti ons. They found that “hubs” in t he EBV int eract ome

where si gnificantl y more essential to yeast survi val than prot ei ns wit h a s mall number of

interacti ons. More specifically, they found cancer devel opment is likel y a result of “hub”

interacti on. However, even wit h si gnificant evi dence for t he pat hogenesis of disease as a

result of prot ei n “hubs” the necessary research is not bei ng funded and perfor med.

Si nce EBV- associated diseases are not as common as ot her i nfecti ous diseases,

many argue t hat research is not a pri orit y. While onl y a s mall popul ati on devel op

mali gnanci es as a result, the potential for more EBV- associated disease cases is vast. As

our environment becomes more poll uted and less resourceful, it is inevitable t hat humans

wi ll be exposed t o har mful ext ernal fact ors t hat may reacti vat e t he virus i n infect ed

indi vi duals. Very little is known about how or why the virus reacti vat es, however, it is

obvi ous t hat dangerous toxi ns or changes have t he pot ential to do so.

Ne w controversial research has also suggest ed t he role of EBV i n the

devel opment of breast cancer. Some st udi es have shown traces of EBV viral mat erial in

51 % of t he breast cancer tissue sa mpl ed, while ot hers failed t o det ect EBV in any tissue

sa mpl es. The variance i n st udy results may be a result of different screeni ng techni ques,

the EBV prot ei ns and RNAs st udied, or i n breast cancer itself. However, wi t h an

esti mat ed 192, 370 ne w breast cancer cases i n 2009 al one, it is i mport ant to understand

the potential rol e of EBV and t he pat hogenesis of thi s disease as well. The linkage

bet ween EBV and breast cancer see ms probabl e after anal yzi ng t he staggering number of

breast cancer cases and the large popul ati on t hat serve as hosts for t he virus.

Understandi ng if the virus is invol ved, and t he role it plays if so, in oncogenesis woul d be

beneficial for all patients and healt hcare professi onals.

Treat ment of EBV- associ ated mali gnanci es

Si nce t he discovery of EBV- associated mali gnanci es, treat ment opti ons have

pr oven relati vel y unsuccessful. Anti viral agents, immune- based t herapies, and specific

monocl onal anti body options are currentl y bei ng expl ored and have shown pr omi si ng

results. Anti herpesvirus and anticyt omegal ovirus agents, such as ganci cl ovir,

fa mcycl ovir, acycl ovir, val aci cl ovir, foscarnet, and ci dofovir, have been applied i n a

cli nical setti ng, but all vary in t heir effecti veness. Ma ny anti viral treat ment opti ons are

used t o i nhi bit EBV replicati on, i nitiate t he apopt osis of EBV- positi ve lympho ma cells,

or bl ock EBV anti gen activati on. Regardl ess of t he encouragi ng clinical results, anti viral

agents are unabl e t o treat the i mmunodeficiency issues t hat all ow for t he devel opment of

EBV- associated mali gnanci esl i i i

.

I mmunot herapy procedures, utilizi ng EBV-specific cyt ot oxi c T l ymphocytes

( CTLs), have also been successful i n treating patients wit h EBV-rel ated t umors, while

addressi ng i mmunodeficiency issues. The treat ment opti on has been most successful i n

patients wit h Hodgki n’s di sease, hel pi ng reestablish i mmunoco mpet ence. EBV- specific

CTLs can be taken from a seropositi ve donor and directl y i nfused i nt o a patient, or

expanded furt her i n vivo then i nfused i nt o the patientl i v

. Whil e t he success rate for t he

i mmunot herapy procedures re mai ns mi ni mal, the abilit y for t he procedure to fi ght

i mmunodeficiency may be successful i n combati ng t he cell ular pat hway disrupti on

caused by EBV.

The useful ness of EBV-specific CTLs is bei ng used i n t he efforts t o devel op and

EBV vacci nati onl v

. Vacci nes efforts are under way to hel p prot ect agai nst initial EBV

infecti on. If proven successful, the vacci ne woul d have t he pot ential to eradicate

nu mer ous EBV- associ ated diseases. Wit h the majorit y of t he worl d’s population i nfect ed

wit h t he virus, a vacci ne woul d undoubt edl y be beneficial. Therefore, it is vital that

cli nical st udies are funded and support ed t o ensure t hat efforts are conti nued and

resources are availabl e. Lastl y, the vacci ne, once devel oped and approved, must be

availabl e i n all countries, regardl ess of t he cost. It woul d not be effecti ve if onl y certai n

countries had access because EBV- associ ate diseases affect indi vi duals worl dwi de.

Whi l e opti ons for EBV treat ment are availabl e, the success rate of anti viral dr ugs

and i mmunot herapy re mai ns i nadequat e when l ooki ng at the number of cases of EBV-

associ ated diseases and mali gnanci es. In order t o successfull y treat disease, a better

understandi ng of t he virus and its ability t o disrupt nor mal prot ei n functi on is required. A

nu mber of st udi es have focused on t he interacti ons bet ween EBV and human prot ei ns and

the possi bl e result on cells and t he human body. However, the fi ndi ngs of the Y2H

syste m have not been further anal yzed as a result of i nsufficient fundi ng for furt her

research. Indi vi dual protei ns have been i ndentified and t heir roles i n EBV infecti on have

been t heorized, however, much is still vague or unknown. More research needs t o be

perfor med t o not onl y i dentify additi on EBV- hu man i nteract ors, but also to furt her

anal yze known i nt eract ors role i n EBV i nfecti on, lat ency, and reacti vati on.

Understandi ng t he abilit y of EBV t o disrupt prot eins t hat hel p carry out vital cellular

functi on may provi de i nsight int o t he devel opment of numer ous diseases. Once t he virus

is better underst ood, researchers can focus on t he devel opment of a vacci ne t hat woul d

pr ot ect indi vi duals agai nst initial infecti on and other treat ment opti ons for indi vi duals

already i nfect ed.

Fut ure Di recti ons:

Thi s st udy ended at DFCI in t he earl y anal ysis stage. Once t he candi dat es are

sequenced and sent back to CCSB at DFCI t hey need t o be anal yzed by bi oinfor matics t o

det er mi ne whi ch i nteractions were det ect ed i n t he screen. Any ne w i nt eractions shoul d be

not ed and redundant i nteracti ons shoul d not be disregarded. If the sa me i nteracti ons are

observed i n numer ous screens they very well may be “hub” prot ei ns or essential to EBV

functi on. Speci al attenti on shoul d be focus on redundant i nteracti ons, especi ally t hose

observed all three EBV Y2 H screens.

Aft er t he i nteracti ons are det er mi ned, retesti ng needs t o be perfor med t o ensure

that those interacti ons were act uall y observed. Once t he retesti ng sequences are anal yzed,

interacti ons that were observed i n t he first screen and agai n i n t he retesti ng shoul d be

added t o t he current EBV interact ome map. An expanded i nt eract ome map woul d be

hel pful i n understandi ng the virus’ effect on host cells and i nfect ed i ndi vi duals.

Next, efforts need t o be made t o i dentify and i ncorporate t he four unknown EBV

pr otei ns i nt o t he current EBV ORFeo me. The readil y availabl e EBV ORFeome cont ai ns

85 of 89 known EBV genes. It is possi bl e t hat t he four unknown EBV protei ns may be

the most i mport ant in t he virus’ ability t o establish latency and cause t he devel opment of

EBV- associated diseases. In expandi ng t he current EBV ORFeo me t o i ncl ude all possi bl e

interact ors, more thorough and effecti ve screens coul d be perfor med.

In additi on, efforts t o expand hORFeo me v5. 1 li brary shoul d be consi dered to

ensure t hat t he maxi mu m number of i nteracti ons is screened, tested, and analyzed. By

expandi ng t he human gene li brary t o i ncl ude numerous ORFs additi ons, a wi der range of

pot entiall y vital interactions may be identified.

Once t he EBV ORFeo me and hORFeo me libraries are expanded, additi onal

screens must be perfor med. Expanded ORFeo me libraries woul d allow for the most

effecti ve, accurat e, and groundbreaki ng screens to be perfor med. These screens woul d

have t he potential to provide insi ght i nt o unknown interacti ons occurri ng between EBV

and human prot ei ns, EBV- EBV protei ns, and human- hu man prot ei ns, which may be

detri ment al to disease devel opment.

V. Concl usi on

In concl usi on, the Y2H hybri d syste m is an effective means for understanding

pr ot ei n i nteracti ons and their role i n disease develop ment. The fiel d of syste ms bi ol ogy

uses t he Y2H syst e m t o devel op i nt eract ome maps, whi ch depi ct all known interacti ons

occurri ng wit hi n a cell ular net wor k. Because protei ns play vital rol es i n life processes, it

is i mport ant to understand how ext ernal fact ors can disrupt nor mal i nteractions i n

indi vi duals, havi ng fatal consequences.

A l ong-ter m goal of t he CCSB at DFCI is understand t he effect of EBV on a

hu man host and t he virus’ role i n oncogenesis. Two screens have been successfull y

compl et ed, identifyi ng 381 i nteracti ons bet ween 49 EBV prot ei ns and 219 human

pr ot ei ns. This st udy has been t he first screeni ng of EBV- hu man prot ei ns since t he

expansi on of t he human ORFeo me library. The results of t his screen have the pot ential to

expand t he current EBV- hu man i nteract ome, provide insi ght i nt o t he virus’ ability t o

hijack cell ular net wor ks, and i dentify vital unknown prot ei n i nteracti ons in i nfect ed

indi vi duals.

It is i mport ant that additional screens are perfor med and efforts are conti nued t o

compl et e t he EBV ORFeo me and expand t he current human ORFeo me. In doi ng so,

infect ed i ndi vi duals would have safer and more effecti ve treat ment opti ons, and ot hers

may be fort unat e t o be prot ect ed agai nst initial EBV i nfecti on.

VI. Acknowl edge ments

It is wit h great pleasure that I thank t he many peopl e i n t he Cent er for Cancer

Syst e m Bi ol ogy Depart ment at Dana Farber Cancer Instit ute for wel comi ng me i nt o t heir

tea m and showi ng me t he i mportance of hard wor k and dedi cati on. Their endl ess hel p

and understandi ng made my experience surel y unfor gettabl e. I woul d li ke to especi all y

thank Davi d Hill for welcomi ng me i nt o the Marc Vi dal Laborat ory and maki ng sure I

was comf ortabl e wit h t he concepts and me mbers of the tea m. I woul d also like t o t hank

Dr. Jennifer Roeckl ei n- Canfiel d for all owi ng me to be a part of her research and hel pi ng

me realize t he i mport ance of EBV- associ ated diseases. It is wit h most i mportance t hat I

thank Lila Gha msari for bei ng t he first person t o wel come me i nt o the lab, havi ng t he

patience t o teach me i mportant concepts, and allowi ng me t o take part in her personal

research.

I woul d also li ke t o t hank my professor, ment or, and friend, Marl ene Sa muelson

for her support throughout t he years. Thank you for blessi ng me wit h this a mazi ng

experience, challengi ng me t hroughout t he years, and encouragi ng me t o fulfill my

drea ms.

To all the me mbers of t he Curry College Bi ol ogy Depart ment I woul d li ke to

thank you for provi di ng me wit h an a mazi ng support syste m t hroughout t he years. Thank

you for bei ng more t han just professors, but dedi cated i ndi vi duals who believe i n t heir

st udents. I am forever grat eful for everyt hi ng each one of you has done for me and I will

mi ss you dearl y.

To my cl osest friends, Jai me Callanan and Amanda Leger, I woul d li ke to thank

you for maki ng my years at Curry College more than me morabl e. Thanks for t he laughs,

love, and support.

Lastl y, and most i mport antl y, I woul d li ke to t hank t he me mbers of my fami l y. To

my mot her and fat her I thank you for l ovi ng and supporti ng me unconditionall y. I

appreci ate every sacrifice you bot h had t o make t o ensure t hat I was gi ven the means t o

make my drea ms come true. To my brot her, Matt, I thank you for al ways being t here for

me t hrough t he good and the bad. To my grandmother, I thank you for bei ng li ke a

second mot her t o me t hroughout my life. Thank you for your selflessness, endl ess l ove,

and support. To all ot her me mbers of my fa mil y, I woul d li ke to t hank you for some of

the best ti mes i n my life. I love you all.

VII. Appendi x:

Yeast Medi a:

1. YEPD

*per liter

20g of pept one

10g yeast extract or 20g for soli d plates

50 ml of 40 % gl ucose

. 15 ml of adeni ne

2. Synt hetic Co mpl ete dropout medi a

*per liter

1. 3g of a mi no aci d powder, cont ai ni ng adeni ne

1. 7g of yeast nitrogen base

5g of a mmoni um Sulfate

500 ml of distilled wat er

10 M Na OH was used t o reach a pH of 5. 9

Sel ecti on Medi a

1. 10X TE Buffer

*per liter

100 ml 1 M Tris- HCl buffer

20 ml 500 mM EDTA

880 ml distilled wat er

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