Newfoundland Studiesin ial

CIIARACTERIZATION OF A ,DNA ENCODING A NOVEL TRANSCRIPTION FACTOR REGULATING EXPRESSIONOF

IIUMAN NEUROTROP ICJCVIR US

Dy

OLnxminarayanaR.Devireddy,M.V.Sc.

Athesis submittedtoTheSchoolofGraduate Studiesin partialfulfilmentof the

requirementsforthedegreeof Mesterof Science

DivisionofBasic MedicalSciences Faculty ofMedicine Memorial Universityof Newfoundland

November, 1995

St. John's Newfoundland

1.1

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ISBN 0-612-13893-3

Canada

Thisthesis isdedicat edto mypar ent s Smt. D.Jayalaaml

and Sri. D.Premo Reddy

AB8'l'RAC'l'

JC virus (JCV) is a ubiquitous human neurotropic polyomavirus and is the etiological agent of the human delllyelinating disease, progre8sive tIlultifocal leukoencephalopathy (PML). clinically, PML is found when immunodeficiency is caused by neoplasia, chronic disease, the illlllunosuppressive chemothe.rar-y used for organ transplantation and, more recently, AIDS. JCV is closely related to two other polyomaviruses,BKV and SV40.Unlike BKV and SV40, JCV has a narrow tissue-tropism to glial cells. Several lines of evidence were used to attribute the JCV neurotropism to thd viral regulatory region, which is organized as two 98 bp repeats.

Previousstudies from our laboratory demonstratedglial cell-"Ipecltic expression of JCV in differentiating P19 embryonal carcinoma cells. The specificity was shown to be conferred by the NF-l Dlotlfa preaent in the JCV regulatory region (Nakshatri I I Al., 1990a; KumarAt41., 1993).A eDNA encoding a factor that binds ee the JCV HF-l motif was cloned from a P19 glial cell eDNA library (KWllar, 1994).

The objective for this thesb was to characterize this eDNA to understand its role in the expression and lifecycle of JCV. First, the eDNA sequence was determined. The analysis of the sequence of the eDNA revealed a significant homology both at protein and nucleic acid levels to a recently characterized

fact o r inte r a c ti ngwith BcI-2 prot o-o nc oq en e pr ota i n called BAG··l. However, the cDNA isolated in cur laboratoryhas a different 3'-end (nt 710-973).Thereforethe cDNAwa s naaed 1z.A9.=2. The 3'-e nd of the eDNAappearedto be altornativClly spUced,becauae thepointof divergenceof theeDNA se qu e nc e froa that ofbAs::.lVllSa consensus splice IIlte.

The ~ eDNA encoded a major 30 kDa prote i n , as deterllli ne dby ~transla tion. The ~txQ- t ra ns la ted BAG-2proteinwas bound specifica llytothe JCV NF-l II/III oli gon uc l e ot i de and to the JCV enhan c e r,

.s

det ertlli ne d by mobil ity shift llnd Sout hwe s te r n blot assays. Fur t he r, the tr llns fected~-::DHAspeci!ic a Uy tranaa c t ivate d JCV early (JCV_f) and la t e (JCVL) pro Doters in non-qUal HeLa cell. and the tr a nsactivatlon requiredthe integrity of theJCV NF-l motifs.The recombinantBAG-2 producedin bacteria stiaulated JCV_f tr a n s c r i p t i o n..I..n...Y.1.by3-fo ld , furthersU9gesting that BAG-2controls transcription.InterClstingly.overexpressionof BAG-2inP19 911alcells inh i bited theactivated ,butnotthe basal, levelof tr an s c r i p t i o n of bothJCV.andJCVLin a dose dependent llIanne r. Howeve r, Buch an inh ib i to ry effect wa e not observed inU87HG human gUo b lastolll&cells .These re s u lts are re':Ilin lsc e nt of tho squ el c hing obse rved for eUka ryotic trsnsc ript ionfactors. The~and2IIRNAewere 8xp res8edin mouseembryonalcarcino lllil andhumancervicalcells butno t in U87 HG cell s. However , the BAC· 2 specific c-terml nus was expres s ed only inPig ce lls .

11

Deletio nanal ysis of BAG- 2 revealed thatth e C-ter1llinus (a.a 19S-229 ) va a ea a e ntial for tr a na a c t i va t i o n . while the cent ral a-h elic a l req i o n waa importa nt for DNA-binding'.

ThoUg'h,the BAG-2C-t.~inuawaa obaervedinPl9 VD. glialand .usclecelh". only in PI9 glialcella waa BAG-2 tra ns l oc a t e d into the nuc l e ua.This was furtherconfirmed by Southwestern blot analys iswith nuclearextractatrollthese cells.

Th/l BAG-I ia importa nt formodulating ce ll death. sinc e SAG-2 showed a significanthomology to BAG-I, I test e d thCl elfact of BAG-2on pS3promoter to delineate its possible.role in apoptosis. The BAG- 2neg atively re gUl ated the mouse p5J prollloter, as revealed by CATusays. Thu a . the BAG- 2 migh t prote c t vi r u a- i n f e c t e d cella frompSJ - induced apop tosis by decreas ingthe express ion le vel s of p5J. A model wa sproposed to explain the latency of JCY out_Ide the central nervous syate.(CNS )and. :ICY 911alcell-apacificexpressionin CNS.In the model. BAG-2 ia a nov e l tranacription factor that JDOdulat es the expression ofJCY differentially in glial and non-qlialceUa. In the former it activatesthe expressionof JCV as atr a ns c r i pt i on factorinthe nucleus, explalnln9the neurotropism of

scv,

Inthela tterit inh i b i ts the apoptosis induc e d by p5J thu a. allowing the vi rus to pers i st for pro longe d time.

i i i

ACDOWLEDQB.XEH'1'8

I alii deeply i",debted to Illy supervisors Drs. Alan and Mary Pater, who, despite othel.- students and ongoing projects.

accepted me as a student. Iam also thankful to them for their impl!ccable guidance and steadfast encouragement. A pal-t of this work is dedicated to Dr. Mary Pater, who passed away on November . 2, 1994.

I wish to express my gratitUde to Drs. Gary Paterno and Ken Kao for their timely sug98stions, advice and help as supervisory committee members. I am grateful to Dr. verna Skanes, assistant dean, Faculty of Medicine, for her moral support and help. I also wish to express my gratitUde to Dr.

hartin Mulligan for his help in analyzing the eDNA.

I am thankful to Dr. xotIo Kumar, who introduced me into this exciting field of transcriptional regUlation with his

"promoter-bashing" techniques, he virtually provided me with another leg for a one-legged soldier. I am grateful to Dr.

Harlkrishna Nakshatrl and Dr. Belaguli Swamy for their stimUlating discussions and help in writing literature.

It is my pleasure to thank my colleaguGs, Mr.Ming F9ng, lorbeing a patient and helpfUl neighbour at the work-bench and Dr. Nakao for sharing his reagents and his help in Northern blot analysis. I also owe a lot to Dr. Suvarnalatha Khare for her discussions, care, affection and being a patient host for my 1Il.ockpresen~-'jtions . I a180 thank Drs. Nagendra

tv

Prasad, Satish, HQgdQ, Raghuram, Balaji, sanjay, Khetmalas.

Prabhu and Denduluri for their company.

Acknowledgements are also due to a host of others for helping lIIe directly or indirectly. I alii highly thankful to Drs. John Reed and Gehring for communicating the resuts prior to publication .

Spec!al thanks are due to Prof. M.S. ShaHa, Prof. a, Raghavan and Dr. Ramachandran for their incessant encouragement.

My grateful thanks are due to Ms.sylvia Ficken and Dr.

Varada P. Rao for their cooperation andpain~takingpatience in making the graphs and cartoons. The technical assistance provided by Masuma Rahimtulla, Ge Jin and Lynne Ryan is gratefully acknowledged. special thanks to Lynne Ryan for proof-reading the thesi8.

Finally, my salutationa to Illy parents and deep appreciation for my sister, brother-in-law and brother, for their care, affection and extreme forbearance. The financial assIstance providedby School of Graduate stUdies and FaCUlty of Medicine,Memorial Universityof Newfoundland was of great help to me and is highly acknowledged.

TABU

or

COlI';lUI'l'l

ABSTRAC'l' • • • • ACItNOWLBOOBK!ml TABLE01' CON'1'EN'l'B LIST01'FIOURES • LIST OF ABBREVIAT!ONS CHAPTER1 IN'1'RODOC'I'ION

iv vi

•ix xii

1.1 GENERAL IHTRODUC'l'IOIl • • • • • • • • • • • • • • 1 1.2 JCV... • • • • • • • • • • • • • • • • • • • • • 6 1.2.1 Genome. •• • • ••• •• • •• • ••• 6 1.2.2 Regulatory region •• • . • • • • ••• 11 1.2.3 IDlnI.-acting factors binding promoter-

enhancer sequences• • •• • . • • • . • 13 1.2.4 Reatricted growth in glial cell cultures 18

1. 2.5 LatentJCVinfection • • 22

1.3 EUJtARYO'I'IC 'l'RANSCR.lP'l'IOIl ••• •. . . . • •. . 28 1.3.1 Promoters and Enhancers • ••• • ••• 28 1.3.2 RNApolymerase II •• •• . •• • • . . 29 1.3.3 General transcription factors • • • •• 30 1.J. 4 sequence-specific transc.riptionfactors J3 1.3.5 Mechanism of transcriptional activation

by trar.scriptionfactors • • •• • •. . J4 1.3 .5.1 Iuteraction withGTFs • • • • 35 1.3.5.2 Interaction with TAFs/

Coactivators • • • • • • •• 36 1.3.5.3 Interaction with adaptors and

accessory proteins • • • . 39 1.3.6 Transcriptional regulationof tissue-

specificgene expression. • • 41 1.'" OLIAL CELL-BPECIJ'IC EJ:PREBSIOIIorJctV • 42

1.5 HYPOTHESI. aND SPECIFICAIXB 52

1.5.1 Hypothesia. •• • 52

1.5.2 specific aim8 • . 5]

CHAPTER2 MATERIALSAm)kBTBOD8

'.1

Kat.rial • • . . • • • • • • 54

2.2 lIethods. . .. . . . 2.2.1 eDNA sequence analysis • • •• • 2.2.2 Deletionanalysisof~cDNA

vi

55 55 56

2.2 .3 Cell culture, differentiation of EC cells and transfeetion• • • • •• 56 2.2.4 Chloramphenicol acetyl transferase

(CAT)assay • • ••• ••• • • • 60 2.2.5 Plas.ids. • ••. • . • . •• . 61 2.2.6 Nuclear extract preparation • • 62 2.2.7 Whole cell extract preparation . 63 2.2.8 lD....Y..1tDltranslation • • . . • . 64 2.2.9 Productionat BAG-2 in bacteria 64 2.2.10Gel mobility shift assay. • •• 65 2.2.11southwoliltern blot analysia • . • 65 2.2.12Far-Western blot analysis • • • 66 2.2.13RNA preparation and analysis. • • 67 2.2.13 .1Northern hlot analysis . 67 2.2.13.2 Primer extension analysis 68

2.2 .14~transcription assay. 68

2.2.15I1lllllunofluorescenee assays 69 CHAPTER 3 RESULTS

3.1 Sequenceanalysis of1a9=.2.cDNA • 70 3.2 Structural features of the deduced amino acid

sequence •. • . • ••• ••• •• • • 73 3.3 BAG-2 protein subcellular distribution . . 79 3.3.1 I1lllllunotluorescence analysis • • • • 79 3.4 Tissue-specific distribution of BAG-2 • •• 83

3.4.1 Northern blot analysis of totalRNA frolll P19, HeLa and UB7 MG cells • • 83 3.4.2 5^1-endanalysis of RNA from unique

cell types• ••• •• •. • . • •• 86

3.5 .In....Yi:tl:2bindinq studies of BAG-2 . . • • • 89

3.5.1 ~-translationof~eDNA. 89 3.5 .2 Mobility shift assays of BAG-2

protein probed with JCVNF-1 II/III oligonucleotide • • . • •• . • • •• 92 3.5.3 Mobility shift assays of BAG-2 protein

probed with JCV enhancer••. • •••, 95 3.5 .4 Southwestern blot analysis• •• • • •• 98

3.5.4.1 Southwestern blot analysis of nuclear extracts from P19 glial, P19 Illuscle, HeLa and U87 MG cells . • • •• • •99 3.5.4.2 Southwestern blot analysisof

~-translatedBAG-2 98 3.6 Transcriptional activity of BAG-2 . • . . . • • 103 3.6.1 BAG-2 trannctivation of NT JCV promoters 103 3.6.2 BAG-2 transactivation requirement of

vii

intactJCVNF-l1II0titS• • ••••• . . 106 3.6.3 BAG-2transa ctivation ofJCVpromot a rs

in BAG-2-de ficie nt U87MGca lls • .•• 109 3.6.4 Over&xpr esaion of BAG-2 exhibits

squelching. . •. • •. .. ••• .. . 109 3.6. 5 Bacterially producedBAG-2 stim ul a tio n

of1n....'l1t111transcription froll JCVe pr omote r innon-glial He La cells 1U 3.7 Domainsof BAG- 2requ ired for DNA-.,inding and

tr an s activatio n •.. • • . •••• . . •. . 118 3.7 .1 Delet ion ana l ys isofW=.Z.t:DNA • •. 118 3.7.2 BAG- 2-S pe c i f i c c-termi nuBi8eBsent ial

for trllnsactivlltion •••••• • • 118 3. 7.3 DNA-bi ndingactivityof thece nt ral

coiled-coil regionof BAG-2 •. 123

3.8 BAG-2 int era c t s withBcI-2 126

3.9 Effe ctof BAG-2on murinep53promote r •. 129 3.9.1 Tra ns criptionalrepress ion of lIlouse

p53 pr omote rby BAG-2 129

CHAPTER 4 DISCOB8IOJ!. • • • CHAPTER5 rOTORSDIRECTIONS CHAPTERIi REFERENCES

vi ii

133 151 157

Fiqure 1.1 J'iqllre 1.2

Fiqure2. 1 Fiqure3.1

Figure3.2

Figure3.3

riqure 3.41

I'iqur.3.5

Piqure3.6

Figure3.7

Figure3.8

I'iqur.3. t I'iqure:;:. 10

LIST OJ' rlaUDS

ci rcu lar mapof theJCVgeno me .••• •• •• •••• • • e JCV regulato ry regionindicatingill- elements and their ttAni.-actingfa c t o r s •.• •19 Deletion ana l ys is of~eDNA..•••.•••.••57 Nuc leotide sequ e nceanddeduced aminoacid se qu e nc e ofmousela9-:.leDNA••.. .•••• ••• .• •71 ProteinsequencehomologybetweenBAG-1 and BAG-2..•• •.• •...•••• • •• ••••••.• •... •. ••74 structuraldetails ofh.a9.=ZeDNA and BAG-2 protein••• • • ••.•••.•••••.•.••••.••• ••77 Sequence and function ofBAG-2 nuclear localizationsignal•• •• ••••••• .• .••• ..• ••••80 Northern blot analysisofbAsl=2.RNA fro m different cell types•••.••••• • • •.•.•• •84 Primer extendon analysis of total RNA froDl Pl9 glial and DO cells 87 SDS-PA GEanalysis of .In...Yi..tJ:-synthesized BAG- 2 pr o t e i n ••••••.••.••••• .••••••••••• • ••90 Specific~bindingof BAG-2 to NF-I II/IIIoligonucleotide in gel mobility shift assays•••• •.• •• ••• • • •••••• • • • • • •• ••••93

~bi nd i ng of BAG-2 to JCV enhancer•.96 Southwestern blot analysis of nuclear ex t rac t s probed with JCV NF-l II/III

1x

Fiqure 3.11

Fiqure 3.12

Fiqure3.13

Piqure3.U

Figure 3.15

Figure 3.16

Piqure 3.17

Fi gu r e 3.18

Figure 3.19

Figure 3.20

Figure 3.21

01 igonucleotide••• • • ••• •••.•• •..•.. •• ••• •. • 99 Southwesternblotanalysisof.i.n...Yi..tx.2- translatedBAG-2 proteinprobed withJCV NF-l II/IIIoligonucleotide..•• •••.••• • ••• 101 TransactivationotJCVpromotersby BAG-Z••• •.• •• •••••••• • •••. .••••.••..••... . 104 Transactivation ofJr:v prollloters by BAG-Z requirementot intactNF-l

II /IIImotifs..• •.... ••..• • •••. .•... . ..• ••107 TransactivationofJCVl byBAG-2

in US7^MGcells•.••••.•••. . .••.. .•• •• •.. . .110 Trans a cti vationand squelching of_JCVEby BAG-ZinPIg qUalcells •.. • ••... .•...112 Production of BAG-2in bacteriaand transcriptional function of purified BAG-2for_JCVr••• •••• ••• •• • •••• •••••• •••••116 SOS-PAGEanalysisof ~-synthesiz ed truncated RAG-2 proteins •. ..•• . .• .... ... . .119 Domains of BAG-2 required for

transactivation••• •• •• • •• • ••••.••.•. . .•...121 I.n....Y.1..tI.binding toHF-lII/III

oligonucl eotideof truncated BAG-2 proteinsin gel mobility shift assa y••••••124 I.n....Y.1..tI.interaction between BAG-2 and Bel-2••• ••••• •• ••••• ••••••••••••• ••• •••• • •121 Effect of BAG-2 on murine p53 promoter.. . .130

J'lqure 4.1 Moda l forrequlationof a xp r e . sion of JCV by BAG-2•• ••• •••• • ••••••••• • • • ••••••• •••• •145

xi

A

AdHLP aHEM AIDS AP-l AT' AT' BAG-l Bcl-2 OKV bp ORL

CAT CCG-1 eDNA CI '

CMV CNS c.p . e cpo CREB

LISTorUBUVIATIO.,.

Adenine

•• ino acid poaition Adenovirus aajar late promoter Alpha modification ofEag l e 's mediullI Acquired immunodeficiencysyndrome Activatorprotein-l

Activatingtran s cri pt i o n fac tor Adenosinetriph o sp h a t e sea-a aasociatedatha n oqe ne- l B-cell lYlllphoma/ leUka Qmlll-2 BKvi ru..

Base pair.

Bethesda ResearchLaboratories Cytosine

Chloramphenicolacetyl tra nsfera s e Cell cycle98no-1

complementary Deoxyribonucleicacid calfint e s ti na lalkalinephosphatase Cyto megalov Irus

Centralnervoussystem cytopathIce!feet ,"cunta per mInute

CyclicAMPresponse .hmant binding proteIn

xli

C-terminus CTD CTF CTL DAB complex DB complex OMSO DHA DNaseI l1l'T ElA E1B EBV

sc EDTA EGTA

G GBPi GFAP CTF HEK HEPES

HIV HNF-l

carboxy-terminus carboxyterminaldomai n CCAAT transcription factor cytotoxic T lymphocyte TFIID-TFIIA-TFIIB complex TFIID-TFIIB complex Dimethylsulfoxide Deoxyribonucleic acid DeoxyribonucleaseJ:

Dithiothreitol

Adenovirus early regien 1 A Adenovirusearly regien.1 B Epstein-Barr virus Embryonal carcinomacells Ethylene diamine tetracetic acid Ethylene qlycol-bis·(p -aminoethyl ether) N,N'-tetraceticacid Guanine

GC-rich binding protein-induced Glial Fibrillary Acidic Protein General transcription factor Human eJlbryonal kidneycells N-(2-hydroxy ethyl) perzine-NI-(2- ethanesulfonic acid)

HUlDan i1lllllunodeficiency vi ru s HepatOClyte nuclear factor-l

xiii

HPV IE Ig IKB Inr JCV JCV, JCV, kDa LeE LCP-l LTR Mad-l MAG MBP MMTV

mRNA MS HE NF-l NF-xB

NP-40 nt N-terminus oee-r

ORF

HWII3n papilloma virus Immediate early Immunoqlobulln Inhibitor ItB Initiator element JCvirus

JCV early promoter-enhancer JCV lat. promoter-enhancer Kilodalton

Lytic control _lemont LCE bindinq protein-l Long terminal repeat JCV Madison strain-l Myelin Associated Glycoprotein Myelin basic protein Mouse mammary tumor virus Messenger ribonucleicacid Multiple sclerosis Nuclear extract Nuclearfactor-l Nuclear factor-JeD Nonidet P-40 Nucleotide position Amino-tenalnus Octamer binding protein-l Open reading frame

xlv

PAGE PBS PeD PCR PHFG PLP prc PML

PHSP Pol POU-domain Pur a RA RNA RNAP II RNasin rpm RSV SRF 50s SV40

t-antigen T-antigen TAF

Polyacrylalllide gel el.ectrophores!s Phosphate buffered saline Progralllllled cell death Polyaerase cha!n reaction Primary hUlllan tetal glial cells Proteo 1 ipid protein Pre initiation complex

p r o g r e s s i v e m u l t i f o c a l leukoencephalopathy

Phenylmothylsulfonylfluoride Polymerase

Pit, OCt andUNc-domain Purina-rich binding protein a:

Retinoic acid Ribonucleic acid RNApolymerase II Ribonuclease inhibitor Revolutions per minute Rous sarcoma virus Serull Response Factor Sodium dodecyl sulfate sildan virus 40 Thymine Small tumor antigen Large tumor antigen TBP a1180ciated factor

tr anac ripti o n TAR

TOP TFII

TOF"

TLC TNFQ Tst-1

UAS UD USA USF WeE YB~ l

Tat activat i ng- reg ion 'rATAbox binding pr ot e i n Tra nscri pt ion factorII:

Tran8fo~inqqrowt h factorfJ Thi n layer chrollla.t oq:raphy Tumorn"cr asisfa ctorQ

Testes-spec if ic factor-l

upstreamacti vating sequence Undifferentiated

ups t ream sti mulatory ac tivi ty Upst reamsti lllulator yfa ctor Wholecel l extr act Y·boxbi ndinqprote i n-1

xvi

1.1 GeneralIntroduotion

'rhe overall objective of bioloqical research is to understand the basicproce ssesof life. This knowledgecan then be ap p lied in learning new s trategies fort"~rrecting anomalies in the physioloqical mi l ieu andto control the invasion by microbes, in an effort to preserve homeostasis .

"Life"can be derinedas a complexmosaic of organ, tissue, cellular and subcellular orqanizatiol' endowed with the propensity of qenetic continu itythrough self-replication.

Therefore, the study of viruseslios close to the heart of biology,because the study of virusesnotonly encompasses the studyof life processes but alsoent w i nes pathological processes.

studying vi ruses tounde r s t a n d the complex process or cell regulationis notan unfami liarconcept. Si mi an virus40 (SV40) isan excellentmodelforst u d yi ng DNAre p lica t i on and tr anscr i pt i o n. Also, the human ne u r ot r op i c JC vi ru s (JCV) represents a 900d model to understand brain-specific qen e expression. JC'/ isa member of the polyomaviruses of the fully,papovaviridae (Shah, 1990). JCVis the etioloqical aqent tor the sUbacute fatal demyelinatinqdisease called progressive multifocal leukoencephalopathy (PML).The fi r s t description of PKL wa s a8 a "unique and non-class i fiable

process", ....ithdiscrete areas ofdemy elina t ion andbiz a r r e en 1 a rqe d astrocytes in two patients (Hallervord en. 19 3 0 ).

Later, PMLwas described. as a disti nctclinica l entity in chronic lymphocytic leukaemialind Hodqkln's IY1llphoma. pati e n ts on the bas i s of uni quepa t holog i calfe a tu r e s , like extensi ve demyeli.nation , abnormal oli qodendrogl ial nuclei andqiant astrocytes(Astrom

n

41.., 1958).

A vi ral etiology for PML was 8u~pected ....hen inclusion bodies det ect ed in the nucl ei of infected oligodendr ocyte s (ca vanaugh & A1,., 1959). Mor p h ol og i cal evidence to furth er sup p o r t this obs erv ation WA S first obtained....hen part icl e s resemblingpol yomavi r uswer e foundin the nuclei of ol1godend r o cy t e s with inclu sion bodies by el ect r o nmi c r oscopy (ZURheinan d Chou, 1965 ;ZURhe in,1967). Subs e que ntlY, apol y o mavi r uswasisolatedfrolll aPMLpatient an d namedJCV af t er theinitialsotthe patient(Padgett I..t Al,-, 1971 ). Atthe same time, anot h e r vi rus with similar characteri sticsasJCVwas isolatedfro1ll theurineofarenal transplant patient, and named BK virus (BIN ) a.fter the initials ofthe patient (Gardner §tll.,1971).Ut h o uqh both we re assoc iatedwith immunosuppre s si onandfo u nd inurine, BKV wa s never- f"l;,n::1in thebra i nof PMLpat i e nts. Furthermore, BKV was detec t edmore frequent1ythanJCl/inthe urine (Arthur

n

A.l.-, 198.5,19881Berger& Al., 1987). Although an SV40-1ike ag e n t Was also isolated from the PML pa t ient, thQr01.ot this aqent inPMLpatho1oqywas unclear (Wei ner .t..t4.l., 1972) .

Howeve r. overwhelming evidence now indicate sthatJCVis the actual etiological agentof PML (Stoner and Ryschkewitsch, 1991:! izurun Al., 1993).

JC viru s is a ubiqu ito us human symbi ote and se r o epi d e lllio l oqy studies suggestthat theviru s is world wide in distribution, with an overall preval en ceinadu l t s of69' (Wa l ker and Padgett, 1983).The spread ofJC'Iis postulatedto be by oral or respiratory means (Shah, 1990). Soon after enteringthe body ,JCV remainslatent in theki d ne y (Arthur.it.

1.1 . ,1988). Thisconc l u s ion was basedon obse rvationsthatJCV DNA"dSdetectedin the kidneysof normalindividuals by ONA hybridizationme t hod s (Ches te rsfir.Al.,1983 : Gr innel lJ.tSIl., 1983) , sU9ges t i ng that the ki dn e y is animpor t a nt si t e of vi r a l persistence . Persis tence rep rese nts a latent non- productive infecti on . Re a c tiva tionofJCVisbelie vedto occ ur underthe cond i ti ona of immunosup pre ss ionobs,,- rv e d in bone mar r owan d renal transplantrecipients , in pregn antwomen, in elderly individual s , in cancer and in AIDS (Coleman§..t.ill. , 1983;HoqanJ.t. Al.,19831Gardner I I Al., 19841Arthur

n

Jll., 1988; Xi t a lltur a §.tAl., 1990). Furthersupport for the above hypothesis comesfrom the observationthat a proteinthat is inducible by cytokines regulated JCVexpre s sion (Raj and Khalili, 1994).

Interestingly ,JCVDNA was identifiedinspleen B cells, bone marrow and brain parenchyma(HOUff.!1t:AJ.., 1988). This suggests thatreactivatedJCV mayente r thecentralnervous

Bystelll (CNS )via infectedB cells and thiB ent ryuy bethe initial event in PML (revi ewed In Cre enl•• , 19 9 0,MajorR Al., 1992). Aha e ma toq e nouf,lIIodeof spread isalso supported bythe na t ure of the mul ti rocal de. yd inatI nq les i o n s and theirlocatio nnearthe grey-wh ite matter junctions,wheretho arteriole s end (Frls que and White , 1992). Also, thea unintegrated form ofJCV DNA wasdetected Inl ivs r, spleen, lymph node, and lung to further co nfirm the hasmatogenous spread(Gr i nne ll

m.

AI. . 1983).

Most cases of PML are believed to be the consequence of reactivated Infection during illlllunos uppression. Hence, the appearance of JCV in the AIDSpopul atIon is rdati valy frequent. AIDSacc ounts fornear ly 55-85\ ofthe recentcases of PML(Kru ppJ.tAl., 198 5;Berq er.nAl., 1987 ) . supporting the notion that JCVis asso ciated with ilUlun o sup pr es s1o n . Furth ermo r e,dire ctIn te ract i onof a human Immunode fi cienc y vIrus (HI V) .t.1:A.m- requlato ry pr o tein Tat wi th the JCV roqulatory reqion wa s demon s tra ted and thIs int era c tion requlatedthe tra nscriptionofJCV(Tada.c:.t.al., 1990). Recent evidenc eindIcate s that 4-5\of patientswithAIDSdevo!lopPML (BergerJlt. J..l., 1987; Levy ~A1,. , 1988: Quinlivan.tltAl., 1992) . Thus, this formerly rare disease, once regarded as clinical curiosity ,has lately become remarkably common.PHL is the ca u s e ot' death of 2-4\ ot AIDS patient. in North America (Markovitztt.A.l.,19931 .

An assoc 1ationof PML was also observed in lymphatic

leukaemia and HOdgkinIs lympho ma (AstroID.It .sU., 1958).

Importantly, PML, the once rare diseas e, is noW' more common because of widespreaduse of i1lllllunosupprsssive chemotherapyin organ transp1!lnt recipients(GardnerItAl..,198 4 ; Arthur.!It.

A,l.,1988) . R8cently ,JCV was a180 implicatedin the etiology of multiple sclerosil (MS) (stoner, 1991).

Studies on the neurooncoganicity of JCV are sc a nty . However, the existing reports demonstrated that the intracerebral inoculationof hamstersIo'!thJCV resulted in medulloblastomas, astrocyt omas , g1 iobl astol'llas, peripheral neuroblastomas , me ning i om as andre t i nobl a s t oma s (Walker II Al., 1973; zu Rhein, 1983).A similarinoc u latio n of owl and squirrel monkey.Jresulted in astrocytomas and neuroblastomas (LondonItill.,1978, 1983).Thus, JCV,unlike SKV and SV40, is the only pol y o mavi r u s that induces tumors in non-human primates.JCVwas shown to induce gliomasin PML patients (Sima n AJ..,1983) .

Transgenic mice harboring the JCV early region were developed to study virally-induced tumors and PHL. Some animalsdeveloped adr enal neuroblastomaswhich subsequently metastasizedto the brainand digestive system(Small~ill., 1986b) and someanbals suf f e r e d from dysmyelination of the eNS but not of the pBripheralnervous system (PNS) (Small§.t.

Al., 1986a; Haas J.t. Al., 1994). Further studies have dellonstrated that JCVlarCjlB tUJllor (T) antigen was expressed mainly inoliCjlodendrocytes of these transgenic mice (Trapp.It.

Al., 1988).The same study also observed the down-regulation of myelin-specific genes, like myelin basic protein (MBP) , proteolipid protein (PLPl and myelin associated glycoprotoain (MAG). Since T-antigen expression was observed in the cells that produce myelin, it was thought that T-antiqen altered the expression of Ilyelin-specific genes and the maturation or oligodendrocytes (TrappJltAl., 1988). However,the mechanism of dysmyelination remains unclear.

As a group, polyomaviruses served as Illodels for addressing various biological conundrums, ranging frOlll cancer to the regulation of gene expression. continued study of the lllolecular bioloqy of JCV may eventually open new vistas in JCV1s PML pathoqenicity and neurooncogenicity.Specifically, this knowledge may help in finding a cure or prevention strategy for PML.Generally, the information may shed light on the brain disorders that devastatlll the lives of so lIIillion Americans, with an economic impact of $400billion (Scientific American, 1994).

1.2 JCV 1.2.1 a.DOIII.

The genome of JCV is a covalently closed circular, double-stranded DNA molecule consisting of5,130bp (Fig.1.1;

Frisque S!t .A..l.., 1984). The viral genome codes for two regulatory proteins, T and small tumor (t) antigens; three viral structural proteins YPl, YP2, VP3; and a 7.9 kDa protein

called the agnoprotein since its function is not known.

Sequence analysis revealed that the JCV genome was organized as three functional regIon!:'.The early region is proximal to the oriqin of replication, the late regIon Is distal to the origin of replication and the non-coding or regulatory region is located between the early and late regions. The regUlatory region contains the promoter-enhancer signals that important for transcription and the core elements for DNA replication.

The early region codes for two proteins, T- and t- antigens. A sinqle cpen reading frame (ORF) codes for both T- and t- antigens but an alternative splicing event generates two mRNA species. Therefore, these two proteins have same N- terminal end but different c-terminalenda. The JCV T-antlgen has not been studied in detail. Nevertheless, the existing data suggest that it is essential for DNA replication(Lynch and Frisque, 1991), vi r a l - i nd uc e d cellular transformation (Frlsque IIA,l., 1980: Bollagl l l l . ,1989; Haggerty§.t.ill., 1989) and ~-regulatlonof JCV late promoter. The latter determines the transition of transcription from the early to the late stage (Lashagari

n

Al., 1989; Nakshatrl

n

Al,., 1990a; Tada

n

ill., 1990). Despite 92\ hOlllology between JCV and SV40 t-antiqens, JCV t-antigen is not effective in enhancing transforJllation, especially under conditions in which the concentration of T-antigenislimiting (Haggerty

n

li., 19(19).

Fig. 1.1. Circular map of theJC!Vgene•••

The late and early regions are shown in clockwise and anticlockwise directions,respectively. The numbers indicate the nucleotide positions. The locations of core origin of replIcationand regulatory reqion are indicated at the bottom.

The reqionsA and B indicatethe two 98 bp tandem repeats of the requlatory region.The large open arrows inside the circle indicate the open reading frames of larq8 and ama1l tumor antigons, respsctively. Ths wavy line indicates the intron sequence in the large T-antigen coding sequence. The open ar-rowsoutsidethe circle indicatethe open reading framesof viral st ru c tu r a l proteinsand of agnoprotein. The figure is not drawntoscale.

Vl'1

10 The late region codes fo r the Ilgnoproteinand three viral structural proteins, namelyVPl,VP2 and VPJ.The slIlallest ORF is at the 5I end of the late coding region and encodes the agnoprotein, whIch ma y be involved in the vi r al capsid assembly (Hou-Janq

n

Al., 1987)and has DNA bindingca p a c i t y (Jay.I..t.l l ••19B1).The larqest ORrisat the]' end and codes for VPl. the major vi r i on structural protein. Sequences between the 5' and 3I ORrs of agnoprotein and VPl enc ode VP2 andVP3 (see rig.1.1; Frisque

n

ill.,1984).JCVexhIbits an extens i ve70\se quenc ehomology with SV40 and 75\wIth human BKvirus (BKV) and tbe homology isweake stin thenon c cd Inq region (Osbo rn

n

.ill.,19741Howley

n y .,

1976). Unlike BKV and SV40, the JCVMad-l strain isstrictly neurotropic and replicates efficiently only in cultures which are rich in sponqioblasts or astrocytes (PadgettII Al., 1977 ;Major and veeenee,1989).

Most of the JCV isolates were derived from brain tissues, ki d ne ys and urine of PML patients and a few were from urineof transplant recipients or normal indlvfdueLa, The first isolation of JCV was reportedfrom Madison,Wisconsin after cultivationof JCV inprimaryhuman fetal qlial cells (PHFG) (Padgett

n

^{Al,.I 1971).}It was referred to as the Mad-1strain and was designated as the"pr o t ot ype " , since no rearrangements were observed in the viral genome during continuous passage in culture. Mad-l has two 98 bp tand•• repeat. of regUlatory region with a uniquely duplicated TATA box (Martin

n

41..I

11 1985). Subsequent isolates were designated Mad-2, Mad-4 etc.

The genomes of JCV from urine of non-PML patients revealed a 23 bp insertion In the regulatory regionwhich is absent in the Mad-l strain (MartinJlt J.l., 19S3). A 66 bp insertionwas also found in these genom8s between nucleotide position (nt) 80and 98 of theta nd e m repeats (Yoga

n

Al .• 1990).Therefore,the JCV genome with sequence rearrangements containing23bp and 66 bp insertions was suggested to be the llarchetype" genome. Based on these reSUlts, it was suggested that the hypervariability of the JCV genome may be a process of adaption of JCV for replication in the brain (Loeber and OOrries,1988).

1.2 .2 Requ latory reqion

ThoughJCV exists in kidneys, replication and expression on ly efficient in the brain. This rf'lstricted tissue- specificity was attributed to the promoter-enhancerregion, becausethe greatest degree of divergence of DNA orqanization was in the regulatory region of JCV, compared to other papovaviruses such as BKV and SV40 (K'mney

n

^Al.,19 8 4 ). JCV promoter-enhancersignals were identified, based on sequence comparisons with the BKVan d SV40 regUlatory regions (Frisque

~Al., 1984). Sequences between nucleotides 5,014 to 276 representth e non-coding region of JCV.The regulatory region of JCV is bidirectional in function and contains the origin of

12 replication and ill-acting signals for early and late transcription.

The regulatory reqion of JCV has uniquely duplicated TATA boxes. These two TATA boxes were believed to control early and late transcription of the virus. Some contraversy exists reqardinq the location of the 5' terminus of early mRNAs.

Initially, 81 nuclease mappinq was used to show that viral mRNAstart sites forearly gene expression were at nt 122-125 and concluded that the TATA elelllsnt present in the repeat B, distal to origin of replication functioned aa a component of the early promoter (Kenney

n.al.,

1986).Later, transcription initiated at nt 5,112 and 5,082 from the TATA box WAS attributed by primer extension analysis to the repeat A, proximal to the replication origin but not in repeat B (Khali11 .!i!.tA.l., 1987). A more recent report resolved the discrepancy by showing that the early and late genes were initiated at nt 5,115-5,125 and 200-203, respectively(Daniel and Frisque, 1993). Taken together at face value, these resul ts suggest that both TATA boxes are utll hQd for the early and late gene transcription of JCV.

In general, viral enhancers are modular collections of a myriad of ill-acting elements that are bound by a variety of cellular transcription fac~ors to enhance and/or repress transcription. Considerable attention was focussed on theslIiiI elements,because they affect gene expression in a tisSUQ- lind species-specific fashion.

lJ 1.2.3 UAILI.-aotlll.g' faCltore bill.diJl.9 promoter-enhancer

••queue ••

Cell culture studies clarified that the glial 0811- specific expression of JCV was due to a number of intracellular factors that bind to the promoter-enhancar sequencesofJCV (Frisque.I..t.ill., 1979). subs e qu e nt studies delineated the ill-acting DNA signals andtheircognatetnulA- ac t ing- protein factors.

To identity thecellular proteins that interactwith JCV enhancer sequence s , UVcrosslinki nq and qel mobilitysh ift as s a y s wereemploy e dwith HeLa and human fetalbrain nuclear extrar.:ts and complementaryoligonucleotidesthat span the 98 bp tandemrepeats ot JCV. oligonucleotides spann i ng the nt 134-160of98 bp repeat interacted with proteins of'S and 85 kDa trom fetal brain and HeLa cells, respectively. Ol i go nuc l e ot i de s havinqhomoloqy to the 5I and 3I terminal regions of the JCV 98 bp repeatsequenc e interacted with82 and 78-30 kDa proteins (believedto be a TATA-li}ce factor) tram both extracts. A 230 kDa protein which was present in both extracts interacted withse v e r a l regulatory sequ e n c es within the 98 bp repeat(Kha lili§..t

u .,

1988) .

FurtherI three protected regions were observed using DNaae I footprintingass ay awIthlQouse brain nuclear extractsI two were within and one wa s outside the repeats. The two protected sequenclilli within the repeats were shown to be homologous to nucleartactor-l(NF- l ) bindingsites (nt 35-58

14 and 133-156) and thesequenc e s outside the repeat cnt208-229) were shown to be a paeudo NF- l motif (Tamura.It.a.l •• 1988).

Later studies employing mobility shift assays identified binding activitiesin nuclear extracts frolQ A172 human glioma, PHrG and HeLa eel ta. No bindinq was detected withextract.

fromHEKcells.Furthermore, DNase I footprintingstudies were done with the same extracts protected the NF-l sequences similar to those, identified in theabo ve described studies.

Protection of these NF-l sequences was competed by oligonucleotides homologous to the adenovirus replication origin, which contains NF-l bindingsi t e s (Amemlya ~Ai••

19851). Also, aSacI mot i f was reported to overlapeach NF-l site present in the repeats. Howe ve r, the nature of the proteins that bind to thismo ti f was not clarified (Tamura.e.t Al., 1990b). Later, it was shown that this reqion interacted with the cyclicAMPresponse element (CRE) blndinq protein

(CREB) (Kumar, 1994).

Studies from our laboratory identified three protected reqions in the JCV regulatory region by DNase I footprintlnq assays with nuclear extracts fr om P19 glial c911111. The two protected regions present inthe repeats werenamed region II and III. The protected region outside the repeatwall named region I.All these regions contained sequences homologous to NF-1 binding sites (Naksh atri

n

Al., 1990a). Sub s e q ue ntl y , the proteins interacting with these NF-1 motifs were characterized in mobility shift assays (Kumar

n

Al.I 1993).

(Mote: The NF-1 motifs in regions II and III are 6 bp inverted palindromic repeats containing the same sequence. Therefore, the JCV NF-1motifisreferred NF-1 II/III mo t if, throughout the text).

A.45 kDa protein from calf brain that interacted with the JCV B domain (equivalent to NF-1 II/III)was purified by DNA- affinity chrolllatoqraphy from calf brain and was shown to specifically stimulate transcription of JCVr (Ahmed

n

Al., 1990a). Subsequently, a eDNA was cloned from a hUllanbrain eDNA library using domain B 218 a probe that codes for a 45 kDa glial factor-l (GF-l), stimulated the transcription of JCVL and to a limited extent JCV[ (Kerr and Khalili, 1991) .

ree-i , a POU-do maintranscript ionfactor family member, was shown to stimulate JCV transcription and DNA replication.

Therefore, Tst-l was suggested to be one of the factors determining the qlial cell-speclticityof JCV (Wegner

n

Al., 1993).A CRE adjacent to the NF-l region II/IIIwas speculated (Amemiya n .il.l., 1992). Work in our laboratory shOWQd that a 43 kDa protein interacted with this putativeCRE and regulated JCVr gene expreAslon (Kumar, 1994).

By DNase I tootprinting with purified proteins, binding of Jun,a proto-oncogene product to the AP-l site near the NF- l region 'Was shown and the binding suggested a direct interaction between Jun and 8F-l proteins bound to theil.- respective DNA sites(.'->i19mi yaAt .il.l., 1991).

AnAGGGAAGGGA pentanucleotide repeat domain, also called

rs the lytic control element (LeE) (Tada and KhaUU, 1992) is present betwoAn the NF-l region II/IIIand TATA box of each repeat and was shown to have a role in transcription (TadaAt.

11.••1991) and replication (Lynch and Fr!sque, 1990,Chang'I.t Al., 1994). The LeE also binds to a50-52 kDa 81n91e strand LeE binding protein (Lef-I) derived fron glial cell extracts (Tada and KhaliU, 1992). Also, a 56-60 kDa double-stranded DNA- binding protein from glial and ncn-eqk La I cell extracts down-regulated JCVL gene expression(Tada

n

Al.,1991: Sharma and Kumar, 1991).

In the presence of the adjacent NF-l and TATA motifs this pentanucleotide repeat was shown to interact with a protein of 70-80 kDa. This suggested that the interactIon facilitated the transcriptional regUlation of JCV₁promoter (Kumar .Il.t.ill., 1994). Interestingly, a cDNA was isolated from a HeLa cell cDNA library, using a 8 region probe comprising the NF-1 and AGGGAAGGGA motifs (nt 134-160). This eDNA encoded a YB-l protein that bound to the pyrimidine-rich opposIte strand (early strand) of the pentanucleotlde repeat In 81ngle- and double- stranded conformations (Kerr.e..t.

ar..

1994). The same stUdy demonstrated that Pura and LCP-lwere the same proteins.

Subsequent work from the same group showed that Pura (a single-stranded DNA binding protein) interacted with the pentanucleotide repeat of the late strand and activated the transcription of JCV1 (Chen .tit. Al .• 1995 a and b).

The HIV type 1 (HIV-l) UADJi-regulatory proteIn tat was

17 shown to be II potent activator ot JCV late gene expression (Chowdhury Jlt A,l.. 1990; Tada J..tAl... 1990). The tat- responsive region was mapped to nt between 5,112-5,130. Tat responsiveness is mediatedbytwo regions within theJCV~on eithtllr .ide of the transcription initiation start site (ChowdhuryAtAl., 1993). Tat was shown to interact directly withtheJCV RNAtranscript containingthe downstream region, analogous to its interaction with HIV TAR (ChowdhuryJ..t.ill., 1992) •

In another study, two T-antigen binding sites were identified which playa role in JCV DNA replication (Chang

n

41.,1994).The T-antigen binding site I was identified at the early side of the origin of replication While the T-antigen binding site II was in the origin of replication containing four T-antigen binding"&1tell.

Another stUdy speculated that a transcription factor abundant in B celb binde the Oct-l lIotif and the NF-1C.B binding sites in the JCY enhancer (Major !It .ill., 1990).

SUbsequent work characterized the NF-KB site present outside the repEu,ts and showed that i t regulates the transcription of the JCVl (Ranganothan and Khalili, 1993).

Domain D on the late side of the JCV regulatory region interacts with 43-50 kDa range proteins from both glial and ReLa cells. This domain activates the early, but not the late promoter of JCV in brain cells (AhlIledrtAl., 1990b).

Taken together, these data suggest that the cell type-

18 specific expressionotJCVIs regulated by both positive and negative regulatory mechanisms. Though JCV 18 present in II vast majority of the population as a latent infectionof the kidney, only under hununocompromlsing conditions doss a productive infection occur. However, i t is not clear how immunosuppressive conditions would reactivate JC virus.

Recently, .. stUdy identified a noval 81 kDa protein which was inducible by interleukin-l,tl. THF-a, y-interforon and TGF-I1.

The 81 kDa protein was bound to a GGA/C-rich sequence in the viral origin of replication and was shown to act ^a8 a transcriptional repressor of JCV~. This novel inducible protein was named GBPi for ~C-rlch region Rindlng Rroteln inducible. When a change in the cytokine profile occurs, usuallyunder iuunocompromlsing conditions, reactivationot JClI infection follo....s (Raj and Khalili, 1994; Atwood

n

Al., 1995).Fig.1.2 summarizas the JCV .l&ll-actingsignals ilnd the putative transcription factors.

1.2.4 a ••trioted qrowth in qUal oell oulture.

JClI is strictly II h\Ullan neurotropic virus. JCV originally isolated troll cultures of PRFG. Enhanced propagation of JCV was observed in these cells at 39° C rather 37° C (Grinnel .e..t.Al., 1982). The PHFG cell popUlation 1&

heterogeneous, and most studies have shown that a culture containing a high proportion of spongioblasts, the precursors of oligodendrocytes, was necessary for optimum gro....th of JCV

I'ig. 1.2. ^J(!I1r.qul.tory ngoion indioatingm-el . . .nt. and their.tnn.I.~aatingfaator••

The numbers indicate the nucleotide positions. The ~­

acting- elements are indicated by the lo....er case letters.The legend belo.... the figure shows the molecular weights and the cha r a c te r isti c s of the tuM-acting factors that interact with the ili-acting elements. A detailed description of these .t.n.n.s.-acting factonisgiven in the text.The diagram is not drawn to scale .

.;co

21 (Pad!]'ettAtAl., 1977). Virions were detected in the nuclei of the sponqloblasts and rarely in the nuclei of the astrocytes , the second most abundant glial cell population (Padgett

n

Al.., 1971). Contraryto these resUlts, another stUdy showed that JCV can be propaqated in astrocyte-enriched cultures

(Major and Vacante, 1989).

The PHFG cells providedthe best sys t e m for propagation of JCV, because continuous passage of JCV in these cells produced no changes in the qenome, transc riptional activity and length of the lytic cycle of the virus. However, the procurementand cultiv ationof these cells are difficult. In addition , the JCV cytopathic effect (c.p .e) is subtle. An alternate permissive cell system for JCV propagation was sought for. To this end, primary human embryonic epithelial cells originating froUl the kidney, lung, liver and intestine, and fibroblasts, were assessed (Padgett and walker, 19761 HiyamuI"afi.Al.,1980;Beckmanf iAl., 1982). Although JCV was adapted to propagat ionin humanembryonickidney (HEK) cells, the DNA found in the virus was defective.Also, the other cell systems were not found to be useful.

Unlike nueen cells, animal cells were absolutely non- permissive for JCV. So, it was thought that the restricted host range of JCV was at the stages of virus adsorption, penetration or uncoating.To test this hypothesis JCV DNA was transfected into HEX, lung and PHFG cells. Viral DNA was recovered only frolllthePHFG cells, not the other cell types

"

(Frisque.tt.Al..,1979).Thus,it appeared that there s t ricte d cell-specificity ot JCVwa s due to Intracellular factors control linqviral expression andrfil p l1c llt i o n (Frlaque.I..t.al.•

1979:Ki l lern 11•• 1983).

1.2.5 Lat.ent;SaiDfea ti oD

Th e ce ll typ e inwhich JCVremaIn s latentafte r primary infe ctionisel us iv e . Initially, itwa s thoug h t JCV couldbe latent in brain ce l l s . Brainsamples fromthe immunos upp ressed and non-immunosuppre s s ed, but not PMLIndlvidual D. failedto sho wanysigns of viral genomes or vIra l pr otei ns , eithe r by PeRorimmunohistochemistry (Hogan.e.tAl •• 1980; Arthur e.t Ai., 1989; relent! .e..t.Ill., 1990: Henson.Il.tAl. ,1991). This suggest s thatJCVremainslatentoutside the CNS and beeDllleli reactivatedinthe eventof blllllunosupp:~'8 8 i onand gains 4CC8ea to theCNS. Thi. reactivation could have two .equalae.ana is the lytic infection of ol i ljlod e nd r oc y t . s and the other is viruria, in wh i ch intactloua vi rua may be shed in urine

(reviewed inFri8 qu e and White, 19 9 2 ).

The idea th at kidneyi. the main.iteofJCV la tencywas bas e don viruria observed during pregnancy (Col emanI..tAl., 1980),ol dage (Kita mu r a

n

ill., 199 0), cance r (Hog a nI..tAl. , 1983),AIDS(Ma r ko....itzr..tAl., 1980) , an d oth er unclasa U ied condit ions (Arthu r and Sh a h , 198 91 . The JCVge no lll8 ....a.

re ;>orte d inlotof nonal kidney. (Ch••te rs!l.tAl.,19 83 ) . By dot blot hybridization , JCVDNAwas alaofound inth..lu ng'S,

2'

Hvers, lymph nod•• andspleens of PML patients (Grinne ll.itt.

Al •• 19 8 3 ).

Interestingly. JCV DNAwas also found inB cells in the bone aarrow,splee nandbrainparenchyma (HOUff liAl ••1988).

The presence of capaid antiqon.and20 0 copies/cellofviral DIfAin 8 cells, suggeststhat viral replicationhad occurred . Ho r e recently, periphe r al blood leucocytes of normal ind ividuals vClre all10 .ho....n to ha r bo r JCV, th U3 provIding stro nq evid en ce lor the persistenc e of Jr:v in these cel ls (DorrIes

n

Al., 1994) . Take n togeth er, the s ere sults suggest that JCVre ma i n s late ntinBcel lsof bone mar row, and",it h th e loss ofth e integ rated immune respons. , suc h as during cellular immunodeficIency, the B cells JDay expand. Such ac t i vated B cella th at aid in the active phase of vI r a l replicatIon lind transcriptIon, may en t e r the CN'S by the vascularsystemandint ec t thebr a in(Au l t and Stoner, 1994) . The other sitesat the JCVlatencyareun kn own at this time.

Ho....eve e,i tisponible thatotherorqa n s besidesBcells and kidneyacy harbor JCV.

Ho....ever, the exact .echani••ot^JCVla t e ncy is notknovn.

sev er al lIlechan i s ms havebeen proposedtoexpl a i n the late ncy exhib ited bymos t of theDNA tWllo r vi ruses (Ahmedand Stevens, 1990 ) .I ....ould liketolimit my discus s ion to theroleat Bcl - 2 protein in viral late nc y , in part iCU l ar , because this discu s sion is morepertinent to th e resu lts obtai n e d inth is stUd y.

"

801-2stands to r »-s;.elllYmphoaa/leuka811l!a-1.ge ne.Bcl-2 first disc overed throuQ'h it. involvelllent in B cell -al1qnancies, whe re the ~.2.qenei • •eved froa it.no raa l chrollosoaa l location into jurtapoalt1on with powerful pro1lotersof theImmunoqlobulinhEla vych aIn(I9H)gene.Thue, deregulation by the transl ocated bs:.l.=.2 qene lead to the overp roduction of ~ 1IlRNAs and their enc oded proteina (Tsujf..1II0ton .41•• 1985; reviewedin Croce , 1987 : Nowell and Croce, 1987). BcI-2was first shownto prolongcel l survival in i1llllla t ure pre-B-c&11s , where stable tr a ns f er of ~ permitted pro longed cell survival even in the absenc e of lymphoklnes(VauxJl.t. Al••15I8B). Sincelymphokin88 pr ol onq the cellsurvival bypreventlnqapoptoshor programmed cell death (PCD) (Tu shl n sJci A.t.Al•• 1982 =Williallls J..t .1.1..,1990). thue results sugg estthat eer-a Is capableof blocking PCD, and subseque ntly thi s abil ity tODally delllOnstrated (Hockenbuyn 11., 1990) . The pr otei nenc oded by theQs:l:1 gene contai n s no .eque nc e moti fs tha t mig ht sugge st a biochemi ca lfunctionfor acr-z, ThelIlos t notewor thy feature 18 astre t c hot highly conserved 19 hydrophobic amino acids at the c-eerednue, wh ich serve to anchor BcI- 2 to lIIembr a nu (Cazal s -HatemI..t.Al..,1992,Sat o.lt.A.l., 1994) . Ho wever , the mec han islllotactionbywh i chthe BcI-2 prot e inprote ct.celt.

trom apop toticcel ldeath18 no t very clilar. Por oxa lllple , in ce lls protected by Bcl-2 frO ID apo pt oa i a , none of the chara c teristicIlOrpholoqlcdchang'e.,Buchas , cdlshrinkage,

25 chromatin conde nsation and nuclear fragme ntat i on, observed. This indicates that act-a bloc ks a final cOln1llon pathway leadingto apoptosis. Th e participantsin this pathway areno t known. However, one such lIediatorco u l dbe the tumor suppressor p53 based on the observationthatBcl- 2protein interferes withthe p53- indlJcedapoptosiswithout illlpa i ri ng the p53-inducedGl/ Sar r e s t (WangJ..tl i.,19931Ryan.itAl., 1994 ; Selvakumaran

n

J.l. •• 1994). p53 was sho wn to in d uc e apoptosisunderavar i e t y of condi tions (reviewedinSachs and Lotem, 199 31Hoff manandLie berman n, 1994;Lieberman n.itAl., 199 5) and inp53-de f i cienttumorcelllines uponexpressionof wild-typep5 3(Yanish- Rouae h .ll..ti\l.,1991:Shaw .ll..tAJ..,1992 ) . It is notcl e a r hoW' pS3 induces apoptos is . Howeve r , p53 was showntonega t i Vely regUlatethe~gene through III ill- acting" negativeresponse element in its 5^1-untranalatedreqion (Miyashita

n

ill.,1994b). Aleo, p53wasshown to incr e asethe levelot1lnmRNA(Miyashita.!lkAl., 199411I ; set va x a earan

n

.Al., 1994).Baxisa Bcl-2-reiated98ne productthatwa sshown to accelerate se nby torming heterodimers wi thBcI-2, thus inactivating BcI-2 (OltaviitAl., 1993) .Rec e ntly, p53 wa s shown to increase1ulxmRNAlevelsbytrans act i vating the W promoter (Miyashita andReed, 199 5).

Ho we ve r, pS3 is not the wh o lest o ry of ap opt o s is, since p53mediatesap optosis for only a particular sst of signals (Lows.At.Al., 1993). ser-a wa", also shown to inhibit pSJ- inde pe nde nt apoptosis (Se n t man .i..t41., 199 1; Miyas hita and

as Reed, 1992 and 1993). By exploiting the yeast two-hybrid system. (Fields and SonCJ. 1989) to identify interactIng proteins that mediate Bcl-2^{l s}fu.nctions, i t was shown recently that Bcl-2 inhIbits apoptosisthrough a novel protein called 12.cl-2 Associated athanogene 1 (BAG-I) (Takayaman Al., 1995).

Interestingly, BAG-l also does not contaIn any protein motifs that would indicat. II biochemical function. Furthel'1llore, it showed no homology to Bcl-2or 8c1-2-related proteinsand any relationships between BAG-l and p53 are not known at this time.However, gene transfer experiments IIhowed that BAG-l can prsvent PeD (Takayama

n

A.1.••1995).

Latentand/o r persistent infections are II part atthe lifestyles ofma n y virus e s, as the ability toma i n tai n a long- term relationship with the hosts is vital for their survival.

Avarietyof viral lIIechanismshaveevctvea to prolongthelife span of host cellsone such mechanism is by exploiting the anti-apoptotic properties ofacr-a,An example i8 Epstein-Barr virus (EBV) , a DNAtumorvi rus which is ubiquitous and is associated with tumors of B celh and epithelial cella (Magrath, 1990; Shibata and Weiss, 19921. In B lymphocytes, the infection is non-productive or latent, whereas in epithelial cells, the infection is productive (Miller, 1990).

The ilUlediate early protein ofEBV, BHRF-l, shows similarity to acr-z, Bcl-xand Bax (Bc1-2homo1ogs). Liket!..c.l.=2., BHRF-l acts as a "cell survival gene" (Cleary At Al., 1986).

Furth8rtllOra, cells that are negative for EBV, but IlO'lCpress

27 BHRF-l, showed strong resistance to apoptosis induced by growth factor deprivation (Henderson ~ l i., 1993). Furthermore, the EBV latent EBNA-4 gene was shown to increase the expression of BCl-2 (Silins llnd scul l e y, 1995). The importanceof these finding's18 that the virus es can harbor Bcl-2 homologswhIch are capabl e of inhibitingPeD. When taken togetherwi th theobservatIonsthat Bcl-2ca n bloc kordelay apoptotic death of vi rus- inf ected ce l l s and converta lytic viralin f ec tio n intoa non-lyt ic persistent infec t ion(Alnemri At.al., 1992;Levi ne.ittAl. ,1993).these result8suggest that virally-encoded Bcl-2 homologscontrIbute to viral lat encyor allow for persIstentinfectIonsin theabsence of cell lysis,

Analoqous toEBV, the African swinefever virusis a DNA virus whichremains persistent forIllong time in peripheral blood monocyte/lIIacrophages of in f ec t e d pigs (Vinuela, 1987) . TheIMW5-HLprotein of African swi ne fever virus also shows significant si mi l a ri t y to acr- a , sugg esting arole for the viral protein in the su p press i on of apoptosis in vi ru8~

infectedcells (Neilanrtli., 1993).Anothe r vir a l pro t e i n, thep35baculovirusgeneproduct ,hasthe abil.ityto bl ockthe host apoptosls response (ClellJiltAl. , 1991).

More recently , the adenovirusElB 19Kproteinwas also shown to exhibit a modest homoloqy with Bcl-2 and can functionally actIl8D.hOlDOlOtjof Bc1-2 (Chiou

n

A.l.,1994 ).

ElB19K waaahown to inhibitthe apoptosiathatis induced by the ElA by inhibiting the Balt (for .D.cl~2 homol o g ous

2.

,Antagoniat/.killClr) wMch promotes the apoptosia (FarrowA..t.

Al./ 19951KIefer §.tAl •• 1995;Chittenden At.ill.. , 1995).

1.S .w:aryotic'rZOlUl.lori.ptioll

1:haveexpl ained(section 1.2.3) several ce l lula r YInI.- acti n gfact o rsthl'ltbi nd tothe ili-actinq signals inthe JCV enhancer.Before explainingthe role ofthe s e factorsin qUal call-specificexpr ession of JCV,I wouldliketo des cr ibeBODle general fea t u re sofeUka ryot i c transcription .

1.3.1 l'rolll.oterl andEnhanoer.

Most of the pr otein co d in g genes in eUk a ryo tes are transcribedbyRNA polymerue II(RNAPII). Eac h gene orten containstwosetsofelements; thebasalpromotBrellamenta or minimal core pr omo t e r elements and the modulator promot er elementsor enha n c ersequence.,The bnal promoterele ments usually detenine thespec if icityofMAP II and tha di rect basal level of trans cription . On the other hand , mo d u la t o r promoter elements enhance or reduce the basal leve ls ot"

transcription.The bas alpr omoterelements generallyconsist of aTATAmotif,loca t edapproximat ely30nucleotidesupstream of the transcription st art site, and the init iat or (Inr) el ementvhich specifies thestart site (revie....ea inConaway andConaway,1993 ; Zawel andReinberq, 19 9 5 ).

Enhancersare usuallyrecoqnized by proteina vhich ar e modular in nature. containing a DNA-binding'domain and an

29 activationdomain.Enhancers are of two types : inducibleand temporal, or tissue-specific. Inducibleenhancers respond to environmental cues, suchas heatshock, heavy metals, vi r a l infection, growth fac t o r s and hormones for the

• •tallothionein , v-interferon and ~genee (reviewed in ManiatisnAl.,1987) . T!&sue-speciflcenhancers have a vital role in developmentaland in tissue-specIfic98ne expression.

For #xampl. ,the Ig8 geneenhancer expresses only in lymphoid cell. (Maniatis.It41.••1987 ;Mitchelland Tjian, 1989 ).Thus, the efficiency and tissue- spec ificityof gene expressionis determined byth e interplaybetweenenhancer binding' factors and .t.nn.ll-activa t ors (r e vi ewedinTjianand Man iatis, 1994).

1.3.2 RNA polymeraseII

The RNA polymoraslI2II (RNAPII) initia tes trans cription otlIlossengorRNA (lDRNA)at prolllotorslocate d5I ot the codi ng sequence of each gene. HWllan RNAP II is a complex mo l e c ul e ma de up of 10subunitswith apparentmolecular weights ranging from 10-240 kDa. The largest subunit has aun i qu e ca r boxy t e n!nal domain (CTD) with mUltiple se r i ne , threonine , tyrosine and prol ine residues, suggesting tbat i t is phosphorylated at serine, tyrosine and threonine res idue s. Furthermore,phosphorylated and unphosphorylated forms of RNAP II can be resolvedonpolyacrylamidegels (Woyc hikeandYoung, 1990). The role of the CTD in transcription initiation is still elusive.However,thegeneticmanipUlationdata suggest

30 that removal of the repeat. in CTO 11 lethal to~, I.t..A.n lind murine cells (Allison

n.

^{11 •• 1988: Bartolomei}!it Al.. 1988; Edwards ~.Il.. 1991). The role of the CTO in enhancer-driven transcription was recantly demonstrated.

Removal ofmostof the CTD abolishedtransactivationat Illost enhancers (Gerber R Al. , 1995) indicating that CTD is eslitential in lIledlatlnq Ienhancer'-type transcriptional activation. Recently. i t suggested that the unphosphorylated form of the CTD is present in the pre initiation complex while the phosphorylated fOl"lll is present in the elongation complex. Thus,the conversion of CTD from an unphosphorylated to a phosphorylated form may allo\ll the transition from transcriptional initiation to elongation (Laybourn and Dahmus, 19901. Other proposed functions are RNAP II localization, DNAbinding, removal of chromatin proteins from DNAand regulation of RNAP II activity (reviewed in Corden, 1990; Dahmus, 1994).

1.3.3. aeDeral transcription hCltors

The process of transcription initiation requires a concerted interaction betwQan RNAP II and at leallt dght protein factors. These factors are termed general transcription factors (GTFII). A total of eight GTFa were identified in human colI. and they are called TFIIA, B, E, F, H, I,Jand D.Toqether withRNAPII, GTFs are necessary for the basal level Clf transcription. The first step in the

31 a•••ablyof the initiation complex lli1 the binding of TrUD to DNA. TFIIDisthe onlycomponent of basal initiation factors that contaInsDHA~bindln9capacity to the TATA box sequence.

TrII0 is a llluitimeric protein complex containing- the TATA box blndlnq protein (TaP) and TOP-associated factors (TAFs)(Pugh and Tjian. 1990).Human TFIID consists of TBP and at least 8 TAFs with lIIolecular weight.,. rang-ing from30- 2 5 0 kDa (Tanese

n

Al., 1991).TOP requires no other factors for TATA element recognitionand the TBP-TATA element complex subsequently incorporates the factors forbasal transcription,but can not respondto transcriptionalactIvators withoutTA~s (Pugh and Tj!an, 19911Hoey !l..t. Al. , 1993). In the assembly of the multimeric initiation complex the bindinq of TFIID to the TATA box appears to be the first step. The TFIID-DNA complex provides a recoqnition site for the association of other basal factors and RNAP II (BuratowskiAtAl., 19B9) .

TFIIA promotes stable binding ot' TFIID to the core promoter . TFIIA wall also sh o wn to allilociatQwith TBP or the TF1ID complex, even in the absence of DNA(Cortes Jlt.al. , 1992).Several negative regUlatorsof transcriptionwere shown to displace TFI1D from thepromoter and others were shown to block TFIID's interaction with other GTFs (Auble and Hahn, 1993).TFIIA was shown to counteract these negative factors by its direct interaction with TBP (Reinberq

n

Al.,1987).

TFIIB recog:nhes either the TFIID-DNA complex or the TFIID-DNA-TFIIA complex to form DB or DAB complexes,

32 respectively (Bur atovski .e..t.Al.., 19 89) . Rec ently, TrIIB v••

showntobecriti cal for int e racti o nbet w8entheinit iatio n coaplex and upstre allac t ivat o r s (Lin.tl.t1.1.,1991 ).I ti .alao requ ired fo rthe rec ru i tmentofRNAP II. TFIIBalao se rve• • • IIbridge betvee n TFIIDandRNAPII. TfII Bcaninteract with MAP IIin sol ution the r efor e , i tIs ahopo. dble tha t th•••

two compone nb could be pre•••ociabd upon enterinq th e initiationcoaplex (Conaway and Conaway, 1993) .

TrIlF assocfates with RNAP II: even inthe absence of other factor. and also it is an lIlpo r ta nt baesl fac t or.

because i t i. abso lutely ess ential for transcription ini t i ation (Reinb e r q andRoeder ,1987 1.RNAPIIrecrultm~ntla ca rried ou t by the small su b unit of TrIll, possibly vi a int e r actio nwith TrU B (Flores.a..t.1.1••199 1,Ha.Il..t. Jl., 199J).

Besid e s actl nqas an in i tiatio n factor, Trur also affects e10nqati on of MAPU (Price

n

Ai., 1989).

Th~'I, TrUD, A, B, r andRNAI' IItor.a .iniaa1 cOllpl ex or preinit iation cO&ple x (PI C). For a complete ini t i atio n COJlplex, TFIIE and H ar e necessary. However, under some cir c u ms t a nces the. ini mal complex canin i tia te t rans cript ion (Parvin and Sha rp, 1993 ). TFIIE is nece s sary for further incorporationof TrUH (FloresJ.t.ill., 1992).

TFIIHis thelast basalfactor to enterinto the complex.

It is amul t isubu n i tcomp l exwithmanybiochemica l activit1ee.

The importa nt ac tivities ar e DnA hellca se, DNA-depe ndent ATPase and CTD ph osphoryla tio n (serbavan 11., 1993) .

33 Finally, tr a n s crip t i on initiation requires thehydrolysis ofATPordATP(Sa",adogoand Roeder , 1984; GoodrichandTjian, 1994), and this proce ss 18 termed the activation of the initiation complex. TFIIH may be the fact or mediati ng activation, sinc e itpossessesATPase~ctiv ity .

:In TATA-lesspromoters ,TFII-Ibinds toth e znr el ems nts andproillotesTBP binding (Smal e, 1994).

1.3.4 ••queIlCle·.peoitic t.ran.criptioD faotora Besides CTr s , add i t i onal tran scription fac t o rs bindto speci:fic DNA sequence s in the ups t r eam regUlatory region.

These are called transcri ptionalactivatorsor repr e ssors , depending whether tileystimulateor represstranscriptio n. A cl as s ical t ranscription factor has a specific DNA-binding domain, ahingeormult imerh a t i on domain for the format ionof homodimers or hete r odim ers and an activation doma i n. DNA- bindingdomainsar eclas s H'ied aSIthe hel !x-tu r n - he lix (HTH) do.ain, the home odomain, zinc fingerseq:mcmts , tho ste r oi d receptor bindi ngdomai n, the leucine zippe r domain,thehelix- loop-helix domainsand the IS-sheet domain (rev iewedin Pabo and Sauer, 1992).

Several other activation domains are classi f ied as acidic, glutuine rich and proline rich activationdomains (Mitchell andTj i an , 1989). A~ter interaction of Bequence- specifictranscription factors with enha n CClrll , theycontact the basal transcription machineryto directlyorindi r e ctly

3.

stimul ate the rat . of tra nscri ption (r e viewed inpt• •hne and Gann, 1990). No t all acti v ation do_ i na of" II giv e n cl•••

int e ract with the same tarqet. Henc e , there _:Ibe ••ve r aI di s ti nct type. of ac tivato r s act i nqon the a. _activ atio n domain. Ynt ere sti nqly.theimporta nt •• ino acidreaidu•• for act ivatio n ar e not nec e s s a r ily the abundant 91utalllnell, acidic. orprolines. The hyd r ophobic redd ueaint e rsper s ed amo ng theseabundant residuesare important for act iv ation (Gi l l.It.Al •• 1994).

1.3.5 MachaDismof transoriptionl activatioD. by transoriptloll.facton

Gene expr essi on in eUkaryote . require s sequ ence-s peci f i c transcriptionfactorsthatbindto••t. of dJi-actinq elea8nt.

spec ific to each 9Qne (Mitc he ll and Tj ian , 1989: ptaahne, 1988 ).tt..A.nI-actlnqfactors maybeubiqu i tous , tissue -specific or staqe-spec i f i c an dtheycan int era c t eyner qLstic a lly with eac h othe r, thue faninq a un i qu e nuc leoprot ein complex (Sawa doqo and Se ntenac ,1990 ).Several steps in the assembly of the initia tion complex arepote ntial tar qe te for.t..1:A.n.:l- ac t ivato r s.Theset..r.A.M-act ivator slIay facili tateTBP bin d i::"l9 to TATAbo X,whi chinturn accelera testhe recoqni t i o not the promoter bytheGTFs . Alternatively, theymaystabiliz e the bindinq of TBPto weakTATAboxe .. Several ••chan i s_. have been postUlllte d for transcriptionalactivation . The qreate r de ta.ils of the lIechanlsms are dis cus.e d 1n the followinq

35 sections.

1.3 .5 .1 luter.oUollowithQ'1"Pa

TFIID bind I n gto the TATA boxisthe fir s t st epinthe assemblyof th ePIC. TFIIDwas though t tobetheIlOs t likel y tarqetforac tiva to rs . Ye a s t Ga 14 (Horiko s hi

n

li., 1988b).

mamlaUan activati ng transc ri ptio nfactor (ATF) (Horikoshi .!l.t Al., 1988a) andupstreamstimulatory factor CUSF) (Sawa dogo and Roeder, aSS) verfll shown to target TFIID and thIs interaction was shown to activate transcription . other activators,likeherpe ssimplexvirusVP16 (stringer

n

Al •• 1990) , papi lloma vI rus E2protein (DostaniI I Al., 1991)lin d

!:p s tei n - Ba r r vlrull Zta protei n (Liebe rman and Berk, 1991 ) . enh an ce tra nsc riptio nbyinteractIng and8tabil lz inq theTFIIO complex at the promoter. The ps eu do r a b i e s virus IJ:lIlle d l llte ear l y prote i n (IE) sti mulates tr a ns cri pt i onby acc e l era tinq the recruitme nt of TFII D to the TATAbox (Abma yr .i..tAl ••

1988). The tumo r suppressor p5J vasshovn tointeract vith TBP or partiallypurifi ed TFI I D andact i v a ted tra nscript ionbyth e fona.ationof a more ata ble Tr II D- DNA compl ex (Che n AtAl••

1993: Traunt

n

^{Al•• 19 93). The other activator, PU.l , vaa}

sbownto interactwith TBPdire c t l y (Schuetze.e.tAl••1992).

In additionto TrIID, other GTFswe n also likelyto be the ta rg e t s for activators . The VP16 activationdomainvas shown to stimulate tr an s criptionbyre cruit1nqTFIIBto the PIC (Lin and Green , 199 1). CREBwas also sh own toInte rac t directly