19

Bwchlm:ca et Btophyszca Acta, 1032 (1990) 19-37 Elsevter BBACAN 87219

Control of papillomavirus gene expression Rosemary Sousa, Nathalie Dostatm and Moshe Yamv Umtd des mrus oncogdnes, Ddpartement de Bwlogte Moidculmre, URA 152 du CNRS, lnstitut Pasteur, Pans (France) (Recetved 30 August 1989)

Contents Introduction A ClasslficaUon B PVs and cancer C Systems for study

19 20 20 21

Organ~atmn of PV genomes A General properUes B Patterns of transcnpUon 1 BPV1 2 CRPV 3 HPVs

21 21 22 22 23 23

Funcuons of wral protems

2 E7 3 E5 B Plasrmd maintenance funcuons 1 Other genes revolved m vtral rephcauon C Late genes 1 L1 and L2 2 FA D E2 a regulator of PV transcnptmn

24 24 24 25 26 27 27 28 28 28 28

IV

Control of ~ral gene expression A Transcnpttonal regulation by E2 B Role of cellular factors m ]?V transcnptt011 C AddRtona] events reqmred for mahgnan~ ¢,onversmt3?

30 30 32 33

V

Concluding remarks

34

III

A Transfornung functions 1 E6

Acknowledgements

References

/

34

...............

3~

1. Introduction AbbrewaUons PV, papdlomavLrus, HPV1, human PV type 1, BPV1, bovme PV type 1, LCR, long control regton, URIL upstream regulatory region Correspondence M Yamv, Umt6 des VLrUSoncog6nes, D6partement de Btologoe Mol&-ulatre, URA 152 du CNRS, lnsUtut Pasteur, 25, Rue du Docteur Roux, 75724 Pans Cedex 15, France

PapiUomavtruses compnse a famdy of small D N A vtruses whtch reduce eplthehal or fibroeplthehal tumors of the skin or mucosa of htgher vertebrates These tumors, called papdlomas or warts, are generally bemgn, show hmited growth, and frequently regress spon-

0304-419X/90/$03 50 © 1990 Elsevier Sctence Pubhshers B V (Blome&cal Dmslon)

20 TABLE I Charactensncs of selected papdlomavlruses

a

Virus

Host

Sites of mfectton

Lesmn

BPV1 BPV4 DPV

HPV5

cattle cattle deer cottontail rabbit mouse (mulumammate) human human

shn, gemtals esophogeal mucos slon skin slon sl~n skan

HPV6

human

HPV11

human human human human human human human

larynx, anogemtal mucosa gemtal mucosa gemtal mucosa gemtal mucosa skan gemtal mucosa gemtal mucosa gemtal mucosa

fibropapfltoma papilloma fibropapdloma bemgn papdloma, skin carcinoma bemgn papdloma, slon carcinoma plantar warts EV b (benign) EV (squamous cell carcinoma) bemgn lesmns (CIN ¢, LP d, CA e)

CRPV

MnPV HPV1

HPV16 HPVI8

HPV21-25 HPV33

HPV42-44 HPV51- 52

bemgn lesmns (CIN, CA) CIN, cervicalcarcmoma CIN, cervical carcinoma EV (benign) CIN, cervicalcarcinoma bemgn lesions (CIN) CIN, cervicalcarcinoma

a From Refs 171 and 172 EV, epxdermodysplaslaverrucfforrms c CIN, cervacalmtraeplthehal neoplasm d CA, condylomaacummatum e LP, laryngeal papilloma Viruses assocmted with mahgnant tumors are indicated in bold ltahcs

taneously [1,2] However, certain members of the paplllomavlrus family reduce tumors that may progress to malignant eptthehal carcinomas, usually after a long latency penod Indwldual paplllomavlrus (PV) types exhibit a restricted host range, and all PVs are strictly eplthehotropinc However, the range of susceptible epltheha and the type of lesmn differs among varus types, ggvlng rise to distinct chmcal symptoms depending on the infecting virus (Table I) For example, human PV type 1 (HPV1) is responsible for plantar warts of the palms and soles, whereas HPVs 16 and 18 are associated with cerwcal mahgnancy Completion of the productive hfe cycle of the virus, including synthests of capsld proteins and virus assembly, depends on the environment provided by termanally differentiated keratmocytes Although such cells can now be obtained in vitro, the successful propagation of virus m tissue culture has not been attained yet The fact that all PV genomes exinblt a rather similar functional organtzatlon is an appealing way to explain why all members of the family share a common eplthehal specificity, but confounds an attempt to explain why different members should show such large variations in host range and oncogemc potential As we shall see, the diversities of lndlwdual wrus types derive m part from properUes inherent to the viral genome and in part from the specificaty of interacuon between the wrlon and the host cell The very fact that tins common geneUc orgamzatlon can lead to such different effects suggests that we have m papdlomavlruses an Interesting

tool to understand how gene expression is regulated An added bonus is prowded by the wral E2 transcriptional modulator proteins, winch possess the unusual capacity to repress or to activate viral transcription by binding to the same D N A sequence These issues will be detailed in this review, winch describes our current knowledge regarding PV gene expression in light of the many questions posed by tins mterestmg fanuly of wruses, including their stnct species-, tissue- and differentiation-tropisms, thetr ability to maintain their genome as an eplsome in mfected cells, their variable oncogemc potential, and the role that vtral and cellular proteins play m these effects I-A

Classlficatton

Paplllomavlruses (PVs) form a genus of the Papovavlndae family, and consist of lcosahedral particles contalnlng protein, and a single, circular, double-stranded D N A molecule averaggng 7 8 kbp Classification of PVs is based on host range and on D N A homology Less than 50% cross-hybridization of heterologous viral D N A s m hquld phase defines independent types [3] To date, about a dozen ammal PVs and more than 55 human PVs have been identified, and many subtypes and variants have been characterized I - B P V s a n d cancer

The recognition of the oncogemc potential of PVs has provoked growing interest, and the fact that varal

21 D N A and RNA persists m carcmomas has lmphcated a &rect role for these varuses m conversion to and maintenance of the mahgnant state The role of PVs m the etiology of eplthehal neoplasms was first demonstrated m early studies of the cottontail rabbit papdlomawrus (CRPV), where 75% of CRPV-lnduced tumors in domestic rabbits progress to mahgnancy [4,5] Later stud~es revealed an interesting assocmtxon between the ingestion of bracken fern and the malignant transformatxon of ahmentary tract papillomas m cattle [6] However, some ttme passed between these first known cases of a DNA virus causing mahgnancles, and the uneqmvocal demonstration that certain PVs are Implicated m human mahgnanc~es The first association of a human PV (HPV5) with a form of human cancer was reported in pauents w~th epldermodysplasm verruclformls, some of whom are lughly susceptible to cutaneous cancers [7,8] More recently, the causatwe role of HPV types 16, 18, 33 and 35 m the reduction of gemtal cancers has been documented, and viral D N A and RNA has been detected m up to 90% of biopsies from these cancers (reviewed m Ref 9) However, refection of the genxtal epltheha by certain other HPVs (types 6 and 11, for example), rarely leads to mahgnant tumors (Table I) Interestingly, m the case of the oncogemc HPVs, specific sequences of wral D N A are found integrated into the cellular genome,

whereas the wral D N A of nonmahgnant types persists as an eplsome Certmn PVs such as bowne papdloma wrus type 1 (BPV-1) are also capable of inducing fibroblasuc tumors m nuce [10], rabbits and hamsters [11], and of transfornung cultured rodent tissue cells in vitro [12-16] Recently, some HPVs have been shown to transform primary rat embryo cells as well as primary human fibroblasts and keratmocytes [17-22] In all these cases viral D N A and R N A persists m the transformed state, frequently as a stable extrachromosomal plasmxd, however only a portion of the viral genome is transcribed

I-C Systems for study Our understanding of the mechanisms of PV gene expression and pathogenesls has been hmtted by the lnablhty to propagate these vxruses in tissue culture Advances in molecular cloning and sequencing techtuques have partmlly circumvented this problem, but at present much of our information comes from the analysis of xn vatro transformation of rodent cells by BPV1, wluch has served as a model for other PVs More recently, analyses of xqral mRNAs from biopsies of HPV-mduced lesions has shed further hght on thxs problem

II. Organization of PV genomes

H-A General properttes 21~1_~ L 0

li I 1000

li

I 2000

I 3000

"

li

"' t

I I 7945

nOI 7000

t • 4000

a 5000

I 3000

I It 4000

I 5000

I 6000

n 7000

t 3000

| 4000

I 5000

I 6000

t |g I 7000 7815

6000

BPV1

3

li

L,. 0

E,

I 1000

I

I 2000

Ii

I |

I

7868

CRPV

2

li L o

~1

t 1000

t 2000

I

HPVla

L2

1

li~i L,,. 0

n 1000

~'

I n 2000

n 3000

li iglg. 4000

n 5000

6000

L,

1 ol0 | 7 0

I 7904

HPV16

Fig 10rgamzatlon of PV genomes For the vLruses BPV1, CRPV, HPVla, and HPV16, the open reading frames (ORFs) in all three phases are depicted E and L stand for early and late regmn ORFs, respectively The verttcal dotted hne represents the hrst ATG codon of each ORF Black carcles show polyadenylatmn sites The bold black lines indicate the long control regmns

Although a rather low degree of sequence homology exasts between human and ammal PV genomes [23,24], they have many properUes m common, which validates the uuhzatton of BPV1 as a model In particular, all PVs exlublt a slmtlar genome organization conslstmg of at least 10 potential protein-coding sequences or open reading frames (ORFs), all on the same strand of D N A (Fig 1) Tins xs consistent with the observation that all viral m R N A transcripts that have been detected are encoded by this strand [25-29], the other D N A strand contains only small unconserved ORFs and is assumed to be noncodmg Tlus arrangement contrasts w~th the genetic organization of other members of the papovavlrus family such as polyoma and SV40, where early and late functions are encoded on different D N A strands [30] PV genomes can be functionally dwlded into two coding regtons separated by a noncodmg segment of 0 4-1 0 kb The E (early) coding region contains the early genes E l - E 8 , including mformatxon necessary for vtral rephcatlon and cellular transformation This defimlaon is denved from stu&es of the E regton of BPV1, a 5 6 kb HmdlII-BamHI fragment compnsmg 69% of the genome, which is sufficient for m vatro transformauon and extrachromosomal rephcalaon [31] The L (late)

22 coding region contains the late genes L1 and L2, which are expressed only m differentiated keratmocytes and wluch code for structural proteins of the virus particle The noncodmg region is located between the end of the L 1 0 R F and the start of the E 6 0 R F and exhibits characteristic features mcludmg AT-rich regions, several R N A polymerase II promoters, constitutive and inducible transcriptional enhancers, binding sites for cellular transcription factors, as well as several copies of the palindrome A C C N 6 G G T known to be the binding site for the viral E2 transcriptional regulatory protein Thus tins region is involved in the control of viral gene expression, it is commonly termed the long control region (LCR) or upstream regulatory region (URR) It will be discussed in more detad below

H-B Patterns of transcription The transcriptional map of PVs is complex, and its understanding has been hampered by the generally low levels of viral mRNAs found m PV-mfected cells Furthermore, PV D N A can erast as stable eplsomes or in some cases as integrated into the cellular genome Productton of viral p a r a d e s proceeds only m Inghly dlfferentaated keratmocytes, m latently infected transformed cells where the virus exists as a plasmld, viral rephcatlon Is possible but the environment is nonpernusswe for production of mature vinons In cases where the virus Is integrated, viral rephcauon proceeds with that of the cellular genome These charactensUcs furnish a number of different opportumtles to analyze viral transcription The particular pattern of viral transcripts found m each case can provide a clue as to the function of the viral ORFs The following section describes the transcnptlonal organization of PVs, including mRNAs detected m bemgn vs mahgnant lesions, and in transformed cells vs productively infected warts Then we will go on to describe how these studaes have been complemented by deletlon/mutataon analyses of viral ORFs, m order to define the roles of the viral protems eDNA and m R N A analyses reveal the usage of multiple promoters, m R N A sphce sites, and polyadenylaUon sites, resulting m the production of a set of mRNAs charactensUc of the state of the mfected cell (wart cells pernusslve for viral rephcatlon vs latently transformed, nonperlmsslve tissue culture cells, for example) These complex patterns of transcription play a role in determanmg the effects of viral infection

H-B 1 BPV1 BPV1 Induces cutaneous fibropapdlomas in cattle and fibroblastle tumors in heterologous hosts including hamsters and mice BPV1 can also transform mouse cells m vitro, and it has been shown that the 69% HmdlII-BarnHI fragment is sufficient for tins effect

[31] In all cases, the viral D N A is maintained as a stable, multxcopy, extrachromosomal plasmld Production of wrus parucles via the expression of the late genes only occurs m the terminally dlfferenuated keratmocytes of the fibropapilloma The entire 7945 bp BPV1 genome has been sequenced [32-34] Transcription of the BPV1 genome m transformed cells and productively infected warts has been analyzed in detail by cDNA clomng, electron microscopy, and S1 nuclease protection [25,27,34-37] Active promotors at nts 7185, 7940, 89, 890, 2443 and 3080 have been identified m BPVl-transformed cells and m dermal fibroblasts of a BPVl-mduced fibropapdloma, in addition to a 'wart-specific' late promoter at nt 7250 that is only utdlzed m the fibropapilloma [37,103] (Fig 2) Multiple m R N A specxes ranging in size from 1 - 4 kb are generated by differential sphcmg from sphce donor sites at nts 304, 864, 1235, 2505, 3764 and 7385 and sphce acceptor sites at nts 528, 3225, 3605 and 5609 [38] All mRNAs originating from the promoters utdlzed in transformed cells share a common polyadenylatlon site at nt 4203 [36] The transcripts from the late promoter PL at nt 7250 utihze a common splice donor site at nt 7385 and a common polyadenylat~on site at nt 7175, winch is specific for warts Thus it appears that the m R N A species found in transformed mouse cells are a subset of the mRNAs found m productively infected papdlomas The P7185, P7940 and P89 'early' promoters are located in the LCR, and these promoters give rise to the transcripts encoding the gene products of the 5' ORFs, including E6, E7 and E1 (Fig 2) In particular, the P89 promoter of BPV1, located just upstream of the E6 ORF, is responsible for expression of the E6 and E7 ORFs Tlus promoter, along with P7940, xs transactwated by the E2 gene product, via an E2-responslve enhancer located in the L C R (see below) Another major BPV1 promoter Is P2443, located in the early coding region just upstream of the E 2 0 R F Two types of m R N A transcripts lmtlatmg from tins promoter have been described Ten percent of these transcripts encode the full length E2 gene products, whale the other 90% are sphced from nt 2505 to 3225 and code for the E5 protein [37] Tins promoter is also responsive to trans-regulatlon by the E2 gene product (and thus E2 is autoregulated) via cts-actmg sequences in the LCR The P3080 promoter is located intemally In the E2 O R F and encodes an N-ternunally truncated E2 protein winch acts as a transcnpUonal repressor [39] (see below) Interestingly, tlus transcript is 10-fold more abundant than the m R N A encodmg the full-length E2 [40,41] The late promoter PL at nt 7250 in the LCR is the only promoter specific to warts and the mRNA transcripts it produces are 10-100-fold more abundant than the levels of m R N A lmUated at any other promoter This promoter produces mRNA representing the major

23 3714 4010

813

2663

3267 3551

L2 449

49

3173 3526

859

501

1699 1479

2561

P7185 P7940 P890

794511

1000

L,

5583/5597

I 7093

3837

P2443 P3080 ,

7000

li

4172

2000

1,

,, 3000

4000

i

I

5000

6000

E6/E7 E6/E4 E6 E7 El/E4 E2 E2

I

and ~ts putatwe E 6 0 R F is almost two-times longer, due to a repeated region [42] (Fig 1) Studies in transplantable VX2 carcinomas, where the CRPV genome is integrated in multiple head-to-tad copies, reveal two major m R N A species transcribed from early region promoters, with a common polyadenylation site at nt 4367 [43,44] A 1 25 kb m R N A species has heterogeneous 5' ends mapped to nt 903-905, winch is close to the first A T G of the E7 ORF and thus tins m R N A most hkely codes for the E7 protein The 5' ends of the 2 0 kb mRNAs map to nt 158, winch is just beyond the first A T G of the E6 ORF and thus tins m R N A would encode a shortened version of the E6 protein A subset of the 2 0 kb mRNAs has 5' ends at nt 87 and is therefore capable of encoding the entire E6 ORF In varus-producmg papillomas of the cottontail rabbit [43] as well as in VX7 carcinomas winch produce intact wrlons [45,46], additional mRNAs are found, winch hkely code for late proteins, and whose 5' ends have not been precisely mapped

E2 ( r e p r e s s o r ) E8/E2

[]

E6

E6

E4

•~

L2

Ftg 2 Transcnpttonal m a p of BPV1 The top part of the figure shows a h n e a n z e d m a p of BPV1, including the ORFs m the three phases, as well as the major promoters and polyadenylatlon sites (black circles) The lower part of the figure shows the structures of various c D N A clones, the first nine are from BPVl-transformed cells and the last s~x are specific to fibropapdlomas Putatwe nontranslated regions are indicated with sohd hnes and sphced sequences by slanted hnes O R F s are shown as open boxes and potentml O R F s expressed for each clone are shown on the right Data from Refs 36, 37 and 103

capsld protein encoded by the L1 ORF [37] However, it ~s interesting that the most abundant m R N A species transcrxbed from PL IS derived from the E 4 0 R F , suggesting that E4 ~s perhaps more appropriately a late viral protein The complex regulation of these promoters contributes to the dffferentml patterns of transcription, and thus the different phenotypes, observed m transformed cells and productively infected warts, and will be discussed in following sections H-B 2 C R P V

The 7868 bp CRPV genome (sequenced by Girl et al [42]) resembles BPV1 in its transcnptlonal orgamzaUon, with the exception that the E 3 0 R F is lacking in CRPV,

H-B 3 H P V s

Information regarding the transcnptlonal organization of HPVs has only recently begun to accumulate Tins section wdl focus on results obtained with HPV16 and HPV18, winch together account for over 80% of cervical cancers [9] An addlUonal caveat for such an analysis involves the fact that the state of viral D N A differs between benign and mahgnant lesions In bemgn or preneoplastlc lesions, HPV D N A persists extrachromosomally as an eplsome, as in the case for BPV1 However, m malignant lesions as well as in cell hnes estabhshed from cervical cancers (Hela, CaSk1, C4-1, SW756, SIHa), HPV D N A is integrated In the cellular genome m single or multiple copies, m some cases integration has been found to occur m the v~cimty of cellular oncogenes [174,178] With regard to the v~ral DNA, tins integration occurs m a rather restricted manner Integration consistently occurs in the E l / E 2 / E 4 / E 5 section of the early region, thereby disrupting a n d / o r deleting these ORFs and consequently joining the 5' part of the early region (ORFs E6 and E7), to downstream host cell sequences Thus the HPV-contaInlng cell hnes differ from mouse cells transformed by BPV1 in that the only sequences available to transcribe derive from the E 6 / E 7 region whereas with BPV1 an additional set of abundant transcripts exists coding for E 2 / E 4 / E 5 As we shall see tins clearly has lmphcatlons for the oncogemc potential of these viruses In C a S h ceils winch contain multiple integrated copws of HPV16 DNA, transcnptlon start sites have been mapped to nt 97, winch is 14 bp downstream from the first A T G of the E6 ORF, (the next A T G is at nt 104) [47] This P97 promoter is responsible for transcnpUon of the early genes E6, E7 and E1 In the cell hnes HeLa, C4-1 and SW56, contalmng integrated copies of

24 HPV18 DNA, an analogous promoter has been mapped to nt 105, and transcription start sites further upstream in the LCR seem to exist as well [48] The P97 and P105 promoters are analogous to the major early promoter P89 of BPV1 Transcription analysis in C a S h cells [49] has also revealed the presence of small transcripts contalnmg only the E2 region winch may be lmtiated from a promoter analogous to the P2443or P3080 promoters of BPV1 [34,36] Thus in C a S h cells the E 2 0 R F may not be entirely interrupted The 3' ends of the major E6 and E7 transcripts of HPV16 and HPV18 terminate in adjacent cellular sequences, since integration of viral D N A and dlsrupuon of the E2 region removes viral processing signals including sphce acceptor sites and a polyadenylatlon sequence [47,48] Since all adjacent cellular sequences looked at contain termanatmn codons m all three reading frames, the generation of a fusion protein seems unhkely [48] Interestingly, HPV16 and HPV18 possess a unique feature which is present neither in the bemgn gemtal viruses HPV6 and H P V l l nor in BPV1 and CRPV [47,48] As a result of a particular sphcing event, the E6 region of HPV16 and HPV18 can code for two versions of E6, the entire protein and also a shorter E6* protein that results from m-frame fusion of the 5' part of the E6 ORF with a small segment of another reading frame [48,50] Most of the cDNAs analyzed in HPV18-mtegrated cell hnes contain this sphced E6* sequence [48] Northern blot analysis [51] revealed different patterns of HPV16 transcription m cervical lntraepithehal neoplastas ('CIN'), where viral DNA exasts as an episome, and lnvaslve carcinomas containing integrated HPV16 D N A Whereas E6 and E7 transcripts were detected m both cases, the E 1 0 R F as well as the E 2 / E 4 / E 5 coding region were only detected m CIN In no cases were late region transcripts found These results support the previous findings that Integration dlsrupts the E 2 / E 4 / E 5 region Recently, primary human keratinocytes and an lmmortahzed squamous cell carcinoma line, transfected by HPV16 DNA, were shown to contain transcripts corresponding to the E 6 / E 7 region [22], and as yet uncharactenzed viral mRNAs as well as the E7 protein itself were detected in HPV16-transformed primary keratlnocytes [21]

III. Functions of viral proteins The persistence of specific viral transcripts in most transformed cells suggests strongly that their corresponding proteins serve a role m the development or maintenance of the transformed state Early studies to identify and assign functions to viral proteins were hampered by the low levels of viral mRNA and protein in transformed cells More recently, the functions of

most of the viral ORFs have been unraveled, primarily by experiments in whtch the ORF of interest is deleted, mutated, or expressed from a surrogate promoter m the absence of other ORFs, to deterrmne its effects of transformation and viral rephcation (Table II)

111-.4 Transforming functions Imual studies with the cloned BPV1 genome defined a 69% subgenormc fragment encompassing the early region ORFs and the LCR, that was sufficient for reducing transformation of mouse C127 or NIH3T3 cells [31] More recently, this region has been subdivided into two independent coding domains that contribute to the transformed phenotype, the E 6 / E 7 region [36,52], and the E 2 / E 4 / E 5 region [53-56] Interestingly, both of these regions are required to obtain the complete transformed phenotype, including anchorage independence and t u m o n g e m o t y m nude mace [36,54] Further analysis identified the E6 protein and the E5 protein as the two independent transformang functions of BPV1 [36,52,55-57] It should be recalled that BPV1 induces both epidermal and dermal prohferatlon, m the case of gemtal HPVs and CRPV whtch act strictly on the eplderrms, the E6 and E7 proteins are responsible for cellular transformation, suggesting perhaps a different mechamsm for ttus effect (see below) The E2 protein has also been lmphcated as playing an indirect role in transformation, as a consequence of tts transcriptional regulation of promoters involved in the expression of the transfornung genes Ttus will be detailed m following sections

III-A 1 E6 The BPV1 E6 gene 1s suffioent by itself for the transformation of mouse C127 cells when expressed from a strong heterologous promoter [36,52] In the context of the entire BPV1 genome, the E6 genome is essential for the fully transformed phenotype [54] TABLE II

Functwns of vtral ORFs ORF

Propertxes

E1 E2 E3 E4 E5

plasrmd maintenance transcriptional modulator of varal promoters unknown 'late' functions, maturauon of vlnon ? BPV1 transforrmng gene, unknown for other v~ruses transforrmng gene transfornung gene (HPVs, CRPV) plasrmd maintenance (BPV1) unknown major capsld protein rmnor capsld protein

E6 E7 E8 L1 L2

See text for detads

25 E6-speclfiC mRNA's are retained and expressed both In fibropapfllomas and in BPVl-transformed cells The E6 protein has been identified m C127 cells by immunoprecip~tation w~th antisera raised against a bactenally synthesized E6 fusion polypeptade [58] The E6 protein (molecular mass 15 5 kDa) comprises 137 amino acids and is locahzed in both nuclear and membrane fractions of the transformed cells The E6 gene is also selectwely retained and IS transcriptionally active In CRPV-lnduced carcinomas [43,44,49] as well as in human cervical carcinomas conraining HPV16 and HPV18 D N A [37,47-49,59] In COS-7 ceils expressing the early CRPV region, two forms of the E6 protein were identified [60], corresponding to the two types of mRNAs coding for the full length (promoter P87) and shortened version (PI58) of E6 [43,44] The full length version (molecular mass 39 kDa) was found In the nucleus while the shorter form (molecular mass 24 kDa) was cytoplasmic and phosphorylated (Recall that the E 6 0 R F of CRPV is about twice as long as that of BPV1 due to a duplicated region, winch explains the difference in the molecular masses of the respective proteins ) As already noted, the integration of gemtal HPV D N A into the cellular genome in mahgnant lesions disrupts the 3' ORFs ( E 2 / E 4 / E 5 ) and gives rise to transcripts derived from the 5' ORFs E6 and E7, suggesting that these viral proteins are involved in the transformed phenotype Indeed, subgenonuc fragments containing the E 6 / E 7 regton of HPV18 were at least as efficient as the entire viral genome in the transformation of NIH3T3 or Rat-1 cells [18] Further analysis has shown that in Immortahzed fibroblasts, both E6 and E7 could independently induce anchorage-independent growth, and the effect of E7 was stronger than that of E6 [20] Recently, the E6 and E7 genes of HPV16 were found to both be required for the transformation of primary human fibroblasts [19], and mRNAs from tins regton were detected in primary human epidermal cells transformed by HPV16 D N A [22] Low levels of E6 protein (molecular mass 11 kDa) were identified by Immunoprecipitation In CaSkl cells winch contain integrated copies of HPV16 D N A [61] Since the complete E 6 0 R F would code for a 19 kDa protein, the authors propose that the E6 protein they detected is generated by a sphclng event Androphy et al [62] detected by Immunoprecipatatlon an 18 kDa HPV16 E6 protein in CaSka and SIHa cells as well as in HPV16-transformed NIH3T3 cells, Interestingly, the HPV16 E6 antisera did not cross-react with the E6 proteins of HPV18 or BPV1 HeLa cells contaimng Integrated copies of HPV18 D N A also express the E6 protein [63] Although m R N A analysis predicts the existence of an E6* protein generated by an internal sphcing event specific to the oncogenic genital HPVs types 16 and 18

[48], such a protein has not yet been identified However, recent studies suggest that tins protein is probably not essential for transformation, at least not in immortahzed rodent fibroblasts [20] or primary human keratlnocytes [175] The E6 proteins of all paplllomavaruses are not Inghly conserved, but they all contain the motif Cys-X-X-Cys repeated 4-times with a conserved pattern of spacing between them, as well as a high percentage of basic armno acids, suggesting that perhaps they share a common function In fact, the conserved cysteine motif is characteristic of the 'zinc finger' type of D N A binding proteins [64], and the E6 proteins of HPVs 16 and 18 and of BPV1 have recently been shown to coordinate zinc ions [65] Furthermore, a strong but nonspeclfiC D N A binding actiwty has been observed wtth E6 [66] However, It should be noted that zinc finger structures may not always be directly involved in specific DNAprotein interactions but rather may play a role In deterrmmng protein conformation or protein-protein interactions essential for D N A binding, as suggested from studies of SV40 large T antigen [67] Therefore, although mutational analysis indicates that each of the four Cys-X-X-Cys repeats is essential for E6 transforming function [68], their precise role - involving D N A binding or protein-protein Interactions - remains to be estabhshed It will be important to further clarify the mechanism(s) by winch E6 exerts its transforming potential III-A 2 E7

The E 7 0 R F encodes a major transforming function of gemtal HPVs E7 m R N A is present in premalignant lesions where HPV D N A exists as an episome as well as in carcinomas and carcinoma-derived cell hnes containing integrated viral D N A In addition, the in vitro transforming functions of HPV16 and HPV18 In rodent cells have been mapped to the E7 gene [20,69-72], whereas both E6 and E7 are required to transform primary human flbroblasts [19] and keratinocytes [175] E7 is In fact the most abundant viral protein in cervical carcinomas and the cell hnes denved from them [47,61,73], whereas an E7 message has not been identified in lesions produced by the bemgn HPVs types la, 6, and 11 [74,75] Interestingly, no direct transcnption initiation site has been mapped to the immediate upstream region of the E7 ORF, instead, the 5' ends of the mRNAs begin directly, or a few nts upstream, of the putative E 6 / E 6 * start codon This arrangement may be a means to regulate synthesis of E6 and E7 by shifting translation to the E7 ClStron, accounting for its abundance in relation to E6 However, even In CRPV, where the mRNAs for E6 and E7 are generated from different promoters [43-45], E7 transcripts are more abundant than E6 transcripts in cancers relative to benign papillomas [43]

26 The E7 protein has been immunopreclpltated from C a S h and SIHa cells containing HPV16 [47,61], from Hela, C4-1 and SW756 cells winch contain HPV18 [61], as well as from human keratmocytes transfected with HPV16 DNA [21] The E7 protein is mainly found in the cytoplasm, is phosphorylated at unspecified serine residues, and exhibits a high heterogeneous sedimentation rate in nondenatunng glycerol gradients, suggesting that it may Interact with itself or with cellular proteins [73,176] It should be noted that hke E6, the E7 protein contains Cys-X-X-Cys motifs winch are important for zinc-binding activity [65] These motifs contribute to the transforming activity of E7 but are not essential [76] The E 6 / E 7 region of oncogenic HPVs (types 16, 18, 33 and 35) but not of HPVs associated wzth benign lesions (HPV6, H P V l l ) , are capable of cooperating w~th the oncogene ras to transform baby rat kidney cells [20,77,78], and more detailed experiments have revealed that at least for HPV16, the E7 protein by itself is sufficient for tins effect [20,78] Transformation of baby rat kidney cells by cooperation with activated ras is also a property of the adenovlrus EIA proteins, and further stu&es have revealed a similarity between the HPV16 E7 protein and adenovlrus E1A in other respects as well [71] In particular, E7 can transact~vate the adenovlrus E2 promoter, and the amino acid sequence of E7 exinblts homologies w~th the conserved domains 1 and 2 of E1A proteins Furthermore, the E7 protein of HPV16, like E1A, brads to the retmoblastoma gene product, and there seems to be a correlauon between the strength of tins binding and the malignant potential among E7 proteins of different viruses [177,179] However, unlike E1A, the HPV16 E7 is also capable of transforming established mouse cells Recent stu&es indicated that a single anuno acid substituuon analogous to an E1A mutation that destroys ~ts transforrmng ability, also destroyed the transforming abihty of E7 [76] Also, it appears that phosphorylation of individual serlne residues of E7 may not be Involved in transformation [76] Clearly, the precise role of E7 in HPV- and CRPVreduced transformaUon remains to be elucidated The relatively high abundance of E7 in relation to E6, as well as the interaction between these two transforming proteins, is of particular interest The observation that E7 alone is sufficient for transformation of certain cell types wl~le for others E6 and E7 are both required, suggests that cell-specific factors may exert an important influence and illustrates the potential drawbacks of extrapolating too much from any one in vitro system In contrast to the above results, the E7 protein of BPV1 does not possess transforming acUvlty but rather is involved in the control of rephcatlon of BPV1 eplsomes to high copy number [79,80] Interestingly, the regions of HPV16 E7 homologous to the EIA protein

are not found m the E7 of BPV1, and the BPV1 E7 protein does not bind the retlnoblastoma gene product [179] As discussed below, the other major transfornung gene of BPV1 apart from E6 is E5 III-A 3 E5

The E5 gene of BPV1 is capable of Independently transforrmng mouse C127 and NIH3T3 cells Mutations winch disrupt the E5 gene downstream from the metinomne codon at nt 3879 strongly lninblt the efficiency of transformation [55,56,81-83] The P2443 promoter, upstream of the E 2 / E 4 / E 5 0 R F s , is required for efficient expression of the E5 gene, and 90% of the m R N A transcripts initiated from P24n3 encode the E5 gene product [36,37] Tins promoter is regulated by the product of the E 2 0 R F , winch transactivates P2443-drlven expression through an E2-dependent enhancer located m the LCR [84,85] The E5 protein has recently been ldenufied in C127 cells transformed by BPV1, using anubodles directed against a synthetic peptlde corresponding to its C-terrmnal region [86] With an apparent molecular mass of 7 kDa (44 anamo acids), E5 is the smallest viral transforming protein yet characterized The protein contains 68% hydrophoblc armno acids (34% leuclnes) in the central and N-terrmnal region, and a Cys-X-Cys motif in the C-terminal Transforming activity is encoded entirely in the C-terminal domain [86,87] Cell fractlonatlon studies localized the protein mainly to cellular membranes, where it is anchored through the hydrophoblc region and where it forms dimers through the cystelnes of the C-terminal, winch is oriented extracellularly [86-88] The E5 protein could not be labeled metabolically with 32P-orthophosphate or fatty acids [86,88] Mutational analysis revealed that all transformation-competent E5 mutants retain the ability to associate with the cellular membrane and to form dlmers Recent results have begun to shed light on the mechanism of transformation by E5, suggesting that E5 may influence the activity of growth factor receptors In particular, E5 acts in synergy with the normal E G F receptor to transform NIH3T3 cells in the absence of exogenously added E G F E5 also increases the transforming activity of the CSF-1 receptor, although no effect is seen with other tyrosine kmase protooncogenes such as c-fes In the case of EGF, E5 lninbits the rapid mternahzatIon or down-regulation of receptor, resulting in the persistence of phosphorylated receptor at the cell surface [180] It is interesting that the E 5 0 R F sequence is well conserved among those PVs winch induce both epidermal and dermal prohferatlon, such as BPV1, whereas the E 5 0 R F s of PVs inducing strictly epidermal prohferations (HPV1, CRPV) show little homology to BPV1 E5 [89,90] This suggests that perhaps the BPVI-llke E5

27 proteins may play a role m stimulating dermal fibroblastlc proliferation and is consistent wath the observation that the BPV1 E5 more efficiently transforms NIH3T3 cells (of fibroblasnc ongm) than C127 cells (of epithelial ong~n) [55] In contrast, for the PVs which do not share E5 homology with BPV1, the E 5 0 R F is not necessary for transformation and indeed its role is completely unknown

I I I - B Plasmld mamtenance functions

BPV1 has again been used as a model to dehneate the hfe cycle of PVs In benign eplthehal and flbroeplthehal lesions and fibroblastic tumors [91], as well as m transformed mouse cells [92,93], the BPV1 genome extsts as a stable multlcopy plasmtd, and integration of the viral genome IS not required for BPV1reduced transformation [93] Only in terminally differentiated keratinocytes does vegetative and exponenhal viral rephcatlon occur, followed by encapsldation [94] Tins part of the viral cycle has been difficult to study due to the absence of a productive propagation system in vatro The fact that BPV1 infection leads to cellular transformation with single hat kinetics [95] and that a high viral plasmld copy number is detected after only a llmated number of cell doubhngs [93,96] suggests art initial mode of rapid viral D N A replication from one or a few copies, followed by another mode of rephcatlon winch allows a stably regulated copy number Genetic analysis reveals a complex network of cts- and transacting viral factors involved in this process Two discrete cts-actmg plasnud maintenance sequences (PMS-1 and PMS-2) have been defined, each of winch can support rephcanon of exogenous plasmads in cells providing BPV1 rephcation functions in trans [96] PMS-1 is located in the LCR region and consists of a domain with enhancer actlvaty and a domain winch overlaps the viral ongm of rephcation [97,98] All PV genomes sequenced to date contain a PMS-1 element at tins conserved position, with at least 50% sequence homology to the BPV1 PMS-1 PMS-2 is located within the E 1 0 R F For a completely permissive rephcatlon system these two regions also require either the distal BPV1 enhancer [99] or a sequence mapped in the BPV1 31% nontransformlng (late) region [97] Mutational studies of single ORFs have revealed several complementation groups that can affect viral plasnud rephcatlon [54,79-81,97,100,101] However, the E 1 0 R F is the only ORF in which mutation completely abohshes transient rephcatlon m C127 cells [54,80,82] Genetic studies Indicate that the E 1 0 R F encodes two complementaUon groups involved m different aspects of viral replication The R gene xs encoded by the 3'

portion of the E 1 0 R F and is absolutely required for early rephcatlon events [97,100] Furthermore, mutations m tins regton always lead to integration of the mutant viral D N A into the host cell genome [54,80,81] In contrast, the 5' portion of the E 1 0 R F encodes a modulator funcnon (M) winch is not necessary for the initial phase of rephcatlon but is required for establishment of the viral genome as a stable nuclear plasrmd [97,100,102] Thus it appears that R encodes a positive factor necessary for the xmtlal amplification of viral D N A soon after infection, while M would be a repressor of amphficatlon or even a positive factor involved in low level replication without amphficatlon [102,103] Recently the M product (23 kDa) was identified by l m m u n o p r e o p l t a n o n m BPVl-transformed cells, where it exists in a phosphorylated state [104] It is interesting that although the E 1 0 R F encodes two distinct, independent gene products, the intact, entire E 1 0 R F is in fact the best conserved early ORF among all the PVs It is possible that perhaps the E1 O R F is used m another way in its entirety, and a recent study suggests that an E1 gene product containing both the M and R sequences Is responsible for repression of BPV1 transcription and ininbltlon of its transformang ability [105] The E1 gene product shows amino acid sequence homology with the SV40 and polyomawrus large T antigens, primarily m regions winch are revolved in nucleotide binding and ATPase activity [106] Tins supports the notion that E1 proteins are involved m DNA replication E1 m R N A has been identified m cervical biopsies containing nonmtegrated HPV16 DNA [51] but surprisingly, a 70 kDa E1 protein was also identified (at low levels) in HeLa cells winch contain integrated HPV18 [61] The fact that such a protein would be found m cases where the viral D N A does not replicate as an extrachrosomal plasmad rmght imply an addmonal function for the E1 protein

I I I - B 1 Other genes mvolved m otral rephcanon

Mutations in the E6 and E 6 / E 7 regions of BPV1 result in viral DNAs winch are able to exast as plasmads but only at low copy number [79,80] In fact the primary role of the BPV1 E 7 0 R F is in the maintenance of Ingh plasnud copy number, in contrast to the oncogemc HPVs where E7 is a major transforming protein In the full genome background, mutanons in the E2 O R F are also defective for replication, and lead to integration of the viral D N A into the host chromosome [54,57,81,82,97,101,102] It is hkely that these effects of E2 are indirect, given the role of E2 in the transcriptional regulation of the 5' ORFs (E6, E7 and El) involved in replication and plasmld maintenance

28 111-C Late genes II1-C 1 L1 and L2 The two large ORFs included in the BPV1 31% nontransforrmng region code for the viral structural proteins These two genes L1 and L2 are only expressed in terrmnally dlfferentmted keratmocytes The L1 O R F is the most highly conserved wral gene and codes for the major capsld protem, with an average molecular mass of 56 kDa [107-111] The L1 protein carries the major antlgemc determinants for group-specific crossreactlwty within different groups of PVs [112,113] It is highly glycosylated, winch is thought to stabilize the virus particle structure [114] The L2 ORF codes for a protein of slnular size but in contrast to L1 it is one of the most poorly conserved viral proteins [115] Thus it is not surprising that the antagemc determinants carned by the L2 protein do not cross-react with antlsera from other viruses even from the same group, but are instead type-specific [116] The L2 protein from HPVla, identified by lmmunopreclpltatlon, represents a minor structural protein whose apparent molecular mass of 76 kDa is different from the 55 kDa molecular mass pre&cted from the nucleotide sequence, but the reasons for tlus are unknown [116] I I I - C 2 E4 The E 4 0 R F totally overlaps with the E 2 0 R F in a different reading frame The role of this 'early' ORF was puzzhng at first, since studies w~th BPV1 showed that it was dispensable for many 'early' functions including transformation and plasmtd rephcatlon [57,117] Further studies showed that in fact E4 represents the major mRNA transcnpt from the BPV1 late promoter PL, winch is active only m warts [37], and that the E4 protein of H P V l a is expressed coordinately with the L1 protein in HPVla-mduced warts [118] These results suggest that E4 should be more appropriately classified as a late function Tins further distinguishes the paplllomavlruses from other members of the papovavlndae farmly in that not only are early and late functions encoded on the same strand of DNA but that a late gene overlaps the early genes The F_A protein of BPV1 has not yet been identified, but the E4 of H P V l a has been isolated as a cytoplasrmc protein and represents up to 30% of total cellular protern in HPVla-lnduced warts [118] In fact, lmmunoprecipitation studies have identified eight polypeptldes corresponding to a 17 kDa E4 protein containing five amino acids from the E 1 0 R F joined onto 120 from the E 4 0 R F , and other products (&mers and degradation products) derived from these [118-120] The function of these E4 gene products has yet to be established, but ~t has been suggested that they may

play a role in productive viral maturation, perhaps as structural or transport proteins [117,118,120] I I I - D E2 a regulator of P V transcrlptton

In a full BPV1 genome background, mutations m the E2 ORF have a strong effect on cellular transformation and plasmld mamtenance [54,57,80-82,101] However, when the expression of E2 alone is directed from a surrogate promoter like SV40, mutations have httle if any effect, and conversely, the transformation- and replication-competent ORFs expressed from surrogate promoters show no requirement for E2 [55,56] These results suggest that E2 plays an redirect role m transformation and rephcatxon, and further stu&es showed that its effects are probably due to E2-medlated transcriptional regulation of promoters involved m directing expression of transformation and replication functions In fact transcriptional activation and repression of viral genes by different versions of the E2 gene product comprises one of the most important regulatory circuits of PV gene expression, and appears to be a property shared by all PVs Again the BPV1 model has been used most extenslvely to characterize the products of the E 2 0 R F , wluch encodes at least three transcriptional modulator proteins that share the same C-terrmnal dommn but possess different biological functions The BPV1 P2443 promotor located just upstream of the E 2 0 R F directs the synthesis of a full length 48 kDa E2 protein, which acts as a transcriptional transactlvator via enhancer elements located in the LCR [121] The P89 and P794o promoters just upstream of the E 6 0 R F , as well as P2443, are responswe to E2 [84,122,123] In addition to the transactlvator, the 3' portion of the E 2 0 R F encodes a 31 kDa protein termed E2-TR, expressed from P3080, winch specifically represses transacUvatlon by the full length E2 gene product [39,124,125] More recently, another functional form of the repressor has been ldentiffed as a 28 kDa E 8 / E 2 fusion protein [34,103,125,126] wtuch appears to be expressed from a novel promoter around nt 890 [103] Proteins corresponding to these three gene products have recently been identified in BPV1 transformed cells [41] Interestingly, the 31 kDa E2-TR is 10-fold more abundant than the full-length E2 transactlvator, and 3-fold more abundant than the 28 kDa E 8 / E 2 fusion protein, consistent with the levels of the corresponding m R N A transcripts [40] The high levels of the repressor version of E2 may explain the low levels of viral transcription m BPVl-transformed cells The E1-TR repressor protein or other truncated versions of E2 have not been identified for any PVs other than BPV1 However, a repressor activity of the Cterminal region of HPV16 and H P V l l E2 has been reported, although the actual protein was not ldenufied m infected cells [124,127]

29 C-TERMINUS

N-TERMINUS hinge region

treneeetlvlltion I. . . .

A1

++

II

A2

DNA binding dlmerlzatlon

II

]

~ region

Fig 3 Secondary structure predictions for E2 The results of a computer deternunaUon of potential secondary structures of ten E2 proteins are summarized Black cyhnders represent conserved ahehces The two long hehces at the N-terrmnus are named A1 and A2 and their overall charges are indicated Grey rectangles represent conserved #-strands Black hnes indicate sequences of undeterrmned structure The zagzag line mdtcates the absence of rehable structural reformation for the internal 'lunge' region

Transcriptional regulation by E2 is mediated by direct binding to viral c l s elements contaimng a short palindromic sequence A C C N 6 G G T [40,122,123,128,129] repeated several times in the control regions of all PVs so far sequenced [130] The number and position of these E2 binding sites differs among virus types, and as shown below this influences the transcriptional effects of E2 The E2 proteins of other PVs have been shown to interact with the ACCN6GGT sequence [131-133], even in a heterologous manner [124,131,132,134] Furthermore, one or two copies of the E2 binding s~tes can serve by themselves as an E2-dependent enhancer when linked in c t s to heterologous promoters of mammalian cells [135-138] and yeast [139] Although the homology between the E2 proteins of unrelated PVs is rather low (35%), ahgnment of 10 amino acid sequences has revealed the presence of conserved motifs defimng distinct structural domains that can be ascribed particular funcuons [140] (Fig 3) As detailed below, mutational analysis revealed that the N-terminal is responsible for transactlvatlon, the Cterminal for D N A binding and dlmenzatlon, and the poorly conserved, prohne-rlch internal domain is consldered to be a flexible 'hinge' region [140,141] The D N A binding dommn of E2 is localized in the 101 C-terminal amino acids [40,131,142], in a small core resistant to pronase digestion [143] C-terrmnal E2 polypeptides synthesized in vitro have been shown to bind to the ACCN6GGT target s~te [40,128,137,142,144] Various studies have indicated that the binding afflmty of E2 vanes greatly among target sites corresponding more or less to the perfectly conserved consensus sequence A C C G N 4 C G G T [137,142,145] Clearly, the existence of sites with different affmmes for E2 In the viral genome may have different influences in mediating E2 effects in wvo The contacts of the BPV1 E2 protein with the palindrome are fully symmetric, and in fact two E2 monomers interact with a single pahndromac target [141,143,146,147] in the form of a pre-exastlng dimer [141] The two monomers contact the major groove of

the D N A helix on the same side [143,147], such that each reverted repeat of the pahndrome acts as a specific anchor The capacity to dlmerlze also resides in the C-terminal domain Although this domain does not exhibit characteristics typical of some D N A binding proteins such as hehx-turn-hehx motifs or zinc fingers, it does contain conserved blocks present in all E2 proteins that harbor conserved hydrophoblc amino acid residues repeated every seventh almno acid These motifs could potentially form a coiled coil structure [148] that resembles the 'leucme zippers' involved in the dlmerlzatlon of certam known D N A binding proteins [149] The actual involvement of such structures in the dlmenzatlon and D N A binding of E2 remains to be established The fact that the full length E2 transactlvator and the shorter E2 repressors share a common C-ternunal suggests that they may share functional actlvitms as well, such as D N A binding and dlmenzatlon Hence it seems hkely that the repressors function either by competitive binding to the target site, preventing binding of the transactlvator, a n d / o r by the formation of Inactive heterodlmers between transactlvator and repressor species [39,141,146] The N-terminal domain is the best conserved among the E2 proteins, and consists of two negative amphipathic a-hehces followed by a small fl-strand [140] (Fig 3) Mutation studies indicated that the two a-helices are essential for transactlvatlon while mutation of the B-region has a less important effect [129,140] The requirement for the negative amphipathic helices is not surprising since these features, common among transactlvator proteins, are thought to be directly revolved in transcription through protein-protein interactions with common transcription factors such as the TATA factor T F I I D or R N A polymerase (Ref 150, reviewed in Ref 151) Experiments in yeast and mammahan cells have shown that a strong synergistic effect in transactlvatlon is observed in the presence of two E2 binding sites, suggesting that an E2 tetramer (two dlmers bound to two sites) is a more optimal transactlvator This capacity to interact synergistically appears to reside in the amphipathic hehx domain in yeast, a fusion protein where an amphipathic helix of E2 has been replaced by a 'model' synthetic amphipathic helix [150], did not exhibit synergy between two binding sites although it was capable of sequence-specific transactlvatton [139] Interestingly, the E2 protein of BPV1 has been reported to activate transcription (albeit to a lesser extent) even in the absence of specific D N A binding to its target sites [122,146,152,1531, and this property has been localxzed to the N-terminal domain [1461 A possible mechanism involves protein-protein Interactions of the N-terminal amphlpathic helix region with transcription factors

30 Enhancers are cts-actlng D N A sequences that medmte transcriptional actwatlon m a manner that xs relatively independent of their orientation and &stance (upstream or downstream) in relation to the transcription start site Although E2 proteins bind with s~mdar afflmtaes to one or two t a n d e m l y r e p e a t e d A C C G N 4 C G G T palindromes in vitro, the nummum functional E2-responswe enhancer requires at least two E2 binding sites in most mammahan cells such as CV1 or HeLa (Refs 135-138 and 153, and Ttuerry, F et al, unpublished data) Furthermore, although the E2 enhancer works relatively independently of orientation, the strength of enhancer activity is dependent on the distance between repeated pahndromes and on the &stance from the promoter (Refs 137 and 153, Thaerry, F et al, unpubhshed data) The low transactlvatlon acUvlty seen for distant sxtes can be augmented by increasing their number, and tins acuvlty can be synergLstically enhanced by the presence of a single E2 binding site (normally not acuve by itself) placed close to the promoter (Thlerry, F et al, unpubhshed data) These observations suggest an interaction between E2 proteins bound to sites near and far from the promoter, perhaps by a mechanism in winch the intervening DNA is looped out Tins looping could bring the distant E2 proteins close to the promoter, enabhng them to Interact with cellular transcription factors bound there The precise mechamsm by winch E2 proteins actwate transcription is unknown Binding of E2 to its target sequence could render the nearby DNA more accessible to the transcription machinery Protein-protein interactions between tins macinnery and E2 could stabilize the

------

7100 I

7200 I P 185

BPVI

L1

7300 I

transcription lmtlatlon complex At present the protein targets of E2 are unknown, but it is likely that they include components of the transcnpt]on machinery such as R N A polymerase II or T F I I D Furthermore, the fact that different cell hnes exhibit different levels of transactivation with the same E2 enhancer constructions, strongly suggests that interactions of E2 with cell- or promoter-specific factors influences enhancer strength

1V Control of viral gene expression All PV genomes contain a noncodlng region located between the stop codon of L1 and the first A T G codon of E6 It ranges m sine between 454 (HPV8) to 979 (HPV1) nts and its sequence is much more variable than that of the ORFs Tins region, called the long control region (LCR) or upstream regulatory region (URR), is responsible for the control of viral D N A rephcatxon and gene expression It contains numerous transcriptional promoters as well as constitutive and reducible enhancers, some of winch are E2-dependent As we shall see below, some of the factors controlhng the species-, tissue- and cell-type tropisms of PVs reside in tins region

IV-A Transcrtptwnal regulatton by E2 Binding sites for the E2 protein have been found m the LCRs of all PVs sequenced to date [130] However, there is a large variation in the number, the position, the responsiveness to E2, and the extent of degeneracy of these A C C G N 4 C G G T pahndromes among the LCRs of

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;

7600 I

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CRPV

E2RE 1

L1

i

4'

I

I

E2RE2

P89

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P87

,

I o IK I I III1

fi

A P97

HPV16

L1

[

A

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A

I

I

G I KI

I

I

~ t ai

t

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constitutive plos HPV18

L1

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x A IKI A

A

AIA

!

A |

E6

constttuhve

HPV11

L1

GI

SlSp leIIKsplIssll ~m"e,, G~ dT 8 A ' I,~il

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~onstttuttve

F~g 40rgamzatxon of xaral long control regions The regulatory regions of selected viruses are shown, with major promoters re&cited by arrows TATA and CAAT boxes are re&cited by closed and open triangles, respectively Binding sites for the viral E2 protein are shown by closed (ACCGN4CGGT) and open (ACCN6GGT) rectangles Homologies to recognmon sequences of known transcrlpt~on factors are indicated as follows, although the presence of a certam sequence does not necessarily imply that specific binding of the factor has been observed Vertical lines, NF1/CTF s~tes, K, ' K sxte', keratznocyte-speclfiC motif, G, glucocortlcold response element, S, Sph motif, O, octamer element, A, AP1 site, X, SV40 enhancer core element, GT and P, motifs found in SV40 enhancer, C, C/EBP site In the case of the HPVs, consUtut~ve, E2-mdependent enhancers are underhned

31 different PVs (Fig 4) Furthermore, at least in the case of BPV1, the E2 O R F can encode different products with antagomstic regulatory functions Thus there is no doubt a great diversity in the responsiveness of viral transcription to E2 proteins The BPV1 model has been a useful tool The BPV1 L C R contains 11 E2 binding sites which exinblt a 300-fold range of affinities for E2 in vitro [145] The strength of the E2 interaction depends not only on the level of h o m o l o g y with the perfect consensus A C C G N 4 C G G T [136,145], but also on the surrounding D N A context [145] The BPV1 L C R can function as an E2-dependent enhancer [121] that can be cross-activated by E2 proteins of other PVs [124,131,132,134] Two different E2-responsive elements have been m a p p e d within the L C R [123] (Fig 4) One of them, E2RE1. is located between nts 7611 and 7806 and contains two pairs of E2 binding sites at each end which act in a cooperative manner [122,123] This element is required for the E2-dependent activation of the promoters P89 and P7940, which give rise to the transfornung gene product E6 as well as the E7 and E1 products involved in plasmtd rephcatlon Furthermore, transcription from the P2443 promoter just upstream of the E2 O R F requires an E2-dependent element in the LCR, possibly E2RE1 [84,85] Tins promoter directs transcription of the E5 transfornung protein as well as the full-length E2 transactlvator [36,37] and thus E2 transcription is autoregulated A single E2 binding site is found just upstream of P2443 but is not sufficient by itself for E2-dependent activation, although Its mutation shghtly reduces activity [84] Thus an interaction between E2 proteins bound at this site and at the E2RE1 seems possible A single E2 binding site is also found just upstream of the P3080 promoter whach codes for the E2 C-ternunal repressor, suggesting a smular interaction, although thas has not been estabhshed The exastence of E2 binding sites outside of the LCR, In the E 2 / E 4 / E 5 coding region, occurs m all PVs except the oncogemc types HPV 16, 18 and 33 Thus it is possible that the expression of E2 in these varuses may not be regulated in the same manner The second E2-responslve element in the BPV1 LCR, E2RE2, is situated between nts 7200-7386 [123], in proximity to the late promoter PL at nt 7250 [37] Tins element is not required for E2-medlated activation of P89 and P7940, but it might be important in the context of the entire L C R [123] Its location near PL suggests a possible role in the expression of the late genes, or perhaps in the passage from early to late transcription in the differentiated keratmocytes of the fibropapdloma The E2RE1 and E2RE2 elements define E2-dependent transcnpUonal enhancers that can activate heterologous promoters in the presence of E2 [123] Interestingly the only other paplllomavlrus to possess

numerous E2 binding sites in the L C R is CRPV [130] (Fig 4) Transcription from the CRPV L C R is transactivated by the homologous and by heterologous E2 gene products, but a basal, E2-mdependent level of transcription, which could be stimulated by E2, was also seen in some cell lines [131] All other PVs studied possess at most four A C C G N 4 C G G T motifs in their LCRs (Fig 4) In all HPVs except H P V l a , these moUfs are arranged in a charactensUc manner Two sites are situated very close to the A T G of the E 6 0 R F , between the putative C A A T and T A T A boxes of the proximal promoter A single site is situated about 100 nt further upstream, in the oncogemc varuses this site is not a perfect pahndrome whale in viruses assocmted with bemgn lesions it is Finally a fourth highly conserved slte is found about 400 nt further upstream The LCRs of all HPVs studied contain an E2-dependent enhancer (as well as constitutive and cell-type specific enhancer activities, see below) The role of each of these sites in E2-responsiveness has been elucidated m certain cases Studies with HPV18 show that the site furthest upstream of the E6 A T G is protected in footprlntlng assays by E2 [154], but m both HPV18 [135] and HPV16 [134] as well as H P V l l [133], this site does not seem to play a role m E2-dependent enhancer activity In HPV18 the imperfect p a h n d r o m e around nt 140 is not footprmted by E2 [154] and Its role is unknown However in H P V l l tins site is a perfect palindrome and is part of an E2-responslve element (which also c o n t a i n s the two p r o x i m a l sites) [127,132,133] The two most proximal sites seem to play the most important role in all HPVs, and it is noteworthy that their response to E2 depends on whether they are m enhancer or promoter configuration When cloned as an enhancer upstream of a heterologous promoter, the region contatmng these sites is responsive to E2-dependent transcriptional activation, and even heterologous E2 proteins such as BPV1 E2 can activate these sites [124,132-135,155,156] This E2-dependent activation requires the presence of at least two E2 binding motifs (Refs 135,155 and 156, and F Thaerry et al, unpubhshed data) In addition, the C-terlmnal E2 repressor can inhibit the activation seen w~th the full length protein [124,127] When the proximal region is cloned in the context of its natural E6 promoter, transcription is repressed by homologous and heterologous full length E2 proteins [155-157] Thus the full-length E2 protein can either actwate or repress transcnpUon, depending on the locauon of its target sites in relation to the promoter F o o t p n n t m g experiments have directly demonstrated the binding of E2 to the two proximal motifs [133,154] Thus it is hypothesized that since in HPVs these two sites are situated very close to the E6 promoter between

32 the CAAT and TATA boxes, repression ~s due to sterlc interference by E2 with the binding of the transcnpuon factors T F I I D or RNA polymerase Tins charactensUc locaUon of E2 binding sites in HPV LCRs (whereas in BPV1 for example the closest s~te is more than 130 nt upstream from the E6 promoter), suggests that for HPVs, a predormnant role of the full length E2 protein would be as a transcriptional repressor Tins could help to explain the transmon from bemgn to malignant lesions The virus exists as an eplsome m many bemgn lesions, but is found integrated into the host chromosome m most mahgnant lesions [48] Dunng the eplsomal part of the HPV hfe cycle, E2 may keep the expression of the transforming genes E6 and E7 tightly regulated However, lntegrauon usually occurs m a precise way winch disrupts the E 2 0 R F , such that E2 is no longer expressed As a result, the expression of E6 and E7 would be derepressed, leading to mahgnant conversion Thus, mtegraUon of the viral genome, perhaps speoflcally designed to select against E2 expression, may be a crucial step m the pathway to mahgnancy (Recall also that E2 is lmphcated indirectly m plasnud mamtenance and its absence precludes the reestabhshment of the virus m the form of eplsomes ) Clearly, the regulation of PV transcnpUon by E2 Is extremely complex, and the differences between different PVs suggests that no single model ~s sufficient In all cases, it is clear that E2 plays a key role m the expression of the transforming genes as well as in the maintenance of stable eplsomes However, whde the BPV1 LCR is either sUmulated or strictly dependent on E2 and functions m a wide variety of cell types [122,123,131,155], the LCRs of the HPVs possess consututwe enhancer acuvmes that function m only a hnuted number of cell types, m addition to E2-responslve elements As described below, these consUtutwe enhancers harbor bmdmg sites for a wide range of cellular transcription factor (Fig 4), implying an important role for cellular proteins, as well as E2, in HPV regulaUon The actual role of E2 dunng the wral cycle is not yet known It ~s possible that a delicate balance between posmve and negative regulation by E2 is a general feature of PVs but that it is acineved by different mechamsms In the case of BPV1, the balancmg factor would revolve direct competmon between the full length E2 transacUvator and the two shorter, repressor versions, for the same E2 binding sites Tins situation is further comphcated by the fact that the expression of these E2 proteins is itself autoregulated For HPVs, the exastence of truncated E2 proteins has not been estabhshed and it is more hkely that the full length E2 protein would exert an activator or repressor funcUon according to the position of the s~te to wluch ~t brads

IV-B Role of cellular factors m P V transcrtptton Although E2 plays a key role m regulation of PV gene expression, it is clear that numerous cellular factors are also revolved In BPV1, transcnpUon directed by the LCR is acuvated by E2 However further stu&es have revealed a low level consututlve enhancer between nts 7215-7353 [136,153], winch contains numerous sites for transcription factors including an octamer element, SV40 enhancer core element, and N F 1 / C T F 1 sites, but no E2 binding sites (Fig 4) Tins enhancer can be actwated by E2, and activation is increased by association with a single E2 binding site (winch by itself is normally mactwe) These results point to a possible interaction of E2 w~th cellular factors, winch may not be totally dependent on its D N A binding, as suggested previously [122,146] However, there is not direct evidence for the binding of cellular factors to tins region The case of the HPVs is even more complex Unlike the BPV1 LCR, winch depends almost entirely on E2 for acUvaUon, the HPV LCRs have a strong E2-mdependent basal activity, and although E2 can enhance tins actlwty, tins effect ~s not always extremely strong in the context of the entire LCR [127,132,135,155,156] In the case of HPV18, funcuonal studies using fragments of the LCR cloned upstream of heterologous promoters, have defined three chstmct, independent enhancer elements wltlun the LCR [135,156,158] The first element is located proxamal to the E6 cap s~te, contains two E2 binding sites, and is E2-dependent (Recall that these two sites are located between the CAAT and TATA boxes and in the context of the natural E6 promoter, E2 binding to these sites results m repression [155,156] ) A constltuuve enhancer is located 200-400 nt upstream of the E6 cap s~te, it reqmres no HPV18encoded factors for function [135,156,158] (Fig 4) Finally, another enhancer is located about 500 bp upstream of the E6 cap site, and contains a single E2 binding site winch is footpnnted by E2 [154] but is not essentml for enhancer actwlty [135,154] Tins element has been shown to be actwated only m E6-contatmng cells It ~s postulated that m fact tins regton may contmn a transcriptional promoter involved m eplsomal maintenance [135,159] The intact HPV18 LCR exinints a cell-type specificity for HeLa and SWl3 cells [160] and keratmocytes [156], but works poorly m nonmammahan cells [160] The constitutive enhancer element by itself is only active in some eplthehal cell hnes meludmg some but not all cervtcal carcinoma cell hnes and in rodent and human dermal fibroblasts, and is reactive m other noncervical cells except for one neuroblastoma cell hne [135,156,158] Furthermore, the consUtuuve enhancer

33 activity is repressed by adenovarus E1A products [158,160] The orgamzatlon of the HPV16 LCR is similar An E2-dependent enhancer located proxamally to the E6 cap site has been defined [134], analagous to that of HPV18 Further upstream, a 400 bp region (nt 74547854) exinbxts a strong constitutive, E2-1ndependent enhancer actlvaty which Is funcUonal In HeLa cells but not in the human breast carcinoma cell hne MCF-7, even though both are of eplthehal ongm [161] This fragment contains an imperfect pahndromac E2 binding site (Fig 4) Within this same 400 bp region is an independent actlvaty exerted by the presence of a glucocorticold response element (GRE) at nt 7641-7655, winch confers strong mduclbihty by dexamethasone of the P9v promoter in both HeLa and MCF-7 cells [161] In HeLa cells where both elements of this region are active, their combined activity stimulates transcription by a factor of 500 The G R E element is footprmted by proteins in cellular extracts and by purified rat glucocortlcoid receptor protein Tins element is also found in the LCRs of all other PV genomes studied (Fig 4), although its position is not conserved and its function is entirely unknown It is interesting, however, that epidermologxcal studies have revealed an elevated susceptlbdlty of pregnant women to PV Infection The role of tins element in HPV-medlated transformation remains to be elucidated Recall that in cases where the viral genome is integrated into the host chromosome, vlrally-directed expression forcibly anses from these constitutive enhancers, since the E2 protein is absent Thus, these different constitutive and E2-dependent enhancer domains undoubtedly play different roles dunng different stages of the HPV life cycle Unlike HPV16 and HPV18, H P V l l is lmphcated in benign gemtal lesions, but the orgamzatlon of its LCR is similar, including an E2-dependent enhancer winch in the context of the E6 promoter as repressed by E2, probably through stenc interference as described above [127,132,157] In addition, two cell-specific, E2-independent consUtutwe enhancers have been m a p p e d [127,133,157] (Fig 4) One of the most intriguing questions in papdlomavxrus research has been to understand the source of their stnct eplthehal specificity Interestingly, a consensus sequence AANCCAAA termed the K site, winch is present in many keratxnocyte-speclfiC genes [162], is found (m inverted onentatlon T T N G G T T r ) in a similar position upstream of the E 6 0 R F m all genital HPVs sequenced, as well as m BPV1 and CRPV [130,160,163,164] (Fig 4) The fact that the constitutive enhancers of HPV16 [124], HPV18 [154,165], and H P V l l [157] confer cell-specificity and that they each contain tins K site, makes it tempting to infer a direct role for this sequence in the epithelial tropism of PVs

However, tins suggestion remains controversial, and only recently has evadence begun to accumulate that a keratmocyte-speclfic cellular factor can bind specifically to this sequence in the HPV18 constitutive enhancer [165] Furthermore, the observation that a second constitutive enhancer in the H P V l l LCR winch does not contain the K site can also act in a cell-specific manner [157] suggests that there is hkely to be more than one deterlmnant for epithelial cell-specificity The enhancer domains of PV LCRs also contain binding sites for numerous cellular factors, and although their role is not yet known, it seems hkely that they influence the species and tissue-specific tropisms of these viruses Their importance is already evadent in the different response to E2-mediated activation m different cell hnes, as discussed above In vatro footpnntlng wath different cell and tissue extracts has identified eight protected sites along the LCR of HPV18, most of which are present in all the extracts [154] These include two AP1 sites, one In the constitutive enhancer and one in the proximal E2-dependent enhancer (Fig 4) The consUtutlve enhancer regions of the H P V l l LCR contain sequences homologous to the GT-, Sph- and P-motifs found in SV40 and polyomavxrus enhancers, as well as C T F / N F 1 sites [133,157], all of whose functional slgmflcance Is unknown Recently, it was shown that HeLa cell extracts Dye 23 footpnnts that protect 557 bp of the 900 bp HPV16 LCR [166] Seven of the footprints m the HPV16 constitutive cell-specific enhancer element contain recogmtlon sites T T G G C T for C T F / N F 1 (Fig 4), and it is suggested that these sites may have some role in enhancer function [166,167] The complexity of PV transcriptional regulation is by now evident, but m any case it seems clear that transcnptlon of viral genes occurs through the Interaction of many elements, both viral and cellular, which determines In part the species-, tissue- and cell-speclflclties of PVs IV-C Additional events reqmred for mahgnant conversion ~ A further comphcatlon involves the posslblhty that infection by the oncogemc HPVs may actually not be sufficient by itself for the development of cervical cancer Several hnes of evidence suggest that a second event is required First, there is generally quite a long latency penod between mfectaon and the development of mahgnancy Second, in many instances a cofactor for carcinogenesis can be clearly identified For example (0 epldermologmal studies show a 5-10-fold elevated rate of cervical cancer In HPV16- or HPV18-mfected, smoking women over slmdar nonsmokers [168], (n) there is a preferential locahzatlon of cancerous epldermodysplasia verruclformls lesions m areas exposed to

34 U V light [169], and (m) m a h g n a n t conversion of bovine alimentary tract papillomas is seen only in cattle ingesting the c a r c m o g e m c bracken fern [6] Furthermore, HPV16 and HPV18 do not gwe a fully transformed phenotype to rodent cells, although they do immortalize these cells [18] F o r complete cellular transformation, the cooperating r a s oncogene is required [39,77] It is not k n o w n whether these effects are due to the action of these agents directly on control sequences in the viral genome, or to their redirect action on the general state of the cell, winch in both cases could potentiate the oncogemclty of the H P V transforming genes A recent example of the first case [170] involves acuvatlon of HPV18 gene expression b y the t u m o r promoter T P A or b y certmn wral t r a n s c n p t l o n factors encoded b y the herpes simplex virus type 1, b o t h of winch act through specific cts-sequences in the HPV18 LCR

the mteractJon between viral and cellular factors plays a role in these effects Despite all of our recent advances however, m a n y questions r e m a m unanswered, in part due to the htluted systems avmlable for study Continued research should shed m o r e hght on these issues, illustrating the uUllty of the papfllomavlrus model system as well as providing possible approaches for the treatment of PV infection

Acknowledgements Tins work was supported b y grants from the InstltUt National p o u r la Sant6 et la Recherche M6&cale, the Association p o u r la Recherche sur le Cancer, the Llgue Natlonale Franqalse contre le Cancer, and the F o n d a tlon p o u r la Research M6dlcale Franqmse

References V. Concluding remarks It is well recognized that paplllomavlruses represent an important chmcal issue However, recent advances m PV research have given rise to the idea that these viruses also furmsh a n u m b e r of interesting features winch can hopefully help to clarify our understanding of the control of gene expression and the mechanisms of cellular transformation For example, PVs possess at least three transforming proteins, E5, E6 and E7, that seem to work b y different m e c h a m s m s Winle the E6 protein rmght exert its acav~ty through direct binding to particular D N A sequences, (although tins remains to be estabhshed), the transforming activity of E7 does not seem to involve D N A binding but instead functions similarly to the adenowrus E1A protein In a d d m o n , E5 m a y work in yet another way, by lnterfenng wath growth factor receptor regulation at the cell surface Another intriguing feature of PVs concerns the family of E2 transcriptional regulatory proteins W e have learned that a single gene can code for products with antagomstlc regulatory functions (m the case of BPV1), and furthermore, that even the same gene p r o d u c t can e~ther repress or actwate transcription, both b y binding to the same D N A sequence (as m HPVs) These proterns clearly furmsh an interesting model for the study of eukaryotic transcription W e have seen that PV transcnptlonal promoters exlublt a complexity slrmlar to that of tugher eukaryotes, with a vast array of binding sites for wral as well as cellular transcription factors These features, m a d d m o n to specific interactions between the infecting v~rus and the host cell, no d o u b t function as p r i m a r y determinants for the dwergent host- and cell-specificmes of individual virus types, and ewdence was presented that

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