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Functionsof human papillomavirus E6 and E7 oncoproteins Alan 1. Farthing and Karen H. Vousden apillomaviruses are a The identification of certain human 51,52,56,57, 58,59 and 61 group of small DNA papillomaviruses as human tumour viruses (Ref. 7). More recent studies viruses that infect mainly has been paralleled by our understanding are beginning to show that a epithelial tissue of a diverse of how the viral oncoproteins function. further distinction will be range of animals, from the The virally encoded E6 and E7 proteins possible, since some of the chaffinch to the elephant. act, in part, to abrogate the activities of high-risk HPVs appear to be The productive lesions caused the tumour suppressor proteins ~53 and found predominantly in highby these viruses are usually pRB. The interaction between these viral grade but noninvasive lesions, benign, self-limiting hyperproproteins and regulators of cell growth and therefore represent an liferations, which in humans offers targets for future therapeutic intermediate risk group6. are recognized as the cosmetdevelopments. Interestingly, HPV infection ically distressing, but rarely is also associated with the A.J. Farthing and K.H. Vousden are in the life-threatening, wart. The high development of the less comLudwig Institute for Cancer Research, profile enjoyed by the papmon disease anal carcinoma, St Mary’s Hospital Medical School, illomaviruses results almost in which a similar distinction Norfolk Place, London, UK W2 I PG. ._._ _ between high- and low-risk entirely from the observation viral types is found8. that certain viral types show clear oncogenic potential, and human papillomavirus Since it was first suggested by zur Hausen in 19769, a large body of epidemiological and experimental (HPV) infection contributes to the development of an evidence has built up to support a role for HPV infecextremely common cancer in women. tion in the etiology of cervical cancer. Despite this The first association with malignancy was recogstrong association, however, HPV infection alone is nized over 50 years ago, when benign lesions induced not sufficient for the development of invasive disease by the cottontail rabbit papillomavirus were induced and represents only part of the multistep tumorigenic to progress to invasive carcinoma’. More than 65 process. Up to 20% of the normal population carry types of HPVs have been identified, some of which are genital HPVs, an incidence that decreases with age present in a variety of malignancies. HPV-associated lesions in the rare disorder epidermodysplasia verruciafter a peak in early adulthoodlOJ1. Clearly not all formis can give rise to squamous-cell carcinomas2, HPV infections result in malignancy, and progression -but our most detailed understanding of the mechof virally induced lesions requires additional events, anisms by which these viruses play a role in the which may be both genetic and epigenetic. carcinogenic process in humans comes from the study of cervical cancers, over 90% of which contain HPV HPV-encoded oncoproteins DNA3,4. The first indication that some of the viral proteins Many types of HPV can infect the female genital themselves may participate in the development, and tract, but epidemiological studies show a clear distincpossibly also the maintenance, of malignancies was tion between the low-risk viruses, associated predomiprovided by studies of the viral genome in benign and nantly with benign disease, and the high-risk viruses, malignant disease. In productive lesions, the HPV which are most prevalent in malignant lesion+. DNA is maintained episomally in the nucleus of the cell, often in large amounts. Although infection Genital warts (condyloma acuminata), which have long occurs in the basal epithelial cells, probably after been recognized to be caused by infection with HPV, wounding or trauma, viral replication occurs in the are found to contain mostly HPV types 6 and 11, and, more highly differentiated layers of the epithelium12, although these lesions represent abnormal proliferand virion production is critically dependent on ations of the epithelium, they only rarely show eviterminal differentiation of the infected ce1113. Maligdence of malignant transformation. Many of these nant conversion of host epithelium is clearly not part low-risk HPV types, which also include HPV types of the normal viral life cycle, and cancer cells, which 13,32,34,40,42,44,53,54,55 and 63 (Ref. 7), have do not undergo normal differentiation, cannot proalso been identified in low-grade cervical dysplasias, duce virus. In many malignant cells the viral DNA which frequently regress spontaneously. However, the high-risk genital HPV types are found in almost all becomes integrated, frequently with loss of various parts of the viral genome i4-16. There is no convincing high-grade cervical dysplasias and invasive cancers. The most common of these is HPV-16, but this group evidence that integration is consistently specific to also includes HPV types 18, 30, 31, 33, 35, 39, 45, any part of the host genome, although some reports

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have revealed integration in the vicinity of cellular oncogenes l’. Virus integration usually leads to the disruption of the E2 gene, which encodes the major proteins controlling viral transcription, and the only region of the virus consistently retained and expressed is that encoding the E6 and E7 proteins1*-20. These observations suggest that the integration event Zinc loop Zinc loop contributes to the oncogenic process by disrupting the strictly regulated expression of E6 and E7. E7 The importance of E6 and E7 in oncogenesis has been supported by further studies showing that each of these viral proteins El A/LT similarity displays immortalizing and transforming activities when expressed in cells in culture. Although E7 is the principal HPV-encoded oncogene when assayed in rodent cells2’, in UU pRB CKII Zinc loop primary human genital keratinocytes, cooperation between E6 and E7 is required for plO7 efficient immortalization2*. Interestingly, these p130 cells differentiate in culture to resemble the Fig. 1. Schematic representation of human papillomavirus type 16 E6 and E7 premalignant cervical lesions termed cervical proteins showing the cysteine-rich zinc-binding regions. E7 shows some structural intraepithelial neoplasia (CIN)23, supporting similarity to adenovirus ElA and simian virus 40 large T antigen (LT) in the epidemiological evidence that these lesions domain responsible for interaction with the pRB protein family. Serine targets for phosphorylation by casein kinase II (CKII) are also shown. are caused by infection with high-risk HPV typesz4. Furthermore, E6- and E7-immortalized human cells are not tumorigenic, although they can be induced to progress after exposure to time for DNA repair, or to cell death through apopcarcinogens or additional oncogenes2s. The corretosis32. Cells lacking this checkpoint function have lation between these in vitro studies and clinical an increased genome instability33, and therefore the observations therefore provides persuasive evidence potential to acquire oncogenic mutations. The importthat E6 and E7 are bona fide viral oncoproteins. ance of the loss of p.53 function in carcinogenesis is confirmed by the discovery of mutations in the gene E6 and E7 are both small nuclear proteins, around encoding ~53 in over 50% of all cancers34. 150 and 100 amino acids, respectively, that bind zinc through cysteine motifs (Fig. 1) and show some The consequence of the interaction between E6 structural similarities to each other26. Their oncoand ~53 is the rapid degradation of the ~53 protein genie potential appears to be related to an ability to through ubiquitin-directed proteolysis3s. E6 asabrogate the activity of cell proteins whose normal sociates with another cell protein, E6-AP, and the complex functions as a ubiquitin ligase36. Consistent function is to negatively regulate cell growth2’. Parwith these in vitro analyses, ticularly interesting are the interactions with proteins E6-expressing cells previously described to be the products of tumour cannot stabilize ~53 in response to DNA damage, suppressor genes. In these respects, HPVs appear and therefore fail to undergo the expected block at to have evolved similar mechanisms of action to the the Gl stage of the cell cycle3’. Several activities of otherwise unrelated adenovirus and simian virus 40 the ~53 protein have been described, including the (Ref. 28). These small DNA viruses cannot encode all ability to regulate transcription of other cellular the proteins necessary for their own replication and genes38, an activity that correlates well with growth so depend on the host replicative machinery. The suppression. Not surprisingly, expression of E6 function of the viral oncogene products in the normal efficiently abrogates this activity3y. Although the viral life cycle is almost certainly to help maintain cell interaction between E6 and ~53 is essential, the division, since viral infection occurs in cells normally subsequent degradation of ~53 is not absolutely redestined for terminal differentiation and ultimately quired to interfere with the transcriptional control death. exerted by ~53 (Ref. 40). This, combined with the observation that the ability of E6 to target proteins EB abrogates p53 activity for degradation is not limited to ~53, offers the possibility that other cell proteins involved in growth The best-understood property of E6 is its ability to interact with the important cell regulatory protein regulation are targets for E6, although such proteins pS3 (Ref. 29). The entire function of ~53 has not been remain to be described. Nevertheless, it seems likely established, but it appears to function as a suppressor that E6 function contributes to the oncogenic poof cell growth in response to stress30. The p53 protein tential of the high-risk HPVs, particularly as HPVaccumulates in response to DNA damage and may associated cervical cancers show an unusually low contribute either to arrest of the cell cycle31, allowing incidence of somatic ~53 mutation4’.

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cell, transcriptionally active E2F is released after phosphorylation of pRB by cyclin-dependent kinases (cdks), leading to cell-cycle progression4’. The interaction of E7 with the negatively regulating pRB complex causes E2F to dissociate and allows unscheduled entry into DNA synthesis48. The pleiotropy of E7 function is enhanced further by its ability to target the pRBrelated proteins, ~107 and ~130 (Ref. 49), which also control cell growth through apparently similar mechanisms. E6 and E7 potentially function in the same pathway Recent studies have suggested the interesting possibility that ~53 and pRB may contribute to the same regulatory pathway. Activation of cellular ~53 in response to DNA damage increases transcription of a cellular gene, termed Wuf-2 (Ref. 50). This gene was independently isolated as CIPZ, encoding a protein that interacts with cdks, SDIZ, a gene active in senescent cells, and ~21, a component of the cyclin-cdk complexes in normal, but not Cell-cycle progression Cell-cycle arrest transformed, cellssl. The ability of Waf-1 to inhibit cdks, which can phosphorylate pRB, provides a mechanism by which p.53 could induce cell-cycle arrest by preventing the expression of E2F-responsive genes (Fig. 2). The HPV oncoproteins would therefore be able to act at two separate points in this cascade, by abrogating either ~53 or pRB function. This model is Fig. 2. A model for ~53 function by arresting cell growth through a pathway that may be targeted by both E6 and E7. The normal cell cycle progresses as pRB is phosphotylated and inactivated supported by several observations, during Gl by cyclin-dependent kinases (cdks). releasing the transcription factor E2F and allowincluding the ability of E7 to overing the expression of genes necessary for entry into DNA synthesis. This process is blocked come the growth inhibitory effects after DNA damage through the activation of ~53, which directs the expression of Waf-1, an inhibiof wild-type ~53 under some cirtor of the cdks responsible for releasing the pRB arrest. Both E6 and E7 potentially function to abrogate the DNA-damage-induced cell-cycle arrest: E6 by targeting ~53 for degradation and E7 cumstancess2, and the detection of by binding pRB, causing transcriptionally active E2F to be released. high levels of ~53 protein in E7expressing cellss3. A simple interpretation is that E7, in acting downstream in the nathwav. allows the cells to mount an E7 targets pRB complexes efficient, if futile, ~53 stabilization response. E7 binds to the product of another tumour suppressor This is clearly an oversimplification of the functions gene, RB-1 (Ref. 42), an interaction that correlates of E6 and E7, which both have additional activities as with the immortalizing functions of the viral protein well as the known interactions with cellular proteins and may even be sufficient for the ability of E7 to described above. For example, mutations in the gene induce cellular DNA synthesis43,44. Unlike ~53, pRB encoding E7 affecting the casein kinase II phosphorylappears to be important in the normal cell cycle, reguation site or the amino terminus of the protein reduce lating progression through Gl by binding and inactitransforming activity without significantly affecting vating a variety of cell proteins, including several the ability to interact with the pRB protein family43,44 of these transcription factors 45. The best understood (Fig. 1). Efficient immortalization of human genital is E2F, which regulates the expression of many genes epithelial cells requires cooperation between E6 and important for cell-cycle progression4h. In the normal I

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E7, suggesting that each contributes a unique activity, and it seems likely that both viral and cellular proteins have multiple functions. Determinants of high and low risk The identification of HPVs with differing oncogenic activities has allowed the dissection of functions that may contribute to malignant progression. At least some of the differences between high- and low-risk viruses depend on the biochemical activities of E6 and E7, since the low-risk proteins have only a low capacity to immortalize cellss4. High-risk virus-encoded E6 and E7 proteins target the cellular tumour suppressor gene products, such as pRB and ~53, with much greater efficiency than do the low-risk virus-encoded proteins, and amino acid substitutions that increase the pRB-binding activity of the HPV-6 E7 protein concomitantly enhance its transforming functionss. Other activities of E6 and E7, as well as their ability to prevent cell-cycle arrest, probably also contribute to oncogenic activity. Human epithelial cells immortalized with high-risk HPV types have chromosomal abnormalitiess6, which may be related to E7 expressions’, and the ability to induce genomic instability after E6 abrogation of ~53 function could be an additional factor that contributes to malignant potential. However, the basis of the differences between the high- and low-risk viral types is likely to be complex, involving several other factors, such as the ability of certain viral types to integrate, a possible predilection of high-risk HPVs for susceptible cells, and variations in oncogene expression and in host determinants, such as immune responses. Despite their association with oncogenesis, malignant progression is not part of the normal viral life cycle for any of the HPVs, and E6 and E7 must also play a role in normal viral replication. Both high- and low-risk E6 and E7 proteins share some ability to target cellular proteinsZ8J9, and abnormal cell proliferation induced by the low-risk HPVs is obvious in the appearance of genital warts. These activities may be necessary to maintain host-cell replication after viral infection and would therefore be expected to be exhibited by all HPV types. Quantitative, rather than qualitative differences in function may be responsible for the observed oncogenic activity, although how these benefit normal viral replication is unknown. Therapeutic possibilities The ability to modify parts of this carcinogenic process has significant therapeutic possibilities, and future research is being directed towards disrupting the activities of the viral oncoproteins. If their binding to pRB or ~53 can be prevented, cell-growth regulation may be restored, preventing or controlling further development of malignancy. Preliminary experiments have shown that small peptides identical to the pRBbinding region of E7 can specifically block the ability of the protein to disrupt pRB-containing complexes60. However, the ability of such peptides to affect the growth of E7-expressing cells has not yet been demonstrated, and there is a danger that they may act

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as agonists, rather than antagonists, of E7 function. Despite these caveats, our increasingly detailed understanding of the functions of a human tumour virus provide a unique opportunity to prevent and treat a common, and often fatal, disease.

Acknowledgements We wish to apologize to the authors of all the excellent papers that contributed to much of the information summarized here, but which we cannot cite because of space constraints.

References 1 Rous, P. and Beard, J.W. (1935)J. Exp. Med. 62,523-548 2 Fuchs, PG. and Pfister, H. (1990) Pupillomauirus Rep. 1, l-4 3 Munoz, N. and Bosch, F.X. (1992) in The Epidemiology of Cervical Cancer and Human l’apillomavirus (Munoz, N. et al., eds), pp. 243-261, IARC ScientificPublications No. 119 4 zur Hausen, H. (1989) Cancer Res. 49,4677-4681 5 De Villiers, E.M. (1989) I. Viral. 63,4898-4903 6 Lorincz, A. et al. (1992) Obstet. Gynecol. 79,328-337 7 von Knebel Doeberitz, M. (1992) Eur. J. Med. 1,415-423 8 Wells, M. (1990) Papillomavirus Rep. 1, 1-2 9 zur Hausen, H. (1976) Cancer Res. 36,794 10 Melkert, P.J.W. et al. (1993) ht. J. Cancer 53,919-923 11 de Sanjose, S. et al. (1992) in The Epidemiology of Cervical Cancer and Human Papillomavirus (Munoz, N. et al., eds), pp. 75-84, IARC ScientificPublications No. 119 12 Broker, T.R. and Botchan, M. (1986) Cancer Cells 4,17-36 13 Kurman, R.J. et al. (1984) in Advances in Immunohistochemistry (DeLellis, R.A., ed.), pp. 201-221, Masson 14 Durst, M.A. et al. (1985) 1. Gen. Viral. 66,1515-1522 15 Cullen, A.P. et al. (1991) J. Viral. 65,606-612 16 Das, B.C. et al. (1992) 1. Gen. Virol. 73,2327-2336 17 Durst, M. et al. (1987) Proc. Nutl Acud. Sci. USA 84, 1070-1074 18 Schwarz, E. et al. (1985) Nature 314,111-113 19 Choo, K-B. et al. (1987) Virology 161,259-261 20 Wilczynski, S.P. et al. (1988) Virology 166,624-627 21 Vousden, K.H. (1991) Semin. Viral. 2,307-317 22 Hawley-Nelson, P. et al. (1989) EMBOI. 8,3905-3910 23 Hudson, J.B. et al. (1990) 1. Viral. 64,519-526 24 Koutsky, L.A. et al. (1992) N. Engl. J. Med. 327,1272-1278 25 Klingelhutz, A.J. et al. (1993) Oncogene 8,95-99 26 Danos, 0. and Yaniv, M. (1987) Cancer Cells 5,145-149 27 Vousden, K.H. (1993) FASEB].7,872-879 28 Levine, A.J. (1990) Virology 177,419-426 29 Werness, B.A. et al. (1990) Science 248,76-79 30 Lane, D.P. (1992) Nature 358,15-16 31 Kastan, M.B. et al. (1992) Cell 71,587-597 32 Lowe, S.W. et al. (1993) Cell 74,957-967 33 Yin, Y. et al. (1992) Cell 70,937-948 34 Hollstein, M. et al. (1991) Science 253,49-53 35 Scheffner, M. etal. (1990) Cell 63,1129-1136 36 Scheffner, M. et al. (1993) Cell 7.5,495-505 37 Kessis, T.D. et al. (1993) Proc. Nut1Acud. Sci. USA 90, 3988-3992 38 Vogelstein, B. and Kinzler, K.W. (1992) Cell 70,523-526 39 Mietz, J.A. et al. (1992) EMBO J. 11,5013-5020 40 Crook, T. et al. (1994) Oncogene 9,1225-1230 41 Crook, T. et al. (1992) Lancet 339,1070-1073 42 Dyson, N. et al. (1989) Science 243,934-937 43 Barbosa, M.S. et al. (1990) EMBO I. 9, 153-160 44 Banks, L. et al. (1990) Oncogene 5,1383-1389 45 Wiman, KG. (1993) FASEB ].7,841-845 46 Nevins, J.R. (1992) Science 258,424-429 47 Hollingsworth, R.E. et al. (1993) Curr. Opin. Cell Biol. 5,

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Retrovirusinsertioninto herpesviruses Robert J. Isfort, Richard Witter and Hsing-Jien Kung

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etroviruses are naturally occurring, self-propagating pathogens and mutagens of humans and animals. They exert their mutagenic effects by integrating into the host genome, which is a natural step in their replication cycle (for a review, see Refs 1,2). Integration is catalysed by a viral integrase, and is an efficient process with little sequence specificity of the integration site3v5. These properties, together with their high efficiency of replication and infection, make retroviruses powerful insertional mutagens for .eukaryotes. Retroviral integration can inactivate host genes by disrupting the genome and activate host genes by providing a viral promoter and translation signa11p2. This can result in cancer and developmental abnormalities at the level of the organism. A novel type of retroviral insertion has been described recently in which the minichromosome of DNA viruses serves as an insertional target for retroviruse+‘. In this article, we focus on retrovirus insertion into herpesviruses and discuss other related phenomena in this context. Retroviral integration into herpesviral genomes The discovery that retroviruses can alter herpesviruses genetically came from our studies of the chicken retrovirus, reticuloendotheliosis

Recent studies have indicated that retroviruses can integrate into and mutate the genomes of herpesviruses during co-infection. This interaction has the potential to change the host range and pathogenicity of both viruses and result in novel infectious agents and diseases. This phenomenon also allows genetic material to be exchanged between these viruses and their hosts. R.J. lsfort is in the Human Safety Dept of The Procter and Gamble Company, Miami Valley Laboratories, PO Box 398707, Cincinnati, OH 45239-8707, USA; R. Witter is in the USDA ARS Regional Poultry Research Laboratory, 3606 E. Mount Hope, East Lansing, MI 48823, USA; and H-1. Kung is in the Dept of Molecular Biology and Microbiology, Case- Western Reserve University, School of Medicine, Cleveland, OH 44106, USA.

virus (REV), and the chicken herpesvirus, Marek’s disease virus (MDV). These viruses have a common disease pathology, including the induction of T cell lymphomas, and have similar tissue and cellular tropisms both in vivo and in z~itro~~~. An initial unexpected result was that an REV long terminal repeat (LTR) probe crosshybridized with the MDV genome6. Direct sequencing of the relevant regions confirmed that there are several regions of REV LTR similarity (70-85% identity in stretches of 20-30 nucleotides) near the junctions of the 0

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unique long region (U,) and the repeat flanking U, (RL) of the oncogenie MDV serotype I genome, but not in nononcogenic serotype#. This suggests that the similar sequences are the remnants of REV integrations that occurred in the distant past after the divergence of the different serotypes of MDV. An attenuated strain of serotype I MDV that harbors newly integrated REV LTRs was subsequently identified6. These new insertions are at different sites from the ancestral insertions, and apparently arose from laboratory contamination of the MDV cultures with REV during an attempt to develop an MDV vaccinelO. Encouraged by these results, we have attempted to integrate REV proviruses into the MDV genome by co-infection of duck embryo fibroblasts under controlled conditions in vitro. Integration of REV proviruses into the MDV genome can be seen by the fourth copassage (i.e. 4 weeks after infection)6. This experiment has now been extended and reproduced in two other systems where REV integrates into herpesvirus of turkey (HVT; MDV serotype III) and avian leukosis virus (ALV), a retrovirus unrelated to REV, integrates into MDV. In all cases, retroviral integration can be detected between the second and fourth copassage (R.J. Isfort et al., unpublished). Retroviral proviruses thus appear to integrate rather

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