Antiviral Therapy 5:229–242

Review Searching for antiviral drugs for human papillomaviruses

Mark R Underwood1, Lisa M Shewchuk2, Anne M Hassell2 and William C Phelps1*

1Department of Virology, GlaxoWellcome, N.C., USA 2Structural Chemistry, GlaxoWellcome, N.C., USA

*Corresponding author: Tel: +1 919 483 9242; Fax: +1 919 315 5243; E-mail: [email protected]

The human papillomaviruses (HPVs) are ubiquitous cervical carcinoma. The absence of a simple monolayer human pathogens that cause a wide variety of benign cell culture system for analysis and propagation of the and pre-malignant epithelial tumours. Of the almost has substantially retarded progress in the devel- 100 different types of HPV that have been characterized opment of diagnostic and therapeutic strategies for to date, approximately two dozen specifically infect HPV infection. In spite of these difficulties, great genital and oral mucosa. Mucosal HPVs are most progress has been made in the elucidation of the mole- frequently sexually transmitted and, with an incidence cular controls of virus gene expression, replication and roughly twice that of virus infection, are pathogenesis. With this knowledge and some important considered one of the most common sexually trans- new tools, there is great potential for the development mitted diseases throughout the world. A subset of of improved diagnostic and prognostic tests, prophy- genital HPVs, termed ‘high-risk’ HPVs, is highly associ- lactic and therapeutic , and traditional antiviral ated with the development of genital including medicines.

A serious and mysterious disease

Unlike most sexually transmitted diseases (STDs), or mucosal . However, a small proportion of genital human papillomavirus (HPV) infection is not a genital infections with high-risk HPVs result in reportable disease, so precise epidemiological numbers progression to invasive cervical . Although HPV are difficult to obtain [1]. Nonetheless, genital HPV infection is not sufficient for the induction of cancer, infection is clearly one of the most prevalent sexually the causal association between high-risk HPV infection transmitted diseases in the world today. The US Center and is very strong. The odds ratio for for Disease Control has estimated that there are nearly HPV infection (particularly HPV-16) and cervical 5.5 million new cases per year in the USA alone cancer is actually higher than that between cigarette resulting in >20 million active cases [2]. Worldwide, smoking and lung cancer [7,8]. there may be >500 million people infected with genital During the 1930s, cervical cancer was the most HPV. In an alarming study of college-age women, 43% common cause of cancer death in the USA [9]. The PAP converted from HPV-negative to HPV-positive during (Papanicolaou) screen, introduced almost 50 years ago, the 3-year period of study [3], reflective of a very high has become the most effective and frequently used incidence of infection and risk of exposure for students method for the detection of cytopathic effects due to on college campuses. It has been suggested that up to HPV infection of cervical . Between 80% of sexually active women will acquire an HPV 1947–1984, there was a 70% decline in US mortality infection at some point during their lifetimes [4]. due to cervical cancer, which is directly attributed to Furthermore, the use of condoms does not appear to early detection and ablation of HPV infected, pre- offer protection against the transmission of HPV to malignant tissue [10]. However, the PAP screen is not partners [5]. The financial burden is high; during 1994 commonly used in developing countries, and about the estimated cost of treating HPV infection in the US 200000 deaths per year worldwide are attributed to was $3.8 billion, and the cost for treatment of cervical cervical cancer, ranking it as the second leading cause cancer was $737 million [6]. of cancer death in women behind breast cancer. The majority of HPV infections induce benign, often Despite the prevalence and serious sequelae of HPV self-limiting or transient epithelial lesions – cutaneous infection, HPV remains a mysterious to

©2000 International Medical Press 1359-6535/00/$17.00 229 MR Underwood et al.

most. In a recent survey, 70% of US women were expression of the E6 and E7 viral oncoproteins. These unaware of an aetiological link between virus infection viral genes are known to disrupt two critical cell cycle and cervical cancer, and 76% had never even heard of regulatory pathways featuring the and the HPV (2000 National Cervical Cancer Public Education retinoblastoma (pRB). The deregulation of Campaign). As awareness of HPV-associated disease controls on cellular proliferation and differentiation is increases in the coming years, there is very likely to be thought to directly contribute to the hyperplastic a concomitant rise in pressure for improved diagnostics phenotype characteristic of pre-malignant lesions [17]. and effective therapies. Advances during the past High-risk HPV infection is clearly not sufficient to decade in our understanding of the HPV lifecycle have induce cervical cancer, as only a subset of infected provided important insights into novel therapeutic women will develop cancer, and progression to malig- opportunities. The intent of this review is to focus on nancy requires 10–20 years. These observations potential therapeutic targets for the identification of highlight a potential role for somatic mutations and new antiviral agents that will selectively and effectively suggest that a defining risk factor might be persistence treat HPV disease in the clinic. of high-risk infections [8]. The development of invasive For more detailed descriptions of papillomavirus cervical cancer typically occurs by progression through molecular and cellular biology, as well as immunolog- a series of neoplastic stages characterized by increasing ical and epidemiological aspects of the virus and dysplasia and severity [cervical intraepithelial neoplasia associated diseases, the reader may refer to a number (CIN I–III) or SIL I–II]. Cytological progression is a of other reviews [11–15]. likely sentinel for the accumulation of genetic alter- ations, which endow the tumour cell with various Clinical and therapeutic growth advantages in the local tissue environment. Since HPV-negative cervical carcinomas are relatively Plurality of HPV types rare (<5%), it is commonly believed that the continued More than 80 HPV types have been characterized to presence and expression of the viral genome is critical date, based on nucleotide sequence comparison with to maintenance of the dysplastic or malignant pheno- around another 100 that have been only partially char- type. Additional risk factors for progression to cancer acterized [13]. A discrete HPV type is defined as having may include a genetic predisposition of the host. Studies less than 90% nucleotide sequence homology within to characterize host genetic influences on HPV infec- the L1 region of all other known types. The biological tion, clearance and disease progression are currently at and genetic factors that are responsible for the remark- an early stage, although they may be substantially accel- able plurality of HPVs are not well understood. HPV erated as the human genome sequence is completed. infection is strictly epitheliotropic, and different HPV types are commonly categorized according to their Low-risk infection tissue specificity as either mucosal or cutaneous HPVs. The most familiar and commonly encountered HPV infection in the population results in the growth of High-risk infection benign cutaneous warts. Infection is very common, Approximately 30 types of HPV have been found to although cutaneous warts are somewhat more preva- infect mucosal epithelium, with most genital infections lent in children than in adults. It has been estimated being associated with HPV types 6, 11, 16, 18, 31, 33 that 7–10% of the population has benign cutaneous and 45. HPV types are commonly divided into two warts [18]. Common, plantar and palmar warts are groups, ‘low-risk’ and ‘high-risk’, based upon their typically caused by HPV types 1, 2, 3, 4 and 10 [12], relative association with malignant progression. and are thought to be transmitted by person-to-person Infection of the genital mucosa by HPV types 6 and 11 contact with infected tissue. Though strictly benign causes genital warts that are considered benign and and primarily a cosmetic nuisance, cutaneous warts ‘low-risk’ because of a poor association with progres- can be painful and may persist for years before spon- sion to invasive cancer. In contrast, high-risk HPV taneous regression occurs. types, HPV-16, -18, -31, -33 and -45, are highly asso- Benign genital warts, or condyloma acuminata, are ciated with persistent and progressive squamous typically caused by infection of the genital mucosa with intra-epithelial lesions (SIL) and with a relatively high- HPV types 6 and 11. Genital warts is a STD and, with risk for the development of cervical cancer. High-risk an estimated incidence of 1–3 million new cases per HPV types have been associated with 80–98% of cases year in the USA alone, it ranks as one of the most of invasive cervical cancer [16]. In addition to the prevalent STDs in the world today [1]. Papillary genital strong epidemiological association, high-risk HPV- warts often grow in clusters and can be readily DNA is frequently found to be integrated into the detected with the unaided eye. They are typically found cellular genome in tumour cells, and to maintain on the penis, vagina, perineum and around the anus.

230 ©2000 International Medical Press Antiviral drugs for human papillomavirus

Genital warts can regress without therapeutic interven- The determinants of this exclusive host and tissue tion or persist for years; however, progression to specificity are not well understood. Several well char- carcinoma is rarely observed [19]. Occasionally during acterized animal models are available including rabbit, pregnancy, condyloma will increase in both size and bovine, canine and the more recently described non- number perhaps due to stimulatory effects of steroid human primate systems [27]. Propagation of HPV in hormones on HPV transcription [20]. As an STD, animal models requires xenotransplantation of human genital warts are socially stigmatizing and engender tissue to immune deficient mice [28,29]. Although embarrassment in patients; furthermore, they can be outside the scope of this review, these animal models painful and are highly infectious to sexual partners. can be used as appropriate surrogates in preclinical HPV types 6 and 11 also cause a relatively rare but development of antiviral compounds and vaccines. potentially life-threatening disease, recurrent laryngeal The is an elegant organ and many people spend . These occur most frequently in children and vast amounts of time and money on its preservation can present in infancy where it has been postulated that and presentation. It functions as an effective barrier to vertical transmission occurred from genital warts of the access by most small pathogenic invaders, and prevents mother [21]. Direct measurements of HPV transmission passive fluid loss to the external environment. The following vaginal birth have yielded mixed results to epidermis contains both basal proliferating date [22,23]. Laryngeal papillomas can grow rapidly, and post-mitotic terminally differenti- resulting in obstruction of the trachea and respiratory ating cells at the more superficial layers. As the cells distress. Endoscopic surgical removal, thus far the stan- differentiate they migrate to the exterior surface where dard of care, may be required as often as bi-weekly to they are continually discarded to make way for maintain a patent airway in otherwise healthy children. succeeding layers of differentiating cells. Host cell DNA replication is localized within stem cells in the Therapy basal layer (and so DNA replication does not normally As described by Thomas [24], warts commonly found occur in the upper layers). In the suprabasal compart- on the hands and feet seem strangely sensitive to ment, terminal differentiation switches gene expression placebo effects, perhaps explaining why there are so from genes associated with active replication to genes many unusual folk remedies for treatment (for that ensure the epidermis will be sturdy, impervious example, Tom Sawyer’s remedy, rubbing with a dead and pliable. It is exclusively within this epithelial tissue cat). The conventional therapies in use today are more environment that HPVs have adapted to conduct their technically sophisticated although still largely non- productive life-cycle. specific. Side-effects of current therapies include pain It is believed that infection of the epidermis by HPV and scarring at the site, and disease recurrence at an occurs as a result of microtrauma that allows infection undesireably high rate. Therapies essentially fall into of the basal cells. The HPV genome is maintained at a two classes, ablative and immune-based; no efficacious low copy number within the basal layer, perhaps 1–100 anti-HPV compounds have been described to date. copies/cell, and increases in copy number in the upper Diseased tissue can be removed using cytotoxins layer of the epidermis. The HPV DNA in the basal (such as, podophyllotoxin, 5-fluorouracil, bleomycin), layer replicates in synchrony with the host DNA using chemical agents (salicylic acid, trichloroacetic acid) or the viral E1 and E2 proteins together with the host surgical techniques including laser vaporization, elec- DNA replication machinery. In normal uninfected trocautery or cryosurgery (for example, liquid nitrogen) basal cell tissue the basal cells divide, and daughter [25]. Since one of the hallmarks of HPV infection is the cells exit the cell cycle and differentiate as they migrate observation that many lesions will eventually undergo to the epithelial surface. In contrast, the differentiation immune-mediated regression, immune stimulation has programme of HPV-infected keratinocytes is delayed as been an attractive approach to therapy. Interferons, viral gene products including E6 and E7 are expressed. both alpha and beta, have been used with some success The viral are thought to transiently inhibit [26]. Recently, the US Food and Drug Administration differentiation through perturbation of approved the use of a topical, small-molecule immunos- the RB and p53 pathways, resulting in the hyperplasia timulant, Imiquimod (3M Pharmaceuticals) for the and acanthosis characteristics of papillomavirus infec- treatment of external genital warts. tion [30]. Late gene expression and production are induced in the most superficial layers of the HPV life-cycle epidermis during completion of terminal cellular differ- entiation. The increase in viral genome copy number in In addition to a stringent host specificity (HPVs only advance of virion packaging may be achieved, in part, infect humans), HPVs are strictly epitheliotropic, through a switching from a bi-directional to a rolling infecting only mucosal or cutaneous epidermal tissues. circle mode of DNA replication [31,32]. In addition,

Antiviral Therapy 5:4 231 MR Underwood et al.

more efficient DNA replication could be facilitated by Figure 1. Human papillomavirus genome organization higher E1 and E2 levels, which can result from induc- tion of keratinocyte differentiation [33]. It is thought E6 that the obligatory link between keratinocyte differen- tiation and HPV gene expression and replication has E7 retarded the development of a simple, productive LCR tissue-culture system for propagation of the virus. L1 However, several promising in vitro systems have been developed, which offer a glimpse of the complete life- cycle [34]. HPV E1 ~8000 bp The HPV genome and potential molecular targets

Genome organization L2 E2 All HPV genomes are dsDNA, covalently closed circles of around 8 kb in size, which reside and are replicated E4 E5 within the nucleus of infected cells. The HPV circular genome (Figure 1) is organized into three regions; a Oncogenesis regulatory region known as the long control region Replication (LCR) or upstream regulatory region (URR), and two coding regions encoding early (E) proteins and late (L) Transcription proteins. The LCR is around 1 kb in length and Late function contains numerous binding sites for host transcription The circular dsDNA genome is shown as the internal double-ring structure. factors as well as the HPV origin of replication. Within Surrounding the circular genome are arrows representing the open reading the early region are E1 and E2 viral genes encoding the frames (ORFs) and the direction of transcription. The letters E and L before each number represent the original early and late gene function designations. major viral replication factors. The E6 and E7 genes The arrows are patterned in four groups representing the functions associated encode the major viral oncogenes of the HPVs and are with the ORFs: oncogenesis, replication, transcriptional regulation and late functions. The LCR region is approximately 1 kb in length, and contains the thought to be involved in stimulation of cell prolifera- viral origin of replication and transcriptional regulatory elements. The arrow tion and inhibition of keratinocyte differentiation. The for the E2 combines two patterns along the length of the arrow indicating that E2 possesses replication and transcription functions, and that late region encodes the two structural capsid proteins, those functions require both the N- and C-terminal regions. The E4 ORF L1 and L2, which when expressed together can overlaps the E2 ORF in a different reading frame, and although E4 has an early assemble into virus-like particles (VLPs) that are being designation, it is functionally thought to be a late protein. utilized for trials and as serological reagents [35]. produce a normal isoform of p53. High-risk HPV E6 proteins have been shown to interact with a 100 kDa Oncogenes as targets cellular protein, E6-AP, which is a E3 ubiquitin-protein Viral oncogenes are virus-expressed proteins, which ligase [39]. E6 and E6-AP form a ternary complex with subvert the normal cellular controls on growth and p53, which facilitates its rapid proteolytic degradation. differentiation to benefit the virus’ lifecycle. Outside Additional recent studies have shown that high risk E6 the context of virus infection, expression of these viral can interact with the CBP/p300 co-activator and inter- genes induces properties associated with cellular trans- fere with p53 mediated transcriptional activity, formation including an extended life span and effectively blocking p53 at two discrete points [40]. defective differentiation. For high-risk HPVs, the E6 Another, less well understood target of HPV E6 is and E7 genes encode the primary transforming activi- hDLG (human homologue of the Drosophila discs ties [36]. Expression of HPV E5 may also lead to large tumour suppressor protein), which complexes growth stimulation as data suggests that E5 can be with the adenomatous polyposis coli (APC) tumour weakly oncogenic. suppressor protein [41,42] that is frequently mutated Mucosa-associated, high-risk HPV E6 proteins (for in colon cancer. HPV E6 can interact with ERC-55 example, HPV-16 and HPV-18) have been shown to [43], a putative Ca2+ binding protein that interacts with inactivate the p53 cell cycle regulatory pathway the vitamin D nuclear receptor [44], and E6 can through stimulation of ubiquitination and accelerated interact with paxillin that mediates attachment of cells proteolytic degradation of p53 [37,38]. Inactivation of to the extracellular matrix [45]. Although the conse- p53 is a common feature of human tumours, as quences of HPV E6 associations are not fully approximately 50% lack a wild-type allele and fail to understood, many of the latter interactions may target

232 ©2000 International Medical Press Antiviral drugs for human papillomavirus

pathways associated with both signal transduction and HPV E7 is cyclin-dependent regulation of the cell cycle, cell-to-cell contact, which are commonly disturbed in inhibitors of cyclin-dependent kinases may have thera- transformed cells [46]. Several other E6 associated peutic utility for HPV infections. Obviously, the major interactions that may contribute to the survival of shortcoming associated with targetting HPV-associated HPV-infected cells in vivo include the stimulation or cellular pathways is the likelihood of accompanying induction of telomerase activity [47], inhibition of Bak toxicity to uninfected tissues. [48] and binding to IRF-3, an interferon regulatory factor which transactivates the IFN‚ promoter [49]. Replication targets The best understood activity of the HPV E7 protein The most direct approach for generating therapeutic is high-affinity binding to the retinoblastoma protein agents for HPV is to target the apparatus responsible (pRb) and disruption of E2F-mediated transcriptional for replicating the viral DNA. Two modes of viral regulation [50]. DNA tumour , including the DNA replication are thought to be employed during papovaviruses and adenoviruses, target the retinoblas- the papillomavirus life-cycle [31]. Papillomavirus toma family of cell cycle regulators to subvert normal genome replication in the basal layer of the skin main- cellular control of G1/S transition and cell proliferation. tains low genome copy number and occurs in The pRb/E2F complex acts as a transcriptional synchrony with host DNA replication. As the virus repressor that negatively regulates S-phase progression. migrates into the suprabasal layers (where host DNA Through association with pRb, HPV E7 can displace replication does not normally occur) genomes are the E2F transcription factor [51] leading to transcrip- amplified in a ‘vegetative’ mode to make the viral DNA tional activation of a number of S-phase-specific for packaging into . Viral-specific DNA replica- cellular genes involved in DNA biosynthesis [52]. As tion inhibitors would be likely to, ideally, inhibit both with HPV E6, several other interactions have been iden- of these modes of replication. tified for HPV E7 and mechanistic details remain As small DNA viruses, the papillomaviruses encode incomplete. Specific interactions have been described a limited number of proteins, and enlist the aid of the for E7 with cyclin A [53,54], cyclin E [55], p21cip1 and host replication apparatus for viral DNA replication. p27kip1 [5658], AP-1 [59], TBP [60], TAF-110 [61], the Only two virally encoded proteins are required, the E1 S4 subunit of the 26S proteosome [62] and hTid-1 [63]. helicase and the E2 DNA binding protein [68]. The E1 Two broad strategies could be employed for thera- and E2 proteins interact transiently and cooperatively, peutically targetting E6 and E7 with a conventional forming a complex at the viral origin of replication to low molecular weight drug. Theoretically, a small facilitate changes in the ori structure, and to recruit the molecule could prevent the productive interaction host polα:primase complex to the viral origin [69]. The between E6 or E7 and one or more of its binding part- minimal origin of replication is located at the 3′ end of ners mentioned above. For example, if a small the LCR, and includes an AT-rich region, consensus E2 molecule were able to bind avidly and specifically to binding sequences and a partially conserved E1 binding HPV E7 and block its ability to bind pRB, we would site [70,71]. anticipate that cell proliferation would be arrested and Both E1 and E2 are relatively well conserved in the epithelial differentiation programme restored sequence and in function. The E1 and E2 proteins from without attendant virus replication. Likewise, if a small animal and human papillomaviruses, in heterologous molecule were able to interfere with the formation of combinations, can support replication of viral DNA the E6/E6AP/p53 ternary complex, it would be containing various origins [72,73]. One important expected that HPV-infected cells would be prone to implication of this functional conservation is that small apoptosis. Unfortunately, in spite of substantial molecules that inhibit HPV replication through screening efforts by the pharmaceutical industry, there binding to E1 (or E2) are likely to bind to E1 proteins are very few drugs that can successfully block such from many different HPV types, and thus have a good protein–protein interactions, although some intriguing chance of being effective in the clinic against a wide advances have been made recently [64–66]. range of low- and high-risk HPVs. Alternatively, drugs could be sought that target Both E1 and E2 are required for efficient initiation enzymes or receptors in the signalling pathways critical of viral DNA replication, although only E1 is required to the activities of E6 and E7. For example, focal adhe- for elongation [74]. The E2 protein binds to E2 sion kinase (FAK) is upregulated in cervical carcinoma binding sites in the origin region and through direct cell lines [67], and since paxillin, which interacts with association with E1, recruits the E1 helicase to its E6, is a substrate for tyrosine phosphorylation by FAK, binding site (Figure 2). At a more detailed level, forma- inhibition of the kinase activity may have a therapeutic tion of the initial E1:E2:DNA complex is thought to benefit in HPV-infected cells. Furthermore, since it is occur in a two-step mechanism involving DNA clear that one of the relevant pathways targetted by bending and a dual interaction between E1 and E2

Antiviral Therapy 5:4 233 MR Underwood et al.

Figure 2. HPV E1 and E2 protein functions in viral DNA simplex virus (HSV) UL5 helicase was recently replication reported [86]. These compounds showed good antiviral activity in cell-culture against HSV without HPV origin of replication obvious cytotoxicity. Unfortunately, however, viral resistance arose very rapidly during passage indicating

E2 BS that these particular compounds would not be clini- cally useful. E1 BS The strand displacement activity of helicases can be E2 readily assayed in vitro using various biochemical tech- E1 niques [87,88], many of which are amenable to high throughput screening. Strand displacement activity for BPV [78,80,89] and HPV E1 [82] has been demon- strated, although purification of active enzyme has been problematic [90]. The biochemical activity of HPV E1 may also be assayed under more complex, and biologically relevant Host DNA replication Viral DNA conditions in which E1 and E2 support origin-depen- factors replication dent in vitro replication [74,91–93]. This cell-free assay format requires purified E1 and E2 proteins, carefully prepared cytoplasmic cell extracts, and The schematic diagrams from top to bottom represent the currently posited events [66] whereby the E1 and E2 proteins facilitate viral DNA replication. The plasmid DNA containing an HPV origin of replication. E2 and E1 binding sites reside within the HPV origin, which in turn is located Although technically complex and not readily within the LCR. As described in the text, E2 binds first and recruits E1 to the E1 binding site. E1 multimerizes to form a hexamer at the E1 binding site, E2 amenable to high throughput screening, this assay does departs and host replication factors are engaged to elongate the viral DNA. not require highly purified E1 [94]. A potential diffi- culty associated with using in vitro replication as a [75]. In the current model, E1 is proposed to assemble primary screening tool is that many non-specific DNA on the E1:E2:DNA complex to generate the enzymati- binders or DNA polymerase inhibitors would surface cally active E1 hexamer. E2 is subsequently displaced as false positives. from the origin in an ATP-dependent process [69]. The polα:primase is recruited to the complex [76] through Cell culture assays. Although technically more difficult association with E1 to facilitate initiation of DNA than enzymatic assays, it is also possible to screen for replication. inhibitors of E1 by measuring HPV replication in cell culture. For papillomaviruses, both transient and The E1 protein stable replication assays are available. Transfection of E1 protein is a nuclear phosphoprotein and the only naive cells in culture can result in transient HPV repli- enzymatic gene product encoded by the virus. E1 has cation [72,73], which can be assayed under relatively ATPase and DNA helicase activities [77–82] that facil- high throughput conditions [95,96]. Following itate unwinding of the DNA helix in front of the primary identification of inhibitors, mechanism of advancing replication fork. Inhibition of this action studies are required to demonstrate that lead unwinding activity should effectively block viral compounds are targetting a viral function and not genome replication. The E1 protein has structural and adversely affecting the growth of the cell. A wide functional homology to the well-characterized SV40 variety of cytotoxic or cytostatic compounds can large tumour antigen helicase [83,84]. E1 is the virally obscure screening results due to broad or indirect encoded component that interacts with the host effects on DNA replication. polα:primase complex and recruits it to the viral origin The need to transfect cells to carry out screening can for efficient viral DNA replication. be avoided through the use of one or more stable cell lines that have recently become available [97–100]. E1 biochemical assays. The helicase activity of E1 is an These cell lines have been derived from low-grade appealing target for antiviral inhibitors since the HPV-positive intra-epithelial lesions which maintain conservation among family III helicases is only modest HPV genomes as unintegrated episomes. Just as in the and restricted to three small regions of the enzyme transient transfection assays, inhibition of viral DNA [85]. Consequently, it should be possible to identify replication can be measured directly through quantifi- inhibitors specific for the viral helicase that do not cation of viral DNA copy number. interfere with related host functions. As a proof of Finally, it has recently been shown that high-risk concept, a 2-amino thiazole inhibitor of the herpes genomes can be transfected into primary keratinocytes,

234 ©2000 International Medical Press Antiviral drugs for human papillomavirus

Figure 3. Papillomavirus E2 DNA binding domain structural comparisons (a)

DNA binding helices

(b)

(a) The ribbon diagrams show that the dimers for the four published E2CT structures closely overlap. On the left ribbon diagram the DNA recognition helices run diagonally from top left to lower right. In the right ribbon diagram the dimer is rotated horizontally approximately 90° (such that the DNA recognition helices are on the left side of the dimer. Ribbon diagrams were generated using QUANTA. (b) Shown in the lower section is a comparison of the surface structures for all four E2CT proteins. Green and grey shading indicates convex and concave regions, respectively. The red arrow above the HPV31 E2CT structure indicates the binding location for the Abbott E2/DNA binding inhibitors described in the text. The orientation of the surface structures is the same as that of the left-side ribbon diagram in part A. Surface structures were generated using GRASP. transiently selected with neomycin, and induced to be an important system to support mechanism of differentiate resulting in late gene expression and action studies for candidate antiviral inhibitors. limited virus particle production [34,101,102]. While infection of naive cells with HPV in cell culture The E2 protein remains inefficient, this system represents an important HPV E2 is a multifunctional nuclear protein that regu- advance, since it enables us to directly examine the lates viral gene transcription, promotes localization of genetic and biochemical determinants that control the viral E1 helicase protein to the viral origin of repli- virus expression and replication in the context of cation, governs viral genome segregation during cell keratinocyte differentiation. For example, using this division and may play a role in virion packaging. To technique it has been shown that mutation of the E6 carry out all of these essential functions, the E2 protein and E7 oncogenes interferes with episomal mainte- must bind as a dimer to one or more copies of a nance even though these viral genes are not required consensus 12 bp palindromic DNA sequence within for transient replication [103]. In the future, this may the HPV genome [104], and thus disruption of DNA

Antiviral Therapy 5:4 235 MR Underwood et al.

binding would be expected to have a strong and between the E2 proteins and EBNA-1, the structural pleiotropic antiviral effect. The consensus E2 binding conservation is likely to be a result of functional simi- site is ACCGN4CGGT (Figure 3), with the N=AT-rich larities and covergent evolution to a common DNA preferred among the HPVs [105]. E2 binding sites are binding motif. present at several locations within the genome, with The conserved N- and C-terminal domains are sepa- four conserved sites located near the viral origin of rated by a poorly conserved hinge region. The hinge replication and a few less well-conserved sites near region is highly variable in both amino acid content other transcriptionally regulated regions. Sequence and in length, and is generally considered to lack struc- variability within the four internal Ns results in a range ture, perhaps instead acting as a flexible ‘linker’ for the of E2 binding affinities while changes to the conserved functions of the N and C terminal domains. A number nucleotides (ACCG… CGGT) can result in a complete of phosphorylation sites are contained within the hinge loss of binding [106,107]. Such variability in affinity region [125], which at least for BPV E2 appear to be may be involved in choreographing transcriptional important for E2-directed segregation of HPV genomes activity in association with different binding sites, and to daughter cells during cell division [126]. Very little in facilitating appropriate delivery of E1 to the viral information is available regarding the role of phospho- origin of replication. rylation of E2 proteins from HPVs.

E2 organization and function. Based upon an analysis of E2 and structure. Most small-molecule pharmaceuticals the predicted primary sequence of all E2 proteins, two act through inhibition of enzymes or membrane-bound conserved regions are recognized: the ~200 amino acid receptors. Enzyme inhibition occurs through binding at N-terminal domain and a ~90 amino acid C-terminal or near the catalytic site, or via an allosteric mecha- domain [108]. The N-terminal domain is primarily nism. Since E2 has no known enzymatic activity, and responsible for both the transcriptional regulatory and has not been shown to be allosterically regulated, it replication-associated activities attributed to E2. These falls into a class of targets that are classically difficult conclusions are well supported by extensive mutational to inhibit, such as those that function via macromolec- data characterizing amino acid determinants for E2 ular interactions [127]. Macromolecular interactions binding to host transcription and replication factors are typically mediated across large, featureless surfaces [109–113], and to the viral E1 protein [114–116]. Very >600 Å2 in area. To increase the chance of success, a recently, the structure of the N-terminal domains of crystal or solution structure can facilitate inhibitor HPV-16 E2 [117] and a protease-resistant sub-frag- development using structure-assisted drug design to ment of HPV-18 were published [118]. guide improvements in binding affinities. The conserved E2 C-terminal (E2CT) DNA binding domain is necessary and sufficient for dimerization, E2 inhibitor examples. A variety of approaches have and for binding to the palindromic E2 binding sites been used to generate therapeutic agents targetting E2, [119,120]. The E2CT structures for a number of papil- although none have been clinically effective against lomaviruses have been solved including BPV1 [121], HPV infection. Phosphorothioate antisense oligonu- HPV-31 [122] and HPV-16 [123]. Overall the structure cleotides were designed to prevent translation of is very well conserved among the papillomavirus E2CT HPV-6 and -11 E2. The in vitro optimized oligonu- structures solved so far. The E2CT proteins form cleotide, ISIS 2105, was shown to inhibit E2 dimeric β-barrels, with each monomer possessing an α transactivation in a cell-based assay [128], although it helical DNA recognition sequence. The recognition failed to demonstrate efficacy in the clinic. More helix makes direct contacts with the four conserved recently, a peptide nucleic acid (PNA) was designed to bases of the E2 binding site (E2BS), and the successive bind to a dsDNA BPV E2 binding site. When the PNA major grooves of the E2-BS engulf the recognition was pre-bound to a plasmid containing an E2-BS, viral helices as the DNA curves around the barrel. With one DNA replication was inhibited in a cell-culture-based intriguing exception, the E2CT DNA binding structure assay [129]. To target interactions of E2 with E1, a 15 is unique to the papillomavirus E2 proteins. The amino acid peptide derived from a region of HPV-16 EBNA-1 protein from the evolutionarily unrelated E2 required for E1 interaction was synthesized and oncogenic virus Epstein–Barr virus (EBV) shares this shown to prevent in vitro DNA replication [130]. No structural organization [124]. EBNA-1 is a dimeric follow-up data regarding activity in cell culture models origin binding protein that is functionally similar to the was reported. E2 protein. EBNA-1 activates and regulates viral DNA Primarily due to constraints associated with drug replication and is involved in segregation of viral delivery – size-related pharmacokinetic and pharmaco- genomes to daughter cells during cell division. As there dynamic properties – most drug molecules are are no apparent amino acid sequence homologies relatively small (<600 MW). With the goal of identi-

236 ©2000 International Medical Press Antiviral drugs for human papillomavirus

Figure 4. E2 protein organization and components of the solved for both crystal forms. TR-FRET DNA binding assay Like other E2CT structures solved so far, the HPV- 11 E2CT protein forms a dimeric β-barrel with surface α E2 Dimer λ340 DNA-binding -helices. Despite the moderate sequence conservation between E2s of different viral

N-terminal domain Eu strains (30–65% identity), all E2CT structures solved λ to date (HPV-11, -16, -31 and BPV) share the same 615 tertiary structure, with only subtle differences in Hinge region subunit orientation. Conserved residues are concen- C-terminal domain λ trated at the dimer interfaces and the DNA recognition 665 A C T helices, which are of very similar length and position. T C G G G N C G A HPV-11 E2CT is most similar in conformation to BPV G N N N C C N G C N N N complexed with DNA. An overlay of HPV-11 E2CT Reader from other viruses is shown in Figure 3a. Although the tertiary structure of the E2CT protein Eu Europium-labelled mouse anti-E2 is highly conserved, the topology of the DNA-binding surface of the protein is greatly influenced by the Streptavidin-Cy5 fluorophore amino acid side-chains that are directed out towards the solvent. Due to the moderate conservation between Biotin linked to dsDNA E2 binding site oligonucleotide E2s, the surfaces presented for DNA binding appear surprisingly different (Figure 3b). For small molecule The diagram shows the general E2 domain organization (N-terminal, C- inhibitors of E2:DNA binding to successfully target a terminal and hinge regions) and E2 binding to the 12 bp E2BS palindrome. broad spectrum of HPV types, these data would Shown also are the components of the TR-FRET assay described in the text. The Eu-labelled anti-E2 mouse monoclonal antibody binds specifically to the E2 suggest that binding needs to be directed within a protein. The E2 BS is conjugated with biotin/streptavidin/Cy5. When E2 binds conserved pocket in the E2 structure. In this regard, the the E2BS, Eu fluorescence is transferred to the Cy5 fluorophore which emits light at 665 nM. binding site for the HPV-31 E2CT small molecule inhibitor identified by Fesik et al. [131] lies within a fying a conventional small molecule antiviral inhibitor region where amino acid conservation is low. As a of E2, Fesik et al. sought to take advantage of the rela- consequence, such compounds are only weak tively small size of the DNA binding domain of E2 and inhibitors of HPV-11 E2 DNA binding and could not the stable structure. Using HPV-31 E2, they developed be co-crystallized (data not shown). a structure−activity relationship (SAR) for candidate small molecule inhibitors by the iterative use of NMR E2 screening. As the master regulator, E2 binds to co-structures [131]. In this ‘SAR by NMR’ approach, specific viral DNA sequences to mediate multiple func- they first identified compounds through random tions, and thus inhibition of E2:DNA binding should screening, which weakly bound particular regions of theoretically provide a pleotropic antiviral effect inter- the E2 DNA binding domain. Using traditional medic- fering with virus transcription, replication, episomal inal chemistry, the NMR-derived binding information, segregation and perhaps DNA packaging. A variety of and combinations of different regions from active methods can be used to screen for compounds that analogues, they were able to optimize mM inhibitors inhibit E2 function either in cell-culture, or in solution. µ and generate compounds with IC50s=10 M in a filter E2-mediated transactivation can be readily screened in binding assay. Unfortunately, no functional, cell-based a cell-culture system in either a transient or stable data is available regarding the compounds activities reporter-based assay. As mentioned above, cell-based against E2-mediated transcriptional control or HPV assays carry a number of limitations such as a rela- replication. tively high technical complexity and a low-to-moderate compound throughput. Biochemical screens are typi- Additional crystallography. In related studies, two crystal cally simpler and more robust. For example, in support forms of E2CT were generated for the HPV-11 E2CT. of the ‘SAR by NMR’ studies, an E2 nitrocellulose One was grown in the absence of any inhibitor (apo), filter binding assay was utilized [131]. and one was grown in the presence of a peptide To identify E2:DNA binding inhibitors, we estab- inhibitor (S-J Deng, in preparation). Crystals grown in lished a highly robust Time-Resolved-Fluorescent- the presence of the inhibitor were obtained from 2 M Resonance-Energy-Transfer (TR-FRET) assay (Leitner, magnesium sulphate and diffracted to ~2Å. Apo crys- in preparation) (Figure 4). A mouse anti-E2 mono- tals were grown from 2–4 M sodium formate and clonal antibody modified with a chelated europium diffracted to ~3Å. Three-dimensional structures were moiety forms one side of a sandwich with the E2

Antiviral Therapy 5:4 237 MR Underwood et al.

protein in the middle; E2 in turn binds to a double decade, tremendous progress has been made in under- stranded oligonucleotide with biotinylated nucleotides standing how HPV controls expression and virus at the ends. The biotinylated DNA binds to strepta- replication. Cell-culture systems although not yet vidin that is conjugated to Cy5. Europium is excited robust, model substantial and relevant segments of the with 340 nm light and fluoresces with a peak in the virus life-cycle that are amenable to compound 615 nm range. If the europium is held proximal to the screening and drug testing. Since HPV does not encode Cy5 as shown in Figure 4, the 615 nm light is absorbed clinically precedented enzymes or receptor targets that by Cy5 which will subsequently fluoresce at 665 nm have yielded marketed drugs in the past, drug for signal detection. E2:DNA binding inhibitors will discovery leaders in industry will have to champion decrease the amount of europium proximal to CY5 new drug targets for a technically challenging virus. and thus correspondingly reduce light emitted at 665 nm. In our hands, this assay is highly sensitive, demon- References strates a low well-to-well variability and is technically amenable to commonly used robot workstations. 1. Koutsky L. Epidemiology of genital human papillomavirus Using the TR-FRET assay, we were readily able to infection. American Journal of Medicine 1997; 102:3–8. screen >500000 compounds for inhibition of DNA 2. Cates W. Estimates of incidence and prevalence of sexually transmitted diseases in the United States. Sexually binding by HPV E2. Transmitted Diseases 1999; 26:S2–S7. 3. Ho GYF, Bierman R, Beardsley L, Chang CJ & Burk RD. Future directions Natural history of cervicovaginal papillomavirus infection in young women. New England Journal of Medicine 1998; 338:423–428. Without question, there is a substantial clinical need 4. Syrjanen KJ. Natural history of genital human papillo- for safe and effective therapies for HPV-associated mavirus infections. In Papillomavirus Reviews Edited by C Lacey. 1996; pp. 189–206. Leeds: Leeds Mecical diseases. HPVs are very common in the population and Information. typically cause benign, eventually self-limiting diseases. 5. Munk C, Svare EI, Poll P, Bock JE & Kjaer SK. History of Although many of these lesions will resolve over the genital warts in 10,838 women 20 to 29 years of age from the general population. Sexually Transmitted Diseases course of months and years, there remains a great need 1997; 24:567–572. for more sophisticated and more specific therapeutic 6. Eng TR & Butler WT. The hidden epidemic: confronting options. For a minority of patients who can not as yet sexually transmitted diseases. 1997; p. 59. Washington DC: be identified with a prognostic tool, persistence of National Academy Press. 7. Franco EL. Cancer causes revisted: human papillomavirus benign lesions is the harbinger of malignant conversion and cervical neoplasia. Journal of the National Cancer to squamous carcinoma. As patients become more Institute 1995; 87:779–780. aware of HPV infection and its associated diseases, 8. Burk RD. Pernicious papillomavirus infection. New they will undoubtedly demand a broader catalogue of England Journal of Medicine 1999; 341:1687–1688. 9. Schoell WMJ, Janicek MF & Mirhashemi R. Epidemiology treatment options. and biology of cervical cancer. Seminars in Surgical One of the most promising opportunities in the Oncology 1999; 16:203–211. short term is the availability of a prophylactic vaccine 10. Richart RM & Wright TC. Controversies in the manage- ment of low-grade cervical intraepithelial neoplasia. Cancer for HPV [132]. Encouraging results have been seen in 1993; S71:1413–1421. animal vaccine models demonstrating good protection 11. Howley PM. Papillomaviruses: the viruses and their repli- from experimental infection with the species appro- cation. In Fields Virology. Edited by BN Fields, DM Knipe & PM Howley. 1996; Vol. 2, pp. 2045–2076. Philadelphia: priate animal PVs [133–135]. A number of different Lippincott-Raven. antigens and delivery stategies are being pursued, 12. Shah KV & Howley PM. Papillomaviruses: the viruses and including purified proteins/peptides, assembled virus- their replication. In Fields Virology. Edited by BN Fields, DM Knipe & PM Howley. 1996; Vol. 2, pp. 2077–2109. like particles and naked DNA [136]. Some of these Philadelphia: Lippincott-Raven. initiatives have progressed to Phase II clinical trials; 13. Zur Hausen H. Papillomaviruses in human cancers. therefore, some efficacy data should be available over Proceedings of the Association of American Physicians the next few years. One of the most significant chal- 1999; 111:581–587. 14. Stubenrauch F & Laimins LA. Human papillomavirus life lenges associated with HPV vaccines is that protection cycle: active and latent phases. Seminars in Cancer Biology is likely to be type-specific. Thus, polyvalent vaccines 1999; 9:379–386. or modifications to antigens and vaccine delivery 15. Crum C. Contemporary theories of cervical : the virus, the host, and the stem cell. Modern Pathology methods, which enhance a broader cell-mediated 2000; 13:243–251. immune response, will be favoured. 16. Walboomers JMM, Jacobs MV, Manos MM, Bosch FX, The development of HPV antivirals has been slow Kummer JA, Shah KV, Snijders PJF, Peto J, Meijer CJL & Munoz N. Human papillomavirus is a necessary cause of due to the absence of a conventional and robust cell invasive cervical cancer worldwide. Journal of Pathology culture system for propagation of the virus and reca- 1999; 189:12–19. pitulation of the virus life-cycle. During the past 17. Alani RM & Munger K. Human papillomavirus and asso-

238 ©2000 International Medical Press Antiviral drugs for human papillomavirus

ciated malignancies. Journal of Clinical Oncology 1998; 37. Huibregtse JM & Beaudenon SL. Mechanism of HPV E6 16:330–337. proteins in cellular transformation. Seminars in Cancer 18. Laurent R & Kienzler JL. Epidemiology of HPV infections. Biology 1996; 7:317–326. Clinical Dermatology 1985; 3:56–70. 38. Scheffner M, Romanczuk H, Munger K, Huibregtse JM, 19. Zur Hausen H & Schneider A. In The Papovaviridae. Mietz JA & Howley PM. In Current Topics in Edited by Salzman N & Howley PM. 1987; Vol. 2, pp. Microbiology and Immunology. Edited by zur Hausen H. 245–263. New York: Plenum Press. 1994; Vol. 186, pp. 83–99. Berlin-Heidelberg: Springer- Verlag. 20. Chan W-K, Klock G & Bernard H-U. Progesterone and glucocorticoid response elements occur in the long control 39. Scheffner M, Huibregtse JM, Vierstra RD & Howley PM. regions of several human papillomaviruses involved in The HPV-16 E6 and E6-AP complex functions as a ubiq- anogenital neoplasia. Journal of Virology 1989; uitin-protein ligase in the ubiquitination of p53. Cell 1993; 63:3261–3269. 75:495–505. 21. Kashima HK & Shah K. Recurrent respiratory papillo- 40. Zimmermann H, Degenkolbe R, Bernard H-U & matosis: clinical overview and management principles. O’Connor MJ. The human papillomavirus type 16 E6 Obstetrics and Gynecology Clinics of North America oncoprotein can down-regulate p53 activity by targeting 1987; 14:581–588. the transcriptional coactivator CBP/p300. Journal of Virology 1999; 73:6209–6219. 22. Sedlacek TV, Lindheim S, Eder C, Hasty L, Woodland M, Ludomirsky A & Rando RF Mechanism for human papil- 41. Lee SS, Weiss RS & Javier RT. Binding of human virus lomavirus transmission at birth. American Journal of oncoproteins to hDlg/SAP97, a mammalian homolog of the Obstetrics and Gynecology 1989; 161:55–59. Drosophila discs large tumor suppressor protein. Proceedings of the National Academy of Sciences USA 23. Watts DH, Koutsky LA, Holmes KK, Goldman D, Kuypers 1997; 94:6670–6675. J, Kiviat NB & Galloway DA. Low risk of perinatal trans- mission of human papillomavirus: Results from a 42. Kiyono T, Hiraiwa A, Fujita M, Hayashi Y, Akiyama T & prospective cohort study. American Journal of Obstetrics Ishibashi M. Binding of high-risk human papillomavirus E6 and Gynecology 1998; 178:365–373. oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein. Proceedings of the 24. Thomas L. On warts. In The Medusa and the Snail 1979; National Academy of Sciences USA 1997; pp. 76–81. New York: Viking Press. 94:11612–11616. 25. Krebs HB. Management strategies. In Clinical Obstetrics 43. Chen JJ, Reid CE, Band V & Androphy EJ. Interaction of and Gynecology. Edited by Pitkin RM & Scott JR. 1989; papillomavirus E6 oncoproteins with a putative calcium- Vol. 32, pp. 200–213. Philadelphia: JB Lippincott. binding protein. Science 1995; 269:529–531. 26. Trofatter KF. Interferon treatment of anogenital human 44. Imai T, Matsuda K, Shimojima T, Hashimoto T, Masuhiro papillomavirus-related diseases. Dermatologic Clinics S, Kitamoto T, Sugita A, Suzuki K, Matsumoto H, 1991; 9:343–352. Masushige S, Nogi Y, Muramatsu M, Handa H & Kato S. 27. Stanley MA, Masterson PJ & Nicholls PK. In vitro and ERC-55, a binding protein for the papilloma virus E6 animal models for antiviral therapy in papillomavirus oncoprotein, specifically interacts with vitamin D receptor infections. Antiviral Chemistry and Chemotherapy 1997; among nuclear receptors. Biochemical and Biophysical 8:381–400. Research Communications 1997; 233:765–769. 28. Kreider JW, Howett MK, Wolfe S, Bartlett GL, Zaino RJ, 45. Tong X & Howley PM. The bovine papillomavirus E6 Sedlacek TV & Mortelk R. Morphological transformation oncoprotein interacts with paxillin and disrupts the actin in vivo of human uterine cervix with papillomavirus from cytoskeleton. Proceedings of the National Academy of condylomata acuminata. Nature 1985; 317:639–641. Sciences USA 1997; 94:4412–4417. 29. Bonnez W, Darin C, Borkhuis C, Jensen KM, Reichman 46. Kubbutat MHG & Vousden KH. New HPV E6 binding RC & Rose RC. Isolation and propagation of human proteins: dangerous liaisons? Trends in Microbiology 1998; papillomavirus type 16 in human xenografts implanted in 6:173–175. the severe combined immunodeficiency mouse. Journal of 47. Klingelhutz AJ, Foster SA & McDougall JK. Telomerase Virology 1998; 72:5256–5261. activation by the E6 gene product of human papillo- 30. Laimins LA. Human papillomaviruses target differentiating mavirus type 16. Nature 1996; 380:79–82. epithelia for virion production and malignant conversion. 48. Thomas M & Banks L. Inhibition of Bak-induced apop- Seminars in Virology 1996; 7:305–313. tosis by HPV-18 E6. 1998; 17:2943–2954. 31. Flores ER & Lambert PF. Evidence for a switch in the 49. Ronco LV, Karpova AY, Vidal M & Howley PM. Human mode of human papillomavirus type 16 DNA replication papillomavirus 16 E6 oncoprotein binds to interferon regu- during the viral life cycle. Journal of Virology 1997; latory factor-3 and inhibits its transcriptional activity. 71:7167–7179. Genes & Development 1998; 12:2061–2072. 32. Dasgupta S, Zabielski J, Simonsson M & Burnett S. 50. Münger K & Phelps WC. The human papillomavirus-E7 Rolling-circle replication of a high-copy BPV-1 plasmid. protein as a transforming and transactivating factor. Journal of Molecular Biology 1992; 228:1–6. Biochimica et Biophysica Acta 1993; 1155:111–123. 33. Klumpp DJ & Laimins LA. Differentiation-induced 51. Phelps WC, Bagchi S, Barnes JA, Raychaudhuri P, Kraus V, changes in promoter usage for transcripts encoding the Munger K, Howley PM & Nevins JR. Analysis of trans human papillomavirus type 31 replication protein E1. activation by human papillomavirus type 16 E7 and aden- Virology 1999; 257:239–246. ovirus 12S E1A suggests a common mechanism. Journal of 34. Frattini MG, Lim HB & Laimins LA. In vitro synthesis of Virology 1991; 65:6922–6930. oncogenic human papillomavirus requires episomal 52. Nevins JR. E2F: A link between the Rb tumor suppressor genomes for differentiation-dependent late expression. protein and viral oncoproteins. Science 1992; Proceedings of the National Academy of Sciences, USA 258:424–429. 1996; 93:3062–3067. 53. Dyson N, Guida P, Münger K & Harlow E. Homologous 35. Kirnbauer R, Booy F, Cheng N, Lowy DR & Schiller JT. sequences in adenovirus E1A and human papillomavirus Papillomavirus L1 major capsid protein self-assembles into E7 proteins mediate interaction with the same set of virus-like particles that are highly immunogenic. cellular proteins. Journal of Virology 1992; 66:6893–6902. Proceedings of the National Academy of Sciences, USA 1992; 89:12180–12184. 54. Arroyo M, Bagchi S & Raychaudhuri P. Association of the human papillomavirus type 16 E7 protein with the S-phase 36. Münger K, Scheffner M, Huibregtse JM & Howley PM. specific E2F-cyclin A complex. Molecular and Cellular Interactions of HPV E6 and E7 with tumour suppressor Biology 1993; 13:6537–6546. gene products. Cancer Surveys 1992; 12:197–217. 55. McIntyre MC, Ruesch MN & Laimins LA. Human papil-

Antiviral Therapy 5:4 239 MR Underwood et al.

lomavirus E7 oncoproteins bind a single form of cyclin E Journal of Virology 1992; 66:5949–5958. in a complex with cdk2 and p107. Virology 1996; 215:73–82. 74. Liu J-S, Kuo S-U, Broker TR & Chow LT. The functions of human papillomavirus type 11 E1, E2, and E2C protein in 56. Funk JO, Waga S, Harry JB, Espling E, Stillman B & cell-free DNA replication. Journal of Biological Chemistry Galloway DA. Inhibition of CDK activity and PCNA- 1995; 270:27283–27291. dependent DNA replication by p21 is blocked by interaction with the HPV16 E7 oncoprotein. Genes & 75. Gillitzer E, Chen G & Stenlund A. Separate domains in the Development 1997; 11:2090–2100. E1 and E2 proteins serve architectural and productive roles for cooperative DNA binding. EMBO Journal 2000; 57. Jones DL, Alani RM & Munger K. The human papillo- 19:3069–3079. mavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by 76. Chow LT & Broker TR. Papillomavirus DNA replication. abrogating p21Cip1-mediated inhibition of cdk2. Genes & Intervirology 1994; 37:150–158. Development 1997; 11:2101–2111. 77. Sun S, Thorner L, Lentz M, MacPherson P & Botchan 58. Zerfass-Thome K, Zwerschke W, Mannhardt B, Tindle R, MR. Identification of a 68 kilodalton nuclear ATP-binding Botz JW & Jansen-Durr P. Inactivation of the cdk inhibitor phosphoprotein encoded by bovine papillomavirus type 1. p27KIP1 by the human papillomavirus type 16 E7 onco- Journal of Virology 1990; 64:5093–5105. protein. Oncogene 1996; 13:2323–2330. 78. Yang L, Mohr I, Fouts E, Lim DA, Nohaile M & Botchan 59. Antimore MJ, Birrer MJ, Patel D, Nader L & McCance MR. The E1 protein of bovine papillomavirus 1 is an ATP- DJ. The human papillomavirus type 16 E7 gene product dependent DNA helicase. Proceedings of the National interacts with and trans-activates the AP1 family of tran- Academy of Sciences, USA 1993; 90:5086–5090. scription factors. EMBO Journal 1996; 15:1950–1960. 79. Thorner LK, Lim DA & Botchan MR. DNA-binding 60. Massimi P, Pim D, Storey A & Banks L. HPV-16 E7 and domain of bovine papillomavirus type 1 E1 helicase: adenovirus E1a complex formation with TATA box Structural and functional aspects. Journal of Virology binding protein is enhanced by casein kinase II phosphory- 1993; 67:6000–6014. lation. Oncogene 1996; 12:2325–2330. 80. Seo Y-S, Muller F, Lusky M & Hurwitz J. Bovine papil- 61. Mazzarelli JM, Atkins GB, Geisberg JV & Ricciardi RP. loma virus (BPV)-encoded E1 protein contains multiple The viral oncoproteins Ad5 E1A, HPV16 E7 and SV40Tag activities required for BPV DNA replication. Proceedings bind a common region of the TBP-associated factor-110. of the National Academy of Sciences, USA 1993; Oncogene 1995; 11:1859–1864. 90:702–706. 62. Berezutskaya E & Bagchi S. The human papillomavirus E7 81. Bream GL, Ohmstede CA & Phelps WC. Characterization oncoprotein functionally interacts with the S4 subunit of of human papillomavirus type 11 E1 and E2 proteins the 26S proteasome. Journal of Biological Chemistry 1997; expressed in insect cells. Journal of Virology 1993; 272:30135–30140. 67:2655–2663. 63. Schilling B, De Medina T, Syken J, Vidal M & Munger K. 82. Hughes FJ & Romanos MA. E1 protein of human papillo- A novel human DNAJ protein, hTid-1, a homolog of the mavirus is a DNA helicase/ATPase. Nucleic Acids Research Drosophila tumor suppressor protein Tid56, can interact 1993; 21:5817–5823. with the human papillomavirus type 16 E7 oncoprotein. 83. Clertant P & Seif I. A common function for polyoma virus Virology 1998; 247:74–85. large-T and papillomavirus E1 proteins. Nature 1984; 64. Hajduk PJ, Meadows RP & Fesik SW. Discovering high- 311:276–279. affinity ligands for proteins. Science 1997; 278:497–499. 84. Mansky KC, Batiza A & Lambert PF. Bovine papillo- 65. Bogan AA & Thorn KS. Anatomy of hot spots in protein mavirus type 1 E1 and simian virus 40 large T antigen interfaces. Journal of Molecular Biology 1998; 280:1–9. share regions of sequence similarity required for multiple functions. Journal of Virology 1997; 71:7600–7608. 66. Way JC. Covalent modification as a strategy to block protein-protein interactions with small-molecule drugs. 85. Koonin EV. A common set of conserved motifs in a vast Current Opinions in Chemical Biology 2000; 4:40–46. variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of 67. McCormack SJ, Brazinski SE, Moore JL, Werness BA & eukaryotic DNA replication. Nucleic Acids Research 1993; Goldstein DJ. Activation of the focal adhesion kinase 21:2541–2547. signal transduction pathway in cervical carcinoma cell lines and human genital epithelial cells immortalized with 86. Spector FC, Liang L, Giordano H, Sivaraja M & Peterson human papillomavirus type 18. Oncogene 1997; MG. Inhibition of replication by a 2- 15:265–274. amino thiazole via interations with the helicase component of the UL5-UL8-UL52 complex. Journal of Virology 1998; 68. Ustav M & Stenlund A. Transient replication of BPV-1 72:6979–6987. requires two viral polypeptides encoded by the E1 and E2 open reading frames. EMBO Journal 1991; 10:449–457. 87. Raney KD, Sowers LC, Millar DP & Benkovic SJ. A fluo- rescence-based assay for monitoring helicase activity. 69. Sanders CM & Stenlund A. Recruitment and loading of the Proceedings of the National Academy of Sciences, USA E1 intiator protein: an ATP-dependent process catalysed by 1994; 91:6644–6648. a transcription factor. EMBO Journal 1998; 17:7044–7055. 88. Eggleston AK, Rahim NA & Kowalczykowski SC. A heli- case assay based on the displacement of fluorescent, nucleic 70. Lu JZJ, Sun YN, Rose RC, Bonnez W & McCance DJ. acid-binding ligands. Nucleic Acids Research 1996; Two E2 binding sites (E2BS) alone or one E2BS plus an 24:1179–1186. A/T-rich region are minimal requirements for the replica- tion of the human papillomavirus type-11 origin. Journal 89. Sedman J & Stenlund A. The papillomavirus E1 protein of Virology 1993; 67:7131–7139. forms a DNA-dependent hexameric complex with ATPase and DNA helicase activities. Journal of Virology 1998; 71. Russell J & Botchan MR. cis-Acting components of human 72:6893–6897. papillomavirus (HPV) DNA replication: linker substitution analysis of the HPV type 11 origin. Journal of Virology 90. Rocque WJ, Porter DJT, Barnes JA, Dixon EP, Lobe DC, 1995; 69:651–660. Su J-L, Willard DH, Gaillard R, Condreay JP, Clay WC, Hoffman CR, Overton LK, Pabel G, Kost TA & Phelps 72. Chiang C-M, Ustav M, Stenlund A, Ho TF, Broker TR & WC. Replication-associated activities of purfied human Chow LT. Viral E1 and E2 proteins support replication of papillomavirus type 11 E1 helicase. Protein Expression and homologous and heterologous papillomaviral origins. Purification 2000; 18:148–159. Proceedings of the National Academy of Sciences, USA 1992; 89:5799–5803. 91. Kuo S-R, Liu J-S, Broker TR & Chow LT. Cell-free replica- tion of the human papillomavirus DNA with homologous 73. Del Vecchio AM, Romanczuk H, Howley PM & Baker viral E1 and E2 proteins and human cell extracts. Journal CC. Transient replication of human papillomavirus . of Biological Chemistry 1994; 269:24058–24065.

240 ©2000 International Medical Press 92. Liu J-S, Kuo S-R, Makhov AM, Cyr DM, Griffith JD, 109. Li R & Botchan MR. The acidic transcriptional activation Broker TR & Chow LT. Human Hsp70 and Hsp40 chap- Domains of VP16 and p53 bind the cellular replication erone proteins facilitate human papillomavirus-11 E1 protein-A and stimulate in vitro BPV-1 DNA replication. protein binding to the origin and stimulate cell-free DNA Cell 1993; 73:1207–1221. replication. Journal of Biological Chemistry 1998; 273:30704–30712. 110. Breiding D, Sverdrup F, Grossel MJ, Moscufo N, Boonchai W & Androphy EJ. Isolation of a BPV1 E2 transactivation 93. Dixon EP, Pahel GL, Rocque WJ, Barnes JA, Lobe DC, domain binding factor required for both transcriptional Hanlon MH, Alexander KA, Chao S-F, Lindley K & Phelps activation and DNA replication. Virology 1997; WC. The E1 helicase of human papillomavirus type 11 221:34–43. binds to the origin of replication with low sequence speci- ficity. Virology 2000; 270:345–357. 111. Li R, Knight JD, Jackson SP, Tjian R & Botchan MR. Direct interaction between Sp1 and the BPV enhacer E2 94. Bonne-Andrea C, Tillier F, McShan GD, Wilson VG & protein mediates synergistic activation of transcription. Clertant P. Bovine papillomavirus type 1 DNA replication: Cell 1991; 65:493–505. the transcriptional activator E2 acts in vitro as a specificity factor. Journal of Virology 1997; 71:6805–6815. 112. Steger G, Ham J, Lefebvre O & Yaniv M. The bovine papillomavirus 1 E2 protien contains two activation 95. Plimpton M, Sharp NA, Liddicoat LH, Remm M, Tucker domains: one that interacts with TBP and another that DO, Hughes FJ, Russell SM & Romanos MA. A high functions after TBP binding. EMBO Journal 1995; capacity assay for inhibitors of human papillomavirus 14:329–340. DNA replication. Biotechnology 1995; 13:1210–1214. 113. Rank NM & Lambert PF. Bovine papillomavirus type 1 E2 96. Clark PR, Roberts ML & Cowsert LM. A novel drug transcriptional regulators directly bind two cellular tran- screening assay for papillomavirus specific antiviral scription factors, TFIID and TFIIB. Journal of Virology activity. Antiviral Research 1998; 37:97–106. 1995; 69:6323–6334. 97. Stanley MA, Browne HM, Appleby M & Minson AC. 114. Yasugi T, Benson JD, Sakai H, Vidal M & Howley PM. Properties of a non-tumorigenic human cervical Mapping and characterization of the interaction domains keratinocyte cell line. International Journal of Cancer of human papillomavirus type 16 E1 and E2 proteins. 1989; 43:672–676. Journal of Virology 1997; 71:891–899. 98. Bedell MA, Hudson JB, Golub TR, Turyk ME, Hosken M, 115. Winokur PL & McBride AA. The transactivation and Wilbanks GD & Laimins LA. Amplification of human DNA binding domains of the BPV-1 E2 protien have papillomavirus genomes in vitro is dependent on epithelial different roles in co-operative origin binding with the E1 differentiation. Journal of Virology 1991; 65:2254–2260. protein. Virology 1996; 221:44–53. 99. Jeon S & Lambert PF. Integration of human papillomavirus 116. Storey A, Piccini A, Massima P, Bouvard V & Banks L. type 16 DNA into the human genome leads to increased Mutations in the human papillomavirus type 16 E2 protein stability of E6 and E7 mRNAs: implication for cervical identify a region of the protein involved in binding to E1 cancer. Proceedings of the National Academy of Sciences, protein. Journal of General Virology 1995; 76:819–826. USA 1995; 92:1654–1658. 117. Antson AA, Burns JE, Moroz OV, Scott DJ, Sanders CM, 100. Hall WS, Goto-Mandeville R, Shih HA, Shank PR & Bronstein IB, Dodson GG, Wilson KS & Maitland NJ. Braun L. Molecular analysis of episomal human papillo- Structure of the intact transactivation domain of the mavirus type 16 DNA in a cervical carcinoma cell line. human papillomavirus E2 protein. Nature 2000; Virus Research 1997; 51:183–195. 403:805–809. 101. Meyers C, Mayer TJ & Ozbun MA. Synthesis of infectious 118. Harris SF & Botchan MR. Crystal structure of the human human papillomavirus type 18 in differentiating epithelium papillomavirus type 18 E2 activation domain. Science transfected with viral DNA. Journal of Virology 1997; 1999; 284:1673–1677. 71:7381–7386. 119. McBride AA, Byrne JC & Howley PM. E2 polypeptides 102. Flores ER, Allen-Hoffmann BL, Lee D, Sattler CA & encoded by bovine papillomavirus type 1 form dimers Lambert PF. Establishment of the human papillomavirus through the common carboxy-terminal domain: type 16 (HPV-16) life cycle in an immortalized human Transactivation is mediated by the conserved amino foreskin keratinocyte cell line. Virology 1999; terminal domain. Proceedings of the National Academy of 262:344–354. Sciences, USA 1989; 86:510–514. 103. Thomas JT, Hubert WG, Ruesch MN & Laimins LA. 120. Prakash SS, Grossman SR, Pepinsky RB, Laimins LA & Human papillomavirus type 31 oncoproteins E6 and E7 Androphy EJ. Amino acids necessary for DNA contact and are required for the maintenance of episomes during the dimerization imply novel motifs in the papillomavirus E2 viral life cycle in normal human keratinocytes. Proceedings transactivator. Genes and Development 1992; 6:105–116. of the National Academy Sciences, USA 1999; 96:8449–8454. 121. Hegde RS, Grossman SR, Laimins LA & Sigler PB. Crystal structure at 1.7 Å of the bovine papillomavirus-1 E2 DNA- 104. McBride AA, Romanczuk H & Howley PM. The papillo- binding domain bound to its DNA target. Nature 1992; mavirus E2 regulatory proteins. Journal of Biological 359:505–512. Chemistry 1991; 266:18411–18414. 122. Liang H, Petros AM, Meadows RP, Yoon HS, Egan DA, 105. Alexander KA Phelps WC. A fluorescence anisotropy study Walter K, Hozman TF, Robins T & Fesik SW. Solution of DNA binding by HPV-11 E2C Protein: A hierarchy of structure of the DNA-binding domain of a human papillo- E2-binding sites. Biochemistry 1996; 35:9864–9872. mavirus E2 protein: evidence for flexible DNA-binding 106. Li R, Knight J, Bream G, Stenlund A Botchan M. Specific regions. Biochemistry 1996; 35:2095–2103. recognition nucleotides and their DNA context determine 123. Hegde RS, Wang AF, Kim SS & Schapira M. Subunit the affinity of E2 protein for 17 binding sites in the BPV-1 rearrangement accompanies sequence-specific DNA genome. Genes & Development 1989; 3:510–526. binding by the bovine papillomavirus-1 E2 protein. Journal 107. Thain A, Webster K, Emery D, Clarke AR & Gaston K. of Molecular Biology 1998; 276:797–808. DNA binding and bending by the human papillomavirus 124. Bochkarev A, Barwell JA, Pfuetzner RA, Bochkareva E, type 16 E2 protein: Recognition of an extended binding Frappier L & Edwards AM. Crystal structure of the DNA site. Journal of Biological Chemistry 1997; binding domain of the Epstein–Barr virus origin-binding 272:8236–8242. protein, EBNA1, bound to DNA. Cell 1996; 84:791–800. 108. McBride AA & Myers G. Section III. In Human 125. McBride AA, Bolen JB & Howley PM. Phosphorylation Papillomaviruses. Edited by G Myers, C Baker, K Munger, sites of the E2 transcriptional regulatory proteins of bovine F Sverdrup, AA McBride, H-U Bernard & J Meissner. papillomavirus type 1. Journal of Virology 1989; 1997; Section III, pp. 54–73. Los Alamos, N.M., USA: Los 63:5076–5085. Alamos National Labortories. 126. Lehman CW & Botchan MR. Segregation of viral plasmids

Antiviral Therapy 5:4 241 MR Underwood et al.

depends on tethering to chromosomes and is regulated by mavirus E2 protein. Journal of Medicinal Chemistry 1997; phosphorylation. Proceedings of the National Academy of 40:3144–3150. Sciences, USA 1998; 95:4338–4343. 132. Lowy DR. HPV Infection: future prospects. Clinical 127. Huber HE, Koblan KS & Heimbrook DC. Protein–protein Dermatology 1997; 15:299–302. interactions as therapeutic targets for cancer. Current Medicinal Chemistry 1994; 1:13–34. 133. Jansen KU, Rosolowsky M, Schultz LD, Markus IIZ, Cook JC, Donnelly JJ, Martinez D, Ellis RW & Shaw AR. 128. Cowsert LM, Fox MC, Zon G & Mirabelli CK. In vitro Vaccination with yeast-expressed cottontail rabbit (CRPV) evaluation of phosphorothioate oligonucleotides targeted virus-like particles protects rabbits from CRPV-induced to the E2 mRNA of papillomavirus: potential treatment for papilloma formation. Vaccine 1995; 13:1509–1514. genital warts. Antimicrobial Agents and Chemotherapy 1993; 37:171–177. 134. Suzich JA, Ghim S-J, Palmer-Hill FJ, White WI, Tamura JK, Bell JA, Newsome JA, Jenson AB & Schlegel R. 129. Kurg R, Langel U & Ustav M. Inhibition of the bovine Systemic immunization with papillomavirus L1 protein papillomavirus E2 protein activity by peptide nucleic acid. completely prevents the development of viral mucosal Virus Research 2000; 66:39–50. papillomas. Proceedings of the National Academy of 130. Kasukawa H, Howley PM & Benson JD. A fifteen amino Sciences, USA 1995; 92:11553–11557. acid peptide inhibits human papillomavirus E1-E2 interac- 135. Kirnbauer R, Chandrachud LM, O’Neill BW, Wagner ER, tion and human papillomavirus DNA replication in vitro. Grindlay GJ, Armstrong A, McGarvie GM, Schiller JT, Journal of Virology 1998; 72:8166–8173 Lowy DR & Campo MS. Virus-like particles of bovine 131. Hajduk PJ, Dinges J, Miknis GF, Morlock M, Middleton papillomvirus type-4 in prophylactic and therapeutic T, Kempf DJ, Egan DA, Walter KA, Robins TS, Shuker SB, immunization. Virology 1996; 219:37–44. Holzman TF & Fesik SW. NMR-based discovery of lead 136. Breitburd F & Coursaget P. Human papillomavirus inhibitors that block DNA binding of the human papillo- vaccines. Seminars in Cancer Biology 1999; 9:431–445. Received 26 June 2000; accepted 6 September 2000

242 ©2000 International Medical Press