The highest prevalence of genital HPV infection is found in sexually active women younger than age 25 years. Polymerase chain reaction analysis of genital specimens from women attending university infirmaries indicated that as many as 40% had some evidence of HPV infection [6]. A hybridization analysis for viral DNA found that 15% of adolescent females attending family planning clinics had evidence of an HPV infection and that 76% of the HPV-positive females had a normal Papanicolaou smear result [7]. A separate comparative analysis of 454 women attending a sexually transmitted diseases clinic and 545 college women showed that 11% of the patients with a sexually transmitted disease but only 2% of the college students presented with visible genital warts. In this study, however, analysis of cervical specimens from women without visible lesions showed that 10% of the patients with a sexually transmitted disease and 11% of the students were positive for HPV DNA or antigen [8].

Although the prevalence of asymptomatic or clinically inapparent disease is uncertain, clearly a substantial proportion of infected persons progress to condylomata or to cervicovaginal dysplasia and cervical cancer. The greatest prevalence of high-grade cervical dysplasia is found in women older than age 25 years, with the peak incidence of cervical cancer occurring after age 35 years [9]. Because most cases of cervical dysplasia and carcinoma (approximately 90%) are associated with HPV infection, the increasing prevalence of genital warts in young women may portend a future epidemic increase in the rate of premalignant disease and cervical cancer.

Despite the high prevalence of HPV infection, no available drug therapy effectively eliminates HPV infection and replication or prevents HPV-associated malignant progression. As a consequence, there is a striking unmet medical need for the development of safe and effective therapies for HPV-associated disease. Current treatment relies on excision or ablation of dysplastic or malignant tissue and is associated with substantial rates of recurrence, discomfort, and expense [10, 11]. Although papillomaviruses cannot yet be routinely cultured in the laboratory, the tools of molecular virology have been used extensively in the last 10 years to study the functions of the 8 to 10 individual viral genes. Several of the viral gene products represent attractive targets for the development of antiviral inhibitors. We examine the current state of antiviral investigations and the prospects for the development of potent and clinically useful therapies for inhibiting HPV replication and pathology.

General Properties of the Human Papillomaviruses

The papillomaviruses are members of the Papovaviridae family and infect most vertebrate species with exclusive host and tissue specificity. The virion is a small, nonenveloped icosahedral capsid that contains a single molecule of supercoiled, double-stranded, circular DNA about 8000 base-pairs in length. Viral DNA is replicated in the nuclei of infected cells in association with low-molecular-weight histones and is organized into nucleosomal structures that are reminiscent of host cellular chromatin. The genomes of many of the animal and human papillomaviruses have been completely sequenced. The arrangement of the open-reading frames, which are translated into viral proteins, and the nucleotide and amino-acid sequences of certain domains of the viral proteins have been well conserved during evolution of the animal and human viruses. As shown in Figure 1, the viral genome can be bisected into early (E1 to E7) and late regions (L1 and L2) separated by a 1-kb long control region (LCR), which contains transcriptional control elements and the origin of viral DNA replication. By convention established for other DNA viruses, viral genes designated as early encode functions that are important for the establishment of infection and for the initiation of viral DNA replication. The late genes encode the viral capsid proteins, the expression of which is normally delayed until after viral DNA is amplified [12].

View larger version (35K): [in this window] [in a new window]   Figure 1. A circular map of the double-stranded genome of human papillomavirus type 11 (HPV-11) (229). The open-reading frames are shown as shaded boxes and are labeled as E for early and L for late. Transcription is completely derived from one strand (proceeding clockwise). The long control region (LCR) is an approximately 1-kb region of DNA that contains promoter and enhancer elements and the origin of viral DNA replication. The different roles of the early and late viral proteins are shown at the bottom of the figure. bp equals base-pair.

For many years, it was assumed that all infectious human epidermal lesions were caused by a single human wart virus. It was not until the mid-1970s that restriction-endonuclease mapping and hybridization analysis provided the earliest indication of the remarkable plurality of HPV genotypes. Approximately 70 different HPV types are currently recognized and have been isolated from various mucosal and cutaneous epithelial lesions (Table 1). Human papillomaviruses are normally grouped according to their pathologic associations and tissue specificity as cutaneous or mucosal [13]. The 23 mucosal-associated HPVs can be further subdivided into those at high risk for malignant progression and those at low risk for progression [14].

Nonmalignant Human Papillomavirus Disease

Cutaneous Warts

The most common and familiar result of HPV infection is the development of a localized, benign epithelial neoplasm or wart. Cutaneous palmar and plantar warts are widespread in the population and are typically found to contain HPV types 1, 2, 3, 4, or 10. Cutaneous warts can persist or grow slowly for years and then abruptly disappear without apparent cause or therapy. The incidence of benign warts is highest in children and young adults and decreases later in life, presumably because of the development of systemic immunity. Plantar and palmar warts are considered to be exclusively benign. Considering the ubiquity of common skin warts in humans and the relation of other types of HPV to cancer, it is remarkable that the reported incidence of malignant conversion of skin warts is essentially 0. In addition, the sites of infection, commonly the hands and feet, are frequently exposed to mechanical trauma or other potential cocarcinogenic factors—humans are often in contact with the environment through bare hands and feet. These observations show and support a seminal theme in the pathobiology of HPV disease: The potential for malignant conversion of HPV-associated lesions is, at least in part, determined by the infecting HPV genotype and its target tissue.

Condyloma Acuminata

In the United States, the incidence of HPV-associated genital warts is increasing dramatically. Ranking only behind chlamydial infection and gonorrhea, sexually transmitted genital warts are one of the fastest growing sexually transmitted diseases in the United States, with an estimated incidence two to three times that of genital herpes. Clusters of exophytic condylomata generally develop on the penis, vagina, vulva, and perineum and around the anus. Only rarely do condylomata develop on the cervix. More than 90% of the lesions examined are associated with infection by the low-risk HPV types 6 and 11, the same two virus types associated with respiratory papillomatosis [15-17]. Condylomata may spontaneously regress or persist for years. Progression to invasive carcinoma is seen at a relatively low frequency [14]. During pregnancy, condylomata have been observed to increase in both size and number, possibly because of the stimulatory effect of steroid hormones on HPV transcription [18, 19] or because of a decrease in host-cell-mediated immunity.

Benign condylomata are often resistant to various forms of therapy, which results in high recurrence rates (30% to 90%). In addition to the social stigma associated with them, genital warts can be painful, may spread locally in an individual patient, and are infectious to sexual partners. Approximately two thirds of the sexual partners of patients with condylomata have been observed to develop genital warts during a 1- to 8-month observation period [5]. Although condylomata are not generally considered life-threatening, without effective antiviral therapy they represent a substantial problem in therapeutic and epidemiologic management.

Laryngeal Papillomas

Laryngeal or recurrent respiratory papillomas are benign epithelial tumors that usually develop on the larynx and are associated with infection by HPV types 6 and 11 [20, 21]. Although spontaneous malignant conversion is rare [22], therapeutic irradiation of juvenile laryngeal papillomas has been associated with a high incidence of carcinoma 5 to 40 years after treatment [23]. More recently, HPVs have been identified in lesions of the nose and oral cavity and have shown a differential propensity for malignant development [24, 25].

Two forms of laryngeal papillomatosis are clinically recognized: juvenile-onset, which initially develops in children younger than 5 years of age, and adult-onset, in which symptoms normally arise after age 20 years. Human papillomavirus infection in juvenile laryngeal papillomatosis is thought to occur during passage through an infected birth canal [26-28]. More than half of mothers of children with juvenile-onset disease have been reported to have a clinical history of genital warts; delivery by cesarean section reduces risk for transmission [29]. The coincidental presence of the same virus types, HPV types 6 and 11, in both the genital tract and in the larynx supports the notion of vertical transmission and suggests that adult-onset disease may be caused by infection from oral-genital contact or from acquisition at birth with a prolonged latency period.

Laryngeal papillomatosis is relatively uncommon. Previous estimates suggested that in- the United States, approximately 1500 cases were diagnosed each year [30]. More recent data suggest that the incidence is similar to that of neonatal HSV infections (1/2500 to 1/10 000 cases per year) [31]. The major challenges in the management of laryngeal papillomatosis are recurrence and rapid growth of papillomas, which can obstruct the trachea. The disease is usually resistant to most available therapies; thus, in moderate to severe cases, endoscopic excision can be required as many as 20 times per year to maintain a patent airway. Unfortunately, many patients with laryngeal papillomatosis have this painful and debilitating disease throughout their lives.

Malignant Human Papillomavirus Disease

Epidermodysplasia Verruciformis and Skin Cancer

During the last 20 years, Orth [32] and Jablonska and colleagues [33] have intensely studied a group of patients highly susceptible to cutaneous warts. Epidermodysplasia verruciformis is a rare, hereditary, and lifelong disease that is clinically characterized by disseminated flat or pityriasis-like skin lesions or reddish macules on the trunk and upper extremities [34]. The underlying genetic basis is poorly understood but appears to be a manifestation of a subtle defect in cell-mediated immunity or natural killer cell activity [35].

Although only about 1000 patients throughout the world are available for analysis, the study of this group has led to two important observations. First, adult patients with epidermodysplasia verruciformis have a high incidence for the development of cutaneous malignant lesions (30% to 50%) on areas of the skin exposed to the sun, such as the forehead. This suggests that in this patient population, sunlight or ultraviolet light may play a cocarcinogenic role for malignant conversion of HPV-infected cutaneous tissue. Second, patients with epidermodysplasia verruciformis are infected by more than 20 different HPV types that are not generally found in the normal population. Thus, these patients have a selective, congenital susceptibility to infection by a group of otherwise harmless or clinically inapparent HPV types. Analyses of infected tissues have shown that only a subset of the HPVs associated with epidermodysplasia verruciformis, in particular HPV types 5 and 8, are found in the malignant lesions; this finding again emphasizes the prognostic importance of the HPV genotype [36-39]. The biological and temporal relations among the HPV type, a cocarcinogen (sunlight), and a subtle immune defect are not yet well understood; however, epidermodysplasia verruciformis is clearly an important model system for the study of the role of viruses in the multistep development of cancer.

Cervical and Anogenital Cancer and Intraepithelial Neoplasia

Several epidemiologic studies had implicated a venereally transmitted agent in the cause of human cervical cancer. These suggestions led to the evaluation of many sexually transmitted viral and bacterial agents, including herpes simplex virus type 2 [40, 41]. The seminal observation to suggest a role for HPV in cervical cancer was the recognition that the cytologic abnormalities that are the diagnostic basis for the Papanicolaou screen are cytopathic effects of HPV infection [42, 43]. During the latter half of the 1980s, molecular hybridization analysis of biopsy tissues strengthened the potential etiologic relation between infection with certain types of HPVs and cervical cancer. Recent analyses of cervical carcinoma tissues have shown that approximately 85% of the lesions maintain and express HPV DNA sequences. In general, it is a subgroup of HPVs, the high-risk types, that are most frequently associated with genital malignancies. Specifically, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, and 56 are found in cases of moderate to severe dysplasia and invasive carcinoma of the cervix, vulva, anus, and penis [44-46]. Convincing epidemiologic confirmation of a causal role for HPV infection in the development of cervical cancer is beginning to emerge from prospective case–control studies of large cohorts of women with HPV infection [47].

Invasive genital cancer appears as the latter stage of a series of progressive cytologic abnormalities of the mucosal epithelium. The precursor lesions are normally identified as intraepithelial neoplasias of specific anatomical sites (for example, the cervix, vagina, penis, and perianal region). Lesions are further classified histologically as mild to severe (grades I to III) according to the degree of dysplasia. Human papillomavirus genomes have been identified in most intraepithelial neoplasias; nearly all genital HPV types are represented in mildly dysplastic lesions. In contrast, in invasive cancers and high-grade dysplasias, high-risk HPV types predominate; HPV type 16 is found in 50% to 60% of invasive cancers, and HPV type 18 is found in 10% to 20%. The low-risk HPV types 6 and 11 are much more frequently found in exophytic condylomata, and only rarely in high-grade dysplasia [48].

Despite the strong epidemiologic association between infection with certain HPV types and the development of anogenital cancer, only a subset of patients infected with high-risk HPVs progresses to invasive squamous carcinoma [47]. Further, it has been suggested that primary infection and progression to invasive cancer may normally be separated by a latency period of 20 to 50 years [49]. These observations clearly indicate that virus infection alone is not sufficient for the development of genital malignancy. Infection with a high-risk HPV type may be an initiating event, whereas other somatic alterations are required to support malignant progression of benign neoplasms. Thus, a goal of antiviral management for papillomavirus infection is the elimination of the initiating or potentiating component in the multistep process of malignant progression. Successful inhibition of virus replication in the benign lesion (wart) or premalignant lesion (intraepithelial neoplasia) should preclude future development of invasive cancer.

Current Therapies

Ablative Therapy

The goal of the various forms of ablative therapy used for treatment of warts is the eradication of the population of infected cells [50]. Such therapy includes either physical destruction of tissue or chemically induced cytotoxicity. None of the treatments currently used is antiviral in mechanisms of action because there is no substantial selectivity in the cytodestruction of virus-infected cells. However, cytolysis may cause the release of viral antigens, which leads to a beneficial immune stimulation.

Surgical techniques that have been used with varying degrees of success include local excision, cryotherapy with liquid nitrogen or dry ice, CO₂ laser vaporization, and electrocautery [51]. Such therapies can be painful, disfiguring, and, in patients with disseminated disease, impractical. In addition, it is not clear to what extent frequent recurrences are caused by reactivation from subclinical or latent infections of normal-appearing epithelium that has been left untreated.

Many chemical and cytotoxic agents have been examined in the past. Simple organic acids such as trichloroacetic acid, bichloroacetic acid, or salicylic acid, and protein cross-linking reagents such as glutaraldehyde and formalin have shown some success for localized topical treatment. Other cytotoxic and antiproliferative drugs that have been used include antimitotic agents such as podophyllin or colchicine and antimetabolites such as bleomycin, cantharidin, and 5-fluorouracil. These agents have shown efficacy for topical or intralesional therapy because of their inhibition of the cellular hyperproliferation characteristic of HPV infection. However, as with physical ablation, chemical cytotoxicity lacks specificity for virus-infected tissues.

Immunomodulation

A hallmark of HPV-induced disease is the ability of benign lesions to spontaneously regress because of a systemic immunologic response in immunocompetent persons. For this reason, stimulation of the immune system has been an appealing treatment. Reagents such as autogenous vaccines, levamisole, dinitrochlorobenzene, and bacille Calmette–Guérin have been used with limited success. In addition, both lymphoblastoid () and fibroblast (ß) interferon have been used with some success for systemic treatment of genital and laryngeal warts [52, 53]. Unfortunately, the efficacy of interferon varies, and the side effects during prolonged therapy can be pronounced.

The present catalog of available therapies for HPV-associated disease is both unsophisticated and unsatisfactory. With the possible exception of interferon, no true antiviral therapy exists. State-of-the-art therapy is defined only by the method used to most effectively eliminate the infected tissue rather than through biological or pharmacologic inhibition of virus replication.

Profile of an Effective Antiviral Agent

Important principles of antiviral drug discovery have emerged from the many disappointments and the few notable successes in the last 25 years of research. The empiric lessons of the past should help to guide future investigations of potential antipapillomavirus chemotherapeutic agents. To begin discussing antiviral screening and drug discovery for inhibitors of the papillomaviruses, it is useful to briefly examine some of the antiviral agents that have been discovered and developed in the last decade.

Nucleoside Analogs

The most successfully exploited antiviral targets have been viral enzymes, specifically the viral nucleotide kinases and polymerases. In infected cells, viral kinases cause the selective enzymatic activation of the nucleoside analogs acyclovir and ganciclovir to their respective monophosphate derivatives [54, 55]. In the subsequent conversion of the monophosphates to diphosphates and triphosphates, host-cell nucleotide kinases are used. Acyclovir triphosphate acts as an effective competitor of deoxyguanosine triphosphate for incorporation by the HSV DNA polymerase into viral DNA. Incorporation of acyclovir leads to termination of the nascent DNA chain and inactivation of the template bound enzyme [56]. A high therapeutic index is achieved with acyclovir because it is preferentially used by two different viral enzymes during nucleotide metabolism.

The most effective antiretroviral drugs inhibit the viral RNA-directed DNA polymerase or reverse transcriptase. Zidovudine is one of the first and still most widely used nucleoside analogs for HIV therapy [57]. Kinetic studies have shown that zidovudine triphosphate is a potent competitive inhibitor of HIV reverse transcriptase. The antiviral activity is caused by preferential incorporation of zidovudine instead of thymidine into the nascent chain, leading to premature termination of viral DNA replication [58, 59]. A second nucleoside analog developed for treatment of HIV is dideoxyinosine [60]. This drug is converted intracellularly to dideoxyadenosine triphosphate, which is incorporated in place of deoxyadenosine triphosphate into the growing viral DNA strand; this process leads to chain termination. Antiviral selectivity is thus determined by the degree of preference shown by HIV reverse transcriptase for the nucleoside triphosphate analogs.

Ribavirin, another nucleoside analog, was originally synthesized in the 1970s [61] and has been shown to have activity against many DNA and RNA viruses [62, 63], including the rabbit papillomavirus [64]. Although its mechanism of antiviral activity is not fully understood, ribavirin perturbs cellular purine metabolism by inhibiting inosine monophosphate dehydrogenase [65], which leads to a decrease in guanosine triphosphate pools [66, 67]. In contrast to acyclovir, zidovudine, and dideoxyinsine, ribavirin relies on inhibition of a cellular enzyme; this reliance undoubtedly contributes to the toxicologic profile of the compound. Nonetheless, ribavirin has been clinically used as an antiviral agent delivered by aerosol for severe respiratory syncytial virus infections, and it was recently recommended for intravenous treatment during the hantavirus outbreak in the southwestern United States [68]. In addition, ribavirin is currently being studied in phase II clinical trials for the treatment of laryngeal papillomatosis [69].

Non-nucleoside Antiviral Agents

Amantadine and rimantadine are weakly basic adamantane compounds that accumulate in acidic vacuoles of eukaryotic cells. In humans, these compounds are used for prophylaxis and treatment of influenza A infections. The mode of antiviral activity is thought to be caused by interference with acid-dependent fusion of the virion coat to vacuolar membranes, which results in the inhibition of the uncoating process [70, 71].

Recently, several non-nucleoside inhibitors of the HIV reverse transcriptase have been identified, including nevirapine [72] and TIBO [73]. In contrast to the nucleoside analogs, these inhibitors are not substrate mimics. Instead, they bind noncompetitively to an adjacent allosteric site on the functional enzyme complex, thereby inhibiting DNA polymerization [74].

Determination of the crystal structure of the HIV protease by many investigators [75-78] has stimulated the development of several peptidomimetic [79-82] and nonpeptide inhibitors of the protease [76, 83]. Inhibition of the HIV protease results in the abrogation of polyprotein processing, which leads to the accumulation of noninfectious, morphologically immature virions.

Clinical Utility

At this point in the discovery and preclinical development of antiviral agents for papillomavirus infections, the most important consideration is the toxicity of candidate compounds. Acyclovir is an exceptionally safe compound because its activation requires a virus-encoded enzyme and because incorporation of acyclovir triphosphate by the viral DNA polymerase is favored over deoxyguanosine triphosphate. In contrast, the broad-spectrum utility of ribavirin is limited by cellular toxicity. The target for inhibition is a cellular rather than a viral enzyme, inosine monophosphate dehydrogenase, which is not a viral enzyme; thus, clinical toxicity can be related to enzyme inhibition in certain tissues. Although anabolism is accomplished by cellular enzymes, the antiviral activities of nucleoside analogs such as zidovudine, dideoxynosine, and dideoxycytosine each rely on selective utilization by a viral enzyme, the HIV reverse transcriptase, and incorporation into the growing nucleic acid chain during virus replication.

As discussed above, successful strategies for the development of antiviral compounds have been directed against virus-specific enzymes (kinases, polymerases, proteases) and functions (uncoating). Unfortunately, few enzymes are encoded by the human papillomaviruses, and none are kinases, polymerases, or proteases. For virus reproduction, the HPV expropriate many host-cell functions, including the host DNA polymerase and the machinery for nucleotide metabolism. Therefore, development of antiviral agents that are selectively incorporated into HPV DNA may be extremely difficult.

Potential Targets in the Virus Life Cycle

Conventional antiviral assays have relied on the growth of viruses in cell culture. Antiviral compounds have been identified by their ability to inhibit growth of the virus in vitro as evidenced by yield reduction, plaque reduction, or abrogation of cytopathic effects in the presence of test compounds. One of the reasons that many of the first antiviral agents to be developed (for example, iododeoxyuridine and trifluorothymidine) were specific to herpes simplex is that the virus can be easily manipulated in cell culture. Consequently, HSV has been amenable to the development of high-throughput assays in many laboratories. In addition, HSV encodes at least 72 distinct viral proteins [84]. Among these are factors involved in temporal regulation of expression; at least seven proteins that function in viral DNA replication, including a DNA polymerase; and several enzymes for nucleotide metabolism (such as ribonucleotide reductase and thymidine kinase). In effect, each compound that is screened for inhibition of virus growth in cell culture is simultaneously evaluated for activity against as many as 70 different HSV functions.

Papillomaviruses cannot yet be grown in cell culture in a manner that would permit conventional antiviral assays. Thus, individual viral functions must be isolated as discrete molecular events, and compounds must be evaluated for activities in the modulation of each of these individual steps in the life cycle of the virus (Figure 2). In contrast to HSV, the papillomaviruses encode only 9 to 10 distinct proteins and do not have a viral protease, a DNA polymerase, or enzymes involved in nucleotide metabolism.

View larger version (22K): [in this window] [in a new window]   Figure 2. The cycle of human papillomavirus infection of epithelial cells. Virus infection is believed to occur within the basal keratinocytes, and early transcription of the viral genome is thought to be regulated by the E2 protein. Cellular proliferation and perturbation of keratinocyte differentiation is induced by the E6 and E7 proteins. Stimulation of cells into the S phase of the cell cycle leads to induction of host enzymes for DNA synthesis. Viral DNA replication is initiated, and double-stranded progeny episomes are accumulated. Capsid proteins, L1 and L2, and a late-associated protein, E4, are synthesized, and virus particles are assembled during terminal stages of keratinocyte differentiation. Progeny virus particles are released as dead keratinocytes are sloughed from the surface of the epithelium.

Selection of appropriate antiviral targets requires that the candidate functions be unique to the virus and be absolutely required for continued replication. For the purpose of assay development or inhibitor design, the molecular targets for consideration may consist of viral enzymes, protein-nucleic acid interactions such as those critical to transcriptional modulation, or protein-protein interactions such as the association between the viral capsid and a membrane receptor. The merits and challenges for study of many potential molecular targets of the HPVs are discussed below.

Replication

During productive infection, the papillomaviruses replicate their viral DNA as an extrachromosomal element physically separate from the host-cell genome. Genetic and biochemical analyses have indicated that only two viral gene products are necessary for origin-dependent replication, the E1 and E2 proteins [85-90]. Deoxyribonucleic acid polymerase and most other associated replication factors are supplied by the host cell.

The E1 protein is a 70- to 80-kd nuclear phosphoprotein with adenosine and guanosine triphosphatase and DNA helicase activity [85, 91-94]. The E1 protein is a DNA-binding protein, and, like other viral replicative proteins such as SV40 T antigen and HSV UL9, it binds to the origin of viral DNA replication [90, 95-98]. In addition, the E1 protein forms a specific protein:protein complex with the HPV E2 protein. Through localization of the viral DNA sequences sufficient for origin-dependent replication, several important sequence elements were identified [97]. The origin region contains an inverted repeat, an AT-rich region, and a binding site for the E2 protein. Complexation between E1 and E2 markedly stimulates the DNA-binding activity of the E1 protein at the origin of replication, possibly through allosteric alteration of the E1 protein [99, 100]. Origin binding by E1 and E2 is thought to be rate-limiting and may lead to the recruitment of other factors for the initiation of replication.

The E1 protein encodes several functions potentially amenable to antiviral intervention. The adenosine triphosphatase-guanosine triphosphatase and helicase activities are attractive targets for large-scale screening of molecules that might inhibit these enzymatic functions. Because HPVs do not encode a viral nucleotide kinase, compound screening would include various nucleotide analogs of purine triphosphates or phosphorylated mimics such as the nucleoside phosphonates [101]. Because of the favorable pharmacologic profiles of several nucleoside inhibitors, many such analogs exist in the chemical libraries of the pharmaceutical industry. Success in the discovery of specific inhibitors of the viral nucleoside triphosphatase (for example, adenosine triphosphatase) should hinge on the degree of similarity to related cellular enzymes. Examination of the amino acid sequence of many nucleic acid-dependent adenosine triphosphatases shows that although certain positions are invariably conserved across three distinct structural motifs, amino acid sequence varies considerably between the E1 proteins and other viral and cellular adenosine triphosphatases [102]. Because nucleoside triphosphatase activity is a prevalent mechanism for supplying energy to various cellular processes, the inherent specificity of any candidate antiviral inhibitor for the viral enzymes is critical to the suppression of widespread cellular toxicity.

In contrast to the targeting of the nucleoside triphosphatase activity, conceptual leads for the specific inhibition of the helicase functions are less obvious, in part because of a relatively poor understanding of the physicochemical mechanism of DNA unwinding. Deoxyribonucleic acid binding or intercalating compounds may be useful as the development of structure or sequence-specific binding chemistry improves [103].

Another potential avenue for antiviral intervention would be in the development of compounds that interfere with the ability of E1 to interact with either E2 or the viral DNA. Biochemical and genetic localization of the polypeptide regions involved in DNA binding and E1:E2 complexation are still incomplete, but such information will contribute to the search for such inhibitory molecules. These studies may initially focus on peptidomimetics as lead compounds for inhibition of complexation or DNA binding.

Transcription

Few small-molecule inhibitors of eukaryotic transcription have been found to have specificity for a particular class of messenger RNA. Classical nonspecific and cytotoxic transcriptional inhibitors such as actinomycin D and -amanitin, both of which inhibit RNA polymerase II, appear to inhibit synthesis of all classes of messenger RNAs. In contrast, only a few specific, small-molecule transcriptional inhibitors have been discovered thus far. The best examples are the immunosuppressive agents cyclosporine and FK506, which, although structurally unrelated, are potent inhibitors of cis-trans prolyl isomerase. In addition, these drugs inhibit transcription of the interleukin-2 gene by interfering with specific transcriptional activating factors [104].

The HPV E2 protein plays a pivotal role in the life cycle of the virus; it coordinates temporal control of viral RNA transcription and possibly couples viral expression to viral DNA replication. Experimental mutations of the E2 gene are pleiotropic and result in disruption of transformation, replication, and transcriptional control functions [105, 106]. The E2 protein can act both as an activator and repressor of transcription [107-109] through sequence-specific DNA binding to a 12-nucleotide palindrome, ACCN₆GGT [110, 111]. The binding of E2 results in modulation of transcriptional initiation, probably through interaction with components of the RNA polymerase II complex such as Sp1 [112] and TATA-binding protein [113].

A mixture of genetic and biochemical data divides the E2 protein into three distinct domains [114, 115]. The N-terminal region of the protein (approximately 200 amino acids long) encodes two acidic amphipathic helices that are required for transactivation [116]. The second distinct segment of the protein is an internal, poorly conserved hinge domain that separates the N- and C-terminal regions. Site-specific DNA binding and dimerization are encoded within the 85 amino acids of the C-terminal region. For transcriptional activation, the E2 protein binds to the palindromic DNA site as a homodimer [117-122].

Discovery of compounds that inhibit the transcriptional modulatory functions of E2 may be effective for blocking both early gene functions and viral DNA replication. E2-mediated transactivation can be readily adapted to large-scale pharmaceutical screening programs for the identification of inhibitors of E2. Alternatively, the E2 protein is an appropriate target for structure-based rational design of candidate inhibitors. Rational drug design has recently seen some practical success with the identification of various pharmacologically active candidates, including inhibitors of HIV protease [76], thymidylate synthetase [123, 124], carbonic anhydrase [125], and purine nucleoside phosphorylase [126, 127]. The crystal structure of the C-terminal fragment of the bovine papillomavirus E2 protein has recently been solved [128], thereby providing insight into the molecular dynamics of the highly conserved dimerization and DNA binding domains. This structure and x-ray crystallographic data for the HPV E2 proteins will be useful in the initiation of rational design efforts for inhibitors of E2 transcriptional modulation.

Human Papillomavirus Cellular Transformation

Three early papillomavirus proteins have cellular-transforming or -immortalizing functions: E5, E6, and E7 [129-132]. In the context of the virus life cycle, these proteins alter the growth properties of the infected cell, permitting or enhancing viral DNA replication. High-level or temporally and spatially inappropriate expression of such proteins is associated with properties of cellular transformation and are thought to be important for HPV-associated malignant progression. Therefore, development of inhibitory compounds that target such functions may have utility in the treatment of both benign warts and malignant carcinomas.

Most of the experimental work with the E5 protein has focused on the bovine papillomavirus polypeptide. Expression of the E5 protein of bovine papillomavirus type 1 is sufficient for morphologic transformation of certain rodent cells in culture [133]. With only 44 amino acids, the bovine papillomavirus E5 polypeptide is the smallest transforming protein yet described [134-136]. Studies of the mechanism of the transformation of bovine papillomavirus E5 have indicated that the E5 protein can perturb normal processing and turnover of the platelet-derived growth factor receptor, resulting in constitutive activation of the receptor [137]. Receptor activation may require other cellular proteins, such as the 16-kd subunit of the vacuolar H⁺-adenosine triphosphatase, which has been shown to form a complex with bovine papillomavirus E5 [138]. Recent studies with the HPV E5 proteins, which have weaker transforming abilities [139], have suggested that these viral proteins may perturb cycling of the epidermal growth factor receptor [140-142].

Although the E5 protein, with a molecular weight of 10 kd, should be amenable to structural studies, it may not be a suitable drug target. Deletion of the E5 open-reading frame in the rabbit papillomavirus animal model was shown to have only a modest quantitative effect on the development of benign warts [143]. Thus, expression of the E5 protein and targeting of growth factor receptors may not be absolutely required for development of benign, proliferative lesions. However, epidermal growth factor is an important mitogen for epidermal keratinocytes and is essential for growth of primary keratinocytes in culture [144]. Moreover, the receptor for epidermal growth factor is overexpressed in most severe cervical dysplasias [145], suggesting that mitogenic signaling may play an important role in malignant progression. As the molecular basis for the activity of the HPV E5 protein is elucidated, its potential as a target for antiviral or antitumor intervention may be enhanced.

In high-grade HPV-associated carcinomas, viral DNA is frequently integrated into the host-cell genome [146-148]. Although integration within the cellular genome appears to be essentially random, integration preferentially disrupts the E1 and E2 regions of the HPV genome [149-151]. An important consequence of this pattern of integration is that only the E6 and E7 genes remain unaffected. Transcriptional analysis of cervical carcinoma tissues and derived cell lines has shown that the E6 and E7 viral genes are always expressed [152, 153], whereas the expression of other viral genes varies. Because these proteins have growth-promoting or immortalizing properties in human keratinocytes, continued expression in tumor tissues suggests that these viral genes play a direct role in maintenance of the malignant phenotype [154, 155]. The E6 and E7 viral proteins are the only virus-specific gene products routinely found in HPV-associated cervical cancer and thus are the premier targets for the development of novel antitumor compounds.

The HPV E6 oncoprotein of the high-risk, mucosal-associated HPVs (for example, types 16, 18, 31, and 33) cooperates with the HPV E7 protein for the efficient immortalization of primary human keratinocytes [156-162]. The E6 protein associates with the tumor suppressor protein p53 [163, 164] and promotes the proteolytic destruction of the protein [165]. Mutational inactivation of the p53 gene is the most common, specific genetic alteration found in human cancer [166], strongly suggesting that loss of this function is a key event in malignant conversion of a cell. For replication of the virus, the selective degradation of a cell-cycle regulatory or tumor suppressor protein may alleviate the block to S phase, which develops during normal keratinocyte differentiation, and thus may permit the induction of the host catalytic machinery that is required for viral DNA synthesis.

The HPV E7 protein is a multifunctional oncoprotein that complexes with the product of the retinoblastoma susceptibility gene (pRB) [167-172]. Association of HPV E7 with pRB inactivates the cell-cycle restriction function of the pRB protein. A consequence of this interaction is the dissociation of the E2F transcription factor from pRB [173-175]. The E2F transcription factor modulates the expression of several cellular genes that are important for cell-cycle progression and DNA synthesis, including dihydrofolate reductase, ribonucleotide reductase, DNA polymerase-, thymidylate synthetase, thymidine kinase, epidermal growth factor receptor, c-myc, and c-myb [176-179].

It is possible to devise assay systems that target the respective association between the viral oncoproteins E6 and E7 and their cellular targets, p53 and pRB. The HPV E6 and E7 proteins have been successfully expressed in various experimental systems to examine the specificity and relative affinities of the protein-protein interactions [129, 159, 180-185]. For HPV16 E7, Jones and colleagues [186, 187] have defined the sequence of peptides that specifically and avidly interfere with E7:pRB interaction. Such studies may be an effective starting point for the development of pharmacologically active peptidic or nonpeptide inhibitors. A similar assay system may be used to identify compounds that target the interface between HPV E6 and p53. An alternative approach to random screening is to use the three-dimensional structure of the oncoproteins in the rational design of inhibitors. Both E6 and E7 are small metal-binding proteins and should be good candidates for structural determination by x-ray crystallography and multidimensional nuclear magnetic resonance spectroscopy.

Epithelial Differentiation

As previously mentioned, the papillomaviruses cannot yet be propagated in conventional tissue culture systems. The coordinate program of papillomavirus replication and expression are intimately tied to progressive vertical differentiation, which occurs during epidermal maturation. Although mammalian keratinocytes can be grown in culture, faithful epithelial differentiation cannot yet be fully achieved in vitro. Recent work with organotypic culture techniques [188, 189] has led to limited expression [190] and replication [191, 192] of certain HPVs in vitro. These systems, although cumbersome, offer great promise as future screening systems for the in vitro evaluation of inhibitors of papillomavirus replication.

Infection of keratinocytes by papillomaviruses results in a substantial perturbation of normal epithelial differentiation. The characteristic hyperplasia and acanthosis of warts is thought to be induced by the early gene products of the virus, including E6 and E7. What is characteristically observed, however, is not a complete inhibition of epithelial differentiation but a temporal delay that results in an increase in the thickness of the spinous layer of the epidermis. This association between epithelial differentiation and the life cycle of the papillomavirus offers opportunities for therapeutic intervention. In situ analyses of infected tissues indicate that a low level of viral DNA is found in the suprabasal and basal layers of the epidermis. Active viral DNA replication, as shown by a high content of papillomavirus DNA, is found exclusively in the more superficial layers of the epidermis, the stratum granulosum and the stratum corneum. Induction of vegetative viral DNA replication may therefore require certain conditions or biochemical factors that are exclusively found in specific layers of the epidermis. Potential therapeutic targets in the future could be directed to the re-establishment of the normal program of epithelial differentiation. Much effort is currently focused on unraveling the control mechanisms for cellular differentiation, senescence, and apoptosis. These studies may lead to the development of new classes of therapeutic agents that could stimulate normal cellular processes of terminal differentiation. Retinoids have been examined for many years because of their powerful effects on epithelial differentiation. Both in vivo and in vitro studies have shown that retinoids can inhibit normal epithelial maturation and keratinization [193]. A few studies have examined the effects of various retinoids on papillomavirus-infected cells or tissues and suggest that retinoic acid can down-regulate HPV transcription in cell culture [194-197].

Several antitumor agents have been used clinically for the treatment of papillomavirus-infected lesions. The characteristic hyperproliferation of warts can be inhibited by antineoplastic drugs, including microtubule inhibitors such as podophyllotoxin [11] and metabolic analogs such as 5-fluorouracil [51]. These agents are effective in suppressing cellular proliferation, which is thought to be required for virus replication. Restraint of hyperproliferation of infected cells inhibits viral DNA amplification through inhibition of the S phase of the cell cycle, potentially resulting in apoptosis of infected cells before virion assembly. However, it is not clear what effect, if any, such inhibition would have on latently infected basal cells of the epidermis.

Infection and Virion Assembly

There is substantial medical and pharmaceutical interest in the development of HPV vaccines for prophylactic immunization against HPV infection. Several laboratories have recently expressed the HPV capsid proteins L1 and L2 in surrogate eukaryotic protein expression systems and have observed spontaneous self-assembly of virus-like particles [198, 199]. The principal goal in many of these studies is the preparation of intact noninfectious virions that do not contain viral DNA for use in human and animal vaccination studies. A by-product of such studies is the availability of a controlled protein assembly process that could be used to screen for compounds that inhibit virion assembly or uncoating. X-ray crystallographic studies of the capsid could be used for rational design of novel inhibitors or to guide and complement compound screening efforts. This approach has been successfully used for the isolation of the inhibitors of picornavirus uncoating [200, 201], in which radiographic studies have defined a binding pocket for the WIN (Sterling-Winthrop) series of inhibitors [202].

Host-Cell Targets

Skin warts frequently regress spontaneously. In nearly 70% of persons with cutaneous warts, the warts disappear without therapy within 2 years. In contrast, almost one third of infected persons have warts that persist, sometimes for decades. The immunologic mechanism resulting in wart regression is poorly understood. It has been noted that in many instances, warts regress simultaneously at multiple sites, a process that suggests a systemic immune response.

Many lines of evidence suggest that cellular rather than humoral immunity is of primary importance for the restraint of papillomavirus-infected lesions [203]. A substantial increase in the incidence of warts and squamous-cell carcinoma has been noted in immunosuppressed patients, including renal transplant recipients [204-207] and HIV-infected patients [208, 209]. In the rare syndrome epidermodysplasia verruciformis (discussed above in detail), a congenital defect in the immune system is associated with a life-long incidence of disseminated wart disease. Clearly, the normal mechanisms that account for tumor rejection and immune surveillance are impaired in these populations, which thereby leads to an enhanced susceptibility to disseminated benign disease and a higher probability for progression to malignancy.

Therapeutic immunomodulation is an attractive area for future development of antiviral treatments [210-212]. Under normal circumstances, infected persons have an effective means for the immunologic elimination of virus-infected cells. Spontaneous regression of viral warts is associated with acute inflammation and infiltration with T cells and macrophages, which promote localized cytodestruction of infected keratinocytes [213]. The antigenic targets of activated T cells, which are critical to the immune response, have yet to be completely defined. Identification of the viral antigens, which participate in immune rejection, might lead to the development of clinically useful, epitope-specific immunostimulatory therapies to promote systemic rejection of infected tissue. Such immune modulatory therapy would presumably be ineffective in immunocompromised persons. Some evidence suggests that persistent papillomavirus-infected lesions are deficient for antigen presentation, in part because of a reduced number of Langerhans cells [214] or down-regulation of major histocompatibility complex expression [215-220]. Alternatively, immunologic unresponsiveness may be due to presentation of viral antigens in a tolerogenic form on infected keratinocytes or to the induction of a focal T-cell suppression [221]. Viral antigens are clearly at least transiently accessible to the immune system; serologic studies have detected circulating antibodies to various HPV proteins or peptides in serum samples of infected patients. The development of antibodies to the high-risk HPV E6 [222] and E7 proteins has recently been found in patients with invasive cervical cancer [223-228]. Future studies that help to clarify the nature of the immune response during persistence and regression of HPV-infected lesions should stimulate the design of therapeutic strategies that rely on induction of natural antiviral defenses.

Additional Considerations

The ideal antiviral drug for papillomavirus infections would eliminate existing lesions, eradicate latent populations of viral DNA, and permit the development of natural immunity to the virus. Examination of these three desirable drug characteristics provokes consideration of several unique facets of HPV-associated disease.

Unlike infections with many human pathogenic viruses, including HIV, cytomegalovirus, influenza, and hepatitis B and C, HPV infections are usually focal and develop on accessible cutaneous or mucosal surfaces. Superficial and localized infections should therefore be amenable to some forms of topical therapy. This becomes an important issue when candidate antiviral agents are assessed for in vivo toxicity. Because HPVs are highly dependent on normal cellular enzymes to complete the virus life cycle, there is potential for substantial host-cell toxicity from agents active against HPV DNA replication and expression. Thus, the ability to treat warts topically can be used to localize the collateral toxigenic effects of the compound and to minimize systemic exposure. However, the highly keratinized nature of HPV-infected lesions may engender additional formulation challenges to achieve desirable transdermal delivery. Alternatively, a more convenient systemic therapy would have to have low overall toxicity, be nonteratogenic for use in a sexually active population, and have favorable pharmacokinetic properties such that therapeutic plasma levels could be maintained to facilitate drug transport across the basement membrane to epidermal keratinocytes.

A substantial therapeutic goal of anti-HPV therapy is the elimination of latent populations of HPV DNA from basal keratinocytes. Concepts of viral latency have been framed primarily by studies with the herpesviruses, wherein HSV infection of the skin or mucous membranes is followed by retrograde axonal migration of the virus to the sensory ganglion, where a latent infection is established. Latent HSV infection of neurons is characterized by maintenance of a few copies of the viral genome with limited gene expression. After months or years, HSV replication can be reactivated through various stimuli, resulting in lytic replication and infectious virion production in epidermal cells.

The latent phase of the HPV life cycle occurs in the basal keratinocytes, where a few copies of the viral genome are maintained under conditions of limited viral expression. Induction of productive virus replication is due in part to terminal differentiation of epidermal keratinocytes. Induction of latent HPV may also be affected by hormone or growth factor changes or may relate to physical trauma and wound healing. With benign cutaneous or mucosal papillomavirus infection, it is difficult to physically or temporally distinguish a distinct latent phase of the infection. In contrast, infection with the high-risk HPVs is more clearly associated with a latent infection that is clinically characterized by a series of premalignant lesions (for example, cervical intraepithelial neoplasia grades I, II, and III), in which the viral DNA is maintained and expressed but few virus particles are produced. As described above, these premalignant lesions can persist or progress to malignant carcinoma; however, it is not clear that these latently infected cells can be readily induced to produce lytic virus particles.

In the future, antiviral therapies that do not inhibit latent replication should be evaluated for potential effects on malignant progression. This is not a trivial exercise, because the factors responsible for malignant progression of a subset of infected lesions are still poorly understood, and the time to progression in patients may be 20 to 50 years. Promotion of malignant progression is an unusual consideration for preclinical development of antiviral compounds. However, similar issues might be appropriate to the development of antiviral compounds for hepatitis B and C with regard to their potential for promotion of hepatocellular carcinoma.

Finally, HPVs show a rather remarkable plurality in that approximately 70 distinct genotypes have been described. One of the preeminent concerns for the development of an HPV vaccine is the induction of broad-spectrum HPV immunity. The activity of any HPV-specific antiviral compound must also be evaluated against several HPV types. To identify an antiviral compound that has inhibitory activity against a broad group of HPV types, it is important to screen for inhibitors of highly conserved targets such as the E1 and E2 functions. One would expect that inhibitors of either of these activities would have broad utility for many HPV types.

Summary

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Human papillomavirus infections underlie substantial human morbidity and mortality throughout the world. Current epidemiologic studies suggest that the prevalence of HPV infection in developed countries may be increasing dramatically, and that if current trends continue, we can expect a proportional increase in the prevalence of cervicovaginal carcinoma in the future. Cervical carcinoma is currently the second leading cause of cancer death in women in developing countries. Available therapies for symptomatic HPV disease are directed toward destruction or excision of the infected tissue. Such methods are expensive and painful and are associated with a high recurrence rate. No effective antiviral chemotherapeutic agent is currently available for HPV infection.

The problems faced in the development of effective agents for HPV infections are unique. More so than in many other viral infections, the life cycle of the virus is inextricably linked to tissue differentiation and cellular maturation. This is due in large part to the austere composition of the virus. Consisting of only 8 to 10 viral proteins, papillomaviruses must rely heavily on host-cell functions to complete the cycle of infection. Thus, the number of potential viral targets is relatively small. Nonetheless, there are distinct phases in the HPV infection cycle in which antiviral agents could be therapeutically useful. These steps include HPV entry into cells, HPV transcriptional regulation, HPV-driven cellular proliferation, HPV replication, and virion assembly.

In the last 15 years, study of the papillomaviruses has led to a marked increase in our understanding of viral transcription, cell proliferation, and human oncogenesis. As our knowledge of these areas grows, prospects for the development of effective agents for treatment of HPV infections are enhanced. Successful development of antiviral therapies for HPV infections will continue to be important both for clinical relief to the many infected persons and for the epidemiologic control of the transmission of a human tumor virus.

Many academic and industrial laboratories have accepted the challenge for the design and development of novel antiviral therapies for the HPVs. The rapid advances in techniques in molecular virology, rational drug design, and robotic compound screening offer tangible incentives for optimism about the discovery of effective medicines to restrain or eradicate HPV-associated disease.

Dr. Alexander: Department of Pediatrics, Duke University Medical School, Durham, NC 27710.