Molecular Targets for Human Papillomaviruses: Prospects for Antiviral Therapy

Phelps, William C.; Barnes, J. Steven; Lobe, DC

doi:10.1177/095632029800900501

Cited by 47 publications

(25 citation statements)

References 212 publications

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“…The genome of HPV6 and HPV11 is present in episomal form in ϳ90% of genital warts (42,43), and inhibition of the E1-E2 interaction is expected to provide therapeutic benefits (44). In this respect, it is worth noting that E1 and E2 are likely to be expressed at much lower levels in natural infections than in transfected cells, a fact that could lead us to underestimate the true potency of these inhibitors.…”

Section: Discovery Of Inhibitors Of the E1-e2mentioning

confidence: 99%

Inhibition of Human Papillomavirus DNA Replication by Small Molecule Antagonists of the E1-E2 Protein Interaction

White

Titolo

Brault

et al. 2003

Journal of Biological Chemistry

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Human papillomavirus (HPV) DNA replication is initiated by recruitment of the E1 helicase by the E2 protein to the viral origin. Screening of our corporate compound collection with an assay measuring the cooperative binding of E1 and E2 to the origin identified a class of small molecule inhibitors of the protein interaction between E1 and E2. Isothermal titration calorimetry and changes in protein fluorescence showed that the inhibitors bind to the transactivation domain of E2, the region that interacts with E1. These compounds inhibit E2 of the low risk HPV types 6 and 11 but not those of high risk HPV types or of cottontail rabbit papillomavirus. Functional evidence that the transactivation domain is the target of inhibition was obtained by swapping this domain between a sensitive (HPV11) and a resistant (cottontail rabbit papillomavirus) E2 type and by identifying an amino acid substitution, E100A, that increases inhibition by ϳ10-fold. This class of inhibitors was found to antagonize specifically the E1-E2 interaction in vivo and to inhibit HPV DNA replication in transiently transfected cells. These results highlight the potential of the E1-E2 interaction as a small molecule antiviral target.Papillomaviruses are a family of small double-stranded DNA viruses that induce benign and malignant hyperproliferative lesions of the differentiating epithelium (reviewed in Refs. 1-4). Approximately 25 types of human papillomavirus (HPV) 1 infect the anogenital region. These HPV types have been classified as low risk or high risk types depending on whether they cause benign warts or lesions that can progress to invasive cancer, respectively. Current therapies to remove HPV-induced lesions include a variety of ablative or cytodestructive procedures and the use of immunomodulatory molecules such as imiquimod (5, 6) to stimulate a host immune response. Small molecule antivirals for the treatment of HPV infections do not currently exist.The life cycle of HPV is coupled to the cellular differentiation program that occurs in the epithelium (7). Maintenance of the viral genome in infected cells of the basal layer is essential for the viral life cycle and the ensuing pathology. Maintenance of the HPV episome in primary keratinocyte cultures depends on the function of E1 and E2, a 3Ј-5Ј helicase (8) and a sequencespecific DNA-binding protein (9), respectively, which are required for replication of the genome (10). HPV DNA replication is initiated by the co-operative binding of E1 and E2 to specific DNA sequences within the viral origin (11-14). Formation of this E1-E2-ori ternary complex is dependent on the interaction of both proteins with DNA but also on a critical interaction between the N-terminal transactivation domain (TAD) of E2 and the C-terminal enzymatic domain of E1 (15-22). Assembly of this initial E1-E2-ori complex serves as a starting point for the recruitment of additional E1 molecules (23, 24) and their assembly into hexamers and double hexamers that have ATPase and helicase activity (25,26).Any of the protein-pr...

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Section: Discovery Of Inhibitors Of the E1-e2mentioning

confidence: 99%

Inhibition of Human Papillomavirus DNA Replication by Small Molecule Antagonists of the E1-E2 Protein Interaction

White

Titolo

Brault

et al. 2003

Journal of Biological Chemistry

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“…E1, a DNA helicase, is the only PV protein that possesses enzymatic activity (5) and is also the most highly conserved of the PV proteins. For these reasons, E1 has been considered as the most attractive molecular target for the development of antiviral agents (6).…”

Section: From the Department Of Biological Sciences Boehringer Ingelmentioning

confidence: 99%

Characterization of Recombinant HPV6 and 11 E1 Helicases

White

Pelletier

Brault

et al. 2001

Journal of Biological Chemistry

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To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K m (ATP) of 12 M for HPV6 E1 and 6 M for HPV11 E1. Interestingly, the value of K m (ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.The papillomaviruses (PVs) 1 are small, nonenveloped DNA viruses that infect and replicate in the cutaneous or mucosal epithelia of humans and other mammals. There are over 100 types of human papillomavirus (HPV), which cause conditions ranging from plantar warts (HPV1) and genital warts (HPV6 and -11) to cervical cancer (HPV16, -18, and -31). HPV6 and -11 are also responsible for laryngeal papillomatosis, a rare but very serious infection of the respiratory tract (1). Antiviral agents capable of specifically inhibiting PV replication could play an important role in the treatment of these diseases, but none exist at this time.Despite their host and tissue specificity, all of the PV types share a common genomic organization. The closed, circular genome of ϳ8000 base pairs codes for only 10 proteins: eight early proteins termed E1-E8 and two late proteins, L1 and L2, that make up the viral coat (2). E1 and E2 are the only viral proteins required for HPV DNA replication (3, 4). E2 is a sequence-specific DNA-binding protein that serves to regulate both transcription and DNA replication. E1, a DNA helicase, is the only PV protein that possesses enzymatic activity (5) and is also the most highly conserved of the PV proteins. For these reasons, E1 has been considered as the most attractive molecular target for the development of antiviral agents (6).During the initiation of PV DNA replication, E2 serves as a specificity factor to enhance binding of E1 monomers to the origin, which by themselves bind to double-stranded DNA with little sequence specificity (4, 7-11). E2 dimers bind with high specificity to DNA sequences within the origin and recruit to it E1 monomers, through direct protein-protein interactions (12, 13). Upon binding to DNA, E1 monomers assemble into hexamers (14, 15), but the E1-E2 interaction is not maintained (16,17). Thus, the role of E2 is to catalyze the assembly of hexameric E1 complexes specifically at the origin. ATP hydrolysis is also required for E1 hexamers to distort the orig...

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“…Papillomaviruses do encode E1, a helicase-ATPase, but it has so far proven a difficult enzyme to develop for classic drug screening. As such, successful development of anti-HPV therapies may require identification of novel antiviral targets, such as viral transcription factors and replication proteins (2,21,25) such as the HPV-encoded E2 protein.…”

mentioning

confidence: 99%

Identification of Peptides That Inhibit the DNA Binding,trans-Activator, and DNA Replication Functions of the Human Papillomavirus Type 11 E2 Protein

Deng¹,

Pearce²,

Dixon³

et al. 2004

J Virol

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Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPVassociated diseases.Present estimates are that 24 million Americans are infected with human papillomavirus (HPV) and that 5 million new cases occur annually. As there are no specific antiviral therapies for HPV infection, there is a clear unmet need for the development of safe and effective therapies for HPV-associated disease.Papillomaviruses lack the enzymes generally targeted by most currently available antiviral agents (e.g., virus-encoded DNA polymerases, reverse transcriptases, and proteases). Papillomaviruses do encode E1, a helicase-ATPase, but it has so far proven a difficult enzyme to develop for classic drug screening. As such, successful development of anti-HPV therapies may require identification of novel antiviral targets, such as viral transcription factors and replication proteins (2, 21, 25) such as the HPV-encoded E2 protein.The 50-kDa E2 protein is comprised of three functional domains (19). The amino-terminal domain of E2 is necessary for viral trans-activation and for direct association with E1. The smaller carboxyl-terminal domain (E2C) encodes the DNA binding and dimerization functions. Linking the amino-and carboxyl-terminal domains is a small, poorly conserved hinge region. E2 function requires binding to a 12-bp palindromic nucleotide sequence, ACCN 6 GGT, that is present at several locations throughout the HPV genome and is repeated several times near the viral origin of replication. Once associated with DNA, E2 interacts with a variety of host cell transcription factors to modulate viral transcription. The papillomavirus E2 protein is also required for origin (ORI)-specific viral DNA replication. Because association of E1 with E2 enhances the affinity of both proteins for the viral ORI, DNA sequencespecific binding by E2 effectively mediates sequence-specific DNA binding by E1 (6).Since the E2-DNA interaction is central to the coordination of the essential papillomavirus transcription and replication processes, disruption of E2-DNA binding should inhibit HPV replication. To this end, we constructed a filamentous bacteriophage 10-mer random peptide library and screened for sequences that disrupted the E2-DNA association by binding to the E2 protein.Phage display selection of E2-DNA binding inhibitors. We used peptide phage display (reviewed in reference 16) to identify peptide binders to bio-E2C, a biotinylated E2 fusion protein consisting of 88 carboxyl-terminal amino acids of the HPV type 11 (HPV-11) E2 DNA binding domain fused to the 99-residue polypeptide substrate of Es...

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Molecular Targets for Human Papillomaviruses: Prospects for Antiviral Therapy

Cited by 47 publications

References 212 publications

Inhibition of Human Papillomavirus DNA Replication by Small Molecule Antagonists of the E1-E2 Protein Interaction

Inhibition of Human Papillomavirus DNA Replication by Small Molecule Antagonists of the E1-E2 Protein Interaction

Characterization of Recombinant HPV6 and 11 E1 Helicases

Identification of Peptides That Inhibit the DNA Binding,trans-Activator, and DNA Replication Functions of the Human Papillomavirus Type 11 E2 Protein

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