The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by singlestranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.Papillomaviruses are small DNA viruses that are the etiological agents of benign and malignant lesions of the differentiating mucosal and cutaneous epithelium (reviewed in references 6, 20, 45, and 61). The life cycle of these viruses is closely associated with the differentiation program that occurs in the epithelium. Soon after infection, the viral genome is established as a low-copy-number extrachromosomal episome in the nuclei of infected basal cells. As these infected cells start to differentiate from the basal layer toward the upper portion of the epithelium, the viral episome is replicated and maintained at approximately 50 copies per cell. When the infected cells reach the suprabasal layers, amplification of the viral episome occurs to a high copy number, probably as a result of a change in the mode of DNA replication from a bidirectional theta mode to a rolling-circle mode (11). In these upper layers, capsid proteins are synthesized and viral particles are assembled, and they are e...

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BI 201335 is a hepatitis C virus (HCV) NS3-NS4A (NS3 coexpressed with NS4A) protease inhibitor that has been shown to have potent clinical antiviral activity. It is a highly optimized noncovalent competitive inhibitor of full-length NS3-NS4A proteases of HCV genotypes 1a and 1b with K i values of 2.6 and 2.0 nM, respectively. K i values of 2 to 230 nM were measured against the NS3-NS4A proteases of HCV genotypes 2 to 6, whereas it was a very weak inhibitor of cathepsin B and showed no measurable inhibition of human leukocyte elastase. BI 201335 was also shown to be a potent inhibitor of HCV RNA replication in vitro with 50% effective concentrations (EC 50 s) of 6.5 and 3.1 nM obtained in genotype 1a and 1b replicon assays. Combinations of BI 201335 with either interferon or ribavirin had additive effects in replicon assays. BI 201335 had good permeability in Caco-2 cell assays and high metabolic stability after incubation with human, rat, monkey, and dog liver microsomes. Its good absorption, distribution, metabolism, and excretion (ADME) profile in vitro, as well as in rat, monkey, and dog, predicted good pharmacokinetics (PK) in humans. Furthermore, drug levels were significantly higher in rat liver than in plasma, suggesting that distribution to the target organ may be especially favorable. BI 201335 is a highly potent and selective NS3-NS4A protease inhibitor with good in vitro and animal ADME properties, consistent with its good human PK profile, and shows great promise as a treatment for HCV infection. Chronic hepatitis C virus (HCV) infection affects 130 to 170 million individuals worldwide (14). The etiologic agent is a small enveloped single-stranded RNA virus belonging to the Flaviviridae family, Hepacivirus genus (32). Although present in human populations for thousands of years, it was discovered only 20 years ago as the causative agent of non-A, non-B hepatitis (6). The HCV genome consists of approximately 9,600 bases, encoding a single polyprotein of approximately 3,000 amino acids, flanked by conserved 5Ј and 3Ј untranslated regions (UTRs). The viral polyprotein comprises four structural proteins followed by six nonstructural (NS) proteins that play essential roles in viral replication (25).One of the best-studied nonstructural proteins is NS3, a bifunctional protein that consists of an N-terminal protease domain and a C-terminal helicase domain (9). The protease domain has a trypsin-like fold with a flat and solvent-exposed substrate binding site (11,21). The central portion of the NS4A protein is integrated into the protein fold of the NS3 protease domain and is required for full activity (3). The NS3-NS4A (NS3 coexpressed with NS4A) protease plays a critical role in the maturation of the viral polyprotein precursor and was recognized early on as potential target for antiviral drugs (2). Indeed, the first direct acting antiviral agent to be studied in humans was the protease inhibitor BILN 2061, and two other protease inhibitors, telaprevir and boceprevir, are currently in phase III trials (10,1...

Interaction between the E2 protein and E1 helicase of human papillomaviruses (HPVs) is essential for the initiation of viral DNA replication. We recently described a series of small molecules that bind to the N-terminal transactivation domain (TAD) of HPV type 11 E2 and inhibits its interaction with E1 in vitro and in cellular assays. Here we report the crystal structures of both the HPV11 TAD and of a complex between this domain and an inhibitor, at 2.5-and 2.4-Å resolution, respectively. The HPV11 TAD structure is very similar to that of the analogous domain of HPV16. Inhibitor binding caused no significant alteration of the protein backbone, but movements of several amino acid side chains at the binding site, in particular those of Tyr-19, His-32, Leu-94, and Glu-100, resulted in the formation of a deep hydrophobic pocket that accommodates the indandione moiety of the inhibitor. Mutational analysis provides functional evidence for specific interactions between Tyr-19 and E1 and between His-32 and the inhibitor. A second inhibitor molecule is also present at the binding pocket. Although evidence is presented that this second molecule makes only weak interactions with the protein and is likely an artifact of crystallization, its presence defines additional regions of the binding pocket that could be exploited to design more potent inhibitors.Human papillomaviruses (HPVs) 1 are the etiological agents of malignant and benign lesions of the differentiating squamous or mucosal epithelium, notably of cervical cancer. Approximately 25 HPV types replicate in mucosal tissues of the anogenital tract. HPV16, -18, and -31 are the most prevalent "high-risk" types found in pre-cancerous or malignant lesions of the cervix. HPV6 and -11 are the most common "low-risk" types, which cause benign genital warts (condyloma acuminata), a less serious condition but one of the most common sexually transmitted diseases (1). Currently, no specific antivirals are available for the treatment of HPV infections.The small circular double-stranded DNA genome of papillomavirus is actively maintained as a multicopy episome in the nucleus of infected epithelial cells. This process is dependent on replication of the viral genome by the viral E1 and E2 proteins, in conjunction with the host DNA replication machinery. E2 is a sequence-specific DNA-binding protein that has a number of functions in the viral lifecycle. In addition to its role in the initiation of viral DNA replication, E2 is involved in regulating the transcription of viral genes (2-7), and in the segregation of the viral genome during cell division (8, 9). As a replication initiation factor, E2 binds with high affinity to specific sites located within the viral origin (ori) to help recruit it to the E1 helicase (10 -13). Formation of a ternary complex between E1, E2, and the origin relies not only on the interaction of E1 and E2 with specific DNA sequences at the origin but is also critically dependent on a direct interaction between these two proteins (14 -18).The 40-kDa E2 prote...

The E1 helicase of papillomaviruses is required for replication of the viral double-stranded DNA genome, in conjunction with cellular factors. DNA replication is initiated at the viral origin by the assembly of E1 monomers into oligomeric complexes that have unwinding activity. In vivo, this process is catalyzed by the viral E2 protein, which recruits E1 specifically at the origin. For bovine papillomavirus (BPV) E1 a minimal DNA-binding domain (DBD) has been identified N-terminal to the enzymatic domain. In this study, we characterized the DBD of human papillomavirus 11 (HPV11), HPV18, and BPV E1 using a quantitative DNA binding assay based on fluorescence anisotropy. We found that the HPV11 DBD binds DNA with an affinity and sequence requirement comparable to those of the analogous domain of BPV but that the HPV18 DBD has a higher affinity for nonspecific DNA. By comparing the DNA-binding properties of a dimerization-defective protein to those of the wild type, we provide evidence that dimerization of the HPV11 DBD occurs only on two appropriately positioned E1 binding-sites and contributes approximately a 10-fold increase in binding affinity. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds DNA with little sequence specificity, similarly to a dimerization-defective DBD. Finally, we show that the amino acid substitution that prevents dimerization reduces the ability of a longer E1 protein to bind to the origin in vitro and to support transient HPV DNA replication in vivo, but has little effect on its ATPase activity or ability to oligomerize into hexamers. These results are discussed in light of a model of the assembly of replication-competent double hexameric E1 complexes at the origin.Papillomaviruses are a family of pathogenic viruses that induce benign and malignant hyperproliferative lesions of the differentiating squamous and mucosal epithelium (reviewed in references 13, 28, 51, and 68). Among the best-characterized human papillomaviruses (HPV) are those types that infect the anogenital region and are associated with the development of benign warts (HPV type 6 [HPV6] and -11; low-risk types) or cancerous lesions (HPV16, -18, and -31; high-risk types).The life cycle of papillomaviruses is tightly coupled to the cellular differentiation program that occurs in the epithelium (for a recent review, see reference 52). These viruses infect the basal cell layer where they establish their small doublestranded DNA genome, 7.9 kbp in length, as a circular extrachromosomal element in the nucleus of infected cells. Maintenance of the viral genome in the infected cell is central to the life cycle of papillomaviruses and their associated pathologies. Hence, interfering with this process has been considered a valuable strategy for the development of antiviral therapeutic agents.Maintenance of the viral genome in infected cells requires the activity of E1 and E2, the two viral proteins necessary for replication of the HPV genome in conjunction with the host cell DNA replication machinery (reviewed...