DNA replication of the papillomaviruses is specified by cooperative binding of two proteins to the ori site: the enhancer E2 and the viral initiator E1, a distant member of the AAA+ family of proteins. Formation of this prereplication complex is an essential step toward the construction of a functional, multimeric E1 helicase and DNA melting. To understand how E2 interacts with E1 to regulate this process, we have solved the X-ray structure of a complex containing the HPV18 E2 activation domain bound to the helicase domain of E1. Modeling the monomers of E1 to a hexameric helicase shows that E2 blocks hexamerization of E1 by shielding a region of the E1 oligomerization surface and stabilizing a conformation of E1 that is incompatible with ATP binding. Further biochemical experiments and structural analysis show that ATP is an allosteric effector of the dissociation of E2 from E1. Our data provide the first molecular insights into how a protein can regulate the assembly of an oligomeric AAA+ complex and explain at a structural level why E2, after playing a matchmaker role by guiding E1 to the DNA, must dissociate for subsequent steps of initiation to occur. Building on previously proposed ideas, we discuss how our data advance current models for the conversion of E1 in the prereplication complex to a hexameric helicase assembly.[Keywords: Crystal structure E1E2 complex; papillomavirus; helicase structure; AAA+ protein; DNA replication] Papillomaviruses (PVs) are small DNA viruses that maintain latency in the stem cells of various epithelial tissues (for review, see Howley 1996). They are the etiological agents of a variety of benign lesions of the epithelium, and certain high-risk variants of the human papillomavirus (HPV), such as HPV-16, 18, and 31, are associated with cervical carcinomas (Bosch et al. 1995). The virus is maintained as an extrachromosomal nuclear plasmid that replicates in S phase with the host genome. Because PVs have few open reading frames (∼8 ORFs), the viral life cycle depends heavily on interaction with the host cell for enzymes and ancillary factors for its own gene expression and replication programs. The PVs have provided important novel insights into the regulation of cell proliferation and cancer etiology; for example, the targeted degradation of p53 directed by the HPV16 E6 protein first revealed how key cellular regulatory proteins could be specifically ubiquitinated by E3 ligases for destruction by the proteosome (Scheffner et al. 1993). The PVs also have provided models to understand how eukaryotic transcription factors coassociate to form loops between segments of DNA (Li et al. 1991) and to explore assembly of specific replication factories on defined origins of replication (Stenlund 2003b).The first two universal steps in the formation of a prereplication complex are origin recognition and subsequent distortion and separation of duplex DNA strands. The simplicity of the PV prereplication complex offers advantages for mechanistic and structural dissection of these steps. Only t...
Many DNA viruses that are latent in dividing cells are noncovalent passengers on mitotic chromosomes and require specific viral-encoded and cellular factors for this activity. The chromosomal protein Brd4 is implicated in the hitchhiking of bovine papillomavirus-1 (BPV-1), and the viral protein E2 binds to both plasmids and Brd4. Here, we present the X-ray crystal structure of the carboxy-terminal domain of Brd4 in complex with HPV-16 E2, and with this information have developed a Brd4-Tat fusion protein that is efficiently taken up by different transformed cells harboring HPV plasmids. In cells treated with these fusion proteins for only 2 hr and arrested in metaphase, the HPV DNA, either HPV-16 or -31, is displaced from mitotic chromosomes. Mutant Brd4 peptides are deficient in ablating this association. We suggest that such peptides may lead to the development of inhibitors of latency for many, if not all, papillomaviruses.
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