Amino acids critical for the functions of the bovine papillomavirus type 1 E2 transactivator

111

DNA replication of papillomaviruses requires the viral E1 and E2 proteins. These proteins bind cooperatively to the viral origin of replication (ori), which contains binding sites for both proteins, forming an E1-E2-ori complex which is essential for initiation of DNA replication. To map the domains in E2 that are involved in the interaction with E1, we have used chimeric bovine papillomavirus (BPV)/human papillomavirus type 11 (HPV-11) E2 proteins. The results from this study show that both the DNA binding domain and the transactivation domain from BPV E2 independently can interact with BPV E1. However, the roles of these two interactions are different: the interaction between E1 and the activation domain of E2 is necessary and sufficient for cooperativity in binding and for DNA replication; the interaction between E1 and the DNA binding domain of E2 is required only when the binding sites for E1 and E2 are adjacent to each other, and the function of this interaction appears to be to facilitate the interaction between E1 and the transactivation domain of E2. These results indicate that the cooperative binding of E1 and E2 to the BPV ori takes place via a novel two-stage mechanism where one interaction serves as a trigger for the formation of the second, productive, interaction between the two proteins.DNA replication in mammalian cells has been studied mainly by using DNA viruses as model systems, and simian virus 40 has been of particular importance (8,49). From studies of DNA replication of many different replicons, it is now evident that transcription factors play a role in DNA replication in virtually all eukaryotic replication systems (for a review, see reference 21). Frequently, binding sites for transcription factors flank the binding sites for the initiator protein. For example, the simian virus 40 and polyomavirus origins of replication (oris) are flanked by nearby enhancer elements which stimulate viral DNA replication in vivo (for a review, see reference 13). Transcription factors other than the ones that naturally bind to the enhancer can in some cases substitute for this stimulatory function, provided that binding sites for these factors are present in the ori (3, 10, 18). These results demonstrate that the requirement for transcription factors in replication in most cases show only a limited degree of specificity. The function of these transcription factors in DNA replication is unclear; it has been suggested however, that the bound transcription factors can function to reverse nucleosomal repression (9,10,30). It has also been demonstrated that the activation domains of certain transcription factors can interact directly with the single-stranded DNA binding protein replication protein A (RPA) and possibly serve to recruit RPA to the ori (20, 29). A different function for transcription factors in replication, which has attracted less attention, has been demonstrated for adenovirus DNA replication. Binding of the adenovirus preterminal protein/DNA polymerase to the viral origin of replication is fac...

Section: Discussionsupporting

confidence: 85%

mentioning

confidence: 99%

Functional interactions between papillomavirus E1 and E2 proteins

Berg

Stenlund

1997

111

“…Notwithstanding the HPV1a exception, as a general rule the E2 transactivator is required for viral DNA replication. Genetic analysis has localized determinants for replicative function within the N-terminal part of the BPV1 E2 molecule, a domain that is also required both for E1 binding and for transcriptional activation (1,5,13,15,16,57). Targeted mutagenesis has allowed us to clearly distinguish between these three functions, the binding of E1 being a likely prerequisite to the replicative function of E2.…”

mentioning

confidence: 99%

Bovine papillomavirus type 1 DNA replication: the transcriptional activator E2 acts in vitro as a specificity factor

Bonne-Andréa

Tillier

McShan

et al. 1997

We previously devised cell-free conditions supporting efficient replication of bovine papillomavirus type 1 (BPV1) DNA (C. Bonne-Andréa, S. Santucci, and P. Clertant, J. Virol. 69:3201-3205, 1995): the use of highly active preparations of viral initiator protein E1, together with extract from a particular cell source, allowed the synthesis of complete DNA circles through successive rounds of replication; this occurred in the absence of the viral transcriptional activator E2, required in vivo for the replication of viral genomes. We now report that adding E2 to cell-free assays produced only slight effects both on the yield of E1-dependent DNA synthesis and on the quality of newly made DNA molecules when a template carrying a wild-type BPV1 replication origin (ori) was used. The performance of mouse cell extracts, unable to sustain efficient BPV1 DNA replication in the presence of E1 only, was likewise not improved by the addition of E2. In a proper in vitro environment, E1 is thus fully capable of efficiently initiating viral DNA synthesis by itself, an activity which is not enhanced by interaction with E2. An important effect, however, was detected: E2 totally suppressed the nonspecific replication of ori-defective DNA templates, otherwise observed in high E1 concentrations. We examined the requirements for building a minimal DNA sequence behaving in vitro as a specific ori sequence under stringent recognition conditions, i.e., in the presence of both E1 and E2. Only two elements, the 18-bp E1 binding palindrome and an AT-rich sequence, were required in cis to allow specific cell-free DNA replication; there seemed to be no need for an E2 binding site to ensure discrimination between specific ori templates and other DNA molecules, even in the presence of E2. This suggests that during the initiation of BPV1 DNA replication, at least in vitro, E2 acts as a specificity factor restricting the action of E1 to a defined ori sequence; this function, likely not demanding the direct binding of E2 to cognate DNA sites, might primarily involve protein-protein interactions.

“…This N-terminal domain of E2 also functions as a transcription activation domain required for expression of the viral early genes (reviewed in reference 37). Although encoded by the same domain, the replication and transcription functions of E2 can be uncoupled by mutations (1,8,9,12,15,21,43). Recently, a 15-amino-acid peptide derived from HPV-16 E2 was shown to inhibit the interaction between E1 and E2 (25).…”

mentioning

confidence: 99%

Role of the ATP-Binding Domain of the Human Papillomavirus Type 11 E1 Helicase in E2-Dependent Binding to the Origin

Titolo

Pelletier

Sauvé

et al. 1999

Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37°C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.