Bovine papillomavirus E1 protein binds specifically DNA polymerase alpha but not replication protein A

Bonne-Andréa, Catherine; Santucci, S; Clertant, Philippe; Tillier, F

doi:10.1128/jvi.69.4.2341-2350.1995

Cited by 66 publications

(42 citation statements)

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“…Biological materials. The cell lines employed for preparing soluble extracts for cell-free replication, the recombinant baculoviruses used for expressing the BPV1 proteins into Sf9 cells, and the antibodies able to recognize these proteins were described previously (4,42,44).…”

Section: Methodsmentioning

confidence: 99%

“…Expression and purification of viral proteins. BPV1 E2 and the glutathione S-transferase-E1 fusion protein (GST-E1) were expressed by infecting Sf9 cells with the appropriate recombinant baculoviruses, as reported previously (4,43). In order to purify the E1 protein, GST-E1-infected cells were lysed by hypotonic shock and the nuclear fraction was extracted in a selective manner by a combination of a high concentration of salt, a high pH, and a high concentration of Mg, as described previously (3).…”

Section: Methodsmentioning

confidence: 99%

“…Soluble extracts for cell-free replication were prepared as described previously (23). Assays were performed as already described (3,4) by incubating cell extract of various sources (200 g of total proteins) and template DNA (either 0.15 g of pSKori-and pUC-derived constructs or 0.25 g of pSV-BPVϩ DNA) together with viral proteins (usually 50 ng of E2 and 150 ng of E1) in a volume of 50 l under standard conditions (50) at 37°C for the indicated times (60 min or more). The extent of DNA synthesis was monitored by counting the incorporation of [ 32 P]dCMP into acid-insoluble material.…”

Section: Methodsmentioning

confidence: 99%

“…Cell-free systems were sufficient, however, to show that E2 could be replaced by other acidic transcriptional activators, helping to tether the replication protein A to the origin of replication (24); their use also helped illustrate how prior binding of E2 to ori DNA could prevent the inhibition of replication exerted by the establishment of nucleosomal structures in vitro (25). We recently reported conditions sustaining much higher levels of cell-free BPV1 DNA synthesis, satisfying both the criteria of semiconservation and bidirectionality on either side of the origin (3,4). Under these conditions BPV1 DNA synthesis is as efficient as that of SV40 DNA in vitro and proceeds through successive rounds of replication.…”

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confidence: 99%

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

Bonne-Andréa

Tillier

McShan

et al. 1997

J Virol

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Bonne-Andréa

Tillier

McShan

et al. 1997

J Virol

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“…Recently it has been suggested that E2TA might facilitate the stepwise assembly of E1 into a multimeric complex akin to the hexameric form of simian virus 40 (SV40) large tumor antigen (T-ag), which assembles at the SV40 origin (5,7,9,22,24,35). Like T-ag, E1 is able to bind ATP (23,40), act as an ATPase-dependent DNA helicase (36,47), and bind DNA polymerase alpha (4), all properties thought to be important for E1's role in viral DNA replication.…”

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confidence: 99%

Bovine papillomavirus type 1 E1 and simian virus 40 large T antigen share regions of sequence similarity required for multiple functions

Mansky

Batiza

Lambert

1997

J Virol

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The full-length product of the bovine papillomavirus type 1 (BPV-1) E1 translational open reading frame is required for viral DNA replication in vivo and in vitro. E1 is a multifunctional protein whose properties include ATP binding, acting as an ATPase-dependent DNA helicase, DNA binding to the BPV-1 origin of viral DNA replication, and association with the E2 transcriptional transactivator, E2TA, a second viral protein involved in DNA replication. All of these properties are thought to be important for E1's role in replicating the viral genome. In addition BPV-1 E1 can inhibit activation of the viral P 89 promoter by the BPV-1 E2TA. E1 has amino acid homology with eight regions of SV40 large tumor antigen (T-ag), a DNA helicase that is essential for the replication of the SV40 DNA genome. These eight regions of similarity lie within the domain of T-ag that confers DNA helicase activity. We created a series of missense mutations in BPV-1 E1 at codons 295, 344-345, 446, 464, 466, 497-498, 523, and 542, which encode amino acids of identity in seven of the eight regions of similarity between E1 and T-ag, and at codon 370. The activities of these mutant E1 genes were compared to wild-type E1 in multiple assays that measured DNA replication, inhibition of E2TA-dependent transcription, DNA binding, ATP binding, and protein expression. Based upon these analyses, the following conclusions were made: (i) at least five of the eight regions in E1 that are similar to regions in T-ag are functionally important in viral DNA replication; (ii) specific E1 missense mutants, themselves defective for supporting DNA replication, could act in trans to suppress the replication function of wild-type E1; (iii) certain regions of similarity with T-ag that are important for E1's ability to support DNA replication are not necessary for its capacity to inhibit E2TA-dependent transcription; and (iv) efficient DNA binding by E1 is not essential for E1 to inhibit E2TA-dependent transcription.

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Structure Elucidation of the First Inhibitors of Human Papillomavirus Type 11 E1E2 ProteinProtein Interaction

Yoakim

Goudreau

McGibbon

et al. 2003

Helvetica Chimica Acta

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A novel series of inhibitors of the HPV11 E1ÀE2 proteinÀprotein interaction was identified. These inhibitors, which were discovered as a result of high-throughput screening, feature an indandione system spirofused onto an appropriately substituted tetrahydrofuran ring. Early stability studies indicated, surprisingly, that this particular series of compounds were readily converted, in binding assay buffer, to the corresponding carboxylates. NMR and mass spectrometry techniques were used to elucidate the structures of these products and the mechanism by which they are produced.Introduction. ± There are more than 100 human papillomavirus (HPV) types, associated with a broad spectrum of disease ranging from cutaneous and genital warts to anogenital cancer. Each of these different HPV types infects specific tissues and is responsible for particular pathologies. For example, HPV1 infections cause plantar warts on the feet, HPV6 and 11 cause genital warts, and HPV16 is the major cause of cervical cancer [1].The HPV genome encodes eight well-characterized proteins, only one of which, the E1 helicase, has enzymatic activity. The others either serve structural roles or are involved in interactions with host-cell factors [2]. In addition to E1, only one other viral protein, E2, is required for replication of the genome [3] [4]. Viral-DNA replication is initiated by the cooperative binding of E1 and E2 to the HPV origin. Assembly of this E1ÀE2Àorigin ( E1ÀE2Àori) complex is dependent on the binding of E2 to highaffinity sites in the origin as well as on a critical proteinÀprotein interaction between E1 and E2 [5] [6]. E1 and E2 also recruit to the origin host-cell proteins required for replication, including the polymerase a primase [7].Currently, genital warts are treated by a variety of ablative and cytodestructive therapies including the use of liquid nitrogen or trichloroacetic acid or by topical administration of the immunostimulator imiquimod [8]. These treatments are far from ideal, and there clearly remains a need for specific inhibitors of viral replication. The essential proteins E1 and E2 are attractive targets for such antiviral chemotherapy [9].Recently, we described a series of compounds that inhibit the cooperative binding of E1 and E2 to the viral origin of replication [10] [11]. These inhibitors, which were discovered as a result of high-throughput screening, feature an indandione system

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Bovine papillomavirus E1 protein binds specifically DNA polymerase alpha but not replication protein A

Cited by 66 publications

References 53 publications

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

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

Bovine papillomavirus type 1 E1 and simian virus 40 large T antigen share regions of sequence similarity required for multiple functions

Structure Elucidation of the First Inhibitors of Human Papillomavirus Type 11 E1E2 ProteinProtein Interaction

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