Proteins encoded by the bovine papillomavirus E1 open reading frame: expression in heterologous systems and in virally transformed cells

Santucci, S; Androphy, Elliot J.; Bonne-Andréa, Catherine; Clertant, Philippe

doi:10.1128/jvi.64.12.6027-6039.1990

Cited by 36 publications

(29 citation statements)

References 53 publications

(40 reference 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%

“…Two viral proteins, E1 and E2, are normally required for replicating PV DNA (54). E1 is similar to another well-studied viral initiator protein, SV40-polyomavirus large T antigen (10): E1 is a nuclear phosphoprotein (42,51) with DNA helicase and ATPase activities (30,44,46,59) that is able to recognize and unwind an 18-bp palindrome within the viral replication origin (47,49,55,56,59). PV origins are short sequences (less than 100 bp) comprising mainly an E1 binding site and important flanking elements (see, for instance, references 14 and 53) such as AT-rich sequences and one or several binding sites for the second viral protein, transactivator E2.…”

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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%

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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

J Virol

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“…Amino acid similarities suggested that El, like SV40 large-T antigen, may be a DNA helicase/ATPase involved in initiating viral DNA replication (3). Subsequently, BPV-1 El protein was shown to be a nuclear 68-72 kD ATP-binding phosphoprotein (4,5,6). A mutation in El (Pro-434 to Ser), analogous to that in the SV40 large-T antigen mutant tsA209 that impairs ATPbinding, renders BPV-1 defective for DNA replication and abolishes ATP-binding by El (6).…”

Section: Introductionmentioning

confidence: 99%

E1 protein of human papillomavirus is a DNA helicase/ATPase

1993

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Replication of human papillomavirus (HPV) DNA requires the viral proteins E1 and E2. Amino acid similarities to SV40 large-T antigen had suggested that E1 is a DNA helicase/ATPase involved in initiating viral DNA replication, and this has recently been shown for bovine papillomavirus type 1 (BPV-1) E1 protein. However, in vitro analysis of HPV E1 has been hampered by the inability to produce purified protein using heterologous expression systems. We have succeeded in demonstrating ATPase and DNA helicase activities in purified HPV E1, expressed in E. coli as a maltose-binding protein fusion (MBP-E1), for the first time. As further confirmation that the ATPase and DNA helicase activities are due to E1 and not contaminating E. coli enzymes, we have shown that a fusion protein containing an amino acid change (E1 Pro-479 to Ser), predicted to inactivate ATP-binding, has impaired activities. We have carried out a structure prediction analysis which suggests that E1 may form two domains: a relatively open N-terminal domain (residues 1-125), and a highly structured C-terminal domain (170-649), with an intermediate region (125-170) predicted to form an inter-domain linker. This is consistent with the proteolytic susceptibility of MBP-E1 at a site 15-20 kD from the N-terminus of E1, and the accumulation of a 58 kD C-terminal fragment of E1. We speculate that the N-terminal domain is involved in DNA-binding, while the C-terminal 58 kD may constitute a distinct enzymatic domain. HPV E1 is of interest as a therapeutic target and the availability of pure enzyme will be invaluable in the search for antiviral compounds.

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“…It is therefore likely that the differences between SV40 and BPV1 lie with the proteins involved in the initiation of replication. The E1 protein, which shares a number of biochemical properties with the T antigen, has been clearly established as the initiator of BPV1 replication (33,35,36,43,52). The role of the E2 transactivator protein in BPV1 replication is less clear.…”

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confidence: 99%

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

Bonne-Andréa

Santucci

Clertant

et al. 1995

J Virol

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Extracts prepared from either mouse cells or monkey cells were examined for the ability to support in vitro bovine papillomavirus type 1 (BPV1) DNA replication, and they were used in parallel as a source of host replication proteins for affinity chromatography. DNA synthesis exhibited an absolute requirement for BPV1 E1 protein. In contrast to previous observations, we found that low levels of E1 were highly efficient in initiating DNA replication in the absence of the BPV1 transcription factor E2. Surprisingly, COS-1 cell extract allowed a high rate of BPV1 DNA replication, supporting an efficient production of mature circular DNA molecules, whereas in mouse cell extracts, the replication products mostly consisted of replicative intermediates. Submitting the extracts to affinity chromatography allowed specific binding of DNA polymerase ␣-primase to E1 protein, up to a total depletion of the extract, regardless of the origin of the cell extract. Furthermore, replication protein A was not retained on E1 affinity columns, even when E2 was complexed with E1. These data confirm that the interactions between E1 and DNA polymerase ␣-primase do not exhibit cell-type specificity, as had already been suggested by data from in vivo and in vitro replication assays, but they imply that other cellular proteins may affect the level of E1-dependent replication.

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Proteins encoded by the bovine papillomavirus E1 open reading frame: expression in heterologous systems and in virally transformed cells

Cited by 36 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

E1 protein of human papillomavirus is a DNA helicase/ATPase

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

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