Cellular Topoisomerase I Modulates Origin Binding by Bovine Papillomavirus Type 1 E1

ABSTRACT In addition to viral proteins E1 and E2, bovine papillomavirus type 1 (BPV1) depends heavily on host replication machinery for genome duplication. It was previously shown that E1 binds to and recruits cellular replication proteins to the BPV1 origin of replication, including DNA polymerase α-primase, replication protein A (RPA), and more recently, human topoisomerase I (Topo I). Here, we show that Topo I specifically stimulates the origin binding of E1 severalfold but has no effect on nonorigin DNA binding. This is highly specific, as binding to nonorigin DNA is not stimulated, and other cellular proteins that bind E1, such as RPA and polymerase α-primase, show no such effect. The stimulation of E1's origin binding by Topo I is not synergistic with the stimulation by E2. Although the enhanced origin binding of E1 by Topo I requires ATP and Mg2+ for optimal efficiency, ATP hydrolysis is not required. Using an enzyme-linked immunosorbent assay, we showed that the interaction between E1 and Topo I is decreased in the presence of DNA. Our results suggest that Topo I participates in the initiation of papillomavirus DNA replication by enhancing E1 binding to the BPV1 origin.

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Interactions of the Papovavirus DNA Replication Initiator Proteins, Bovine Papillomavirus Type 1 E1 and Simian Virus 40 Large T Antigen, with Human Replication Protein A

Journal of Virology ◽

10.1128/jvi.73.6.4899-4907.1999 ◽

1999 ◽

Vol 73 (6) ◽

pp. 4899-4907 ◽

Cited By ~ 62

Author(s):

YuFeng Han ◽

Yueh-Ming Loo ◽

Kevin T. Militello ◽

Thomas Melendy

Keyword(s):

Dna Replication ◽

Protein A ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Bovine Papillomavirus ◽

Replication Proteins ◽

E1 Protein ◽

Cellular Dna

ABSTRACT Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase α-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase α-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia colias a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.

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Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

Journal of Virology ◽

10.1128/jvi.78.4.1605-1615.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1605-1615 ◽

Cited By ~ 68

Author(s):

Yueh-Ming Loo ◽

Thomas Melendy

Keyword(s):

Dna Replication ◽

Protein A ◽

Replication Protein A ◽

T Antigen ◽

Replication Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Physical Interaction ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Papillomavirus Dna

ABSTRACT With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.

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p53 Protein Is a Suppressor of Papillomavirus DNA Amplificational Replication

Journal of Virology ◽

10.1128/jvi.72.8.6822-6831.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6822-6831 ◽

Cited By ~ 27

Author(s):

Dina Lepik ◽

Ivar Ilves ◽

Arnold Kristjuhan ◽

Toivo Maimets ◽

Mart Ustav

Keyword(s):

Cell Cycle ◽

Transcriptional Activation ◽

P53 Protein ◽

Protein A ◽

Intrinsic Activity ◽

Bovine Papillomavirus ◽

Suppressor Activity ◽

Barr Virus ◽

Epstein Barr ◽

Papillomavirus Dna

ABSTRACT p53 protein was able to block human and bovine papillomavirus DNA amplificational replication while not interfering with Epstein-Barr virus oriP once-per-cell cycle replication. Oligomerization, intact DNA-binding, replication protein A-binding, and proline-rich domains of the p53 protein were essential for efficient inhibition, while the N-terminal transcriptional activation and C-terminal regulatory domains were dispensable for the suppressor activity of the p53 protein. The inhibition of replication was caused neither by the downregulation of expression of the E1 and E2 proteins nor by cell cycle block or apoptosis. Our data suggest that the intrinsic activity of p53 to suppress amplificational replication of the papillomavirus origin may have an important role in the virus life cycle and in virus-cell interactions.

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Multiple Signals Direct the Assembly and Function of a Type 1 Secretion System

Journal of Bacteriology ◽

10.1128/jb.00178-10 ◽

2010 ◽

Vol 192 (15) ◽

pp. 3861-3869 ◽

Cited By ~ 20

Author(s):

Muriel Masi ◽

Cécile Wandersman

Keyword(s):

Atp Hydrolysis ◽

Protein A ◽

Secretion System ◽

Secretion Systems ◽

Tight Coupling ◽

Secretion Signal ◽

Abc Protein ◽

C Terminus ◽

Wide Range

ABSTRACT Type 1 secretion systems (T1SS) are present in a wide range of Gram-negative bacteria and are involved in the secretion of diverse substrates such as proteases, lipases, and hemophores. T1SS consist of three proteins: an inner membrane ABC (ATP binding cassette) protein, a periplasmic adaptor, and an outer membrane channel of the TolC family. Assembly of the tripartite complex is transient and induced upon binding of the substrate to the ABC protein. It is generally accepted that T1SS-secreted proteins have a C-terminal secretion signal required for secretion and that this signal interacts with the ABC protein. However, we have previously shown that for the Serratia marcescens hemophore HasA, interactions with the ABC protein and subsequent T1SS assembly require additional regions. In this work, we characterize these regions and demonstrate that they are numerous, distributed throughout the HasA polypeptide, and most likely linear. Together with the C-terminal signal, these elements maximize the secretion of HasA. The data also show that the C-terminal signal of HasA triggers HasD-driven ATP hydrolysis, leading to disassembly of the complex. These data support a model of type 1 secretion involving a multistep interaction between the substrate and the ABC protein that stabilizes the assembled secretion system until the C terminus is presented. This model also supports tight coupling between synthesis and secretion.

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