Calcein represses human papillomavirus 16 E1-E2 mediated DNA replication via blocking their binding to the viral origin of replication

ABSTRACTTo replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination.IMPORTANCEHuman papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted.

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Interactions of the cellular CCAAT displacement protein and human papillomavirus E2 protein with the viral origin of replication can regulate DNA replication

Virology ◽

10.1016/j.virol.2006.01.047 ◽

2006 ◽

Vol 350 (2) ◽

pp. 302-311 ◽

Cited By ~ 12

Author(s):

Janaki Narahari ◽

John C. Fisk ◽

Thomas Melendy ◽

Ann Roman

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

Origin Of Replication ◽

Viral Origin ◽

E2 Protein ◽

Ccaat Displacement Protein

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Human TATA Binding Protein Inhibits Human Papillomavirus Type 11 DNA Replication by Antagonizing E1-E2 Protein Complex Formation on the Viral Origin of Replication

Journal of Virology ◽

10.1128/jvi.76.10.5014-5023.2002 ◽

2002 ◽

Vol 76 (10) ◽

pp. 5014-5023 ◽

Cited By ~ 16

Author(s):

Kelly A. Hartley ◽

Kenneth A. Alexander

Keyword(s):

Transcription Factors ◽

Human Papillomavirus ◽

Dna Replication ◽

Dna Binding ◽

Tata Binding Protein ◽

Keratinocyte Differentiation ◽

Origin Of Replication ◽

Viral Origin ◽

Hpv Dna

ABSTRACT The human papillomavirus (HPV) protein E2 possesses dual roles in the viral life cycle. By interacting directly with host transcription factors in basal keratinocytes, E2 promotes viral transcription. As keratinocyte differentiation progresses, E2 associates with the viral helicase, E1, to activate vegetative viral DNA replication. How E2's major role switches from transcription to replication during keratinocyte differentiation is not understood, but the presence of a TATA site near the viral origin of replication led us to hypothesize that TATA-binding protein (TBP) could affect HPV replication. Here we show that the C-terminal domain of TBP (TBPc) is a potent inhibitor of E2-stimulated HPV DNA replication in vitro (50% inhibitory concentration = 0.56 nM). Increasing the E1 concentration could not overcome TBPc inhibition in replication assays, indicating that TBPc is a noncompetitive inhibitor of E1 binding. While direct E2-TBPc association could be demonstrated, this interaction could not fully account for the mechanism of TBPc-mediated inhibition of viral replication. Because E2 supports sequence-specific binding of E1 to the viral ori, we proposed that TBPc antagonizes E1-ori association indirectly through inhibition of E2-DNA binding. Indeed, TBPc potently antagonized E2 binding to DNA in the absence (Ki = 0.5 ± 0.1 nM) and presence (Ki = 0.6 ± 0.3 nM) of E1. Since E2 and TBPc cannot be coadjacent on viral sequences, direct DNA-binding competition between TBPc and E2 was responsible for replication inhibition. Given the ability of TBPc to inhibit HPV DNA replication in vitro and data indicating that TBPc antagonized E2-ori association, we propose that transcription factors regulate HPV DNA replication as well as viral transcription.

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DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication

Viruses ◽

10.3390/v8060175 ◽

2016 ◽

Vol 8 (6) ◽

pp. 175 ◽

Cited By ~ 8

Author(s):

Molly Bristol ◽

Xu Wang ◽

Nathan Smith ◽

Minkyeong Son ◽

Michael Evans ◽

...

Keyword(s):

Dna Damage ◽

Human Papillomavirus ◽

Dna Replication ◽

Human Papillomavirus 16

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Correction for Morgan et al., “Werner Helicase Control of Human Papillomavirus 16 E1-E2 DNA Replication Is Regulated by SIRT1 Deacetylation”

mBio ◽

10.1128/mbio.01635-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 3

Author(s):

Dipon Das ◽

Molly L. Bristol ◽

Nathan W. Smith ◽

Claire D. James ◽

Xu Wang ◽

...

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

Human Papillomavirus 16

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An Interaction between Human Papillomavirus 16 E2 and TopBP1 Is Required for Optimum Viral DNA Replication and Episomal Genome Establishment

Journal of Virology ◽

10.1128/jvi.01002-12 ◽

2012 ◽

Vol 86 (23) ◽

pp. 12806-12815 ◽

Cited By ~ 34

Author(s):

M. M. Donaldson ◽

L. J. Mackintosh ◽

J. M. Bodily ◽

E. S. Dornan ◽

L. A. Laimins ◽

...

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

Viral Dna ◽

Viral Dna Replication ◽

Human Papillomavirus 16

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The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication

Journal of Virology ◽

10.1128/jvi.72.4.3436-3441.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 3436-3441 ◽

Cited By ~ 22

Author(s):

Nianxiang Zou ◽

Jen-Sing Liu ◽

Shu-Ru Kuo ◽

Thomas R. Broker ◽

Louise T. Chow

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

Expression Vectors ◽

Atpase Domain ◽

Viral Origin ◽

Hpv 16 ◽

E2 Protein ◽

Amino Terminal ◽

E1 Protein ◽

Carboxyl Terminal

ABSTRACT The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.

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Human papillomavirus 16 L2 inhibits the transcriptional activation function, but not the DNA replication function, of HPV-16 E2

Virus Research ◽

10.1016/j.virusres.2004.07.004 ◽

2005 ◽

Vol 108 (1-2) ◽

pp. 1-14 ◽

Cited By ~ 11

Author(s):

A. Okoye ◽

P. Cordano ◽

E.R. Taylor ◽

I.M. Morgan ◽

R. Everett ◽

...

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

Transcriptional Activation ◽

Activation Function ◽

Hpv 16 ◽

Human Papillomavirus 16 ◽

Replication Function

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The E1 Initiator Recognizes Multiple Overlapping Sites in the Papillomavirus Origin of DNA Replication

Journal of Virology ◽

10.1128/jvi.75.1.292-302.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 292-302 ◽

Cited By ~ 35

Author(s):

Grace Chen ◽

Arne Stenlund

Keyword(s):

Dna Replication ◽

Binding Sites ◽

Double Helix ◽

Origin Of Replication ◽

Recognition Sequence ◽

Viral Origin ◽

Initiator Protein ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Origin Of Dna Replication

ABSTRACT A common feature of replicator sequences from a variety of organisms is multiple binding sites for an initiator protein. By binding to the replicator, initiators mark the site and contribute to melting or distortion of the DNA. We have defined the recognition sequence for the papillomavirus E1 initiator and determined the arrangement of binding sites in the viral origin of replication. We show that E1 recognizes a hexanucleotide sequence which is present in overlapping arrays in virtually all papillomavirus replicators. Binding of the initiator to these sites would result in the formation of a closely packed array of E1 molecules that wrap around the double helix.

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Characterization of the Minimal DNA Binding Domain of the Human Papillomavirus E1 Helicase: Fluorescence Anisotropy Studies and Characterization of a Dimerization-Defective Mutant Protein

Journal of Virology ◽

10.1128/jvi.77.9.5178-5191.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5178-5191 ◽

Cited By ~ 37

Author(s):

S. Titolo ◽

K. Brault ◽

J. Majewski ◽

P. W. White ◽

J. Archambault

Keyword(s):

Human Papillomavirus ◽

Dna Replication ◽

Dna Binding ◽

Fluorescence Anisotropy ◽

Binding Domain ◽

Dna Binding Domain ◽

Sequence Specificity ◽

Viral Origin

ABSTRACT The E1 helicase of papillomaviruses is required for replication of the viral double-stranded DNA genome, in conjunction with cellular factors. DNA replication is initiated at the viral origin by the assembly of E1 monomers into oligomeric complexes that have unwinding activity. In vivo, this process is catalyzed by the viral E2 protein, which recruits E1 specifically at the origin. For bovine papillomavirus (BPV) E1 a minimal DNA-binding domain (DBD) has been identified N-terminal to the enzymatic domain. In this study, we characterized the DBD of human papillomavirus 11 (HPV11), HPV18, and BPV E1 using a quantitative DNA binding assay based on fluorescence anisotropy. We found that the HPV11 DBD binds DNA with an affinity and sequence requirement comparable to those of the analogous domain of BPV but that the HPV18 DBD has a higher affinity for nonspecific DNA. By comparing the DNA-binding properties of a dimerization-defective protein to those of the wild type, we provide evidence that dimerization of the HPV11 DBD occurs only on two appropriately positioned E1 binding-sites and contributes approximately a 10-fold increase in binding affinity. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds DNA with little sequence specificity, similarly to a dimerization-defective DBD. Finally, we show that the amino acid substitution that prevents dimerization reduces the ability of a longer E1 protein to bind to the origin in vitro and to support transient HPV DNA replication in vivo, but has little effect on its ATPase activity or ability to oligomerize into hexamers. These results are discussed in light of a model of the assembly of replication-competent double hexameric E1 complexes at the origin.

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