MECHANISMS OF HUMAN PAPILLOMAVIRUS DNA REPLICATION

Summary

Principal Investigator: LOUISE CHOW
Affiliation: University of Alabama at Birmingham
Country: USA
Abstract: Human papillomaviruses (HPVs) comprise an extended family of medically important human pathogens. These small DNA viruses establish persistent infections of squamous epithelia, typically causing benign hyperproliferative lesions. Infections by higher risk genotypes can progress to dysplasias and carcinomas, notably cervical and penile cancers. The double-stranded, circular genome replicates as extrachromosomal plasmids at low copy number in cycling basal keratinocytes. Productive amplification takes place only in differentiating spinous cells. We and others developed transient and cell-free systems to study the mechanisms of HPV DNA replication and showed that replication requires an origin of replication (ori), the HPV ori recognition protein E2, the viral replicative helicase E1, the cellular DNA replication machinery and cyclin/cdk complexes and that the E1 activity is regulated by multiple kinases. Our preliminary results reveal that a number of cellular proteins known to control chromosomal DNA replication also regulate HPV replication in the cell-free replication system, including Cdt1, geminin, TopBP1, and p53. Geminin inhibits Cdt1, which is necessary for loading the cellular replicative helicase MCM complex onto the cellular chromatin. Our results implicate MCM in controlling HPV replication when E1 concentration is low. TopBP1 is involved in both cellular DNA replication and repair. It interacts with DNA polymerase epsilon, a polymerase with critical but yet-to-be elucidated functions. TopBP1 has been reported to interact with HPV16 E2 and to stimulate transient HPV replication. We now show that TopBP1 enhances HPV cell-free replication. p53 is targeted by HPV E6 protein for inactivation and is known to bind E2 and inhibit viral transient amplification replication. We demonstrate that p53 inhibits HPV replication in the presence or absence of E2, indicative of additional mechanisms. We suggest that, in each case, the cell-free system provides an excellent opportunity to elucidate the mechanisms of regulation for both viral and cellular DNA replication. We propose four specific aims. 1. To test a new model for HPV DNA replication and regulation. This model would account for the maintenance mode and the amplification mode of viral DNA replication in infected tissues. 2. To determine the roles of TopBP1 and DNA polymerase epsilon in HPV DNA replication. 3. To test our hypothesis that p53 inhibition of HPV DNA replication is mediated at least in part by interfering with the activities of recombination proteins that have been hypothesized to be involved in chromosomal DNA replication. 4. To elucidate how E1 phosphorylation modulates its replication functions, a continuation of present research.
Funding Period: 1999-04-01 - 2010-03-31
more information: NIH RePORT

Top Publications

  1. ncbi Simultaneous in situ detection of RNA, DNA, and protein using tyramide-coupled immunofluorescence
    Brian A Van Tine
    Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA
    Methods Mol Biol 292:215-30. 2005
  2. ncbi Dynamic localization of the human papillomavirus type 11 origin binding protein E2 through mitosis while in association with the spindle apparatus
    Luan D Dao
    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA
    J Virol 80:4792-800. 2006
  3. ncbi Conditionally activated E7 proteins of high-risk and low-risk human papillomaviruses induce S phase in postmitotic, differentiated human keratinocytes
    N Sanjib Banerjee
    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA
    J Virol 80:6517-24. 2006
  4. ncbi Mitogen-activated protein kinases activate the nuclear localization sequence of human papillomavirus type 11 E1 DNA helicase to promote efficient nuclear import
    Jei Hwa Yu
    Department of Biochemistry and Molecular Genetics, McCallum Building, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, Alabama 35294 0005, USA
    J Virol 81:5066-78. 2007
  5. ncbi Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein
    Jeonggu Sim
    Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7295, USA
    J Mol Biol 375:1165-77. 2008
  6. ncbi Casein kinase II motif-dependent phosphorylation of human papillomavirus E7 protein promotes p130 degradation and S-phase induction in differentiated human keratinocytes
    Nicholas J Genovese
    University of Alabama at Birmingham, Biochemistry and Molecular Genetics, 1918 University Boulevard, McCallum Building 510, Birmingham, AL 35294 0005, USA
    J Virol 82:4862-73. 2008
  7. ncbi Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes
    Hsu Kun Wang
    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA
    Genes Dev 23:181-94. 2009
  8. ncbi Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6
    Xiaohong Wang
    HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
    RNA 15:637-47. 2009
  9. ncbi A highly efficient system to produce infectious human papillomavirus: Elucidation of natural virus-host interactions
    Louise T Chow
    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
    Cell Cycle 8:1319-23. 2009

Scientific Experts

  • LOUISE CHOW
  • Thomas R Broker
  • Hsu Kun Wang
  • Jei Hwa Yu
  • Nicholas J Genovese
  • N Sanjib Banerjee
  • Brian A Van Tine
  • Xiaohong Wang
  • Jeonggu Sim
  • Jei-Hwa Yu
  • Biing Yuan Lin
  • Luan D Dao
  • Janet S Rader
  • J Philip McCoy
  • Zhi Ming Zheng
  • Craig Meyers
  • Aaron A Duffy
  • Nilam S Banerjee
  • Sezgin Ozgur
  • Jack Griffith
  • Wentao Deng
  • Cheng-Ming Chiang
  • Aaron Duffy
  • Francisco Noya
  • Wei-Ming Chien
  • Shwu-Yuan Wu

Detail Information

Publications9

  1. ncbi Simultaneous in situ detection of RNA, DNA, and protein using tyramide-coupled immunofluorescence
    Brian A Van Tine
    Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA
    Methods Mol Biol 292:215-30. 2005
    ..Finally, we describe the detection of nascent viral RNA transcripts simultaneously with integrated viral genomes or chromosomal domains in single cells or tissue sections...
  2. ncbi Dynamic localization of the human papillomavirus type 11 origin binding protein E2 through mitosis while in association with the spindle apparatus
    Luan D Dao
    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA
    J Virol 80:4792-800. 2006
    ..We conclude that this region of HPV E2C is critical for localization with the mitotic apparatus, enabling the HPV DNA to sustain persistent infections...
  3. ncbi Conditionally activated E7 proteins of high-risk and low-risk human papillomaviruses induce S phase in postmitotic, differentiated human keratinocytes
    N Sanjib Banerjee
    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA
    J Virol 80:6517-24. 2006
    ..These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs...
  4. ncbi Mitogen-activated protein kinases activate the nuclear localization sequence of human papillomavirus type 11 E1 DNA helicase to promote efficient nuclear import
    Jei Hwa Yu
    Department of Biochemistry and Molecular Genetics, McCallum Building, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, Alabama 35294 0005, USA
    J Virol 81:5066-78. 2007
    ..In contrast, E1 proteins mutated in the MAPK docking motifs were completely inactive in transient replication, an indication that additional properties were adversely affected by those changes...
  5. ncbi Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein
    Jeonggu Sim
    Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7295, USA
    J Mol Biol 375:1165-77. 2008
    ..The role of these structures in the modeling and regulation of the HPV-11 ori is discussed...
  6. ncbi Casein kinase II motif-dependent phosphorylation of human papillomavirus E7 protein promotes p130 degradation and S-phase induction in differentiated human keratinocytes
    Nicholas J Genovese
    University of Alabama at Birmingham, Biochemistry and Molecular Genetics, 1918 University Boulevard, McCallum Building 510, Birmingham, AL 35294 0005, USA
    J Virol 82:4862-73. 2008
    ..Collectively, these data support the notion that p130 controls the homeostasis of the differentiated keratinocytes and is therefore targeted by E7 for degradation to establish conditions permissive for viral DNA amplification...
  7. ncbi Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes
    Hsu Kun Wang
    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA
    Genes Dev 23:181-94. 2009
    ..This system will be invaluable for HPV genetic dissection and serves as a faithful ex vivo model for investigating infections and interventions...
  8. ncbi Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6
    Xiaohong Wang
    HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
    RNA 15:637-47. 2009
    ..Together, this is the first time a viral oncoprotein has been shown to regulate cellular miRNA expression. Our data have provided new insights into mechanisms by which high-risk HPVs contribute to the development of cervical cancer...
  9. ncbi A highly efficient system to produce infectious human papillomavirus: Elucidation of natural virus-host interactions
    Louise T Chow
    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
    Cell Cycle 8:1319-23. 2009
    ..We provide additional insights into the viral life cycle and contrast them to conclusions derived from experiments in cell lines...