title: DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells.
abstract: Human bocavirus 1 (HBoV1), an emerging human-pathogenic respiratory virus, is a member of the genus Bocaparvovirus of the Parvoviridae family. In human airway epithelium air-liquid interface (HAE-ALI) cultures, HBoV1 infection initiates a DNA damage response (DDR), activating all three phosphatidylinositol 3-kinase-related kinases (PI3KKs): ATM, ATR, and DNA-PKcs. In this context, activation of PI3KKs is a requirement for amplification of the HBoV1 genome (X. Deng, Z. Yan, F. Cheng, J. F. Engelhardt, and J. Qiu, PLoS Pathog, 12:e1005399, 2016, https://doi.org/10.1371/journal.ppat.1005399), and HBoV1 replicates only in terminally differentiated, nondividing cells. This report builds on the previous discovery that the replication of HBoV1 DNA can also occur in dividing HEK293 cells, demonstrating that such replication is likewise dependent on a DDR. Transfection of HEK293 cells with the duplex DNA genome of HBoV1 induces hallmarks of DDR, including phosphorylation of H2AX and RPA32, as well as activation of all three PI3KKs. The large viral nonstructural protein NS1 is sufficient to induce the DDR and the activation of the three PI3KKs. Pharmacological inhibition or knockdown of any one of the PI3KKs significantly decreases both the replication of HBoV1 DNA and the downstream production of progeny virions. The DDR induced by the HBoV1 NS1 protein does not cause obvious damage to cellular DNA or arrest of the cell cycle. Notably, key DNA replication factors and major DNA repair DNA polymerases (polymerase η [Pol η] and polymerase κ [Pol κ]) are recruited to the viral DNA replication centers and facilitate HBoV1 DNA replication. Our study provides the first evidence of the DDR-dependent parvovirus DNA replication that occurs in dividing cells and is independent of cell cycle arrest.
  
  
  OBJECTIVE
  The parvovirus human bocavirus 1 (HBoV1) is an emerging respiratory virus that causes lower respiratory tract infections in young children worldwide. HEK293 cells are the only dividing cells tested that fully support the replication of the duplex genome of this virus and allow the production of progeny virions. In this study, we demonstrate that HBoV1 induces a DDR that plays significant roles in the replication of the viral DNA and the production of progeny virions in HEK293 cells. We also show that both cellular DNA replication factors and DNA repair DNA polymerases colocalize within centers of viral DNA replication and that Pol η and Pol κ play an important role in HBoV1 DNA replication. Whereas the DDR that leads to the replication of the DNA of other parvoviruses is facilitated by the cell cycle, the DDR triggered by HBoV1 DNA replication or NS1 is not. HBoV1 is the first parvovirus whose NS1 has been shown to be able to activate all three PI3KKs (ATM, ATR, and DNA-PKcs).
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Deng
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.|College of Life Sciences, Central China Normal University, Wuhan, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Xu
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Zou
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Weiran
authors.initials: W
authors.lastname: Shen
authors.affiliation: College of Life Sciences, Central China Normal University, Wuhan, China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Peng
authors.affiliation: College of Life Sciences, Central China Normal University, Wuhan, China.
authors.forename: Kaiyu
authors.initials: K
authors.lastname: Liu
authors.affiliation: Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa, USA.
authors.forename: John F
authors.initials: JF
authors.lastname: Engelhardt
authors.affiliation: Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Ziying
authors.initials: Z
authors.lastname: Yan
authors.affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA jqiu@kumc.edu.
authors.forename: Jianming
authors.initials: J
authors.lastname: Qiu
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics* / Q000378:metabolism; D002455:Cell Division; D004249:DNA Damage; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D016325:HIV Long Terminal Repeat; D006657:Histones / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D056661:Human bocavirus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010322:Parvoviridae Infections / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D000099270:Y-Family DNA Polymerases
chemical_list: D064007:Ataxia Telangiectasia Mutated Proteins; D004279:DNA, Viral; D051747:DNA-Activated Protein Kinase; D004259:DNA-Directed DNA Polymerase; D006657:Histones; D009687:Nuclear Proteins; D051716:Replication Protein A; D017361:Viral Nonstructural Proteins; C468783:H2AX protein, human; C576324:ATM protein, human; C576597:ATR protein, human; C497263:PRKDC protein, human; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C516794:RPA2 protein, human; C112338:Rad30 protein
keywords: DNA damage; DNA replication; parvovirus