scholarly journals The large nonstructural protein (NS1) of the human bocavirus 1 (HBoV1) directly interacts with Ku70, which plays an important role in virus replication in human airway epithelia

2021 ◽  
Author(s):  
Liting Shao ◽  
Kang Ning ◽  
Jianke Wang ◽  
Fang Cheng ◽  
Shengqi Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Nonstructural Protein ◽  
Direct Interaction ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Human Airway ◽  
Damage Response

Human bocavirus 1 (HBoV1), an autonomous human parvovirus, causes acute respiratory tract infections in young children. HBoV1 infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI). HBoV1 expresses a large nonstructural protein, NS1, that is essential for viral DNA replication. HBoV1 infection of polarized human airway epithelial cells induces a DNA damage response (DDR) that is critical to viral DNA replication involving DNA repair with error-free Y-family DNA polymerases. HBoV1 NS1 or the isoform NS1-70 per se induces a DDR. In this study, using the second-generation proximity-dependent biotin identification (BioID2) approach, we identified that Ku70 is associated with the NS1-BioID2 pulldown complex through a direct interaction with NS1. Bio-layer Interferometry (BLI) assay determined a high binding affinity of the NS1 with Ku70, which has an equilibrium dissociation constant (K D ) value of 0.16 μM and processes the strongest interaction at the C-terminal domain. The association of Ku70 with NS1 was also revealed during HBoV1 infection of HAE-ALI. Knockdown of Ku70 and overexpression of the C-terminal domain of Ku70 significantly decreased HBoV1 replication in HAE-ALI. Thus, our study provides for the first time a direct interaction of a parvovirus large nonstructural protein NS1 with Ku70. IMPORTANCE Parvovirus infection induces a DNA damage response (DDR) that plays a pivotal role in viral DNA replication. The DDR includes activation of ATM (Ataxia telangiectasia mutated), ATR (ATM- and RAD3-related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). The large nonstructural protein (NS1) often plays a role in the induction of DDR; however, how the DDR is induced during parvovirus infection or simply by the NS1 is not well studied. Activation of DNA-PKcs has been shown as one of the key DDR pathways in DNA replication of HBoV1. We identified that HBoV1 NS1 directly interacts with Ku70, but not Ku80, of the Ku70/Ku80 heterodimer at a high affinity. This interaction is also important for HBoV1 replication in HAE-ALI. We propose that the interaction of the NS1 with Ku70 recruits the Ku70/Ku80 complex to the viral DNA replication center, which activates DNA-PKcs and facilitates viral DNA replication.

scholarly journals Temporal Regulation of the Mre11-Rad50-Nbs1 Complex during Adenovirus Infection

2009 ◽  
Vol 83 (9) ◽  
pp. 4565-4573 ◽  
Author(s):  
Kasey A. Karen ◽  
Peter J. Hoey ◽  
C. S. H. Young ◽  
Patrick Hearing
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Adenovirus Infection ◽  
Viral Dna ◽  
Terminal Protein ◽  
Viral Dna Replication ◽  
Mrn Complex ◽  
Viral Dnas ◽  
Damage Response

ABSTRACT Adenovirus infection induces a cellular DNA damage response that can inhibit viral DNA replication and ligate viral genomes into concatemers. It is not clear if the input virus is sufficient to trigger this response or if viral DNA replication is required. Adenovirus has evolved two mechanisms that target the Mre11-Rad50-Nbs1 (MRN) complex to inhibit the DNA damage response. These include E4-ORF3-dependent relocalization of MRN proteins and E4-ORF6/E1B-55K-dependent degradation of MRN components. The literature suggests that degradation of the MRN complex due to E4-ORF6/E1B-55K does not occur until after viral DNA replication has begun. We show that, by the time viral DNA accumulates, the MRN complex is inactivated by either of the E4-induced mechanisms and that, with E4-ORF6/E1B-55K, this inactivation is due to MRN degradation. Our data are consistent with the conclusion that input viral DNA is sufficient to induce the DNA damage response. Further, we demonstrate that when the DNA damage response is active in E4 mutant virus infections, the covalently attached terminal protein is not cleaved from viral DNAs, and the viral origins of replication are not detectably degraded at a time corresponding to the onset of viral replication. The sequences of concatemeric junctions of viral DNAs were determined, which supports the conclusion that nonhomologous end joining mediates viral DNA ligation. Large deletions were found at these junctions, demonstrating nucleolytic procession of the viral DNA; however, the lack of terminal protein cleavage and terminus degradation at earlier times shows that viral genome deletion and concatenation are late effects.

scholarly journals Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Wei Zou ◽  
Zekun Wang ◽  
Min Xiong ◽  
Aaron Yun Chen ◽  
Peng Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Parvovirus B19 ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cycle Arrest ◽  
Damage Response ◽  
Cellular Dna

ABSTRACTHuman parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.IMPORTANCEHuman parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.

Hairpin transfer-independent Parvovirus DNA Replication Produces Infectious Virus

2021 ◽  
Author(s):  
Weiran Shen ◽  
Zekun Wang ◽  
Kang Ning ◽  
Fang Cheng ◽  
John F. Engelhardt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Hek293 Cells ◽  
Progeny Virus ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Airway Epithelia ◽  
Viral Dna Replication ◽  
Human Airway ◽  
The Right ◽  
Human Airway Epithelia

Parvoviruses package a linear single-stranded DNA genome with hairpin structures at both ends. It has been thought that terminal hairpin sequences are indispensable for viral DNA replication. Here, we provide evidence that the hairpin-deleted duplex genomes of human bocavirus 1 (HBoV1) replicate in human embryonic kidney (HEK) 293 cells. We propose an alternative model for HBoV1 DNA replication in which the leading strand can initiate strand-displacement without “hairpin-transfer.” The transfection of the HBoV1 duplex genomes that retain a minimal replication origin at the right-end ( OriR ), but with extensive deletions in the right-end hairpin (REH), generated viruses in HEK293 cells at a level 10-20 times lower than the wild-type (WT) duplex genome. Importantly, these viruses that have a genome with various deletions after the OriR , but not the one retaining only the OriR , replicated in polarized human airway epithelia. We discovered that the 18-nt sequence (nt 5,403-5,420) beyond the OriR was sufficient to confer virus replication in polarized human airway epithelia, although its progeny virus production was ∼5 times lower than that of the WT virus. Thus, our study demonstrates that hairpin transfer-independent productive parvovirus DNA replication can occur. Importance Hairpin transfer-independent parvovirus replication was modeled with human bocavirus 1 (HBoV1) duplex genomes whose 5’ hairpin structure was ablated by various deletions. In HEK293 cells, these duplex viral genomes with ablated 5’/hairpin sequence replicated efficiently and generated viruses that productively infected polarized human airway epithelium. Thus, for the first time, we reveal a previously unknown phenomenon that the productive parvovirus DNA replication does not depend on the hairpin sequence at REH to initiate “rolling hairpin” DNA replication. Notably, the intermediates of viral DNA replication, as revealed two-dimensional electrophoresis, from transfections of hairpin sequence-deleted duplex genome and full-length genome in HEK293 cells, as well as from virus infection of polarized human airway epithelia are similar. Thus, the establishment of the hairpin transfer-independent parvoviral DNA replication deepens our understanding in viral DNA replication and may have implications in development of parvovirus-based viral vectors with alternative properties.

scholarly journals Cellular Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Mediates Nuclear Import of Human Bocavirus 1 NP1 Protein and Modulates Viral Capsid Protein Expression

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Xiaomei Wang ◽  
Peng Xu ◽  
Fang Cheng ◽  
Yi Li ◽  
Zekun Wang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Capsid Protein ◽  
Nuclear Import ◽  
Nonstructural Protein ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Specificity Factor ◽  
Tract Infections

ABSTRACT Human bocavirus 1 (HBoV1), which belongs to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro, HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid protein (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed that two host mRNA processing factors, DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6; also known as CFIm68), a subunit of the cleavage factor Im complex (CFIm), interact with HBoV1 NP1 independently of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased the expression of capsid protein but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in the nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication. IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens causing acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein (NP1) that plays an important role in the maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor, CPSF6, that directly interacts with NP1, mediates the nuclear import of NP1, and plays a role in the maturation of capsid protein-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism by which a viral small nonstructural protein facilitates the multiple regulation of viral gene expression and replication and reveals a novel target for potent antiviral drug development.

scholarly journals Viral DNA Replication-Dependent DNA Damage Response Activation during BK Polyomavirus Infection

2015 ◽  
Vol 89 (9) ◽  
pp. 5032-5039 ◽  
Author(s):  
Brandy Verhalen ◽  
Joshua L. Justice ◽  
Michael J. Imperiale ◽  
Mengxi Jiang
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Viral Replication ◽  
Dna Damage Response ◽  
Marrow Transplant ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Transplant Patients ◽  
Bk Polyomavirus ◽  
Damage Response

ABSTRACTBK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability.IMPORTANCEPolyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR activation is viral replication. Furthermore, we show that the virus is able to cause host DNA damage in the absence of viral replication and DDR activation. These results suggest an intricate relationship between viral replication, DDR activation, and host genome instability.

scholarly journals Analysis ofcisandtransRequirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome

2016 ◽  
Vol 90 (17) ◽  
pp. 7761-7777 ◽  
Author(s):  
Weiran Shen ◽  
Xuefeng Deng ◽  
Wei Zou ◽  
John F. Engelhardt ◽  
Ziying Yan ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Respiratory Tract Infections ◽  
Nonstructural Protein ◽  
Hek293 Cells ◽  
Human Bocavirus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
The Right ◽  
Tract Infections

ABSTRACTParvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in the dependoparvovirus adeno-associated virus 2 (AAV2) and the protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney 293 (HEK293) cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large nonstructural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriRin vitro, indicating that other viral and cellular components or the oligomerization of NS1 is required for NS1 binding to the OriR.In vivostudies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small nonstructural protein NP1 is required for HBoV1 DNA replication at OriR. NP1 and other viral nonstructural proteins (NS1 to NS4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of NP1 in viral DNA replication at OriR. Overall, our study revealed the characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication.IMPORTANCEHuman bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells and produces progeny virions that are infectious in well-differentiated airway epithelial cells. A recombinant AAV2 vector pseudotyped with an HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified bothcis-acting elements andtrans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin, which contains both NS1 nicking and binding sites, to a 46-nucleotide sequence in the right-end hairpin. The identification of these essential elements of HBoV1 DNA replication acting both incisand intranswill provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin and to design next-generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases.

scholarly journals Werner Helicase Control of Human Papillomavirus 16 E1-E2 DNA Replication Is Regulated by SIRT1 Deacetylation

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Dipon Das ◽  
Molly L. Bristol ◽  
Nathan W. Smith ◽  
Claire D. James ◽  
Xu Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Life Cycle ◽  
Dna Replication ◽  
Homologous Recombination ◽  
Dna Damage Response ◽  
Human Papillomaviruses ◽  
Repair Protein ◽  
Damage Response ◽  
Dna Repair Protein

ABSTRACTHuman papillomaviruses (HPV) are double-stranded DNA viruses causative in a host of human diseases, including several cancers. Following infection, two viral proteins, E1 and E2, activate viral replication in association with cellular factors and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous recombination (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously, we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2-replicating DNA and regulates the level of replication. Here, we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2-replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2-replicating DNA. We present a model in which E1-E2 replication turns on the DDR, stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2-replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.IMPORTANCEHPV16 is the major viral human carcinogen responsible for between 3 and 4% of all cancers worldwide. Following infection, this virus activates the DNA damage response (DDR) to promote its life cycle and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here, we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall, the results suggest a mechanism by which SIRT1 deacetylation of WRN promotes its interaction with E1-E2-replicating DNA to control the levels and fidelity of that replication.

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