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

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.

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Bocavirus Infection Induces a DNA Damage Response That Facilitates Viral DNA Replication and Mediates Cell Death

Journal of Virology ◽

10.1128/jvi.01534-10 ◽

2010 ◽

Vol 85 (1) ◽

pp. 133-145 ◽

Cited By ~ 36

Author(s):

Y. Luo ◽

A. Y. Chen ◽

J. Qiu

Keyword(s):

Dna Damage ◽

Cell Death ◽

Dna Replication ◽

Dna Damage Response ◽

Viral Dna ◽

Viral Dna Replication ◽

Damage Response

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

Journal of Virology ◽

10.1128/jvi.00042-09 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4565-4573 ◽

Cited By ~ 33

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.

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BK Polyomavirus Activates the DNA Damage Response To Prolong S Phase

Journal of Virology ◽

10.1128/jvi.00130-19 ◽

2019 ◽

Vol 93 (14) ◽

Cited By ~ 5

Author(s):

Joshua L. Justice ◽

Jason M. Needham ◽

Sunnie R. Thompson

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

S Phase ◽

Genome Replication ◽

Viral Production ◽

Viral Dna ◽

Bk Polyomavirus ◽

Damage Response ◽

Infected Cells

ABSTRACT BK polyomavirus (PyV) is a major source of kidney failure in transplant recipients. The standard treatment for patients with lytic BKPyV infection is to reduce immunosuppressive therapy, which increases the risk of graft rejection. PyVs are DNA viruses that rely upon host replication proteins for viral genome replication. A hallmark of PyV infection is activation of the DNA damage response (DDR) to prevent severe host and viral DNA damage that impairs viral production by an unknown mechanism. Therefore, we sought to better understand why BKPyV activates the DDR through the ATR and ATM pathways and how this prevents DNA damage and leads to increased viral production. When ATR was inhibited in BKPyV-infected primary kidney cells, severe DNA damage occurred due to premature Cdk1 activation, which resulted in mitosis of cells that were actively replicating host DNA in S phase. Conversely, ATM was required for efficient entry into S phase and to prevent normal mitotic entry after G2 phase. The synergistic activation of these DDR kinases promoted and maintained BKPyV-mediated S phase to enhance viral production. In contrast to BKPyV infection, DDR inhibition did not disrupt cell cycle control in uninfected cells. This suggests that DDR inhibitors may be used to specifically target BKPyV-infected cells. IMPORTANCE BK polyomavirus (BKPyV) is an emerging pathogen that reactivates in immunosuppressed organ transplant patients. We wanted to understand why BKPyV-induced activation of the DNA damage response (DDR) enhances viral titers and prevents host DNA damage. Here, we show that the virus activates the DNA damage response in order to keep the infected cells in S phase to replicate the viral DNA. The source of DNA damage was due to actively replicating cells with uncondensed chromosomes entering directly into mitosis when the DDR was inhibited in BKPyV-infected cells. This study clarifies the previously enigmatic role of the DDR during BKPyV infection by demonstrating that the virus activates the DDR to maintain the cells in S phase in order to promote viral replication and that disruption of this cell cycle arrest can lead to catastrophic DNA damage for the host.

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Host DNA Damage Response Factors Localize to Merkel Cell Polyomavirus DNA Replication Sites To Support Efficient Viral DNA Replication

Journal of Virology ◽

10.1128/jvi.03656-13 ◽

2014 ◽

Vol 88 (6) ◽

pp. 3285-3297 ◽

Cited By ~ 31

Author(s):

S. H. Tsang ◽

X. Wang ◽

J. Li ◽

C. B. Buck ◽

J. You

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Merkel Cell Polyomavirus ◽

Merkel Cell ◽

Viral Dna ◽

Response Factors ◽

Viral Dna Replication ◽

Damage Response ◽

Replication Sites

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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

Journal of Virology ◽

10.1128/jvi.01840-21 ◽

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.

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

Journal of Virology ◽

10.1128/jvi.01881-17 ◽

2017 ◽

Vol 92 (5) ◽

Cited By ~ 9

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.

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