The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K+HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K+HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K+HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K+HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K+HSFs. After DNA damage, appreciably more foci were formed in 55K+HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K+HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability.

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Differential Requirements of the C Terminus of Nbs1 in Suppressing Adenovirus DNA Replication and Promoting Concatemer Formation

Journal of Virology ◽

10.1128/jvi.00900-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8362-8372 ◽

Cited By ~ 40

Author(s):

Seema S. Lakdawala ◽

Rachel A. Schwartz ◽

Kevin Ferenchak ◽

Christian T. Carson ◽

Brian P. McSharry ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Viral Replication ◽

Dna Damage Response ◽

Viral Genomes ◽

Mrn Complex ◽

E4 Region ◽

C Terminus ◽

Damage Response ◽

Cellular Dna

ABSTRACT Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.

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Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

Journal of Virology ◽

10.1128/jvi.00402-19 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 7

Author(s):

Reshma Nazeer ◽

Fadi S. I. Qashqari ◽

Abeer S. Albalawi ◽

Ann Liza Piberger ◽

Maria Teresa Tilotta ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Viral Replication ◽

Signaling Pathways ◽

Dna Damage Response ◽

Replication Fork ◽

Dependent Manner ◽

Damage Response ◽

Adenovirus E1b ◽

Cellular Dna

ABSTRACT Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication. IMPORTANCE Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular 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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Gene co-expression analysis applied to RNASEH2A reveals functional networks associated with DNA replication, DNA damage response, as well as cell cycle regulation

10.26226/morressier.5ebd45acffea6f735881b15e ◽

2020 ◽

Author(s):

Stefania Marsili

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Expression Analysis ◽

Cell Cycle Regulation ◽

Functional Networks ◽

Damage Response ◽

Cycle Regulation

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Faculty Opinions recommendation of Activation of the cellular DNA damage response in the absence of DNA lesions.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1108695.571444 ◽

2008 ◽

Author(s):

Yosef Gruenbaum

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Dna Lesions ◽

Damage Response ◽

Cellular Dna

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A special issue on the DNA damage response and genomic instability

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmw051 ◽

2016 ◽

Vol 48 (7) ◽

pp. 593-593

Author(s):

Xiaochun Yu

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Dna Damage Response ◽

Special Issue ◽

Damage Response

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Targeting Protein-Protein Interactions in the DNA Damage Response Pathways for Cancer Chemotherapy

RSC Chemical Biology ◽

10.1039/d1cb00101a ◽

2021 ◽

Author(s):

Kerry Silva McPherson ◽

Dmitry Korzhnev

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Protein Interactions ◽

Cell Cycle Progression ◽

Protein Protein Interactions ◽

Cycle Progression ◽

Damage Recognition ◽

Damage Response ◽

Cellular Dna ◽

Dna Damage Recognition

Cellular DNA damage response (DDR) is an extensive signaling network that orchestrates DNA damage recognition, repair and avoidance, cell cycle progression and cell death. DDR alternation is a hallmark of...

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Avian Sarcoma and Leukosis Virus Cytopathic Effect in the Absence of TVB Death Domain Signaling

Journal of Virology ◽

10.1128/jvi.79.13.8243-8248.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 8243-8248 ◽

Cited By ~ 7

Author(s):

Sara Klucking ◽

Asha S. Collins ◽

John A. T. Young

Keyword(s):

Dna Damage ◽

Signaling Pathways ◽

Dna Damage Response ◽

Cytopathic Effect ◽

Tumor Necrosis Factor Receptor ◽

Death Domain ◽

Trail Receptors ◽

Damage Response ◽

Cellular Dna ◽

Avian Sarcoma

ABSTRACT The cytopathic effect (CPE) seen with some subgroups of avian sarcoma and leukosis virus (ASLV) is associated with viral Env activation of the death-promoting activity of TVB (a tumor necrosis factor receptor-related receptor that is most closely related to mammalian TNF-related apoptosis-inducing ligand [TRAIL] receptors) and with viral superinfection leading to unintegrated viral DNA (UVD) accumulation, which is presumed to activate a cellular DNA damage response. In this study, we employed cells that express signaling-deficient ASLV receptors to demonstrate that an ASLV CPE can be uncoupled from the death-promoting functions of the TVB receptor. However, these cell-killing events were associated with much higher levels of viral superinfection and DNA accumulation than those seen when the virus used signaling-competent TVB receptors. These findings suggest that a putative cellular DNA damage response that is activated by UVD accumulation might act in concert with the death-signaling pathways activated by Env-TVB interactions to trigger cell death. Such a model is consistent with the well-established synergy that exists between TRAIL-signaling pathways and DNA damage responses which is currently being exploited in cancer therapy regimens.

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