Roles of ATM and ATR-Mediated DNA Damage Responses during Lytic BK Polyomavirus Infection

100

117

bClonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers. Merkel cell polyomavirus (MCV) is the first polyomavirus to be clearly associated with cancer in humans (1). Its genome was recently found integrated into the chromosomes of a highly aggressive skin cancer, Merkel cell carcinoma (MCC) (2). Subsequent analyses of a large number of MCC tumors have revealed that this polyomavirus is associated with at least 80% of all MCC cases (2-4). Integrated MCV genome has also been detected in non-small-cell lung cancer (5). Epidemiological surveys for MCV seropositivity (6, 7) and sequencing analyses of healthy human skin (8) have indicated that MCV represents a common component of the human skin microbial flora.As with other polyomaviruses, the MCV genome contains an early region that encodes the viral tumor antigens. Differential splicing of the early mRNA produces large tumor antigen (LT), small tumor antigen (sT), and 57kT proteins (9, 10). The highly multifunctional LT protein is involved in a variety of processes, including initiation of viral genome replication, as well as manipulation of the host cell cycle through a number of protein-protein interactions. It has been shown that MCV LT interacts with at least some of the same cellular factors as simian virus 40 (SV40) LT (11). SV40 LT interacts with classic partners including heat shock protein 70 (Hsc70) through the LT DnaJ domain and also interacts with retinoblastoma "pocket protein" (Rb) family members through a classic LxCxE motif in the N-terminal region of LT. SV40 LT binding of Rb abrogates its role as a repressor of E2F transcription factors,...

Section: Discussionmentioning

confidence: 99%

Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation

Wang

Díaz

et al. 2013

100

117

“…Indeed, fragile sites and telomeres may be the preferential breakpoints in LT-expressing cells, as replication stress preferentially causes DSBs in hard-to-replicate regions (85). The ATR/Chk1 pathway is activated by LT to cope with replication stress (7,9,11,86), which highlights a therapeutic opportunity that targeted inhibition of this pathway could LT-expressing cells and attempts to link these temporally. Note that in some cases, the relationship between two phenotypes can be bidirectional, which is illustrated by black and red arrows, respectively.…”

Section: Discussionmentioning

confidence: 99%

Replication Stress and Mitotic Dysfunction in Cells Expressing Simian Virus 40 Large T Antigen

Filippakis

Huang

et al. 2013

cWe previously demonstrated that simian virus 40 (SV40) large T antigen (LT) binds to the Bub1 kinase, a key regulator of the spindle checkpoint and chromosome segregation. Bub1 mutations or altered expression patterns are linked to chromosome missegregation and are considered to be a driving force in some human cancers. Here we report that LT, dependent on Bub1 binding, causes micronuclei, lagging chromatin, and anaphase bridges, which are hallmarks of chromosomal instability (CIN) and Bub1 insufficiency. Using time-lapse microscopy, we demonstrate that LT imposes a Bub1 binding-dependent delay in the metaphase-to-anaphase transition. Kinetochore fibers reveal that LT, via Bub1 binding, causes aberrant kinetochore (KT)-microtubule (MT) attachments and a shortened interkinetochore distance, consistent with a lack of tension. Previously, we showed that LT also induces the DNA damage response (DDR) via Bub1 binding. Using inducible LT cell lines, we show that an activated DDR was observed before the appearance of anaphase bridges and micronuclei. Furthermore, LT induction in serum-starved cells demonstrated ␥-H2AX accumulation in cells that had not yet entered mitosis. Thus, DDR activation can occur independently of chromosome segregation defects. Replication stress pathways may be responsible, because signatures of replication stress were observed, which were attenuated by exogenous supplementation with nucleosides. Our observations allow us to propose a model that explains and integrates the diverse manifestations of genomic instability induced by LT.

“…Total cell proteins were harvested, quantified, and immunoblotted as previously described (16). Antibodies were used at previously described concentrations (14). For all the experiments for which representative results are shown in the figures, multiple gels were run in each independent experiment in order to cover all the proteins that were probed.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, we have demonstrated that in the absence of either ATM or ATR, there is an accumulation of host DNA damage in BKPyV-infected cells, as evidenced by the appearance of abnormal nuclei and a dramatic increase in the number of damaged metaphase chromosomes (14). In this study, we aimed to further understand how the DDRs are activated by BKPyV and how the activation might be tied to the host genome stability.…”

mentioning

confidence: 99%

“…Using a primary human renal proximal tubule epithelial (RPTE) cell culture model (13), we have previously shown that BKPyV is able to activate both the ATM-and the ATR-mediated DDRs and that both pathways function to ensure optimal viral replication and infectious progeny production (14). In addition, we have demonstrated that in the absence of either ATM or ATR, there is an accumulation of host DNA damage in BKPyV-infected cells, as evidenced by the appearance of abnormal nuclei and a dramatic increase in the number of damaged metaphase chromosomes (14).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Verhalen

Justice

Imperiale

et al. 2015

Self Cite

BK 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. The BK polyomavirus (BKPyV) is a ubiquitous opportunistic human pathogen which causes severe disease in immunocompromised patients (1). BKPyV is thought to be acquired through the respiratory route during early childhood, and by adulthood, up to 90% of the general population becomes seropositive (2). Following primary exposure, the virus establishes a lifelong, subclinical, persistent infection in the genitourinary tract. BKPyV can reactivate from the persistent state under immunosuppressed conditions, most commonly in kidney transplant patients, resulting in viral shedding in urine or blood and, ultimately, polyomavirus-associated nephropathy, a significant cause of renal dysfunction (3). There are no FDA-approved therapies for BKPyV infection, and the usual treatment is to reduce immu...