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A novel mechanism inducing genome instability in Kaposi's sarcoma-associated herpesvirus infected cells.

Jackson BR, Noerenberg M, Whitehouse A - PLoS Pathog. (2014)

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple AIDS-related malignancies.Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage.Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple AIDS-related malignancies. Like other herpesviruses, KSHV has a biphasic life cycle and both the lytic and latent phases are required for tumorigenesis. Evidence suggests that KSHV lytic replication can cause genome instability in KSHV-infected cells, although no mechanism has thus far been described. A surprising link has recently been suggested between mRNA export, genome instability and cancer development. Notably, aberrations in the cellular transcription and export complex (hTREX) proteins have been identified in high-grade tumours and these defects contribute to genome instability. We have previously shown that the lytically expressed KSHV ORF57 protein interacts with the complete hTREX complex; therefore, we investigated the possible intriguing link between ORF57, hTREX and KSHV-induced genome instability. Herein, we show that lytically active KSHV infected cells induce a DNA damage response and, importantly, we demonstrate directly that this is due to DNA strand breaks. Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage. Moreover, we describe a novel mechanism showing that the genetic instability observed is a consequence of R-loop formation. Importantly, the link between hTREX sequestration and DNA damage may be a common feature in herpesvirus infection, as a similar phenotype was observed with the herpes simplex virus 1 (HSV-1) ICP27 protein. Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX.

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Lytically replicating KSHV cells demonstrate the presence of R-loops.(A) 293T rKSHV.219 cells were left either unreactivated, or reactivated for 36 hours and transfected with Myc-Aly or EGFP-RNaseH1 and alkaline comet assays were performed. (B) Comet tails were scored using CometScore and n- and P-values are represented for all data and error bars show the standard error from the mean.
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ppat-1004098-g006: Lytically replicating KSHV cells demonstrate the presence of R-loops.(A) 293T rKSHV.219 cells were left either unreactivated, or reactivated for 36 hours and transfected with Myc-Aly or EGFP-RNaseH1 and alkaline comet assays were performed. (B) Comet tails were scored using CometScore and n- and P-values are represented for all data and error bars show the standard error from the mean.

Mentions: To examine whether our hypothesis of hTREX sequestration by ORF57 away from cellular transcription to sites of viral transcription leads to R-loop formation during KSHV infection we utilised the HEK 293T based cell line containing the entire KSHV genome, 293T rKSHV.219. We performed a series of alkaline comet assay experiments to determine the levels of DNA strand breaks comparing KSHV latent and reactivated cells (Figure 6). Unreactivated cells showed a tail moment of 0.43, whilst after being reactivated using 20 ng/ml TPA and 1.5 mM sodium butyrate for 36 hours cells showed a significant increase in the tail moment to 5.49 (P = 6.18×10−9) demonstrating a level of DNA strand breaks. This confirms our previous observations in TREx BCBL-Rta cells showing that KSHV lytic replication directly causes DNA damage. To determine whether the observed DNA damage could be attributed, at least in part, to R-loop formation we again utilised a construct expressing EGFP-RNaseH1 and transfected it into the 293T rKSHV.219 cells 8 hours prior to reactivation. Importantly, there was a significant decrease in the tail moment when compared to the reactivated cells without EGFP-RNaseH1 over-expression down to 1.42 (P = 4.31×10−6). This demonstrates that the DNA strand breaks in KSHV lytically replicating cells are as a result of the formation of R-loops. Moreover, to demonstrate that the R-loop formation is as a result of ORF57 sequestering hTREX away from cellular transcription to sites of viral transcription we transfected Myc-Aly into the 293T rKSHV.219 cells. As was observed in the ORF57 over-expression system this again dramatically and significantly reduced the tail moment to 2.06 (P = 1.24×10−4 when compared to reactivated 293T rKSHV.219 cells) indicating that hTREX sequestration by ORF57 is the cause of R-loop formation and DNA strand breaks in lytically active KSHV infected cells.


A novel mechanism inducing genome instability in Kaposi's sarcoma-associated herpesvirus infected cells.

Jackson BR, Noerenberg M, Whitehouse A - PLoS Pathog. (2014)

Lytically replicating KSHV cells demonstrate the presence of R-loops.(A) 293T rKSHV.219 cells were left either unreactivated, or reactivated for 36 hours and transfected with Myc-Aly or EGFP-RNaseH1 and alkaline comet assays were performed. (B) Comet tails were scored using CometScore and n- and P-values are represented for all data and error bars show the standard error from the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4006916&req=5

ppat-1004098-g006: Lytically replicating KSHV cells demonstrate the presence of R-loops.(A) 293T rKSHV.219 cells were left either unreactivated, or reactivated for 36 hours and transfected with Myc-Aly or EGFP-RNaseH1 and alkaline comet assays were performed. (B) Comet tails were scored using CometScore and n- and P-values are represented for all data and error bars show the standard error from the mean.
Mentions: To examine whether our hypothesis of hTREX sequestration by ORF57 away from cellular transcription to sites of viral transcription leads to R-loop formation during KSHV infection we utilised the HEK 293T based cell line containing the entire KSHV genome, 293T rKSHV.219. We performed a series of alkaline comet assay experiments to determine the levels of DNA strand breaks comparing KSHV latent and reactivated cells (Figure 6). Unreactivated cells showed a tail moment of 0.43, whilst after being reactivated using 20 ng/ml TPA and 1.5 mM sodium butyrate for 36 hours cells showed a significant increase in the tail moment to 5.49 (P = 6.18×10−9) demonstrating a level of DNA strand breaks. This confirms our previous observations in TREx BCBL-Rta cells showing that KSHV lytic replication directly causes DNA damage. To determine whether the observed DNA damage could be attributed, at least in part, to R-loop formation we again utilised a construct expressing EGFP-RNaseH1 and transfected it into the 293T rKSHV.219 cells 8 hours prior to reactivation. Importantly, there was a significant decrease in the tail moment when compared to the reactivated cells without EGFP-RNaseH1 over-expression down to 1.42 (P = 4.31×10−6). This demonstrates that the DNA strand breaks in KSHV lytically replicating cells are as a result of the formation of R-loops. Moreover, to demonstrate that the R-loop formation is as a result of ORF57 sequestering hTREX away from cellular transcription to sites of viral transcription we transfected Myc-Aly into the 293T rKSHV.219 cells. As was observed in the ORF57 over-expression system this again dramatically and significantly reduced the tail moment to 2.06 (P = 1.24×10−4 when compared to reactivated 293T rKSHV.219 cells) indicating that hTREX sequestration by ORF57 is the cause of R-loop formation and DNA strand breaks in lytically active KSHV infected cells.

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple AIDS-related malignancies.Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage.Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple AIDS-related malignancies. Like other herpesviruses, KSHV has a biphasic life cycle and both the lytic and latent phases are required for tumorigenesis. Evidence suggests that KSHV lytic replication can cause genome instability in KSHV-infected cells, although no mechanism has thus far been described. A surprising link has recently been suggested between mRNA export, genome instability and cancer development. Notably, aberrations in the cellular transcription and export complex (hTREX) proteins have been identified in high-grade tumours and these defects contribute to genome instability. We have previously shown that the lytically expressed KSHV ORF57 protein interacts with the complete hTREX complex; therefore, we investigated the possible intriguing link between ORF57, hTREX and KSHV-induced genome instability. Herein, we show that lytically active KSHV infected cells induce a DNA damage response and, importantly, we demonstrate directly that this is due to DNA strand breaks. Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage. Moreover, we describe a novel mechanism showing that the genetic instability observed is a consequence of R-loop formation. Importantly, the link between hTREX sequestration and DNA damage may be a common feature in herpesvirus infection, as a similar phenotype was observed with the herpes simplex virus 1 (HSV-1) ICP27 protein. Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX.

Show MeSH
Related in: MedlinePlus