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Activation of DNA Damage Response Pathways during Lytic Replication of KSHV.

Hollingworth R, Skalka GL, Stewart GS, Hislop AD, Blackbourn DJ, Grand RJ - Viruses (2015)

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies.Inhibition of ATM results in the reduction of overall levels of viral replication while inhibition of DNA-PK increases activation of ATM and leads to earlier viral release.Activation of the DDR following KSHV lytic reactivation does not result in a G1 cell cycle block and cells are able to proceed to S-phase during the lytic cycle.

View Article: PubMed Central - PubMed

Affiliation: School of Cancer Sciences, the College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK. rxh291@student.bham.ac.uk.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies. Human tumour viruses such as KSHV are known to interact with the DNA damage response (DDR), the molecular pathways that recognise and repair lesions in cellular DNA. Here it is demonstrated that lytic reactivation of KSHV leads to activation of the ATM and DNA-PK DDR kinases resulting in phosphorylation of multiple downstream substrates. Inhibition of ATM results in the reduction of overall levels of viral replication while inhibition of DNA-PK increases activation of ATM and leads to earlier viral release. There is no activation of the ATR-CHK1 pathway following lytic replication and CHK1 phosphorylation is inhibited at later times during the lytic cycle. Despite evidence of double-strand breaks and phosphorylation of H2AX, 53BP1 foci are not consistently observed in cells containing lytic virus although RPA32 and MRE11 localise to sites of viral DNA synthesis. Activation of the DDR following KSHV lytic reactivation does not result in a G1 cell cycle block and cells are able to proceed to S-phase during the lytic cycle. KSHV appears then to selectively activate DDR pathways, modulate cell cycle progression and recruit DDR proteins to sites of viral replication during the lytic cycle.

No MeSH data available.


Related in: MedlinePlus

Effect of DDR inhibition on production of infectious virus. (A) Expression of late lytic glycoprotein K8.1A in TRE-BCBL-1-RTA cells 24 and 48 h following treatment with DDR kinase inhibitors and doxycycline; (B) Expression of LANA in EA.hy926 cells following infection with supernatants collected from TRE-BCBL-1-RTA cells 24 and 48 h after treatment with DDR kinase inhibitors and doxycycline; (C) Immunofluorescence microscopy analysis of the percentage of EA.hy926 cells expressing LANA following infection with supernatants collected from TRE-BCBL-1-RTA cells 24 and 48 h after treatment with DDR kinase inhibitors and doxycycline. Each column represents the mean of three independent experiments while the error bars represent the standard error of the mean (SEM). A minimum of 500 cells were analysed for each repetition. * p < 0.05; ** p < 0.01; *** p < 0.001 (statistical analyses were performed using a two-tailed and unpaired Student’s t-test); (D) Percentage of TRE-BCBL-1-RTA cells expressing early lytic protein ORF59 24 h following treatment with DDR kinase inhibitors and doxycycline. Each column represents the mean of three independent experiments while the error bars represent the standard error of the mean (SEM). A minimum of 500 cells were analysed for each repetition; (E) Levels of phosphorylated DDR proteins in TRE-BCBL-1-RTA cells 24 h following treatment with DDR kinase inhibitors and doxycycline.
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viruses-07-02752-f003: Effect of DDR inhibition on production of infectious virus. (A) Expression of late lytic glycoprotein K8.1A in TRE-BCBL-1-RTA cells 24 and 48 h following treatment with DDR kinase inhibitors and doxycycline; (B) Expression of LANA in EA.hy926 cells following infection with supernatants collected from TRE-BCBL-1-RTA cells 24 and 48 h after treatment with DDR kinase inhibitors and doxycycline; (C) Immunofluorescence microscopy analysis of the percentage of EA.hy926 cells expressing LANA following infection with supernatants collected from TRE-BCBL-1-RTA cells 24 and 48 h after treatment with DDR kinase inhibitors and doxycycline. Each column represents the mean of three independent experiments while the error bars represent the standard error of the mean (SEM). A minimum of 500 cells were analysed for each repetition. * p < 0.05; ** p < 0.01; *** p < 0.001 (statistical analyses were performed using a two-tailed and unpaired Student’s t-test); (D) Percentage of TRE-BCBL-1-RTA cells expressing early lytic protein ORF59 24 h following treatment with DDR kinase inhibitors and doxycycline. Each column represents the mean of three independent experiments while the error bars represent the standard error of the mean (SEM). A minimum of 500 cells were analysed for each repetition; (E) Levels of phosphorylated DDR proteins in TRE-BCBL-1-RTA cells 24 h following treatment with DDR kinase inhibitors and doxycycline.

Mentions: To determine what effect inhibition of DDR kinases has on production of infectious virus, TRE-BCBL-1-RTA cells were treated with two concentrations of ATR, ATM and DNA-PK inhibitors or an equivalent concentration of DMSO 1 h prior to the addition of 0.5 μg/mL doxycycline. After 24 and 48 h, expression of late lytic glycoprotein K8.1A was initially assessed as an indicator of completed viral replication (Figure 3A). At 24 h, expression of K8.1A was relatively low compared to 48 h but was clearly elevated following inhibition of DNA-PK. After 48 h, expression was similar between the DMSO-treated and DNA-PK inhibitor-treated cells but clearly reduced in the cells treated with the higher concentrations of ATR and ATM inhibitors.


Activation of DNA Damage Response Pathways during Lytic Replication of KSHV.

Hollingworth R, Skalka GL, Stewart GS, Hislop AD, Blackbourn DJ, Grand RJ - Viruses (2015)

Effect of DDR inhibition on production of infectious virus. (A) Expression of late lytic glycoprotein K8.1A in TRE-BCBL-1-RTA cells 24 and 48 h following treatment with DDR kinase inhibitors and doxycycline; (B) Expression of LANA in EA.hy926 cells following infection with supernatants collected from TRE-BCBL-1-RTA cells 24 and 48 h after treatment with DDR kinase inhibitors and doxycycline; (C) Immunofluorescence microscopy analysis of the percentage of EA.hy926 cells expressing LANA following infection with supernatants collected from TRE-BCBL-1-RTA cells 24 and 48 h after treatment with DDR kinase inhibitors and doxycycline. Each column represents the mean of three independent experiments while the error bars represent the standard error of the mean (SEM). A minimum of 500 cells were analysed for each repetition. * p < 0.05; ** p < 0.01; *** p < 0.001 (statistical analyses were performed using a two-tailed and unpaired Student’s t-test); (D) Percentage of TRE-BCBL-1-RTA cells expressing early lytic protein ORF59 24 h following treatment with DDR kinase inhibitors and doxycycline. Each column represents the mean of three independent experiments while the error bars represent the standard error of the mean (SEM). A minimum of 500 cells were analysed for each repetition; (E) Levels of phosphorylated DDR proteins in TRE-BCBL-1-RTA cells 24 h following treatment with DDR kinase inhibitors and doxycycline.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4488719&req=5

viruses-07-02752-f003: Effect of DDR inhibition on production of infectious virus. (A) Expression of late lytic glycoprotein K8.1A in TRE-BCBL-1-RTA cells 24 and 48 h following treatment with DDR kinase inhibitors and doxycycline; (B) Expression of LANA in EA.hy926 cells following infection with supernatants collected from TRE-BCBL-1-RTA cells 24 and 48 h after treatment with DDR kinase inhibitors and doxycycline; (C) Immunofluorescence microscopy analysis of the percentage of EA.hy926 cells expressing LANA following infection with supernatants collected from TRE-BCBL-1-RTA cells 24 and 48 h after treatment with DDR kinase inhibitors and doxycycline. Each column represents the mean of three independent experiments while the error bars represent the standard error of the mean (SEM). A minimum of 500 cells were analysed for each repetition. * p < 0.05; ** p < 0.01; *** p < 0.001 (statistical analyses were performed using a two-tailed and unpaired Student’s t-test); (D) Percentage of TRE-BCBL-1-RTA cells expressing early lytic protein ORF59 24 h following treatment with DDR kinase inhibitors and doxycycline. Each column represents the mean of three independent experiments while the error bars represent the standard error of the mean (SEM). A minimum of 500 cells were analysed for each repetition; (E) Levels of phosphorylated DDR proteins in TRE-BCBL-1-RTA cells 24 h following treatment with DDR kinase inhibitors and doxycycline.
Mentions: To determine what effect inhibition of DDR kinases has on production of infectious virus, TRE-BCBL-1-RTA cells were treated with two concentrations of ATR, ATM and DNA-PK inhibitors or an equivalent concentration of DMSO 1 h prior to the addition of 0.5 μg/mL doxycycline. After 24 and 48 h, expression of late lytic glycoprotein K8.1A was initially assessed as an indicator of completed viral replication (Figure 3A). At 24 h, expression of K8.1A was relatively low compared to 48 h but was clearly elevated following inhibition of DNA-PK. After 48 h, expression was similar between the DMSO-treated and DNA-PK inhibitor-treated cells but clearly reduced in the cells treated with the higher concentrations of ATR and ATM inhibitors.

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies.Inhibition of ATM results in the reduction of overall levels of viral replication while inhibition of DNA-PK increases activation of ATM and leads to earlier viral release.Activation of the DDR following KSHV lytic reactivation does not result in a G1 cell cycle block and cells are able to proceed to S-phase during the lytic cycle.

View Article: PubMed Central - PubMed

Affiliation: School of Cancer Sciences, the College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK. rxh291@student.bham.ac.uk.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies. Human tumour viruses such as KSHV are known to interact with the DNA damage response (DDR), the molecular pathways that recognise and repair lesions in cellular DNA. Here it is demonstrated that lytic reactivation of KSHV leads to activation of the ATM and DNA-PK DDR kinases resulting in phosphorylation of multiple downstream substrates. Inhibition of ATM results in the reduction of overall levels of viral replication while inhibition of DNA-PK increases activation of ATM and leads to earlier viral release. There is no activation of the ATR-CHK1 pathway following lytic replication and CHK1 phosphorylation is inhibited at later times during the lytic cycle. Despite evidence of double-strand breaks and phosphorylation of H2AX, 53BP1 foci are not consistently observed in cells containing lytic virus although RPA32 and MRE11 localise to sites of viral DNA synthesis. Activation of the DDR following KSHV lytic reactivation does not result in a G1 cell cycle block and cells are able to proceed to S-phase during the lytic cycle. KSHV appears then to selectively activate DDR pathways, modulate cell cycle progression and recruit DDR proteins to sites of viral replication during the lytic cycle.

No MeSH data available.


Related in: MedlinePlus