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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

Inhibition of CHK1 signalling at late times during KSHV lytic replication. Levels of phosphorylated CHK1 (S317 and S345) following UV treatment of TRE-BCBL-1-RTA cells treated with doxycycline for varying lengths of time. At each time point following the addition of doxycycline, cells were treated with 20 Jm−2 UV and levels of phosphorylated CHK1 were assessed by western blot.
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viruses-07-02752-f004: Inhibition of CHK1 signalling at late times during KSHV lytic replication. Levels of phosphorylated CHK1 (S317 and S345) following UV treatment of TRE-BCBL-1-RTA cells treated with doxycycline for varying lengths of time. At each time point following the addition of doxycycline, cells were treated with 20 Jm−2 UV and levels of phosphorylated CHK1 were assessed by western blot.

Mentions: While there is evidence of ATR activation during lytic replication, substantial CHK1 phosphorylation is not observed (Figure 1B). To determine whether ATR-CHK1 signalling is functional during lytic replication, TRE-BCBL-1-RTA cells were exposed to 20 Jm−2 UV at several time points following initiation of lytic replication (Figure 4). Expression of RTA and ORF57 were used to confirm lytic reactivation of KSHV. Exposure to UV at 3 and 6 h post-doxycycline treatment led to phosphorylation of CHK1 at S345 and S317 which was comparable to the un-reactivated control. However, when TRE-BCBL-1-RTA cells were treated with UV at 12, 24 and 48 h following addition of doxycycline, levels of phosphorylated CHK1 were noticeably lower than in the un-reactivated control. This suggests that at later times in the lytic cycle, when phosphorylation of other DDR substrates occurs, activation of CHK1 is specifically inhibited by the virus.


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)

Inhibition of CHK1 signalling at late times during KSHV lytic replication. Levels of phosphorylated CHK1 (S317 and S345) following UV treatment of TRE-BCBL-1-RTA cells treated with doxycycline for varying lengths of time. At each time point following the addition of doxycycline, cells were treated with 20 Jm−2 UV and levels of phosphorylated CHK1 were assessed by western blot.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488719&req=5

viruses-07-02752-f004: Inhibition of CHK1 signalling at late times during KSHV lytic replication. Levels of phosphorylated CHK1 (S317 and S345) following UV treatment of TRE-BCBL-1-RTA cells treated with doxycycline for varying lengths of time. At each time point following the addition of doxycycline, cells were treated with 20 Jm−2 UV and levels of phosphorylated CHK1 were assessed by western blot.
Mentions: While there is evidence of ATR activation during lytic replication, substantial CHK1 phosphorylation is not observed (Figure 1B). To determine whether ATR-CHK1 signalling is functional during lytic replication, TRE-BCBL-1-RTA cells were exposed to 20 Jm−2 UV at several time points following initiation of lytic replication (Figure 4). Expression of RTA and ORF57 were used to confirm lytic reactivation of KSHV. Exposure to UV at 3 and 6 h post-doxycycline treatment led to phosphorylation of CHK1 at S345 and S317 which was comparable to the un-reactivated control. However, when TRE-BCBL-1-RTA cells were treated with UV at 12, 24 and 48 h following addition of doxycycline, levels of phosphorylated CHK1 were noticeably lower than in the un-reactivated control. This suggests that at later times in the lytic cycle, when phosphorylation of other DDR substrates occurs, activation of CHK1 is specifically inhibited by the virus.

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