3 years ago

Localisation of double-strand break repair proteins to viral replication compartments following lytic reactivation of KSHV.

Grand RJ, Hollingworth R, Horniblow RD, Stewart GS, Forrest C
Double-strand breaks (DSBs) in DNA are recognised by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases that play key roles in regulating the cellular DNA damage response (DDR). DNA tumour viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently co-localise with viral genomes in replication compartments (RCs) whereas other DSB repair proteins form foci outside of RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impacts on viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in non-homologous end joining repair (NHEJ), enhances amplification of viral DNA. Although the recruitment of DSB sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signalling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA packaging proteins provides a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localisation of DSB repair proteins that would otherwise have a detrimental effect on viral replication.Importance Tumour viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localisation of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in non-homologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall this study extends our understanding of the viral-host interactions that occur during lytic replication of KSHV and provides a deeper insight into the how the DDR is manipulated during viral infection.

Publisher URL: https://www.ncbi.nlm.nih.gov/pubmed/28855246

DOI: PubMed:28855246

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