Localization of Double-Strand Break Repair Proteins to Viral Replication Compartments following Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

Abstract

Double-strand breaks (DSBs) in DNA are recognized by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases, which play key roles in regulating the cellular DNA damage response (DDR). DNA tumor 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 colocalize with viral genomes in replication compartments (RCs), whereas other DSB repair proteins form foci outside RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impact viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in nonhomologous end joining (NHEJ) repair, enhance 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 signaling 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 provide a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localization of DSB repair proteins that would otherwise have a detrimental effect on viral replication.IMPORTANCE Tumor 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 localization 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 nonhomologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall, this study extends our understanding of the virus-host interactions that occur during lytic replication of KSHV and provides a deeper insight into how the DDR is manipulated during viral infection.

Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication Is Independent of Anaphase-Promoting Complex Activity

“…The lytic cells, identified by high ORF50 expression, showed also higher levels of phosphorylated H2AX at S139 (pH2AX-S139) indicating that DDR was stronger in the lytic cells ( Fig. 2 A-B) as previously shown (39)(40)(41).…”

“…This, in turn, allows APC/C-CDH1 to trigger the degradation of important cellular regulators that promote S-phase transition and thereby facilitating the cell cycle block(47). KSHV lytic replication following virus reactivation and during the lytic burst that normally occurs upon primary infection lead to DDR(39)(40)(41)48). In LECs de novo infected with KSHV-Lyt, the mRNA and protein levels of EMI1 and CCNB1 were initially downregulated but increased at later timepoints (48 and 72 h p.i) when compared to uninfected cells.…”

Productive herpesvirus lytic replication in primary effusion lymphoma cells requires S-phase entry

“…We, and others, have previously reported that KSHV lytic replication results in cellular DNA damage and concurrent activation of the DNA damage response (DDR) [8][9][10][11]. As part of our previous report into the effect of DDR kinase inhibitors on KSHV replication efficiency, we observed, through examining relative DNA content, that inducing KSHV lytic replication in a PEL line increased the proportion of these cells in S phase [10].…”

RNA decay during gammaherpesvirus infection reduces RNA polymerase II occupancy of host promoters but spares viral promoters

“…Notably, during latency key lytic promoters like RTA contain both the activating H3K4me3 and the repressive H3K27me3 histone marks, representing a 'poised' state [53]. At later stages of infection, KSHV, EBV and HSV-1 genomes have been shown to be largely nucleosome free, consistent with histones not being packaged in virions [33,35,54,55]. At later stages of infection, KSHV, EBV and HSV-1 genomes have been shown to be largely nucleosome free, consistent with histones not being packaged in virions [33,35,54,55].…”