Fig 2: Contribution of the ATM kinase to efficient KSHV replication. (A) TREx BCBL-1-RTA cells were treated with either DMSO or 5 µM KU-60019 ATM kinase inhibitor prior to induction of lytic replication. The relative viral genome load was then measured after 48 h by qPCR, while Western blots were used to monitor levels of total and phosphorylated CHK2. Expression of viral K-bZIP was used to confirm lytic replication of KSHV. (B and C) Control and AT fibroblasts were de novo infected with KSHV on glass coverslips and treated with sodium butyrate for 72 h. (B) IF analysis was then carried out for expression of SSB and K8.1 lytic proteins. (C) The numbers of control and AT fibroblasts containing viral RCs were then counted across a coverslip in five repeat experiments. To assess release of infectious virus, supernatants from each cell line were then used to infect EA.hy926 cells, and the number of infected cells was determined after 48 h by IF analysis of LANA expression. All data bars represent the mean from five independent experiments, while error bars signify the SEM. *, P < 0.05 (statistical analyses were performed using a two-tailed and unpaired Student t test) (D) Vero and VK219-RTA cells were treated with doxycycline and sodium butyrate for the indicated times, and Western blotting was used to assess the levels of total and phosphorylated ATM, CHK2, and H2AX. VK219-RTA cells treated with either IR or UV were included as positive controls for DDR activation, while blotting for the lytic proteins SSB and K8.1A confirmed viral reactivation in VK219-RTA cells. (E) TREx BCBL-1-RTA cells were treated with doxycycline, while Vero cells were de novo infected with KSHV and treated with sodium butyrate. After 48 h, IF analysis was carried out for nuclear localization of Ku80 and MRE11 in relation to viral RCs marked by the viral SSB protein. Scale bars represent 5 µm.

Fig 3: Effect of depletion of Ku80 and inhibition of DNA-PK activity on viral replication. (A) TREx BCBL-1-RTA cells were treated with either DMSO or 1 µM NU7441 DNA-PK inhibitor prior to induction of lytic replication. The relative viral genome load was then measured after 48 h by qPCR, while Western blots were used to monitor levels of total and phosphorylated RPA32. Expression of viral K-bZIP was used to confirm lytic replication of KSHV. (B) VK219-RTA cells were treated with siRNA against Ku80 or control siRNA, and Western blotting was used to monitor levels of Ku80 and Ku70 after 72 h. Following induction of lytic replication, the relative viral genome load was measured after 72 h by qPCR, while supernatants were then used to infect HEK293 cells, and the percentage of cells expressing GFP was determined after 48 h using flow cytometry. (C) VK219-RTA cells were treated with either DMSO or 1 µM NU7441 DNA-PK inhibitor prior to induction of lytic replication. After 72 h, qPCR was used to assess the relative genome load, while supernatants were then used to infect HEK293 cells, and the percentage of cells expressing GFP was determined after 48 h using flow cytometry. All data bars represent the mean from three independent experiments, while error bars signify the SEM. *, P < 0.05; **, P < 0.01 (statistical analyses were performed using a two-tailed and unpaired Student t test).

Fig 4: Localization of DSB-sensing complexes during lytic replication of KSHV. EA.hy926-RTA cells containing latent KSHV were treated with doxycycline (Dox) for 48 h or left untreated prior to IF analysis of nuclear localization of MRE11, NBS1, RAD50, Ku70, and Ku80 in relation to KSHV RCs marked by the viral SSB protein. Scale bars represent 5 µm.