Kaposi's Sarcoma-Associated Herpesvirus Induces the ATM and H2AX DNA Damage Response Early during De Novo Infection of Primary Endothelial Cells, Which Play Roles in Latency Establishment

Abstract

The DNA damage response (DDR) that evolved to repair host cell DNA damage also recognizes viral DNA entering the nucleus during infections. Here, we investigated the modulation of DDR signaling during de novo infection of primary endothelial cells by Kaposi's sarcoma-associated herpesvirus (KSHV). Phosphorylation of representative DDR-associated proteins, such as ataxia telangiectasia mutated (ATM) and H2AX, was induced as early as 30 min (0.5 h) postinfection and persisted during in vitro KSHV latency. Phosphorylated H2AX (␥H2AX) colocalized at 30 min (0.5 h) with the KSHV genome entering the nuclei. Total H2AX protein levels also increased, and the increase was attributed to a decrease in degradative H2AX Lys48-linked polyubiquitination with a concomitant increase in Lys63-linked polyubiquitination that was shown to increase protein stability. ATM and H2AX phosphorylation and ␥H2AX nuclear foci were also induced by UV-inactivated KSHV, which ceased at later times of infection. Inhibition of ATM kinase activity by KU-55933 and H2AX knockdown by small interfering RNA significantly reduced the expression of the KSHV latency-associated nuclear antigen 1 (LANA-1; ORF73) and LANA-1 nuclear puncta. Knockdown of H2AX also resulted in a >80% reduction in the nuclear KSHV DNA copy numbers. Similar results were also observed in ATMnegative cells, although comparable levels of viral DNA entered ATM-negative and ATM-positive cell nuclei. In contrast, knockdown of CHK1 and CHK2 did not affect ORF73 expression. Collectively, these results demonstrate that KSHV induces ATM and H2AX, a selective arm of the DDR, for the establishment and maintenance of its latency during de novo infection of primary endothelial cells. IMPORTANCEEukaryotic cells mount a DNA damage response (DDR) to sense and repair different types of cellular DNA damage. In addition, DDR also recognizes exogenous genetic material, such as the viral DNA genome entering the nucleus during infections. The present study was undertaken to determine whether de novo Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates DDR. Our results demonstrate that early during de novo infection of primary endothelial cells, KSHV induces a selective arm of DDR signaling, such as the ATM kinase and its downstream target, H2AX, which are essential for KSHV's latent gene expression and the establishment of latency. These studies suggest that targeting ATM and H2AX could serve as an attractive strategy to block the establishment of KSHV latent infection and the associated malignancies.

Murine Gammaherpesvirus 68 LANA and SOX Homologs Counteract ATM-Driven p53 Activity during Lytic Viral Replication

“…MHV68 induces ATM and H2AX phosphorylation, and infected cells exhibit additional DDR-related phosphorylation events during lytic replication in fibroblasts (20,22,(43)(44)(45). However, the kinetics of the MHV68-associated DDR and whether it is associated with p53 activation are not well defined.…”

“…ATM inhibition also does not block ORF59 expression during induced KSHV reactivation (63). Given differential requirements for ATM in GHV replication (20,24,44,63,(67)(68)(69), it is reasonable to suggest that ATM is situationally required for viral replication, perhaps in terminally differentiated cell types. However, since p53 was stabilized and transcriptionally active early during infection, these findings do indicate that viral induction of a DDR can have consequences on the infected cell, especially activation of downstream effectors such as p53.…”

Targeting HGF/c-MET induces cell cycle arrest, DNA damage, and apoptosis for primary effusion lymphoma

“…28 In fact, it has been reported recently that KSHV can activate the DNA damage response during de novo infection of primary endothelial cells and this plays a role in establishing latency. 51 More recently, it has been demonstrated that lytic reactivation of KSHV leads to activation of the ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase DNA damage response kinases. 52 Inhibition of ATM results in the reduction of overall levels of viral replication whereas inhibition of DNA-dependent protein kinase increases activation of ATM and leads to earlier viral release.…”

B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection

“…Similar to other DNA viruses, both human (Epstein-Barr virus [EBV] and Kaposi's sarcoma-associated herpesvirus [KSHV]), and rodent (mouse gammaherpesvirus 68 [MHV68]) gammaherpesviruses benefit from ATM in vitro. Specifically, ATM activity and expression facilitate EBV reactivation from transformed cell lines (8) and the establishment of KSHV latency in primary endothelial cell cultures (9). Further, ATM expression enhances MHV68 replication in primary macrophages, but not mouse embryonic fibroblasts (MEF), in vitro (10), indicating that ATM functions as a proviral host factor in the context of many infected cell types.…”