Epstein-Barr Virus Viral Processivity Factor EA-D Facilitates Virus Lytic Replication by Inducing Poly(ADP-Ribose) Polymerase 1 Degradation

Abstract

PARP1 acts as a negative regulator of lytic replication in EBV. To successfully enter the reactivation cycle, EBV has developed multiple strategies to counteract the host's repressive mechanisms. Gammaherpesviruses, including Epstein-Barr virus (EBV), are important human pathogens because they are associated with various tumors. Poly(ADP-ribose) polymerase 1 (PARP1) is a multifunctional host nuclear protein responsible for poly(ADP-ribosyl)ation (PARylation) of target proteins. While PARP1 acts as a negative regulator that suppresses the lytic replication of gammaherpesviruses, viruses are often equipped with various strategies to overcome PARP1 inhibition. However, the mechanisms of how EBV may modulate a repressive host protein, PARP1, are still elusive. In this study, we found that EBV reactivation induced PARP1 downregulation in EBV-infected cells. EBV DNA polymerase processivity factor EA-D, encoded by the BMRF1 gene, directly interacted with the central automodification domain (AD) of PARP1 and was necessary and sufficient to downregulate PARP1 via K29-linked polyubiquitination. Moreover, knockdown of EA-D in B95.8 cells restored PARP1 levels and abrogated the expression of ZTA (also known as ZEBRA), a switch molecule of the EBV life cycle during reactivation. Interestingly, PARP1 PARylated RTA, another key switch molecule, and decreased RTA transactivation on the promoters of the ZTA, BMRF1, and BMLF1 genes. EA-D alleviated the PARylation of RTA and further enhanced RTA-mediated transactivation of these lytic promoters in reporter assays. Taken together, our results suggest that EBV viral processivity factor plays a key role in facilitating lytic replication by inducing PARP1 degradation via its interaction with the PARP1 AD, which is a highly conserved mechanism among gammaherpesviruses to counteract host repressive activity of PARP1 against viral lytic replication. IMPORTANCE PARP1 acts as a negative regulator of lytic replication in EBV. To successfully enter the reactivation cycle, EBV has developed multiple strategies to counteract the host's repressive mechanisms. In this study, we investigated how EBV manipulated the host repressive factor PARP1 to facilitate lytic replication. The EBV processivity factor EA-D downregulated PARP1 in a proteasome-dependent manner via its direct binding with PARP1 AD. The knockdown of EA-D restored the PARP1 level and inhibited ZTA expression during reactivation. Interestingly, PARP1 PARylated RTA and EA-D reduced the PARylation of RTA, thereby promoting the ZTA promoter activity. These results suggest that EA-D plays a key role in EBV lytic replication by inducing PARP1 degradation in addition to supporting DNA replication as a viral processivity factor. Given that the KSHV processivity factor also induces PARP1 degradation and enhances RTA function, gammaherpesviruses share a conserved molecular mechanism to overcome the inhibitory effects of PARP1, promoting lytic replication.