Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-B at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-B activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts. Author Summary Epstein-Barr virus (EBV) is a human herpesvirus that persistently infects >90% of adults worldwide. One factor underlying the ability of EBV to establish such widespread and lifelong infections is its capacity to escape elimination by the human immune system. Among the first lines of defense against viral infection is the human Toll-like receptor (TLR) system. These receptors can detect the presence of viruses and initiate an intracellular protein signaling cascade that leads to the expression of immune response genes. The activation status of many proteins in this signaling cascade is regulated by the addition of ubiquitin tags. EBV has previously been reported to encode enzymes, called deubiquitinases (DUBs), which are capable of removing such ubiquitin tags from substrate proteins. In our study, we found that one of these enzymes, BPLF1, functions as an active DUB during EBV production in infected cells before being packaged into newly produced viral particles. Furthermore, our study provides insight into the way in which EBV can subvert the human immune response, as we show that BPLF1 can remove ubiquitin tags from proteins in the TLR signaling cascade. This inhibits TLR signaling and decreases the expression of immune response genes.