High-throughput technology is emerging as a powerful tool for discovery of factors involved in pathogen-host interactions (Brass et al., 2009
; Calderwood et al., 2007
; Karlas et al., 2010
; Konig et al., 2010
; Shapira et al., 2009
). Here, we took a protein microarray approach to identify enzyme-substrate interactions for four conserved human herpesvirus kinases, with the hypothesis that the common substrates would reveal host pathways that are critical for replication across the herpesvirus family. By analyzing over 100 shared host substrates we identified the DDR pathway as a central target of the conserved herpesvirus kinases. Mechanistic studies showed that in the absence of external DNA damage cues, the EBV kinase phosphorylated and activated an upstream master regulator of the DDR, the histone acetyltransferase TIP60. TIP60 was additionally integrated to the virus lytic program by recruitment to the viral chromatin where TIP60 activated specific EBV genes critical for viral replication.
TIP60 was originally identified as a partner of the human immunodeficiency virus type 1 (HIV-1) transactivator Tat (Kamine et al., 1996
) and is targeted by several viruses. Human T cell lymphotropic virus type 1 (HTLV-1) p30II enhances Myc transforming activity through stabilizing Myc-TIP60 transcriptional interactions (Awasthi et al., 2005
). TIP60 interaction with viral TAT, E6 and UL27 proteins encoded by HIV-1, human papillomavirus (HPV) and HCMV, respectively, induces TIP60 degradation (Col et al., 2005
; Jha et al., 2010
; Reitsma et al., 2011
), which is believed to enable establishment of viral latency and enhance virus-induced oncogenesis. In the case of HCMV, viruses deleted or mutated for the UL97 protein kinase escape through secondary mutations in the UL27 protein that degrades TIP60 (Chou, 2009
; Reitsma et al., 2011
). A recent study by Nikitin et al. found that the DDR induced upon EBV infection is a robust host anti-viral defense and EBV employs counter-measures to overcome the growth inhibitory effects of the host DDR in order to establish latency (Nikitin et al., 2010
). These authors found that treatment of B cells with an ATM inhibitor increased latency establishment. We find that TIP60 inhibition with shRNA also increases latency establishment implying that TIP60 is an upstream mediator of the DNA damage response induced upon EBV infection. Interestingly, BGLF4 is present in the EBV tegument (Asai et al., 2006
) and is consequently introduced into cells upon EBV infection. BGLF4 would therefore be available to initiate a transient activation of TIP60 and the DDR and the BGLF4/TIP60 partnership may therefore be an important factor in inducing a cellular environment that is hostile to latency establishment.
In counterpoint, we demonstrate that TIP60 plays a positive role in the lytic replication of herpesviruses: TIP60 shRNA significantly reduces virus production from β and γ herpesvirus infected cells. In the case of EBV, TIP60 HAT activity is enhanced via phosphorylation by the EBV-encoded protein kinase BGLF4 at the same sites phosphorylated by CDC2 and GSK3β (Charvet et al., 2011
; Lemercier et al., 2003
) and this interaction is sufficient to trigger the DDR. The DDR plays an important role in the lytic viral life cycle. EBV lytic replication elicits a DDR by triggering ATM autophosphorylation and cativation. Activated ATM phosphorylates its downstream targets, such as H2AX, p53, CHK2 and RPA2, and phosphorylated ATM, RPA2 and Mre11/Rad50/Nbs1 (MRN) complexes are recruited to replication compartments in nuclei during EBV lytic replication (Kudoh et al., 2005
; Kudoh et al., 2009
). However, the mechanism of virus-triggered ATM activation has been elusive. Although γ-HV68 kinase orf36 and EBV BGLF4 have been found to directly phosphorylate H2AX, this phosphorylation was significantly reduced in ATM deficient cells (Tarakanova et al., 2007
) and, as shown in Figure 4B in the present study, in cells treated with an ATM inhibitor. As summarized in , our experiments mechanistically link the viral kinases to ATM and its downstream targets CHK2 and H2AX via TIP60.
Model for conserved herpesvirus kinases in regulating vrial replication through TIP60
We also demonstrate that TIP60 plays a positive role in transcriptional regulation of key lytic viral genes (). BGLF4 has been implicated in facilitating viral egress from the nucleus by phosphorylating lamins (Lee et al., 2008
). Interestingly, we find that TIP60 is recruited to the LMP1 promoters after lytic induction and is needed for achieving normal levels of lytic LMP1 transcription. LMP1 downstream signalling is important for nuclear egress of virions (Ahsan et al., 2005
) and our data suggest that TIP60 mediated activation of LMP1 expression represents another mechanism by which BGLF4 promotes this aspect of infectious EBV production. TIP60's negative role in establishment of latency and positive role in lytic viral replication place TIP60 at the decision point between viral latency establishment and productive lytic replication ( & ).
This work illustrates the value of high throughput, unbiased approaches for the discovery of conserved viral targets. There are few drugs available to treat herpesvirus infections and viral escape mutants develop upon extensive use of this limited repertoire. The herpesvirus protein kinases are attractive anti-viral drug targets. However, developing broadly effectively drugs requires knowledge of their common cellular substrates. The information provided by our common substrate identification will assist in the design of assays for new broadly effective anti-herpesvirus therapeutics.