N6-methyladenosine Modification of Hepatitis B Virus RNA in the Coding Region of HBx.

N6-methyladenosine (m6A) is a post-transcriptional modification of RNA involved in transcript transport, degradation, translation, and splicing. We found that HBV RNA is modified by m6A predominantly in the coding region of HBx. The mutagenesis of methylation sites reduced the HBV mRNA and HBs protein levels. The suppression of m6A by an inhibitor or knockdown in primary hepatocytes decreased the viral RNA and HBs protein levels in the medium. These results suggest that the m6A modification of HBV RNA is needed for the efficient replication of HBV in hepatocytes.

Serine 13 of the human cytomegalovirus viral cyclin-dependent kinase UL97 is required for regulatory protein 14-3-3 binding and UL97 stability.

The human cytomegalovirus (HCMV) UL97 protein is a conserved herpesvirus protein kinase (CHPK) and a viral cyclin-dependent kinase (v-CDK). However, mechanisms regulating its activity in the context of infection are unknown. Here, we identified several cellular regulatory 14-3-3 proteins as UL97-interacting partners that promote UL97 stability. Humans are known to encode seven isoforms of 14-3-3 proteins (ß, ε, η, γ, σ, θ, and ζ) that bind phosphoserines or phosphothreonines to impact protein structure, stability, activity, and localization. Our proteomic analysis of UL97 identified 49 interacting partners, including 14-3-3 isoforms ß, η, and γ. Furthermore, coimmunoprecipitation with Western blotting assays demonstrated that UL97 interaction with 14-3-3 isoforms ß, ε, η, γ, and θ occurs in a kinase activity-dependent manner. Using mutational analysis, we determined the serine residue at amino acid 13 of UL97 is crucial for 14-3-3 interaction. We demonstrate UL97 S13A (serine to alanine substitution at residue 13) retains kinase activity but the mutant protein accumulated at lower levels than WT UL97. Finally, we show both laboratory (AD169) and clinical (TB40/E) strains of HCMV encoding UL97 S13A replicated with WT kinetics in fibroblasts but showed decreased UL97 accumulation. Taken together, we conclude that 14-3-3 proteins interact with and stabilize UL97 during HCMV infection.

Ubiquitination of SARS-CoV-2 NSP6 and ORF7a Facilitates NF-κB Activation.

Patients with severe coronavirus disease 2019 tend to have high levels of proinflammatory cytokines, which eventually lead to cytokine storm and the development of acute respiratory distress syndrome. However, the detailed molecular mechanisms of proinflammatory cytokine production remain unknown. Here, we screened severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genes and found that nonstructural protein 6 (NSP6) and open reading frame 7a (ORF7a) activated the NF-κB pathway. NSP6 and ORF7a interacted with transforming growth factor ß-activated kinase 1 (TAK1), and knockout (KO) of TAK1 or NF-κB essential modulator (NEMO) abolished NF-κB activation by NSP6 and ORF7a. Interestingly, K61 of NSP6 was conjugated to K63-linked polyubiquitin chains by the E3 ubiquitin ligase tripartite motif-containing 13, and this polyubiquitination of NSP6 appeared crucial for recruitment of NEMO to the NSP6-TAK1 complex and NF-κB activation. On the other hand, ring finger protein 121 (RNF121) was required for the polyubiquitination of ORF7a. Knockdown of RNF121 significantly decreased ORF7a binding of TAK1 and NEMO, resulting in the suppression of NF-κB activation. Taken together, our results provide novel molecular insights into the pathogenesis of SARS-CoV-2 and the host immune response to SARS-CoV-2 infection. IMPORTANCE The detailed molecular basis of the induction of proinflammatory cytokines and chemokines by SARS-CoV-2 is unclear, although such induction is clearly related to the severity of COVID-19. Here, we show that SARS-CoV-2 NSP6 and ORF7a lead to NF-κB activation through associations with TAK1. K63-linked polyubiquitination of NSP6 and ORF7a by TRIM13 and RNF121, respectively, appears essential for NF-κB activation. These results suggest that inhibition of the NSP6 and ORF7a gene products may reduce the severity of COVID-19 symptoms by decreasing proinflammatory cytokine levels.

Reduction of severe acute respiratory syndrome coronavirus-2 infectivity by admissible concentration of ozone gas and water.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Because complete elimination of SARS-CoV-2 appears difficult, decreasing the risk of transmission is important. Treatment with 0.1 and 0.05 ppm ozone gas for 10 and 20 hr, respectively, decreased SARS-CoV-2 infectivity by about 95%. The magnitude of the effect was dependent on humidity. Treatment with 1 and 2 mg/L ozone water for 10 s reduced SARS-CoV-2 infectivity by about 2 and 3 logs, respectively. Our results suggest that low-dose ozone, in the form of gas and water, is effective against SARS-CoV-2.

Direct Substrate Identification with an Analog Sensitive (AS) Viral Cyclin-Dependent Kinase (v-Cdk).

Viral cyclin-dependent kinases (v-Cdks) functionally emulate their cellular Cdk counterparts. Such viral mimicry is an established phenomenon that we extend here through chemical genetics. Kinases contain gatekeeper residues that limit the size of molecules that can be accommodated within the enzyme active site. Mutating gatekeeper residues to smaller amino acids allows larger molecules access to the active site. Such mutants can utilize bio-orthoganol ATPs for phosphate transfer and are inhibited by compounds ineffective against the wild type protein, and thus are referred to as analog-sensitive (AS) kinases. We identified the gatekeeper residues of the v-Cdks encoded by Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) and mutated them to generate AS kinases. The AS-v-Cdks are functional and utilize different ATP derivatives with a specificity closely matching their cellular ortholog, AS-Cdk2. The AS derivative of the EBV v-Cdk was used to transfer a thiolated phosphate group to targeted proteins which were then purified through covalent capture and identified by mass spectrometry. Pathway analysis of these newly identified direct substrates of the EBV v-Cdk extends the potential influence of this kinase into all stages of gene expression (transcription, splicing, mRNA export, and translation). Our work demonstrates the biochemical similarity of the cellular and viral Cdks, as well as the utility of AS v-Cdks for substrate identification to increase our understanding of both viral infections and Cdk biology.

Phosphorylation of transcriptional regulators in the retinoblastoma protein pathway by UL97, the viral cyclin-dependent kinase encoded by human cytomegalovirus.

Human cytomegalovirus (HCMV) encodes a viral cyclin-dependent kinase (v-CDK), the UL97 protein. UL97 phosphorylates Rb, p107 and p130, thereby inactivating all three retinoblastoma (Rb) family members. Rb proteins function through regulating the activity of transcription factors to which they bind. Therefore, we examined whether the UL97-mediated regulation of the Rb tumor suppressors also extended to their binding partners. We observed that UL97 phosphorylates LIN52, a component of p107- and p130-assembled transcriptionally repressive DREAM complexes that control transcription during the G0/G1 phases, and the Rb-associated E2F3 protein that activates transcription through G1 and S phases. Intriguingly, we also identified FoxM1B, a transcriptional regulator during the S and G2 phases, as a UL97 substrate. This survey extends the influence of UL97 beyond simply the Rb proteins themselves to their binding partners, as well as past the G1/S transition into later stages of the cell cycle.

Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins.

The human cytomegalovirus (HCMV)-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates the retinoblastoma (Rb) tumor suppressor. Here, we identify the other Rb family members p107 and p130 as novel targets of UL97. UL97 phosphorylates p107 and p130 thereby inhibiting their ability to repress the E2F-responsive E2F1 promoter. As with Rb, this phosphorylation, and the rescue of E2F-responsive transcription, is dependent on the L1 LXCXE motif in UL97 and its interacting clefts on p107 and p130. Interestingly, UL97 does not induce the disruption of all p107-E2F or p130-E2F complexes, as it does to Rb-E2F complexes. UL97 strongly interacts with p107 but not Rb or p130. Thus the inhibitory mechanisms of UL97 for Rb family protein-mediated repression of E2F-responsive transcription appear to differ for each of the Rb family proteins. The immediate early 1 (IE1) protein of HCMV also rescues p107- and p130-mediated repression of E2F-responsive gene expression, but it does not induce their phosphorylation and does not disrupt p107-E2F or p130-E2F complexes. The unique regulation of Rb family proteins by HCMV UL97 and IE1 attests to the importance of modulating Rb family protein function in HCMV-infected cells.

TRF2 recruits ORC through TRFH domain dimerization.

Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain.

Molecular Determinants for the Inactivation of the Retinoblastoma Tumor Suppressor by the Viral Cyclin-dependent Kinase UL97.

The retinoblastoma (Rb) tumor suppressor restricts cell cycle progression by repressing E2F-responsive transcription. Cellular cyclin-dependent kinase (CDK)-mediated Rb inactivation through phosphorylation disrupts Rb-E2F complexes, stimulating transcription. The human cytomegalovirus (HCMV) UL97 protein is a viral CDK (v-CDK) that phosphorylates Rb. Here we show that UL97 phosphorylates 11 of the 16 consensus CDK sites in Rb. A cleft within Rb accommodates peptides with the amino acid sequence LXCXE. UL97 contains three such motifs. We determined that the first LXCXE motif (L1) of UL97 and the Rb cleft enhance UL97-mediated Rb phosphorylation. A UL97 mutant with a non-functional L1 motif (UL97-L1m) displayed significantly reduced Rb phosphorylation at multiple sites. Curiously, however, it efficiently disrupted Rb-E2F complexes but failed to relieve Rb-mediated repression of E2F reporter constructs. The HCMV immediate early 1 protein cooperated with UL97-L1m to inactivate Rb in transfection assays, likely indicating that cells infected with a UL97-L1m mutant virus show no defects in growth or E2F-responsive gene expression because of redundant viral mechanisms to inactivate Rb. Our data suggest that UL97 possesses a mechanism to elicit E2F-dependent gene expression distinct from disruption of Rb-E2F complexes and dependent upon both the L1 motif of UL97 and the cleft region of Rb.

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication.

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27(Kip1) was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF(Skp2) ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF(Skp2) pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.

Hsp90 inhibitor 17-DMAG decreases expression of conserved herpesvirus protein kinases and reduces virus production in Epstein-Barr virus-infected cells.

All eight human herpesviruses have a conserved herpesvirus protein kinase (CHPK) that is important for the lytic phase of the viral life cycle. In this study, we show that heat shock protein 90 (Hsp90) interacts directly with each of the eight CHPKs, and we demonstrate that an Hsp90 inhibitor drug, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreases expression of all eight CHPKs in transfected HeLa cells. 17-DMAG also decreases expression the of the endogenous Epstein-Barr virus protein kinase (EBV PK, encoded by the BGLF4 gene) in lytically infected EBV-positive cells and inhibits phosphorylation of several different known EBV PK target proteins. Furthermore, 17-DMAG treatment abrogates expression of the human cytomegalovirus (HCMV) kinase UL97 in HCMV-infected human fibroblasts. Importantly, 17-DMAG treatment decreased the EBV titer approximately 100-fold in lytically infected AGS-Akata cells without causing significant cellular toxicity during the same time frame. Increased EBV PK expression in 17-DMAG-treated AGS-Akata cells did not restore EBV titers, suggesting that 17-DMAG simultaneously targets multiple viral and/or cellular proteins required for efficient viral replication. These results suggest that Hsp90 inhibitors, including 17-DMAG, may be a promising group of drugs that could have profound antiviral effects on herpesviruses.

Transient increases in p53-responsible gene expression at early stages of Epstein-Barr virus productive replication.

Expression of Epstein-Barr Virus BZLF1, a key protein initiating the switch from latent to lytic infection, is known to cause cell growth arrest by accumulating p53 and p21(WAF1/CIP1) in epithelial cells, but its molecular mechanism remains elusive. We found here that the BZLF1 protein stimulates p53 binding to its recognition sequence. The BZLF1 accelerated the rate of p53-DNA complex formation through the interaction with p53 protein and also enhanced p53-specific transcription in vitro. Furthermore, p53 protein was found to bind to its target promoter regions specifically in the early stages of lytic replication. Overexpression of p53 at the early stages of lytic replication enhanced viral genome replication, supporting the idea that p53 plays an important role in the initiation steps of EBV replication. Taking the independent role of BZLF1 on p53 degradation into consideration, we propose that the BZLF1 protein regulates p53 and its target gene products in two distinctive manners; transient induction of p53 at the early stages for the initiation of viral productive replication and p53 degradation at the later stages for S-phase like environment preferable for viral replication.

CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells.

CDC6, a replication licensing protein, is partially exported to the cytoplasm in human cells through phosphorylation by Cdk during S phase, but a significant proportion remains in the nucleus. We report here that human CDC6 physically interacts with ATR, a crucial checkpoint kinase, in a manner that is stimulated by phosphorylation by Cdk. CDC6 silencing by siRNAs affected ATR-dependent inhibition of mitotic entry elicited by modest replication stress. Whereas a Cdk-phosphorylation-mimicking CDC6 mutant could rescue the checkpoint defect by CDC6 silencing, a phosphorylation-deficient mutant could not. Furthermore, we found that the CDC6-ATR interaction is conserved in Xenopus. We show that the presence of Xenopus CDC6 during S phase is essential for Xenopus ATR to bind to chromatin in response to replication inhibition. In addition, when human CDC6 amino acid fragment 180-220, which can bind to both human and Xenopus ATR, was added to Xenopus egg extracts after assembly of the pre-replication complex, Xenopus Chk1 phosphorylation was significantly reduced without lowering replication, probably through a sequestration of CDC6-mediated ATR-chromatin interaction. Thus, CDC6 might regulate replication-checkpoint activation through the interaction with ATR in higher eukaryotic cells.

Degradation of phosphorylated p53 by viral protein-ECS E3 ligase complex.

p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.

Epstein-Barr virus polymerase processivity factor enhances BALF2 promoter transcription as a coactivator for the BZLF1 immediate-early protein.

The Epstein-Barr virus (EBV) BMRF1 protein is an essential replication protein acting at viral replication forks as a viral DNA polymerase processivity factor, whereas the BALF2 protein is a single-stranded DNA-binding protein that also acts at replication forks and is most abundantly expressed during viral productive replication. Here we document that the BMRF1 protein evidently enhances viral BZLF1 transcription factor-mediated transactivation of the BALF2 gene promoter. Mutagenesis and electrophoretic mobility shift assays demonstrated the BALF2 promoter to harbor two BZLF1 protein-binding sites (BZLF1-responsive elements). Direct binding of the BZLF1 protein to BZLF1-responsive elements and physical interaction between BZLF1 and BMRF1 proteins are prerequisite for the BMRF1 protein up-regulation of the BALF2 gene promoter. A monomeric mutant, C95E, which is defective in homodimerization, could still interact and enhance BZLF1-mediated transactivation. Furthermore although EBV protein kinase phosphorylates BMRF1 protein extensively, it turned out that phosphorylation of the protein by the kinase is inhibitory to the enhancement of the BZLF1-mediated transactivation of BALF2 promoter. Exogenous expression of BMRF1 protein augmented BALF2 expression in HEK293 cells harboring the EBV genome but lacking BMRF1 and BALF5 genes, demonstrating functions as a transcriptional regulator in the context of viral infection. Overall the BMRF1 protein is a multifunctional protein that cannot only act as a DNA polymerase processivity factor but also enhances BALF2 promoter transcription as a coactivator for the BZLF1 protein, regulating the expression level of viral single-stranded DNA-binding protein.

Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase.

The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

Phosphorylation of p27Kip1 by Epstein-Barr virus protein kinase induces its degradation through SCFSkp2 ubiquitin ligase actions during viral lytic replication.

Epstein-Barr virus (EBV) productive replication occurs in an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. The EBV protein kinase (PK), encoded by the viral BGLF4 gene, is a Ser/Thr protein kinase, which phosphorylates both viral and cellular proteins, modifying the cellular environment for efficient viral productive replication. We here provide evidence that the EBV PK phosphorylates the CDK inhibitor p27(Kip1), resulting in ubiquitination and degradation in a proteasome-dependent manner during EBV productive replication. Experiments with BGLF4 knockdown by small interfering RNA and BGLF4 knock-out viruses clarified that EBV PK is involved in p27(Kip1) degradation upon lytic replication. Transfection of the BGLF4 expression vector revealed that EBV PK alone could phosphorylate the Thr-187 residue of p27(Kip1) and that the ubiquitination and degradation of p27(Kip1) occurred in an SCF(Skp2) ubiquitin ligase-dependent manner. In vitro, EBV PK proved capable of phosphorylating p27(Kip1) at Thr-187. Unlike cyclin E-CDK2 activity, the EBV PK activity was not inhibited by p27(Kip1). Overall, EBV PK enhances p27(Kip1) degradation effectively upon EBV productive replication, contributing to establishment of an S-phase-like cellular environment with high CDK activity.

Homologous recombinational repair factors are recruited and loaded onto the viral DNA genome in Epstein-Barr virus replication compartments.

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

Expression of Epstein-Barr virus BZLF1 immediate-early protein induces p53 degradation independent of MDM2, leading to repression of p53-mediated transcription.

The Epstein-Barr virus (EBV) lytic program elicits ATM-dependent DNA damage response, resulting in phosphorylation of p53 at N-terminus, which prevents interaction with MDM2. Nevertheless, p53-downstream signaling is blocked. We found here that during the lytic infection p53 was actively degraded in a proteasome-dependent manner even with a reduced level of MDM2. BZLF1 protein enhanced the ubiquitination of p53 in SaOS-2 cells. The degradation of p53 was observed even in the presence of Nutlin-3, an inhibitor of p53-MDM2 interaction, and also in mouse embryo fibroblasts lacking mdm2 gene, indicating that the BZLF1 protein-induced degradation of p53 was independent of MDM2. Furthermore, Nutlin-3 increased the level of p53 in the latent phase of EBV infection but not in the lytic phase. Although p53 level is regulated by MDM2 in the latent phase, it might be mediated by the BZLF1 protein-associated E3 ubiquitin ligase in the lytic phase for efficient viral propagation.

TORC2, a coactivator of cAMP-response element-binding protein, promotes Epstein-Barr virus reactivation from latency through interaction with viral BZLF1 protein.

Reactivation of the Epstein-Barr virus from latency is dependent on expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical inducers such as 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We found here that Transducer of Regulated cAMP-response Element-binding Protein (CREB) (TORC) 2 enhances Zp activity 10-fold and more than 100-fold with co-expression of the BZLF1 protein. Mutational analysis of Zp revealed that the activation by TORC is dependent on ZII and ZIII cis elements, binding sites for CREB family transcriptional factors and the BZLF1 protein, respectively. Immunoprecipitation, chromatin immunoprecipitation, and reporter assay using Gal4-luc and Gal4BD-BZLF1 fusion protein indicate that TORC2 interacts with BZLF1, and that the complex is efficiently recruited onto Zp. These observations clearly indicate that TORC2 activates the promoter through interaction with the BZLF1 protein as well as CREB family transcriptional factors. Induction of the lytic replication resulted in the translocation of TORC2 from cytoplasm to viral replication compartments in nuclei, and furthermore, activation of Zp by TORC2 was augmented by calcium-regulated phosphatase, calcineurin. Silencing of endogenous TORC2 gene expression by RNA interference decreased the levels of the BZLF1 protein in response to 12-O-tetradecanoylphorbol-13-acetate/ionophore. Based on these results, we conclude that Epstein-Barr virus exploits the calcineurin-TORC signaling pathway through interactions between TORC and the BZLF1 protein in reactivation from latency.