To ensure a successful infection, herpesviruses have developed elegant strategies to counterbalance the host anti-viral responses. Sterile alpha motif and HD domain 1 (SAMHD1) was recently identified as an intrinsic restriction factor for a variety of viruses. Aside from HIV-2 and the related simian immunodeficiency virus (SIV) Vpx proteins, the direct viral countermeasures against SAMHD1 restriction remain unknown. Using Epstein-Barr virus (EBV) as a primary model, we discover that SAMHD1-mediated anti-viral restriction is antagonized by EBV BGLF4, a member of the conserved viral protein kinases encoded by all herpesviruses. Mechanistically, we find that BGLF4 phosphorylates SAMHD1 and thereby inhibits its deoxynucleotide triphosphate triphosphohydrolase (dNTPase) activity. We further demonstrate that the targeting of SAMHD1 for phosphorylation is a common feature shared by beta- and gamma-herpesviruses. Together, our findings uncover an immune evasion mechanism whereby herpesviruses exploit the phosphorylation of SAMHD1 to thwart host defenses.

Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411). Epstein-Barr virus (EBV) is a herpesvirus that is associated with B cell and epithelial human cancers. Herpesviruses encode a protein kinase which is an important regulator of lytic virus replication and is consequently a target for anti-viral drug development. The EBV genome encodes for a serine/threonine protein kinase called BGLF4. Previous work on BGLF4 has largely focused on its cyclin-dependent kinase 1 (CDK1)-like activity. The range of BGLF4 cellular substrates and the full impact of BGLF4 on the intracellular microenvironment still remain to be elucidated. Here, we utilized unbiased quantitative phosphoproteomic approach to dissect the changes in the cellular phosphoproteome that are mediated by BGLF4. Our MS analyses revealed extensive hyperphosphorylation of substrates that are normally targeted by CDK1, Ataxia telangiectasia mutated (ATM), Ataxia telangiectasia and Rad3-related (ATR) proteins and Aurora kinases. The up-regulated phosphoproteins were functionally linked to the DNA damage response, mitosis and cell cycle pathways. Our data demonstrate widespread changes in the cellular phosphoproteome that occur upon BGLF4 expression and suggest that manipulation of the DNA damage and mitotic kinase signaling pathways are central to efficient EBV lytic replication.

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a transcription factor of the IRF family. IRF8 plays a key role in normal B cell differentiation, a cellular process that is intrinsically associated with Epstein-Barr virus (EBV) reactivation. However, whether IRF8 regulates EBV lytic replication remains unknown. In this study, we utilized a CRISPR/Cas9 genomic editing approach to deplete IRF8 and found that IRF8 depletion dramatically inhibits the reactivation of EBV upon lytic induction. We demonstrated that IRF8 depletion suppresses the expression of a group of genes involved in apoptosis and thus inhibits apoptosis induction upon lytic induction by B cell receptor (BCR) stimulation or chemical induction. The protein levels of caspase-1, caspase-3 and caspase-8 all dramatically decreased in IRF8-depleted cells, which led to reduced caspase activation and the stabilization of KAP1, PAX5 and DNMT3A upon BCR stimulation. Interestingly, caspase inhibition blocked the degradation of KAP1, PAX5 and DNMT3A, suppressed EBV lytic gene expression and viral DNA replication upon lytic induction, suggesting that the reduced caspase expression in IRF8-depleted cells contributes to the suppression of EBV lytic replication. We further demonstrated that IRF8 directly regulates CASP1 (caspase-1) gene expression through targeting its gene promoter and knockdown of caspase-1 abrogates EBV reactivation upon lytic induction, partially through the stabilization of KAP1. Together our study suggested that, by modulating the activation of caspases and the subsequent cleavage of KAP1 upon lytic induction, IRF8 plays a critical role in EBV lytic reactivation. Infection with Epstein-Barr virus (EBV) is closely associated with human cancers of both B cell and epithelial cell origin. The EBV life cycle is tightly regulated by both viral and cellular factors. Here, we demonstrate that interferon regulatory factor 8 (IRF8) is required for EBV lytic replication. Mechanistically, IRF8 directly regulates caspase-1 expression and hence caspase activation upon B cell receptor (BCR) stimulation and chemical induction, which leads to the cleavage and de-stabilization of several host factors suppressing lytic replication, including KAP1. Caspase-1 depletion blocks EBV reactivation while KAP1 depletion facilitates reactivation in caspase-1 depleted cells. These results together establish a IRF8/caspase-1/KAP1 axis important for EBV reactivation.

H9N2 avian influenza viruses circulate worldwide in poultry and have sporadically infected humans, raising concern whether H9N2 viruses have pandemic potential. Here, we use a guinea pig model to examine whether serial passage results in adaptive viral changes that confer a transmissible phenotype to a wild-type H9N2 virus. After nine serial passages of an H9N2 virus through guinea pigs, productive transmission by direct contact occurred in 2/3 guinea pig pairs. The efficiency of transmission by direct contact increased following the fifteenth passage and occurred in 3/3 guinea pig pairs. In contrast, airborne transmission of the passaged virus was less efficient and occurred in 1/6 guinea pig pairs and 0/6 ferret pairs after the fifteenth passage. Three amino acid substitutions, HA1-Q227P, HA2-D46E and NP-E434K, were sufficient for contact transmission in guinea pigs (2/3 pairs). The two HA amino acid substitutions enhanced receptor binding to α2,3-linked sialic acid receptors. Additionally, the HA2-D46E substitution increased virus thermostability whereas the NP-E434K mutation enhanced viral RNA polymerase activity in vitro. Our findings suggest that adaptive changes that enhance viral receptor binding, thermostability and replicative capacity in mammalian cells can collectively enhance the transmissibility of H9N2 AIVs by direct contact in the guinea pig model.