Identification of Viral Activators of the HSV-2 UL13 Protein Kinase

Although previous studies reported that the herpes simplex virus 2 (HSV-2) UL13 protein kinase mediates the phosphorylation of elongation factor 1{delta} (EF-1{delta}) in infected cells, we show here that individual expression of UL13 was insufficient to induce phosphorylation of EF-1{delta} in mammalian cells. This led us to hypothesize that HSV-2 UL13 requires viral cofactors for full kinase activity and prompted us to identify such cofactors. Our results were as follows. (i) Co-expression of UL13 with UL55 or Us10 significantly enhanced phosphorylation of EF-1{delta} compared to UL13 alone. (ii) UL13 was co-precipitated with UL55 or Us10 upon co-expression, and its kinase activity was significantly increased in their presence, as demonstrated by in vitro kinase assays. (iii) In HSV-2-infected cells, UL13 was specifically co-precipitated with Us10 and UL55. (iv) The UL55-null mutation significantly reduced phosphorylation of EF-1{delta} in HSV-2-infected cells, whereas the Us10-null mutation had little effect; however, the double-null mutation further decreased the phosphorylation compared to the UL55-null mutation alone. (v) The UL55-null mutation, but not the Us10-null mutation, significantly reduced HSV-2 replication and cell-cell spread in U2OS cells to levels comparable to those observed with the UL13 kinase-dead mutation. These results suggest that UL55 acts as a principal activator of UL13 in HSV-2-infected cells, whereas Us10 serves as an auxiliary activator. Moreover, the role of UL13 kinase activity in HSV-2 replication and cell-cell spread in U2OS cells appears to be largely dependent on UL55. ImportanceHerpesviruses encode conserved protein kinases (CHPKs) that often target cellular cyclin-dependent kinase (CDK) phosphorylation sites. CHPKs from beta-and gammaherpesviruses can exhibit these CDK-like functions even when individually expressed in mammalian cells. In contrast, CHPKs from alphaherpesviruses display these CDK-like functions in infected cells, but not upon individual expression, suggesting that they require additional viral factors to exhibit full kinase activity. In this study, we focused on HSV-2 UL13, an alphaherpesvirus CHPK, and identified HSV-2 UL55 and Us10 as viral activators of UL13. In HSV-2-infected cells, UL55 functions as a principal activator of UL13, while Us10 serves as an auxiliary activator. Importantly, the contribution of UL13 kinase activity to HSV-2 replication and cell-cell spread appears to be largely dependent on the presence of UL55. Our findings uncover a previously unrecognized mechanism of CHPK regulation in alphaherpesviruses and provide new insights into the evolutionary diversification of viral kinase control.