The P proteins of paramyxoviruses are essential for viral RNA synthesis. The P protein has multiple phosphorylation sites and is likely phosphorylated by more than one host kinase. While it is thought that the phosphorylation of P is important for its role in viral RNA synthesis, the function of phosphorylation of P remains an enigma. For instance, it was demonstrated that the putative CKII phosphorylation sites of the P protein of RSV play a role in viral RNA synthesis using a mini-genome replicon system, mutating these putative CKII phosphorylation sites within a viral genome has no effect on viral RNA synthesis, leading to the theory that phosphorylation, at least phosphorylation by CKII, does not play a role in viral RNA synthesis. In preliminary studies, phosphorylation state of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlates with ability of P to facilitate synthesizing viral RNA, indicating that phosphorylation of P does play a role in viral RNA synthesis. Furthermore, host kinase polo-like kinase 1 (PLK1) as well as AKT1 interacts with P and phosphorylates the P protein in vitro. We hypothesize that PLK1 and AKT1 play critical roles in paramyxovirus RNA synthesis through regulation of phosphorylation status of the P protein. We propose to test this hypothesis by focusing on three specific aims. (1) Investigating the mechanism of regulation of viral RNA synthesis by PLK1. In preliminary studies, we have found that phosphorylation of serine residue at 157 of the P protein of PIV5 results in reduced viral genome RNA replication, indicating that phosphorylation of P plays a critical role in regulating viral RNA replication. The Ser residue 157 is within a SSP motif, a known PLK1 binding site. We have found that PLK1 interacts with P in infected as well as transfected cells through its SSP motif and that PLK1 can phosphorylate P in vitro. We have determined that serine residue 308 of P is a phosphorylation site of PLK1. We hypothesize that phosphorylation of P by PLK1 reduces the ability of P to facilitate synthesis of the viral RNA genome. We will investigate the mechanism by which PLK1 regulates viral RNA synthesis. (2) Elucidating the role of AKT1 in viral RNA synthesis. In preliminary studies, AKT1 phosphorylates the P protein of PIV5 in vitro. Furthermore, inhibitors as well as siRNA against AKT1 reduce growth and viral protein synthesis of PIV5 in cells. We hypothesize that AKT1 plays a direct role in viral RNA synthesis by phosphorylating the P protein of PIV5 in infected cells. We will determine the AKT1 phosphorylation site(s) within the P protein utilizing a proteomics approach as well as the in vitro kinase assay using purified P from bacteria in combination with mutational analysis. If the AKT1 phosphorylation site is determined, we will investigate its role in viral RNA synthesis using a mini-genome system as well as recombinant PIV5 containing mutations at AKT1 phosphorylation site. (3) Investigating the activation of AKT1 by virus infection. AKT1 is a kinase that is activated through phosphorylation. AKT1 is often activated in many cancer cells. However, PIV5 is known to infect primary cells, in which AKT1 is not normally activated. In preliminary studies, the L protein of PIV5 activates NF-kB through an AKT1-dependent pathway. Furthermore, expression of L enhances phosphorylation of AKT1, indicating that L can activate AKT1. We hypothesize that the L protein activates AKT1 in infected cells, leading to activation of NF-kB as well as phosphorylation of P, which is critical for viral RNA synthesis. We will map the pathway leading to the activation of AKT1 in infected cells. PUBLIC HEALTH RELEVANCE: Paramyxoviruses include many important human and animal pathogens such as measles virus, mumps virus, human parainfluenza viruses and respiratory syncytial virus (RSV) as well as emerging viruses such as Nipah virus and Hendra virus. The paramyxovirus RNA-dependent RNA polymerase (RdRp) consists of the phosphorprotein (P) and the large protein (L). The P protein is essential for viral RNA synthesis. The P protein has multiple phosphorylation sites and is likely phosphorylated by more than one host kinase. It was thought that the phosphorylation of the paramyxovirus P proteins plays an important role in regulating viral RNA synthesis. However, conclusive data on the role of phosphorylation of the P protein in paramyxoviruses has not been reported. On the contrary, the most recent work seems to indicate that the phosphorylation of the P proteins of paramyxoviruses does not have a role in viral RNA synthesis. It is thought that phosphorylation of viral proteins is carried out by host kinases. Two host kinases, CKII and PKC-? have been identified so far as main host kinases that phosphorylate the P proteins of paramyxoviruses. While the role of CKII in phosphorylation of the P proteins has been studied most, interestingly, due to the nature of CKII (ubiquitous expression and multiple subunits and isoforms) and lack of specific inhibitor against CKII, it has never been shown that CKII is directly involved in paramyxovirus replication in infected cells. Similarly, the role of PKC-? in viral RNA synthesis has not been validated using specific siRNA. Our research into the roles of host kinases in phosphorylation of the P protein and the role of phosphorylation of P in viral RNA synthesis happened somewhat serendipitously. My lab has been very much interested in virus and host interaction, particularly on how PIV5 interacts with host signaling pathways. We have identified host proteins, kinases that interact with viral P protein. Unexpectedly and interestingly, the most direct effects of these kinases are on viral RNA synthesis. We have found that P interacts with PLK1 as well as AKT1. Both PLK1 and AKT1 phosphorylate PIV5 P in vitro. Interestingly, interaction with PLK1 results in lower activity for P whereas interaction with AKT1 appears to be essential for activity of P. We hypothesize that phosphorylation of P is essential for activity of the P protein in viral RNA synthesis and we will test the hypothesis in this proposal. The road to understanding the role of phosphorylation of the P proteins of paramyxoviruses has been challenging. Perhaps, our approach that starts with analyzing a virus (rPIV5-CPI+) derived from a naturally occurring isolate with reduced phosphorylation level of P that has an effect on viral RNA synthesis, then investigates the roles of novel host kinases (PLK1 and AKT1) and ultimately identifies and confirms the roles of phosphorylated sites within P may provide a fresh perspective that may lead to elucidating the roles of phosphorylation of the P protein in viral RNA synthesis. Beyond understanding paramyxovirus RNA replication, the work proposed here may also result in a new paradigm in treating infections caused by these viruses. For many years, tremendous effort has been invested unsuccessfully in developing small molecule drugs that directly target viral proteins as antiviral treatments for paramyxovirus infection. Targeting host protein such as AKT that is required for paramyxovirus replication is a novel approach. With many anti-cancer drugs based on targeting AKT in development, it is exciting to speculate that some of these drugs can be readily applied to treat infections caused by paramyxovirus based on our work that AKT plays a critical role in regulating paramyxovirus replication.