Panel layout is as described in B. the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is usually insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain name III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important. Author Summary Hepatitis C virus (HCV) is usually a life-threatening contamination afflicting some 170 million people worldwide, and current antiviral therapies are only marginally effective in treating these patients. Clearly, more effective anti-viral drugs for HCV are needed. Of paramount importance to this process is usually understanding the detailed mechanisms HCV uses to infect cells, replicate the viral genome, assemble progeny virus, PF-05089771 and exit the cell. Using genetic mapping, we have identified a single amino acid residue of the HCV NS5A protein that is phosphorylated by host cell kinase, and this modification regulates the production of new infectious virus particles. This modification of NS5A results in the release of some of the viral genome from replicative events, thereby making this material available for progeny virus particle production. We have identified genetic and chemical methods to modulate this event, resulting in our ability to control the production of infectious virus particles in the laboratory. The ability to individual the replication of the virus genetic material and the assembly of new viruses allows us a valuable tool to monitor how this process occurs and, potentially, a novel target for the development of much needed anti-viral drugs. Introduction Hepatitis C virus (HCV) chronically infects nearly 3% of the population of the planet [1]. Persistent virus replication in these individuals often progresses to chronic liver disease, including cirrhosis and hepatocellular carcinoma. Since the discovery of HCV as the causative agent of non-A, non-B hepatitis in 1989 [2], considerable progress has been made in therapeutics, but current anti-virals PF-05089771 are still ineffective for the majority of patients. One of the major obstacles to developing new anti-viral strategies is the nebulous nature of many aspects of the HCV lifecycle. One particularly vague area of HCV biology is usually that of the regulation of the transit of RNAs from active replication to virion biogenesis. HCV is usually a member of the family of enveloped, single strand positive sense RNA viruses [3]. The 9.6 kb viral genome contains a single open reading frame encoding PF-05089771 a polyprotein that is cleaved co- and post-translationally to yield ten viral proteins [4],[5]. These include the structural proteins (Core, E1 and E2) and the nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). HCV RNA replication occurs in association with ER-like cellular membranes and requires several viral non-structural (NS) proteins including; NS3, NS4A, NS4B, NS5A, and NS5B, as well as host cell factors [6]. The site of virion assembly is usually unknown, but recent data has proposed the FGF22 recruitment of HCV RNA and non-structural proteins by the HCV core protein from the replicase to lipid droplets as an early event in virion assembly [7]. Viral genomes that lack core, or contain mutations PF-05089771 in NS5A domain name I that PF-05089771 block lipid droplet binding, prevent the production of infectious virions [7]. The regulatory events that control these events are not known, but it is usually clear that productive virus assembly requires the NS5A protein. The complexity of intracellular events associated with HCV contamination is usually staggering, with RNA involved in.