Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein

Abstract

Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within 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 insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain 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. Hepatitis C virus (HCV) is a life-threatening infection 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 understanding the detailed mechanisms HCV uses to infect cells, replicate the viral genome, assemble progeny virus, 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 separate 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.