Lipid metabolism during dengue virus replication

Dengue virus (DENV) is a positive strand RNA virus that causes significant burden to human health with an estimated 50-100 million infections annually worldwide. Despite this prevalence, there is a limited understanding of the basic biology of viral infection. Specifically, the host factors required during viral infection and how the virus manipulates the host during the viral replication cycle are poorly understood. In order to address these questions, we initially used RNA interference (RNAi) technology to silence host factors and looked for defects in viral replication. Our analysis resulted in the identification of three required cellular pathways: actin polymerization, fatty acid biosynthesis, and ER stress/autophagy. We subsequently characterized the roles for autophagy and fatty acid biosynthesis during DENV replication.;Autophagy is a process wherein cellular cytoplasmic material can be degraded in bulk. We showed that the process of autophagy is induced and required for efficient replication of DENV. Further, cellular lipid droplets are targeted for degradation during DENV induced autophagy and the released lipids undergo beta-oxidation at the mitochondria to generate ATP for the cell. Thus, DENV infection up-regulates a lipid catabolic process in order to change the metabolic state of the infected cell to favor rapid viral replication.;Fatty acid biosynthesis is the process in which acetyl-CoA and malonyl-CoA are polymerized into a 16-carbon fatty acid. The proteins responsible for the first two steps in de novo fatty acid biosynthesis are required for DENV replication and inhibition of this pathway not only inhibits DENV but the related yellow fever virus and West Nile virus as well. Further characterization of this pathway revealed that the major protein involved in fatty acid biosynthesis is relocalized to sites of viral replication. The mechanism of this re-localization was shown to be physical binding to one of the viral non-structural proteins (NS3). In addition, NS3 is capable of modulating the activity of FASN.;To further characterize the nature of the NS3/FASN interaction, mapping experiments were performed to define the genetic requirements for FASN relocalization. The interaction between NS3 and FASN was mapped to eight charged amino acids on the surface of NS3. Mutating these amino acids to alanine reduces the affinity of NS3 for FASN by two orders of magnitude. Mutation of NS3 in this manner does not affect the other known enzymatic activities of NS3 significantly, but when these mutations are introduced into the full-length virus, viral replication is severely compromised. Thus, the recruitment of FASN is a novel function of NS3 that is required for viral replication.;In sum, the work presented in this thesis sheds some light on the required host factors during DENV replication. We have shown that lipid metabolic processes, both anabolic and catabolic, are manipulated to facilitate robust viral replication. Further work on these and other cellular processes will not only advance basic science into the mechanisms of viral replication, but may highlight novel targets for anti-viral therapies.