Membrane rearrangements in dengue virus infected cells
EMSL Project ID
33591
Abstract
We are requesting Standard Access, Open Call, for this General Access type proposal with Non-Proprietary Status. The objective of this proposal is to use high-resolution mass spectrometry to characterize the changes in the membrane lipid composition of human and mosquito cells infected with dengue virus. Resources within EMSL, specifically the LC-LTQ-FT mass spectrometry platform will be a great asset to obtain the necessary mass accuracy measurements and high mass resolution power required for this systems level analysis of the cellular lipidome. Dengue virus causes about 50 million infections per year and has recently been classified as one of the most aggressive re-emerging pathogens worldwide. The virus is transmitted by mosquitoes and virus replication within the arthropod host is required for transmission to humans. As in many other enveloped viruses, lipids play a significant role in the life cycle of this virus by regulating the processes of virus entry, genome replication, packaging, assembly and release. They have also been implicated in diversion of host antiviral defense mechanisms during virus infections.
Infection of cells with dengue virus results in enhanced synthesis of new lipid-derived structures and extensive membrane rearrangements. These rearrangements result from altered lipid biosynthesis and trafficking induced by the expression of virus gene products. Thin section electron microscopy and biochemical analyses have indicated that the induced membranes display morphologically distinct characteristics associated with specific functions such as viral polyprotein processing or viral RNA replication. These studies have also succeeded in biochemically separating these membrane structures for functional analysis in vitro. The objective of this proposal is to analyze these membrane structures further by characterizing their lipid constituents that may contribute to the morphologically and functionally distinct features of these membranes. Specifically, we will isolate membrane fractions from uninfected and dengue virus-infected cells and characterize and compare the lipid components of these membranes using high resolution mass spectrometry. We will use both human hepatoma cells and mosquito derived C6/36 cells for these analyses as both hosts are imperative to the virus life cycle. Through this approach we hope to identify the lipid-mediators and indirectly, the signaling events that may be important for a productive dengue virus infection. These studies will support parallel ongoing investigations aimed at systematically analyzing the precise role of lipids in dengue virus infection. These studies include, electron tomography of infected cells to define the three-dimensional architecture and connectivity of the virus-induced membranes, and proteomic and cell biological analyses to identify cellular and viral protein candidates that are responsible for the trafficking, assembly and function of these membrane structures. The scientific questions addressed within this proposal are fundamental to our understanding of how viruses influence their local host environments and will shed light on how biological membranes respond to environmental perturbations such as virus infections.
Project Details
Project type
Exploratory Research
Start Date
2009-04-08
End Date
2010-04-11
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Perera RM, C Riley, GI Mezengie, A Hopf- Jannasch, RJ Moore, KK Weitz, L Pasa-Tolic, TO Metz, J Adamec, and RJ Kuhn. 2012. "Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells ." PLoS Pathogens 8(3):, doi:e1002584