Comparative analysis of the Mosquito Vector and Vertebrate Host of Dengue Virus: Characterizing the Membrane Structures Required for Virus Replication
EMSL Project ID
46895
Abstract
This proposal is submitted in response to EMSL's science theme "Biological Interactions and dynamics." Our objective is to use high-resolution mass spectrometry and time-lapse single-molecule fluorescence imaging to characterize the changes induced in cellular membrane composition and architecture upon virus infection. Specifically, we will carry out a comparative analysis of the host cell lipid composition and cellular protein expression/modification profile of human and mosquito cells infected with dengue virus (DENV). We will also analyze these profiles in its mosquito vector, Aedes aegyptii. Using high resolution time-lapse single-molecule fluorescence microscopy, we will image in real time, the formation of virus induced membranes structures and study the dynamic molecular interactions between viral and host proteins and membrane lipids. Resources within EMSL, specifically the LC- and GC-MS platform will be necessary to obtain the mass accuracy measurements and high mass resolution power required for this systems level analysis of the cellular lipidome and proteome. The high resolution imaging capabilities will significantly advance the currently available low-resolution information regarding viral and host protein and lipid interactions that drive the formation of virus-induced membrane structures. The scientific questions addressed in 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. DENV is the most aggressive re-emerging pathogen worldwide. It causes 50-100 million infections per year. Mosquitoes transmit the virus 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 pathways induced by the expression of virus gene products. Thin section electron microscopy, electron tomography 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. However, the molecular composition of the virus-induced membranes has not yet been characterized.
Using lipidomics we will investigate the temporal regulation of host lipid composition during DENV infection. We will also identify metabolic changes that are unique to DENV infection compared to those induced by cellular stress response pathways such as autophagy. Using proteomics we will identify temporal perturbations in protein expression and post-translational modification during DENV infection and select for protein effectors and signaling pathways that may assist in DENV-induced membrane biogenesis and re-organization. The high-resolution microscopy studies will complement ongoing electron tomography of infected cells aimed at defining the three-dimensional architecture and connectivity of the virus-induced membranes. It will also complement cell biological analyses to identify cellular and viral protein candidates that are responsible for the trafficking, assembly and function of these membrane structures.
Project Details
Project type
Exploratory Research
Start Date
2012-01-09
End Date
2013-02-12
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members