Mechanisms of novel host factors in alphavirus infection

Alphaviruses include important and emerging human pathogens such as encephalitic viruses and the arthritogenic chikungunya virus. In spite of their importance, there are currently no licensed antiviral therapies or vaccines. New approaches and information are needed to develop antiviral strategies. Alphaviruses are small enveloped viruses with highly ordered structures. The nucleocapsid core contains the plus-sense RNA genome packaged within a capsid protein shell. The core is surrounded by the virus envelope, which contains an organized array of the E2 and E1 transmembrane proteins. The structure and function of the virus and viral proteins have been extensively characterized. However, there is comparatively little information on the role of host proteins in the virus life cycle, in particular for steps such as the uncoating of the incoming viral RNA during entry, the assembly and transport of the nascent nucleocapsid, and the budding of virus particles from the plasma membrane. We used an innovative approach to identify host proteins that specifically associate with the capsid/nucleocapsid during infection. The approach is based on biotinylation of the capsid protein in alphavirus-infected cells, chemical crosslinking, specific capsid protein retrieval under denaturing conditions, release of crosslinked cellular proteins, and identification of significant host protein association by mass spectrometry. These candidate host factors were evaluated by siRNA depletion for effects on infection by two different alphaviruses. Based on extensive preliminary results, we propose to characterize 4 host proteins for their previously unidentified pro-viral roles in alphavirus infection. The specific aims are: 1. Define the role of novel host proteins in the alphavirus exit pathway. Depletion of two host proteins inhibited alphavirus spread but did not affect primary infection or envelope protein expression at the cell surface. DARS is the cytoplasmic aspartyl tRNA synthetase, and part of the dynamic multi-tRNA synthetase complex (MSC). Its depletion phenotype suggests that DARS plays a role in steps other than protein synthesis, perhaps involving the MSC. YWHAB is a member of the family of 14-3-3 adaptor proteins, which can regulate protein-protein interactions and protein modifications. We will use our established panel of assays to define the role of DARS and YWHAB in the virus exit pathway. 2. Define the role of novel host proteins in early steps in the alphavirus life cycle. Depletion of two host proteins inhibited both virus spread and primary infection, but did not inhibit viral RNA replication. We will perform mechanistic studies to define the differential roles of Rab1B and b spectrin in virus infection. The results of these studies will provide fundamental information on host proteins involved in the alphavirus life cycle, and potential new targets for antiviral strategies.