Location: Office of The Director
Project Number: 5090-31320-005-011-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2021
End Date: Jun 30, 2023
Characterize the involvement of stress granule formation and function in pathogen response and the establishment of protective immunity in fish cell-lines.
Work will characterize the function, and functional domains of critical rhabdoviral proteins to identify components of virulence and possible viral targets for new vaccine candidates. The following hypotheses will be tested: Hypothesis 1: Testing attenuated viral vaccine candidates in trout cell-lines will provide new approaches to high throughput analysis for future immunization of fish against viral pathogens. Approach: Recombinant virus production was previously coupled with mutational analyses to validate the physiological significance of VHSV structure/function studies to identify targeted changes in key viral proteins (V & M) protein that leads to decreased anti-host function, but retained viral replication support. Hypothesis 2: Stress granules (SGs) play a critical role in host detection and response to rhabdoviral infections in fish. Approach: To assess involvement of SG formation kinetics with virus replication and immune activation, time course experiments with IHNV or VHSV will be conducted in rainbow trout gonad (RTG)-2 cells. After infection with IHNV, VHSV or control for 12-96 h, conditioned medium will be harvested and viral titers determined. mRNA levels of key immune genes will be quantitated by qRT-PCR. In parallel, cells on coverslips will be fixed and stained with anti-G3BP1 to monitor SG formation by immunofluorescence microscopy. Lysates will be analyzed by SDS-PAGE and immunoblotting for phosphorylation of key proteins involved with formation of SGs. Hypothesis 2: Stress granules (SGs) play a critical role in host detection and response to rhabdoviral infections in fish. Approach 3.B.1: To assess SG induction kinetics, and activation of the PRR detection pathway, time course experiments with IHNV or VHSV will be conducted in RTG-2 cells and viral titers determined. Interferon beta (IFN-beta) mRNA levels will be quantitated by qRT-PCR. In parallel, cells on coverslips will be fixed and stained with anti-G3BP1 to monitor SG formation by immunofluorescence and fluorescence intensities quantified by using ImageJ software. Lysates will be analyzed by SDS-PAGE and immunoblotting for phosphorylation of key SG-associated proteins, in the presence or absence of inhibitors, and immunoblots quantified by ImageJ software. Expression plasmids carrying Myc-tagged versions of these viral proteins will be transfected in RTG-2 cells. Cells positive for Myc staining will be scored for SG formation or suppression. Based on the constructs that promote SG formation, we will test mutant M and NV proteins to see if they induce SG formation. To further assess the role of viral proteins in SG induction or suppression, UV-inactivated virus will be used to deliver non-replication viral genomes to host cells, followed by the same SG analyses above. To determine if the SG signals to Type I IFN genes, IFN promoter luciferase assays will be performed in ISIRB treated cells.