The project had 3 scientific objectives: 1) Perform “Proteomics informed by Transcriptomics” (PIT) on infected cells to identify genes and proteins differentially regulated in cells during SFTSV infection; 2) Identify SFTSV N and L protein-host protein complexes in vivo by affinity purification mass spectrometry (AP/MS) analysis; 3) Validate and assess candidate factors to characterize their impact on viral infection in cells, with an emphasis on potential immune factors/pathways.
For objective 1, I developed a methodology to enable RNA sequencing of tick cells by depleting ribosomal RNA, allowing us to collect unprecedented datasets encompassing coding and non-coding tick RNAs, as well as viral RNA. Once this methodology was established, it was applied to the proteomics informed by transcriptomics (PIT) method. Briefly, R.microplus cells were infected (MOI 1) with Dabie bandavirus, RNA and protein were extracted and analyzed by sequencing and mass-spectrometry. By comparing the data between day 3 and day 6 post-infection, we were able to establish the infectious landscape within tick cells. However, due to the large size of the R. microplus genome and the extensive dataset collected, data processing is still underway to enrich our current understanding of the tick transcriptome and proteome.
Regarding objective 2, we encountered insufficient viral protein expression through plasmid transfection. As an alternative approach, we decided to utilize the available N antibody and conducted an affinity purification-mass spectrometry experiment on infected tick cells. Using the de novo proteome generated through objective 1, I successfully identified tick interactors of Dabie bandavirus nucleocapsid protein for the first time.
For objective 3, I selected a subset of identified targets from either objective 1 or objective 2, with a particular emphasis on proteins associated with the antiviral immune response. To induce gene silencing, I developed a transfection methodology utilizing magnetic nanobeads for the delivery of dsRNA into tick cells. Following the infection of these knock downed cells, we identified important viral restriction factors crucial for tick cell infection. As an example, NMD (Non-Sense Mediated Decay) pathway was identified as anti-viral for the first time in arthropod vector. Further studies will help us to understand this anti-viral mechanism.
The results from the 3 objectives will be published as one high impact publication (currently being drafted, and interest for the publication was expressed by PLoS Biology editorial board). Due to the nature of the project, and the large size of the tick cells genome, we had to extend the time allocated to the data analysis. Consequently, this delayed the progress of the papers; however, the first scientific output will be submitted for publication in the prestigious open access journal PLoS Biology. The remaining papers which will relate to different data analysis on the generated datasets (identification of transposable elements, and non-coding RNAs important for the infection) will be written and submitted in 2024.
