Investigating Antiviral Tripartite Motif (TRIM) Proteins in the Duck

• Author / Creator

  Campbell, Lee Kaitlynn

• Ducks are the natural host and reservoir of influenza A virus (IAV). It is currently unknown how ducks can both tolerate and restrict IAV replication. It is likely that ducks have evolved unique transcriptional responses to IAV infection to restrict virus while limiting damage from inflammation, however this is poorly understood. Here we examine global transcriptome changes in tissues from influenza infected ducks to identify differentially expressed genes. We also mined the data for tripartite motif (TRIM) proteins, a group of proteins that arose early in eukaryote evolution of which some members have antiviral functions. TRIM proteins are defined by a conserved N-terminal RING, B-box and coiled-coil domains, and are further subclassified by their variable C-terminal domains. TRIM proteins can inhibit viral replication through either direct targeting of viral proteins, or by augmenting antiviral signaling pathways in the cell. Here we investigate how many TRIM proteins ducks have, which duck TRIM genes change in expression in response to IAV infection or which act as antiviral effectors.
  In Aim 1 of my thesis, I investigated the transcriptional responses in spleen, lung and intestines in ducks infected with a low pathogenic avian influenza (LPAI) strain (BC500) and in the lung and spleen in ducks infected with a highly pathogenic avian influenza (HPAI) strain (VN1203). The results of these experiments found that ducks have 65 genes that share upregulation in all tissues sampled when ducks were infected with HPAI or LPAI and that many of these genes were involved in the RIG-I signaling pathway. Tissues involved with IAV replication (lung and intestine) also saw selective downregulation of certain proinflammatory cytokines. The results of this global transcriptome analysis suggest that global and tissue-specific regulation patterns help the duck control viral replication as well as limit some inflammatory responses in tissues involved in replication to avoid damage. In the second Aim of my thesis, I created a de novo transcriptome assembled from Pekin duck RNA-sequencing reads mined from the SRA database on NCBI. From this transcriptome I identified 57 TRIM genes in the duck. I compared these duck TRIM genes to that of the chicken and documented seven TRIM genes that were found in duck but appear absent in chicken, while chickens had two TRIM genes which appear to be missing in duck. TRIM27L and RNF135 are TRIM proteins found in duck but not chicken which increases RIG-I signaling. I also show that many of the MHC-linked TRIM genes arose in a lineage-specific manner in birds and reptiles, and these genes are predominantly expressed in immune relevant tissues such as lung, intestine and spleen. In Aim 3, I investigated the differential expression of the 57 TRIM genes identified in Aim 2, to both VN1203 and BC500. VN1203 caused much more differential expression of duck TRIM genes than BC500 did in all tissues sampled. I investigated if several duck TRIM proteins were able to restrict IAV replication when overexpressed in both duck and chicken cells. TRIM27L, a TRIM protein found in duck but appearing to be missing in chicken, could restrict IAV replication when overexpressed in duck cells only, while TRIM32 and diaTRIM58 could restrict IAV in both chicken and duck cells. Finally, in Aim 4 I investigated the mechanisms behind TRIM27L IAV restriction. Previous work in our lab found TRIM27L could increase IFN-β promoter activity when cotransfected with constitutively active RIG-I (d2CARD) in chicken cells. I determined that TRIM27L was able to increase this promoter activity when cotransfected with d2CARD and dMAVS in both duck and chicken cells, however when cotransfected with duck IRF7 (which is downstream in the RIG-I signaling pathway) TRIM27L inhibited IFN-β promoter activity. TRIM27L uses its RING domain to inhibit IFN-β downstream of IRF7, and its C-terminal PRYSPRY domain to activate IFN-β promoter activity downstream of RIG-I and MAVS. By increasing our knowledge of the functions of TRIM proteins in influenza infection in ducks we can gain a better understanding of both TRIM protein biology and function in host-pathogen interactions in the reservoir host.

• Subjects / Keywords

  • TRIM protein
  • TRIM gene
  • Influenza A Virus
  • Reservoir host
  • duck
  • TRIM27L

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-zb68-vg63

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.