Abstract

MT Tech

Viral infections occur across the globe, and emerging viral infections can stem from the transmission of viruses out of animal reservoirs into humans.  In Montana, some zoonotic viruses, such as Colorado Tick Fever virus, are unique to this area.  Though transmission of CTFV from ticks to humans is currently rare, it does occur and this suggests the possibility of increased transmission.  Human immune responses, starting with the interferon system, restrict viral host ranges.  As many as 300 proteins are induced by the interferon system and many of these proteins likely have antiviral functions.  Many of these interferon stimulated genes (ISGs) have not been well-characterized.  Here, we will build a research program that is designed to provide a pipeline for analyzing the functional role of ISGs that are members of the tripartite motif (TRIM) family of proteins.  Briefly, we will seek to identify cellular proteins that interact with the TRIM proteins and evaluate the functional outcome of that interaction.  Our hypothesis is that many, if not all, of the TRIM proteins evaluated will have antiviral functions that can be evaluated via the experimental pipeline described in this proposal’s research plan.  Since there are 75 TRIM genes in the human genome with many different TRIM protein isoforms possibly produced from 90% of these genes, success in characterizing several TRIM proteins in this pilot project could lead to an expansion of this project in future years.  This research is relevant and translatable to all viral infections and could specifically lead to a better understanding of host-range breeches by CTFV, a virus that is unique to Montana and the surrounding Rocky Mountain regions.

Specific Aims

Cell-based immunity to viral infections is due, in large part, to the host’s interferon (IFN) system.  Nearly all cells in the human body are capable of detecting the presence of a virus and subsequently “warning” nearby cells that a viral infection is in progress.  As an example, IFNβ is a key cytokine secreted by virally-infected cells that is subsequently recognized by cell-surface receptors on the infected cell and on neighboring cells.  Approximately 300 genes induced by IFN-mediated signaling, known collectively as interferon stimulated genes (ISGs), commonly function in antiviral defenses. Many members of the tripartite motif (TRIM) protein family are ISGs and, accordingly, many TRIM proteins are involved in antiviral defense.

There are 75 TRIM protein family members encoded by the human genome, yet few have been studied in great detail.  Here, we will begin filling the “TRIM protein knowledge gap” in an unbiased manner by using yeast two-hybrid (Y2H) screening assays to identify proteins that interact with individual TRIM family members.  New hypotheses will be formed to describe the function of the TRIM proteins that will be based on published knowledge of the function of the TRIM-interacting proteins.  While it is impossible to know the results of Y2H screens ahead of time, we hypothesize that the TRIM-interacting proteins will implicate unique antiviral mechanisms for most, if not all, TRIM proteins examined.  These antiviral mechanisms will be assessed in cells infected with a virus endemic to Montana, Colorado Tick Fever virus (CTFV).  It is important to note that the follow-up experiments are not entirely dependent upon a successful Y2H screen.  Y2H-based human “interactome” studies have already identified interacting partners for many TRIM proteins, such as melanoma antigen gene (MAGE) family members, but have not further characterized the functional consequences of the interactions (Rolland et al., 2014).  Therefore, we will also confirm and expand upon these Y2H datasets.

Specific aim 1: Identify and characterize TRIM protein interactions with binding partners.  Novel interactions will be identified using Y2H assays.  The novel and previously published interactions will be further characterized using Y2H assays to identify specific motifs or TRIM protein isoforms involved in the interaction.   Finally, using both in vitro- and cell-based assays, we will confirm the protein-protein interaction results with and without the presence of Colorado Tick Fever Virus.

Specific aim 2:Evaluate the role of the TRIM protein interactions.  We will monitor the influence of TRIM protein isoforms and RING finger mutants on the fate of TRIM protein interactions with their partners.  Furthermore, the impact of the interactions on innate immunity-related cell signal transduction pathways will be explored.

Primary Contact

Joel Graff jgraff@mtech.edu