Ifit2 in the Innate Immune Response to Rabies Virus
Development of targeted therapies to rabies virus (RABV) and other nonsegmented negative stranded RNA viruses (NNSV) is limited by the current understanding of the innate immune response to infection. The Ifit (interferon-induced with tetratricopeptide repeats) protein family, which restricts the pathogenicity of a number of RNA viruses, represents a pathway which can potentially be exploited or manipulated to relieve an infection. In order to understand the relationship between RABV and Ifit-dependent immunity, we performed an experimental RABV infection in knockout mouse lines for two Ifit proteins, Ifit1 and Ifit2, as well as a knockout mouse line for the interferon alpha receptor (Ifnar), which controls expression of Ifits and other antiviral interferon-stimulated genes (ISGs). We found that Ifit2 knockout lead to increases in RABV pathogenicity even more dramatic than Ifnar knockout, and that Ifit1 knockout does not significantly increase RABV pathogenicity. We measured RABV mRNA, viral titers, and visualized RABV spread in the brains of infected mice, finding that loss of Ifit2 lead to an increase in overall RABV replication and an accelerated course of infection. We also developed a cell culture model of Ifit2-dependent RABV restriction in mouse neuroblastoma (NA) cells based on siRNA depletion of Ifit2 mRNA. The results of the mouse study were considered in agreement with the outcomes of prior virus infections of Ifit knockout mice, where Ifit2 is considered more important for restricting NNSV infections, whereas Ifit1 is more likely to restrict nonsegmented positive stranded RNA virus (NPSV) infections. Based on prior research into the mechanism of Ifit1-dependent antiviral effects, which involve both translation inhibition and direct binding to specific viral cap structures, we designed and partially carried out a further course of research into the antiviral mechanism of Ifit2. Specifically, we asked whether Ifit2-dependent translation inhibition depends on mRNA cap structure in cell culture, using a RABV minigenome system to recapitulate the unusual mRNA maturation pathway shared by NNSV. Furthermore, we tested the binding of Ifit2 to specific RNA cap structures which may be made by RABV. Our results indicate that Ifit2 likely uses an RNA cap-dependent pathway to restrict RABV infection, but does not necessarily bind directly to a specific cap structure produced by RABV. Based on these results, we suggest strategies to explore the Ifit2 mechanism of RABV restriction in the future, with the goal of understanding a novel pathway of immunity to NNSV.
Davis, Benjamin M, "Ifit2 in the Innate Immune Response to Rabies Virus" (2019). ETD Collection for Thomas Jefferson University. AAI13863816.