Regulation of viral reactivation and cell death by inflammatory cytokine signaling during murine gammaherpesvirus infection

Rachel Suzanne Beard, Purdue University

Abstract

Gammaherpesviruses (γHV) cause multiple cancers in immunocompromised individuals. Due to the species' specificity of human γHV, which include Epstein-Barr and Kaposi's sarcoma herpesviruses, we use murine gammaherpesvirus 68 (MHV68) as an animal model to study these viruses in each stage of the viral life cycle. The goal of this research is to define the role of cytokines involved in the innate immune response in control of lytic infection and reactivation from latency. One of these cytokines is TNFα, which is involved in death receptor signaling. To test the hypothesis that MHV68 regulates TNFα-induced cell death, we treated infected 3T3 cells with TNFα either alone or in combination with cycloheximide (CHX). In uninfected cells, TNFα simultaneously activates pro-survival and apoptotic signaling pathways, resulting in cell activation but not cell death. CHX blocks NF-κB-dependent synthesis of pro-survival proteins, causing apoptosis when administered with TNFα. We found that cells infected with MHV68 for 8 hours underwent rapid TNFα-induced cell death even in the absence of CHX. We also found that a replication-competent virus is necessary for the cell death, suggesting that pro-survival pathways are blocked by early steps in MHV68 infection. We show that anti-apoptotic pathways are intact by demonstrating that the NF-κB subunit p65 translocates to the nucleus and functions as a transcription factor. We have also identified a modification of the anti-apoptotic protein cellular FLICE-like inhibitory protein Raji (cFLIPR) during MHV68 infection, which, we hypothesize, predisposes the infected cells to death upon TNFα treatment in a novel way. To date, no other viruses have been shown to modify cFLIP to induce cell death in this manner. Another innate immune response cytokine known to slow the spread of infection is IFNαβ. We found that mice lacking receptors for IFNαβ (Ifnar1-/-) were able to clear MHV68 infection but experienced a significant increase in reactivation of latent virus ex vivo. To determine which specific cell types respond to IFNαβ to control reactivation in vivo, we used tissue-specific Cre-recombinase-transgenic mice to delete Ifnar1 only in macrophages. Macrophages are a known latency reservoir for MHV68, and this technique allowed us to target the role of macrophages and only in macrophages. Macrophages are a known latency reservoir for MHV68, and this technique allowed us to target the role of macrophages and IFNαβ signaling during latency. Macrophage-specific Ifnar1-/- mice were infected with MHV68 and viral reactivation assessed at 16 days post-infection. We found that removal of IFNAR1 function from macrophages causes a 10-fold increase in reactivation over wild-type in peritoneal cells, as well as a 2.5-fold increase in splenocytes. These data indicates an essential role for IFNαβ signaling in macrophages to control MHV68 reactivation. Determining the role of IFNαβ signaling and the specific cell types that are important for controlling γHV reactivation from latency, as well as understanding how MHV68 modulates cell death pathways, will help guide future research into therapeutics for these diseases.

Degree

Ph.D.

Advisors

Barton, Purdue University.

Subject Area

Cellular biology|Virology|Immunology

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