We will explore the role of a novel host factor in retrovirus replication, heme oxygenase 2 (HO-2), which we discovered binds N-terminally myristoyled proteins and regulates their trafficking to the plasma membrane. We have generated cell lines deficient in HO-2 by shRNA methods, or entirely lacking HO-2 by CRISPR-directed knockout (KO), and documented dramatic increases in retrovirus virion production. We will now characterize the intracellular transport of the myristoylated murine leukemia virus (MLV) Gag protein with and without HO-2 by examination of fixed sections by immunofluorescence, and by live cell imaging. We will study the size of intracellular complexes containing Gag by sedimentation analysis of extracts, followed by Western blots probed with anti-Gag antibodies. We will follow the course of Gag multimerization by co-immunoprecipitation experiments. We will look for alterations in Gag association with its known trafficking partners (including ABCE1, DDX6, and MOV10), and with proteins known to be important in the process of virion assembly and release (notably the ESCRT and CHMP proteins). We will assess the course of Gag interaction with viral RNA by RNA-immunoprecipitation (RIP) assays. These experiments should give us a comprehensive view of the role of HO-2 in controlling Gag localization and function in virion assembly. The work will define a new potential target for antiviral therapies against retroviruses, and other viruses encoding myristoylated proteins. In addition, we will explore the role of HO-2 in trafficking of selected cellular myristoylated proteins. We have developed an extensive hit list of host proteins bound by HO-2 and will focus on two of these proteins: Toll-like receptor adaptor molecule 2 (TRAM), involved in innate immune signaling;? and reticulon 2, a key protein in intracellular trafficking and in promoting membrane curvature. We will characterize the role of HO-2 in controlling the localization of TRAM, the association of TRAM with the TLR4 receptor, and the signaling through TRAM in response to lipopolysaccharide (LPS). We will similarly test for the role of HO-2 in the localization of reticulon 2 to the endoplasmic reticulum. Because of its potential role in membrane bending, we will test for the effect of reticulon 2 KO on ER structure and virion budding. These experiments will reveal new functions of HO-2 in regulating those specific host functions that mediate virus restriction and impact virion assembly. The findings have great potential to reveal entirely new approaches to regulate or inhibit virus replication.
We will explore the functions and mode of action of heme oxygenase 2 (HO-2), which we discovered is a key myristate-binding protein that negatively regulates the functions of both viral and cellular proteins modified by N-terminal myristoylation. We will assess its impact on the intracellular trafficking of the viral Gag protein, and Gag association with host proteins and the viral RNA genome as it moves along the pathway toward the assembly and release of progeny virions. We will similarly examine the effects of HO-2 on the trafficking of myristoylated host substrates such as TRAM, the signaling molecule in inflammatory response to lipopolysaccharide and the reticulons, which regulate membrane bending, ER structure, and replication of many viruses.
