Activation of Protein Ubiquitination by the Antiviral Enzyme, Viperin

Abstract

Viperin (Virus Inhibitory Protein; Endoplasmic Reticulum-associated, Interferon iNducible) is an interferon-stimulated gene that is upregulated as a part of the innate immune response to viral infection. It has been shown to restrict the replication of a broad range of human viruses including influenza, hepatitis C, human immunodeficiency, Dengue, West Nile, Zika, and tick-borne encephalitis viruses. However, the mechanism with which viperin restricts infection varies dependent upon the type of virus. Viperin is a member of the radical S-adenosylmethionine (SAM) enzyme superfamily, and recently was shown to catalyze the dehydration of cytidine triphosphate (CTP) to form the antiviral nucleotide 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP) through a SAM-dependent radical mechanism. ddhCTP acts as a chain terminating inhibitor of viral genome replication of some, but not all, viral RNA-dependent RNA polymerases. These recent findings are exciting but do not fully account for viperin’s antiviral activity against most other viruses. Viperin is also known to play a key role in the Toll-like receptor 7 and 9(TLR-7/9) immune signaling pathways. It recruits signaling proteins to lipid bodies, and thereby facilitates the downstream activation of numerous genes. However, evidence for activation of downstream genes comes from studies conducted with proteins transiently expressed in mammalian cells, and the interpretation of such data is complicated by the potential involvement of other cellular proteins. Therefore, this study focuses on reconstituting viperin’s interactions with two enzymes involved in TRL-7/9 signaling in vitro using purified proteins: TRAF6 (tumor necrosis factor receptor associated factor 6) and IRAK1 (interleukin receptor associated kinase 1). TRAF6 is an E3 ubiquitin ligase that catalyzes K63-linked polyubiquitination of a broad range of substrate proteins which are involved in several signaling pathways including TLR7/9, NF-kB, and MAPK signaling cascades. In addition, TRAF6 itself is auto-ubiquitinated to recruit downstream kinases into signaling complexes. Here, I describe the recombinant expression and purification of various domains of TRAF6, the ‘death’ domain of IRAK1, and an N-terminal truncation of viperin (viperin-ΔN50) from E. coli. This has allowed the interaction between viperin and TRAF6 to be directly demonstrated. It also allowed the auto-ubiquitination activity of TRAF6 to be reconstituted in vitro using purified enzymes. Using this system, viperin was shown to activate TRAF6 ubiquitin ligase activity, which provides a biochemical mechanism to explain viperin’s role in potentiating innate immune signaling. The interaction of viperin with IRAK1 has also been studied. IRAK1 is a serine/threonine kinase that is involved in TLR7/9 pathways. Viperin is predicted to facilitate the ubiquitination of IRAK1 by TRAF6 to activate the production of type I interferons. Using truncated IRAK1 constructs, transiently expressed in HEK293T cells, the interactions of IRAK1 with viperin was localized to the ‘death’ domain of IRAK1. Unfortunately, attempts to express and purify this IRAK1 domain in its soluble form in E. coli to facilitate in vitro studies proved unsuccessful. Lastly, I developed a method to purify the full-length, membrane associated form of viperin using lipid nanodiscs to maintain a membrane-like environment. These experiments represent the first time that full-length viperin has been purified in its active form. This work thus provides a new platform to facilitate structural studies on full-length viperin and study its interaction with other membrane-associated proteins that may contribute to its antiviral activity.