Marsh Lab - Viperin

Viperin

Viperin: A Novel Interferon-inducible Protein that Inhibits Virus Replication

Radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Although SAM is best known as cellular methylating agent, in radical SAM enzymes it is used to generate a highly reactive 5’-deoxyadenosyl radical and methionine by 1-electron reduction from an active site iron-sulfur cluster. Viperin (Virus Inhibitory Protein, Endoplasmic Reticulum associated, INterferon inducible) is one only eight identified radical SAM enzymes in humans. It is highly expressed in response to type I interferons and is one of the few interferon-stimulated genes shown to have direct antiviral activity. Viperin is primarily located on the cytoplasmic face of the endoplasmic reticulum, but may re-localize to other organelles, including mitochondria. Viperin been shown to restrict infectivity of numerous clinically important human viruses including cytomegalovirus, influenza A, HIV, hepatitis C, West Nile, Dengue and tick-borne encephalitis (TBEV) viruses.

Viperin catalyzes the dehydration of CTP to form 3’-deoxy-3’,4’-didehydro-CTP (ddhCTP). This occurs through a radical mechanism, initiated by the 5’-deoxyadenosyl radical generated from SAM removing a hydrogen atom from the substrate, CTP. ddhCTP acts as an antiviral ribonucleotide when it is mis-incorporated by viral RNA polymerases which terminates synthesis of the viral RNA genome. Equally importantly, viperin also interacts with a wide range of cellular and viral proteins, some of which are summarized in the figure above. These interactions are vital to the protein’s broad antiviral properties. Therefore, our lab is focused on understanding how the interactions between viperin and its target proteins contribute to its multifaceted antiviral properties.

Current Research Interests

Characterizing the interaction between viperin, interleukin receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor-6 (TRAF6), which are important in regulating innate immune signaling.
Establishing an in vitro system to characterize viperin protein complexes using electron microscopy and dynamic light scattering.
Identification and characterization of interactions between viperin and fatty acid β-oxidation enzymes.
Consider viperin's impact on mitochondrial function

Purification of the full-length, membrane-associated form of the antiviral enzyme viperin utilizing nanodiscs

Viperin incorporated in a membrane-mimetic environment is a model system for considering protein-protein interactions

Viperin is a radical S-adenosylmethionine enzyme that catalyzes the formation of the antiviral ribonucleotide, 3’-deoxy-3’,4’-didehydroCTP. The enzyme is conserved across all kingdoms of life, and in higher animals viperin is localized to the ER-membrane and lipid droplets through an N-terminal extension that forms an amphipathic helix. Evidence suggests that the N-terminal extension plays an important role in viperin’s interactions with other membrane proteins. These interactions serve to modulate the activity of various other enzymes that are important for viral replication and constitute another facet of viperin’s antiviral properties, distinct from its catalytic activity. However, the full-length form of the enzyme, which has proved refractory to expression in E. coli, has not been previously purified. Here we report the purification of the full-length form of viperin from HEK293T cells transfected with viperin. The purification method utilizes nanodiscs to maintain the protein in its membrane-bound state. Unexpectedly, the enzyme exhibits significantly lower catalytic activity once purified, suggesting that interactions with other ER-membrane components may be important to maintain viperin’s activity.

Recent Publication

A. Patel, K. Koebke, T. Grunkemeyer, C. Riordan, Y. Kim, R. Bailey, E. N. G. Marsh (2022). "Purification of the full-length, membrane-associated form of the antiviral enzyme viperin utilizing nanodiscs" Sci. Reps. 12(1). DOI: 10.1038/s41598-022-16233-z

The Antiviral Enzyme, Viperin, Activates Protein Ubiquitination by the E3 Ubiquitin Ligase, TRAF6

Viperin is shown to bind and activate TRAF6.

Viperin is a broadly conserved radical SAM enzyme that synthesizes the antiviral nucleotide ddhCTP. In higher animals, viperin expression also accelerates the degradation of various cellular and viral proteins necessary for viral replication; however, the details of this process remain largely unknown. Here, we show that viperin activates a component of the protein ubiquitination machinery, which plays an important role in both protein degradation and immune signaling pathways. We demonstrate that viperin binds the E3 ubiquitin ligase, TRAF6, which catalyzes K63-linked ubiquitination associated with immune signaling pathways. Viperin activates ubiquitin transfer by TRAF6–2.5-fold and causes a significant increase in polyubiquitinated forms of TRAF6 that are important for mediating signal transduction. Our observations both imply a role for viperin as an agonist of immune signaling and suggest that viperin may activate other K48-linked E3-ligases involved in targeting proteins for proteasomal degradation.

Recent Publication

A. Patel, E. N. G. Marsh (2021). "The Antiviral Enzyme, Viperin, Activates Protein Ubiquitination by the E3 Ubiquitin Ligase, TRAF6." J. Am. Chem. Soc. 143(13): 4910-4914

Exploring the interaction between viperin, NS5A, and VAP33

Role of Viperin in restricting HCV replication complex formation via interaction with HCV Nonstructural 5A (NS5A) protein through vesicle trafficking host protein VAP-33.

The hepatitis C virus protein NS5A and human vesicle-associated membrane protein A (VAP33) have been shown to interact with viperin, thereby inhibiting HCV replication. We found that viperin directly interacts with both proteins to inhibit the replication complex formed during viral replication while targeting NS5A for proteolytic degradation. Furthermore, viperin’s activity was adversely affected by this interaction, providing evidence for an evolutionary response by HCV to viperin’s inhibition of its replication complex.

Recent Publication

S. Ghosh, A.M. Patel, T.J. Grunkemeyer, K.A. Zegalia, R.T. Kennedy, E.N.G. Marsh (2020). “Interactions between Viperin, Vesicle-Associated Membrane Protein A, and Hepatitis C Virus Protein NS5A Modulate Viperin Activity and NS5A Degradation”. Biochemistry 59 (6), 780-789. DOI: 10.1021/acs.biochem.9b01090

HADHB stimulates Viperin radical-SAM activity

Illustration of Viperin's effect on HADHB thiolase activity, HADHB's effect on Viperin's radical-SAM activity.

We have investigated the nature of viperin’s interaction with the β-subunit of the mitochondrial trifunctional protein, HADHB. Utilizing both in vivo and in vitro studies, we found that viperin, when re-localized to the mitochondrion, forms a complex with HADHB. Interestingly, formation of this complex decreases HADHB thiolase activity while increasing viperin’s specific activity.

Recent Publication

A. B. Dumbrepatil, K.A. Zegalia, K. Sajja, R.T. Kennedy, E.N.G. Marsh (2020). “Targeting Viperin to the mitochondrion inhibits the thiolase activity of the trifunctional enzyme complex”. J. Biol. Chem. 295 (9), 2839-2849. DOI: 10.1074/jbc.RA119.011526

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