Biochemical and Biophysical Research Communications
Structural characterization reveals that viperin is a radical S-adenosyl-l-methionine (SAM) enzyme
Introduction
Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) was originally cloned from interferon treated human macrophages [1], [2]. Subsequent studies reveal that viperin is a highly inducible candidate gene in response to a wide spectrum of viruses and microbial products such as LPS and double-stranded RNA [3], [4]. Viperin is highly conserved across both mammals and lower vertebrates, localized in the endoplasmic reticulum (ER), thus implying its extremely important role. Indeed, viperin has been demonstrated to inhibit a large array of DNA and RNA viruses, such as human cytomegalovirus [1], [2], influenza, hepatitis C virus (HCV) and alphaviruses [5], [6], [7], as well as human immunodeficiency virus [8]. However, it remains largely elusive how viperin is capable of inhibiting such a diverse spectrum of viruses since it is very unlikely that it functions by directly binding to viral proteins.
The human viperin gene encodes a protein of 361 amino acids, and based on the sequence alignment [9], it appears composed of three distinctive regions: an N-terminal variable transmembrane domain approximately from residues 1 to 44, followed by a middle region (45–213) containing a sequence motif found in the superfamily of S-adenosyl methionine (SAM)-dependent radical enzymes, and a C-terminal region (214–361) highly conserved in viperins of all species examined (Fig. 1A). Recently the N-terminal region was experimentally demonstrated to form an amphipathic α-helix which is anchored into the endoplasmic reticulum (ER) membranes to inhibit protein secretion [10], and onto lipid droplets to inhibit HCV [11]. On the other hand, a CX3CX2C sequence motif was previously identified over residues 83–90 of the human viperin (Fig. 1B) [12], which is conserved in all radical SAM enzymes required for the synthesis of molybdopterin (Mao A) (Fig. 1B), heme D1 (NIRJ), and PQQ (PQQIII) [12], [13], [14], [15]. These enzymes with more than 2800 putative members all share one common feature, an unconventional [4Fe–4S] cluster coordinated by three rather than four closely spaced cysteine residues in the motif (Fig. 1C). The unique Fe uncoordinated by a cysteine residue is thus available for binding to the ligand, S-adenosyl methionine (SAM) (Fig. 1D) [12], [13], [14], [15], [16]. As such, viperin has been extensively hypothesized to be a SAM enzyme and indeed, mutation of the cysteine residues in the motif has been found to eliminate its antiviral activity against HCV [9]. However, previous attempts to reconstitute the [4Fe–4S] cluster in viperin have all failed [10].
So far no structural characterization has been reported for viperin. In the present study, we aimed to structurally characterize the human viperin by dissecting it into a set of 12 fragments of different lengths, followed by detailed CD and NMR assessment. Our results provide the first insight into the structural properties of viperin. Most importantly, we successfully identified a buffer-soluble and structured fragment Viperin (45–361), which only has the N-terminal transmembrane helix deleted. Markedly, after many failures under aerobic condition, we have finally succeeded in reconstituting the [4Fe–4S] cluster under anaerobic condition, thus experimentally revealing that viperin is indeed a radical SAM enzyme.
Section snippets
Materials and methods
Cloning, expression and purification of the human viperin fragments. Total RNA was extracted from human Monocytic cells using Qiagen RNeasy Mini kit and then the full-length viperin RNA was reverse-transcribed into cDNA by RT-PCR using One-step RT-PCR kit (Qiagen). Subsequently, by using different pairs of primers, the 361-residue human viperin was dissected into 12 fragments, which include nine with the N-terminus differentially truncated, spanning over residues 11–361, 22–361, 36–361, 43–361,
Structural characterization of the human viperin fragments
In the present study, we have cloned the full-length viperin and its 12 dissected fragments into His-tagged expression vector. However, the entire viperin was found to be not expressed. For other fragments, only five were expressible, which include fragments (43–361), (45–361), (71–361), (81–361) and (214–361). Out of them, the fragments (71–361), (81–361) and (214–361) were only found in inclusion body and could not be refolded by fast dilution or dialysis against various buffers. Viperin
Discussion
Interferons (IFNs) initiate the first line of defense against viral infection by altering the expression of hundreds of genes, out of which viperin is highly inducible by both type I and type II IFNs. The key role of viperin in innate immunity is strongly implicated by its evolutionary conservation across both mammals and lower vertebrates. Recently several studies have successfully revealed that the N-terminal region functions by forming an amphipathic α-helix to target different membranes [5]
Conclusion
Here, for the first time we have characterized the structural properties of different viperin fragments and most importantly obtained a buffer-soluble and structured fragment, Viperin (45–361). This thus led to our successful reconstitution of the [4Fe–4S] cluster under anaerobic condition, thus experimentally confirming that viperin is indeed a radical S-adenosyl-l-methionine (SAM) enzyme. Together with the previous mutagenesis result [9], our present study strongly implies that the radical
Acknowledgments
This study is supported by the Ministry of Education (MOE) of Singapore Tier 1 Grant R-154-000-330-112 and Tier 2 Grant R-154-000-388-112 to Jianxing Song.
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2020, Molecular CellCitation Excerpt :Viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible) is a powerful antiviral effector against a broad array of viruses (Helbig et al., 2005, 2013; Nasr et al., 2012; Rivieccio et al., 2006; Seo et al., 2011; Waheed and Freed, 2007; Wang et al., 2012). Viperin contains an α-helical domain, a radical S-adenosylmethionine (SAM) domain, and a highly conserved C-terminal domain (Shaveta et al., 2010). Viperin can activate a variety of complex mechanisms to exert its antiviral effects (Gizzi et al., 2018; Helbig et al., 2011; Nasr et al., 2012; Wang et al., 2007).