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# A naturally occurring antiviral ribonucleotide encoded by the human genome

### Subjects

An Author Correction to this article was published on 16 June 2020

A Publisher Correction to this article was published on 06 July 2018

## Abstract

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies1. Viperin, a member of the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3ʹ-deoxy-3′,4ʹ-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.

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## Change history

• ### 16 June 2020

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

• ### 06 July 2018

Change history: In the HTML version of this Letter, Extended Data Fig. 4 incorrectly corresponded to Fig. 4 (the PDF version of the figure was correct). This has been corrected online.

## References

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6. Fenwick, M. K., Li, Y., Cresswell, P., Modis, Y. & Ealick, S. E. Structural studies of viperin, an antiviral radical SAM enzyme. Proc. Natl Acad. Sci. USA 114, 6806–6811 (2017).

7. Kennedy, A. D. et al. Complete nucleotide sequence analysis of plasmids in strains of Staphylococcus aureus clone USA300 reveals a high level of identity among isolates with closely related core genome sequences. J. Clin. Microbiol. 48, 4504–4511 (2010).

8. Yokoyama, K., Numakura, M., Kudo, F., Ohmori, D. & Eguchi, T. Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin. J. Am. Chem. Soc. 129, 15147–15155 (2007).

9. Honarmand Ebrahimi, K. et al. The radical-SAM enzyme Viperin catalyzes reductive addition of a 5′-deoxyadenosyl radical to UDP-glucose in vitro. FEBS Lett. 591, 2394–2405 (2017).

10. Lee, H. A Proposed Mechanism for the Radical SAM Enzyme Viperin. BSc thesis, Univ. of Illinois (2017).

11. Giese, B., Beyrich-Graf, X., Erdmann, P., Petretta, M. & Schwitter, U. The chemistry of single-stranded 4′-DNA radicals: influence of the radical precursor on anaerobic and aerobic strand cleavage. Chem. Biol. 2, 367–375 (1995).

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15. Teng, T. S. et al. Viperin restricts chikungunya virus replication and pathology. J. Clin. Invest. 122, 4447–4460 (2012).

16. Wang, B. et al. Viperin is induced following toll-like receptor 3 (TLR3) ligation and has a virus-responsive function in human trophoblast cells. Placenta 36, 667–673 (2015).

17. Jiang, D. et al. Identification of five interferon-induced cellular proteins that inhibit West Nile virus and dengue virus infections. J. Virol. 84, 8332–8341 (2010).

18. Van Slyke, G. A. et al. Sequence-specific fidelity alterations associated with West Nile virus attenuation in mosquitoes. PLoS Pathog. 11, e1005009 (2015).

19. Panayiotou, C. et al. Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation. J. Virol. 92, e02054-17 (2018).

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## Acknowledgements

We thank S. J. Booker for helpful discussions, L. Nordstroem (Chemical Synthesis & Biology Core Facility) for synthesis of ddhC and R. Sharma and J. Perryman for assistance with construction of RdRp expression plasmids and purification of RdRp enzymes. This work was supported by National Institutes of Health (NIH) Grants R21-AI133329 (T.L.G. and S.C.A.), P01-GM118303-01 (J. A. Gerlt and S.C.A.), U54-GM093342 (J. A. Gerlt and S.C.A.), U54-GM094662 (S.C.A.), R01-AI045818 (C.E.C.), Pennsylvania State University Start-Up Funds (J.J.), and the Price Family Foundation (S.C.A.). We acknowledge the Albert Einstein Anaerobic Structural and Functional Genomics Resource (http://www.nysgxrc.org/psi3/anaerobic.html).

## Author information

Authors

### Contributions

A.S.G., T.L.G., J.J.A., C.E.C. and S.C.A. designed the research; A.S.G. and T.L.G. contributed equally; A.S.G. and T.L.G. prepared protein and performed experiments; J.J.A. performed polymerase biochemistry; J.J. performed ZIKV release assays; Q.D. prepared isotopologues; R.K.J. and K.C. provided advice on virologic experiments and performed statistical analysis; S.M.C. performed NMR measurements; S.J.G. prepared HEK293T cells; N.G.D. prepared RAW264.7 cells; all authors analysed data. T.L.G., J.D.L., A.R.B., C.E.C. and S.C.A. supervised research. A.S.G., T.L.G., J.J.A., C.E.C. and S.C.A. wrote the manuscript.

### Corresponding authors

Correspondence to Tyler L. Grove or Steven C. Almo.

## Ethics declarations

### Competing interests

A.S.G., T.L.G., J.J.A., C.E.C. and S.C.A. are co-inventors on a U.S. provisional patent application (No. 62/548,425; filed by S.C.A.) that incorporates discoveries described in this manuscript.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

## Extended data figures and tables

### Extended Data Fig. 1 Purification of rVIP and ddhCTP.

a, Amino acid sequence from a Lacinutrix mariniflava fusion gene product of CMPK2- and a viperin-like protein. b, SDS–PAGE analysis after affinity and size-exclusion chromatography. The protein corresponding to amino acid residues 51–361 has a predicted molecular mass of 38.36 kDa. This construct was chosen because approximately 100 mg of protein could be purified from a 2-l fermentation. In addition, the protein is soluble at concentrations greater than 2 mM. c, UV-visible spectrum of purified rVIP (29.5 μM, UV 280/400 ratio of 4.2). d, Purification of ddhCTP using ammonium bicarbonate pH 9, with an elution gradient (dashed line) from 0.2 M to 0.8 M over 200 column volumes. All results have been repeated at least three times.

### Extended Data Fig. 2 NMR spectroscopy of ddhCTP.

a, 13C–13C COSY spectrum of 13C915N3–ddhCTP. The assignments for the observed correlations of the 13C-connectivities are indicated with the grey dotted lines. b, 31P NMR spectra (300 MHz) of ddhCTP (1 mM) in D2O at 300 K. Three resonance peaks at –19.5 (triplet), −9.5 (doublet) and –3.9 (doublet) p.p.m. correspond to the beta, alpha and gamma phosphates of ddhCTP, respectively. c, 2D-HSQC NMR spectra collected on purified 1 mM ddhCTP in D2O. d, 2D-HSQC NMR spectra collected on 1 mM synthetic ddhC in D2O. All experiments have been repeated twice.

### Extended Data Fig. 3 rVIP produces a 1:1 stoichiometry of 5′-dA and ddhCTP and reacts specifically with CTP.

a, Formation of ddhCTP (red squares) and 5′-dA (blue circles) from CTP and SAM in the presence of dithionite and 100 μM rVIP. ddhCTP is formed at roughly stoichiometric amounts with that of 5′-dA. Data are mean ± s.d. from three replicates. b, Formation of ddhCTP (open triangle,) and 5′-dA (open circle) from CTP and SAM in the presence of the flavodoxin, flavodoxin reductase and NADPH using 100 μM rVIP. Data are mean ± s.d. from three replicates. ddhCTP and 5′-dA are formed at roughly stoichiometric concentrations. The production of ddhCTP by this enzyme-driven reducing system indicates that ddhCTP formation is not the consequence of a side reaction with dithionite. c, High-performance liquid chromatography analysis (0 min, blue trace; 20 min, red trace; 12 h, green trace) showing the generation of a new peak at 1.55 min in the 12 h sample corresponding to a 5′-dA–dithionite adduct in the presence of 100 μM rVIP, 1 mM SAM and 10 mM IPP. d, Corresponding mass spectra in ESI negative mode of the peak occurring at 1.55 min in the 12 h sample. The 5′-dA–dithionite conjugate was calculated to have an exact mass of 315 Da and an m/z of 314.1. These results have been repeated twice. e, The rate of 5′-dA formed by 100 μM rVIP in the presence of 1 mM SAM and 1 mM CTP alone or 1 mM CTP with 10 mM IPP or UDP–glucose. Data are mean ± s.d. from three independent experiments. f, Mass spectrum traces of 5′-dA by ESI+. Reactions were conducted with 100 μM rVIP, 1 mM SAM and 1mM deuCTP with or without 10 mM UDP–glucose. The mass spectrum of 5′-dA produced during these reactions shows only the presence of deuterium, which derives from deuCTP, even when UDP–glucose is present at a tenfold higher concentration. An m/z of 252.1 represents the natural abundance peak of 5′-dA, an m/z of 253.1 indicating the addition of one deuterium. g, Mass spectrum trace showing –m/z of 5′-dA formed by combining 100 μM rVIP with 1 mM deuCTP (dotted blue trace) or 1 mM deuCTP with 1 mM deoxyCTP (red trace). The y-axis of each spectrum was normalized to 100% with arbitrary units (au) to enable direct comparison between each sample. The 5′-dA produced during this reaction has an m/z of 251.1, which is only consistent with rVIP abstracting a deuteron from deuCTP and not acting on the deoxyCTP (that is, lack of m/z 250.1). h, i, Mass spectrum trace showing –m/z of 5′-dA (h) or the new product (i), formed by combining 100 μM rVIP with either 1 mM CTP (dotted blue trace) or 1 mM deuCTP (red trace). When rVIP was incubated with SAM and CTP deuterated at the 3′, 4′, 5′ and 5 positions (deuCTP), the −m/z of 5′-dA increased from 250.1 to 251.1, consistent with the transfer of one deuterium from deuCTP to 5′-dA•. When ddhCTP from the reaction was analysed by mass spectormetry, the product exhibited a −m/z of 468.1, indicating that the deuterium abstracted by 5′-dA during catalysis did not return to the product. The y-axis of each spectrum was normalized to 100% with arbitrary units (au) to enable direct comparison between each sample. These results have been repeated at least twice.

### Extended Data Fig. 4 Viperin abstracts the 4′-H from CTP.

ad, Using CTP with deuterium (2H denoted with a red D) incorporated at either the 2′-2H (a), 3′-2H (b), 4′-2H (c) or 5′-2H2 (d) (left column), we were able to monitor the loss of deuterium from the resulting product (middle column) and gain of a deuterium in the resulting 5′-dA (right column). The 5′-dA –m/z increases by one only in reactions containing CTP with a 4′-2H (c, right column). Natural abundance peaks are denoted with dashed vertical lines. All experiments were repeated twice.

### Extended Data Fig. 5 CMPK2 phosphorylates UDP or CDP and synthetic ddhC can be converted to ddhCTP by cellular machinery.

a, Formation of trinucleotide species (UTP, CTP or ddhCTP) from mono- and diphosphate species (1 mM UMP, UDP, CMP, CDP and ddhCDP) in the presence of either ATP or GTP as the phosphate donor by 5 μM hCMPK2. b, ddhCTP formation in HEK293T cells expressing either Flag–hVIP (N- or C-terminal tags), Flag–hVIP without the N-terminal amphipathic region (delta 1–42), hCMPK2 only, Flag–hVIP (N- or C-terminal tags) and hCMPK2, Flag–hVIP without the N-terminal amphipathic region (delta 1–42) and hCMPK2, a control plasmid or cells only. Only when the tag is on the N terminus of the full-length hVIP is ddhCTP produced at detectable levels. c, ddhCTP concentrations from HEK293T suspension cells that were incubated with synthetic ddhC (0 or 1 mM) for 24, 48 or 72 h (see Supplementary Information for details). d, ddhCTP concentrations from adherent Vero cells that were incubated with synthetic ddhC (0, 0.3 or 1 mM) for 24, 48 or 72 h (see Supplementary Information for details). All experiments were repeated once. nd, not detectable.

### Extended Data Fig. 6 Cellular concentrations of nucleotides are not affected by viperin expression.

ad, HEK293T cells expressing Flag–hVIP (aqua), Flag–hVIP and hCMPK2 (purple) or cells only (dark blue). Samples were taken at 16, 24, 48 and 72 hours post infection (h.p.i.). Extraction performed with a mixture of acetonitrile, methanol and water (40:40:20 and 0.1 M formic acid). Cellular concentrations were determined using 13C915N15–CTP, 13C1015N10–ATP, 13C10N5–GTP and 13C915N2–UTP spiked into the extraction mixture at known concentrations and using equations (1) and (2) in Supplementary Information. Analysis of nucleotides ATP (a), CTP (b), GTP (c) and UTP (d) did not show statistically significant differences (ns) between Flag–hVIP, Flag–hVIP and hCMPK2 or cells only for any time point. Data are from three biologically independent samples. Statistical significance was determined using a two-way ANOVA (Supplementary Tables 12, 13, 14 and 15). e, Ratio of cellular concentrations of ddhCTP to CTP from HEK293T cells expressing Flag–hVIP (aqua) or Flag–hVIP and hCMPK2 (purple); samples were taken at 16, 24, 48 and 72 h post transfection. The overall ratio of ddhCTP to CTP remains constant when only Flag–hVIP is expressed, but the concentration of ddhCTP is boosted significantly relative to CTP when both Flag–hVIP and hCMPK2 are co-expressed (plots are derived from data shown in c and Fig. 3a).

### Extended Data Fig. 7 Nucleotide concentrations are not affected during ddhCTP production.

a, b, Concentrations of ddhCTP (a) and CTP, UTP and ATP (b) in immortalized macrophage cells (RAW 264.7) grown in serum-free medium in the presence of increasing concentrations of murine IFNα (10 ng ml−1, 50 ng ml−1 and 250 ng ml−1). Data are from two biologically independent samples.

### Extended Data Fig. 8 ddhCTP is used as a substrate by dengue virus and WNV RdRp and chain terminates RNA synthesis.

a, Schematic of primer-extension assay for evaluating dengue virus and WNV RdRp activity. b, Dengue virus RdRp-catalysed nucleoside incorporation using CTP, 3′-dCTP or ddhCTP as nucleoside triphosphate substrates. Some full-length product was observed in the presence of ddhCTP (more than 99% pure), which is due to residual contaminating CTP that could not be removed. c, Reaction products resolved by denaturing PAGE containing 40% formamide showing the trace amount of CTP contaminate in the ddhCTP preparation. These experiments were repeated independently at least four times with similar results. d, Longer incubation times and more dengue virus RdRp enzyme does not increase the yield of extended product. e, Dengue virus RdRp-catalysed nucleoside incorporation with increasing concentrations of ddhCTP (0, 1, 10, 100, 200 and 750 μM) at varying concentrations of CTP. This experiment was repeated independently three times with similar results. f, Plot of the percentage inhibition as a function of ddhCTP concentration at varying concentrations of CTP. Data were fit to a dose–response curve to obtain half-maximum inhibitory concentration (IC50) values of ddhCTP of 60 ± 10, 120 ± 20, 520 ± 90 and 3,900 ± 700 μM at 0.1, 1, 10 and 100 μM CTP, respectively. This experiment was repeated at least three times with similar results. The total sample size is 24. The error reported is the standard error from the fit of the data to a dose–response curve. g, Plot of IC50 values as a function of CTP concentration. The data were fit to a line with a slope of 38 ± 1 and an intercept of 91 ± 25. The error reported is the standard error from the fit of the data to a line. h, WNV RdRp-catalysed nucleoside incorporation with increasing concentrations of ddhCTP (0, 1, 10, 100, 200 and 750 μM) at varying concentrations of CTP. This experiment was repeated at least three times with similar results. i, Plot of the percentage inhibition as a function of ddhCTP concentration at varying concentrations of CTP. Data were fit to a dose–response curve to obtain IC50 values of ddhCTP of 20 ± 10, 70 ± 10, 300 ± 40 and 2,700 ± 300 μM at 0.1, 1, 10 and 100 μM CTP, respectively. The total sample size is 24. The error reported is the standard error from the fit of the data to a dose–response curve. j, Plot of IC50 values as a function of CTP concentration. The data were fit to a line with a slope of 27 ± 1 and an intercept of 31 ± 8. Both of these results demonstrate that once ddhCMP is incorporated, it effectively terminates synthesis and that the small amount of extended product is from a trace amount of CTP contamination. The error reported is the standard error from the fit of the data to a line.

### Extended Data Fig. 9 HRV-C and poliovirus RdRp are poorly inhibited by ddhCTP.

a, Schematic of primer extension assay for evaluating HRV-C RdRp activity. b, HRV-C RdRp-catalysed nucleoside incorporation using CTP, 3′-dCTP or ddhCTP as nucleoside triphosphate substrates. These experiments were repeated independently at least four times with similar results. c, Increasing concentrations of ddhCTP does not efficiently inhibit HRV-C RdRp-catalysed RNA synthesis. HRV-C RdRp-catalysed nucleoside incorporation in the presence of increasing concentrations of either ddhCTP or 3′-dCTP. These experiments were repeated independently at least five times with similar results. d, Plot of the percentage inhibition as a function of either ddhCTP or 3′-dCTP concentration. Data were fit to a dose–response curve to obtain IC50 values of 900 ± 300 μM for ddhCTP and 5 ± 1 μM for 3′-dCTP. The total sample size is eight. The error reported is the standard error from the fit of the data to a dose–response curve. e, f, HRV-C (e) and poliovirus (f) RdRp-catalysed nucleoside incorporation with increasing concentrations of ddhCTP (0, 1, 10, 100 and 200 μM) at varying concentrations of CTP. Reactions were performed with the trinucleotide primer, 5′-pGGC, and 20-nt RNA template as described for dengue virus and WNV RdRp to directly compare results with HRV-C and poliovirus RdRp. At the highest concentration of ddhCTP, only approximately 2% inhibition was observed for HRV-C RdRp at 0.1 and 1 μM CTP. The IC50 values at 0.1 and 1 μM CTP are estimated to be approximately 10,000 and 20,000 μM ddhCTP, respectively. These values are three orders of magnitude higher than those obtained for dengue virus and WNV RdRp. Reactions in the presence of 3′-dCTP (200 μM) are shown as a control for inhibition. These experiments were repeated independently at least four times with similar results. g, Efficiency of incorporation and inhibition of viral RdRps. Footnotes: aCalculated for ddhCTP in direct competition with CTP (800 μM) using the linear equations obtained from the fit of the data shown in g and j. For HRV-C, the IC50 value was estimated to be two orders magnitude greater than that calculated for dengue virus and WNV RdRps as evidenced from the data shown in d. bCalculated for a ddhCTP concentration of 350 μM using the following equation: probability = [ddhCTP]/([ddhCTP] + IC50). cCalculated using the following equation: $${\rm{full\; -\; lengthgenome( \% )}}=100\times {(1-{\rm{probability}})}^{{{\rm{C}}}_{n}}$$; in which Cn is the number of cytidine residues in the viral genome with values of 2,200, 2,497, 1,565 and 1,737 for dengue virus, WNV, HRV-C and poliovirus respectively.

### Extended Data Fig. 10 ddhC reduces virus release of three different ZIKV isolates.

Vero cells were treated with different concentrations of ddhC (0, 0.1 and 1 mM) for 24 h. After this 24-h period, the medium over cells was removed and cells were infected with fresh medium that contained the original concentrations of ddhC (0, 0.1 and 1 mM) and one of three strains of ZIKV; African strain MR766 (Uganda 1947), PRVABC59 (Puerto Rico; 2015) or R103451 (Honduras; 2016, GenBank: KX262887). After three hours of ZIKV infection, virus inoculum was removed and cells were treated with fresh medium that contained the original concentrations of ddhC (0, 0.1 and 1 mM). Virus samples were collected and the medium over cells was replaced with fresh medium that contained the original concentrations of ddhC at 24, 48 and 72 h.p.i. Viral titres at 24, 48 and 72 h.p.i. were determined using the plaque assay. ac, Effect of ddhC on three different ZIKV isolates: MR766 (Uganda 1947) (a), PRVABC59 (Puerto Rico; 2015) (b) or R103451 (Honduras; 2016) (c). Analysis of ZIKV titres indicates that 1 mM ddhC inhibits all three ZIKV isolates compared to 0 mM ddhC. However, reduction in virus titre is more prominent at 24 h.p.i. and 48 h.p.i. compared to 72 h.p.i. when using an MOI of 1.0. The antiviral effect of ddhC is more prominent at an MOI of 0.1. Data are mean ± s.d. from three biologically independent samples, P values from a two-way ANOVA with Dunnett’s post hoc analysis.

## Supplementary information

### Supplementary Information

This file contains Supplementary Text and Tables used to generate the data for this study, which includes a list materials, sequences of primers, genes and proteins, LC-MS methods, NMR chemical shift table, and raw output from statistical analyses.

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Gizzi, A.S., Grove, T.L., Arnold, J.J. et al. A naturally occurring antiviral ribonucleotide encoded by the human genome. Nature 558, 610–614 (2018). https://doi.org/10.1038/s41586-018-0238-4

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• DOI: https://doi.org/10.1038/s41586-018-0238-4

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