Article Prokaryotic viperins produce diverse antiviral molecules https://doi.org/10.1038/s41586-020-2762-2 Aude Bernheim1, Adi Millman1, Gal Ofir1, Gilad Meitav1, Carmel Avraham1, Helena Shomar2, Masha M. Rosenberg2, Nir Tal2, Sarah Melamed1, Gil Amitai1 & Rotem Sorek1 ✉ Received: 25 February 2020 Accepted: 27 August 2020 Viperin is an interferon-induced cellular protein that is conserved in animals1. It Published online: xx xx xxxx has previously been shown to inhibit the replication of multiple viruses by producing Check for updates the ribonucleotide 3′-deoxy-3′,4′-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase2. Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modifed ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems. Viperin is an antiviral protein that becomes highly expressed in cells genes involved in defence systems is a strong predictor that the genes in stimulated by interferons1. In humans, this protein has broad antiviral the cluster have a role in phage resistance8,9. We denoted the genes in the activity against DNA and RNA viruses, including human cytomegalovi- defensive cluster pVips (for prokaryotic viperin homologues). As pVips rus, West Nile virus, dengue virus, hepatitis C virus and HIV1,3. Viperin are relatively rare in prokaryotic genomes (164 genes in the cluster), we was recently shown to catalyse the conversion of CTP to ddhCTP2. This performed an online homology search with additional genomes that modified nucleotide lacks a hydroxyl group at the 3′ carbon of the were not included in our original database, retrieving 86 additional ribose, and when the viral polymerase incorporates it into the nas- such genes, resulting in a total of 250 pVips (Supplementary Table 2). cent chain of the viral RNA, it acts as a chain terminator that does not To determine whether pVips can defend against phages, we selected allow further polymerization of the RNA chain2. Thus, ddhCTP directly 59 genes that span the space of the pVip sequence diversity (Supple- inhibits the RNA-dependent replication of RNA viruses such as Zika mentary Table 2) and cloned them in Escherichia coli under the con- virus in vivo. trol of an inducible promoter. Similarly, we also cloned GFP and the Some bacteria and archaea encode genes that have marked sequence MoaA gene from E. coli as negative controls. We then challenged the similarity to vertebrate viperins, although their roles have remained pVip-expressing bacteria with an array of phages spanning several unknown4–6. We set out to examine whether prokaryotic homologues major phage families (Myoviridae: P1; Siphoviridae: lambda, SECphi6, of human viperin participate in defence against phages. We first per- SECphi18 and SECphi27; Podoviridae: T7; Leviviridae: MS2 and Qβ) formed a profile-based search for viperin homologues in a database of (Fig. 1b, c, Extended Data Fig. 1). more than 38,000 bacterial and archaeal genomes. This search yielded Around half of the tested pVips conferred clearly identifiable activ- 1,724 genes (1,112 non-redundant sequences) homologous to the human ity against phages. Most of these protected against T7, as evidenced viperin gene (RSAD2), which we aggregated into 17 clusters on the basis by plaque assays (up to tenfold reduction in T7 plaque sizes) (Fig. 1b) of sequence similarity (Methods) (Supplementary Table 1). and by a delay or absence of culture collapse in T7 infection assays in Viperin is a member of the radical S-adenosyl-methionine (SAM) liquid culture (Fig. 1c, Extended Data Fig. 2a). Mutations in the cysteine family of enzymes, and shows both sequence and structural homology residues predicted to coordinate the iron–sulfur cluster in the Cxxx- to other members of that family, particularly the housekeeping molyb- CxxC motif in pVips resulted in loss of defensive capacity against T7, denum cofactor biosynthesis enzyme MoaA4. To differentiate between suggesting that the catalytic activity of pVips is necessary for defence viperin homologues with housekeeping properties and homologues (Extended Data Fig. 2b). A subset of the pVips also protected against that may participate in defence against phages, we took advantage of P1, lambda, SECphi6 and SECphi18 phages, reducing the observed the fact that, in prokaryotes, genes involved in antiviral activity tend number of plaques by between 10-fold and 10,000-fold (Extended to colocalize next to one another on the genome, forming ‘defence Data Fig. 1). Of note, when the human viperin gene was cloned and islands’7,8. Most clusters of viperin homologues did not show a tendency expressed in E. coli under the same conditions, it protected against T7 to colocalize with defence genes (Supplementary Table 1). However, infection in a similar manner to that observed for many pVips (Fig. 1, in one of the clusters, 60% of the genes were found in the vicinity of Extended Data Figs. 1, 2). CRISPR–cas systems, restriction-modification systems and other bacte- The pVips that we identified are present in phylogenetically dis- rial defence genes (Fig. 1a). This high propensity for colocalization with tant organisms, suggesting an ancient evolutionary origin, frequent 1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel. 2Pantheon Biosciences, Yavne, Israel. ✉e-mail: [email protected] Nature | www.nature.com | 1 Article a Homologues of human viperin pVip2 Type I RM Type IV RM Nucleotide kinase TA TA Chryseobacterium gambrini DSM 18014 Ga0111674_101 10k 20k 30k pVip8 CRISPR–cas type III Psychrobacter lutiphocae DSM 21542 H140DRAFT_ scaffold00013.13 12k 22k 32k pVip68 Type I RM Nucleotide kinase Transposase Vibrio vulnificus YJ016 chromosome I: NC_005139 2206k 2196k pVip147 Type I RM TA Pseudomonas sp. 1-7 Ga0057553_134 153k 163k 173k pVip208 Nucleotide kinase Gabija Aliivibrio sp. 1S128 Ga0166917_1058 133k 143k 153k b 140 ******* *** * ** * * 120 100 80 60 40 T7 plaque size (mm) 20 0 GFP MoaA pVip6pVip7pVip8pVip9pVip1pVip120 pVip15pVip19pVip21pVip27pVip32pVip34pVip3pVip397 pVip42pVip4pVip464 pVip47pVip48pVip50pVip54pVip5pVip576 pVip58pVip6pVip610 pVip6pVip632 Human viperin c Human Viperin pVip9 pVip21 pVip34 Homo sapiens Vibrio porteresiae Lewinella persica Cryomorphaceae bacterium 0.8 0.6 0 0.4 OD60 0.2 0.0 0 100200 0 100200 0 100200 0 100200 Time post infection (min)Time post infection (min)Time post infection (min)Time post infection (min) Control uninfected Control infected Vip uninfected Vip infected Fig. 1 | pVips and human viperin exhibit antiviral activity in bacteria. viperin were grown on agar plates and phage lysate was added onto the plates. a, Representative instances of pVip genes and their genomic neighbourhoods. Graphs show mean of three replicates with individual data points overlaid. Homologues of human viperin are shown in red, genes annotated as nucleotide Asterisks indicate statistically significant differences compared with the GFP kinase are brown, genes known to be involved in defence are yellow and genes negative control. Two-sided t-test; *P < 0.0001 except for pVip46 (P = 0.0034)). with mobile genetic elements are dark grey. RM, restriction modification; TA, c, Growth curves for E. coli strains expressing viperins that were infected with toxin–antitoxin; Gabija is a recently described defence system8. The bacterial phage T7. Negative controls are GFP-expressing cells and are the same in all species, and the accession of the relevant genomic scaffold in the Integrated four graphs. Each growth curve represents the mean of 3 biological replicates, Microbial Genomes (IMG) database20, are indicated on the left. b, Sizes of each with an average of 2 technical replicates, and the shaded area corresponds plaques caused by T7 phage infecting E. coli strains that express different to the 95% confidence interval. viperins. Bacteria expressing pVips, negative controls (GFP or MoaA) or human horizontal gene transfer or both. We found pVips in 176 species overall, during the interferon response2,10. This kinase phosphorylates cyti- belonging to 14 bacterial and archaeal phyla that include Proteobacte- dine monophosphate to CTP, thus generating the substrate for viperin ria, Firmicutes, Cyanobacteria, Actinobacteria, Bacteriodetes, Euryar- activity2. We found that 47 of the 250 pVips (19%) genes were adjacent chaeota and others (Supplementary Table 2). To better understand their to genes annotated as a nucleotide kinase in their genome of origin diversity and phylogenetic distribution, we generated a phylogenetic (Fig. 1a, Fig. 2) and that, in some cases, the kinase was fused to the pVip tree of the viperin family, including pVips and eukaryotic viperins (Fig. 2, gene (Fig. 2, Supplementary Table 2). This further strengthens the Supplementary Table 3). MoaA genes from bacteria and eukaryotes hypothesis that the pVip substrate is a triphosphorylated nucleotide. were added to the tree as an outgroup. We found that pVips are grouped Whereas some pVip-associated kinases were annotated as cytidylate into seven major