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Homologous recombination is an intrinsic defense against antiviral RNA interference

Lauren C. Aguadoa, Tristan X. Jordana, Emily Hsieha, Daniel Blanco-Meloa, John Hearda, Maryline Panisa, Marco Vignuzzib, and Benjamin R. tenOevera,1

aDepartment of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and bViral Populations and Pathogenesis Unit, Institute Pasteur, 75015 Paris, France

Edited by Michael B. A. Oldstone, The Scripps Research Institute, La Jolla, CA, and approved August 16, 2018 (received for review June 14, 2018) RNA interference (RNAi) is the major antiviral defense mechanism . The RNAi machinery was evolutionarily repurposed of and invertebrates, rendering the capacity to evade it a separately in both the and animal lineages to generate defining factor in shaping the viral landscape. Here we sought to microRNAs (miRNAs) (16). A central difference between these determine whether different replication strategies provided two systems, apart from their general function in the , is the any inherent capacity to evade RNAi in the absence of an antagonist. source of the small RNA. In contrast to being of viral origin, as is Through the exploitation of host microRNAs, we recreated an RNAi- the case for antiviral RNAi, miRNAs derive from - like environment in vertebrates and directly compared the capacity encoded hairpins and are generally used to control of positive- and negative-stranded RNA to cope with this and lineage fate (17). As miRNAs do not engage their target selective pressure. Applying this defense against four distinct with perfect complementarity, cleavage does not occur and thus viral families revealed that the capacity to undergo homologous transcriptional regulation is subtle. While this dynamic is not recombination was the defining attribute that enabled evasion of potent enough to be utilized during an acute infection, the long- this defense. Independent of gene expression strategy, positive- term consequences that miRNA-based regulation can impose stranded RNA viruses that could undergo strand switching rapidly make miRNAs an ideal platform for coordinating developmental excised genomic material, while negative-stranded viruses were processes. A reflection of this biology can be inferred from the effectively targeted and cleared upon RNAi-based selection. fact that of the four Argonaut (Ago) effector involved These data suggest a dynamic relationship between host an- in vertebrate miRNA biology, only Ago2 has retained the capacity for tiviral defenses and the biology of virus replication in shaping pathogen prevalence. double-stranded RNA cleavage (17). Ago2 activity and the expression of miRNAs, however, can be exploited by introducing a perfect RNAi | virus polarity | virus | homologous recombination | binding site into a target transcript to enable cleavage (18). Using this miRNA system, which mimics the RNAi response of invertebrates and plants, we set out to determine how different viruses would adapt to evade the same selective pressure in the absence of an antagonist. he capacity to elicit a defense to foreign material is an es- Tsential attribute to maintain life. With respect to antiviral Results defenses, numerous strategies have evolved to inhibit virus rep- Engineering an Artificial Antiviral System. The combination of sheer lication. In and , viruses are subjected to a diversity, rapid mutation, and the evolutionary time scale of life combination of restriction enzymes and clustered regularly inter- on the planet severely limits the capacity to deduce the spaced short palindromic repeat (CRISPR) systems, in addition to other, less defined defense platforms (1, 2). These defenses pre- dominantly target foreign DNA reflecting the composition of Significance the majority of prokaryotic viruses (3, 4). In plants and inverte- brates, the most dominant antiviral defense comes in the form of In an effort to determine whether host defenses can signifi- RNA interference (RNAi) although additional strategies are also cantly influence the prevalence of different virus groups, we employed (5). Despite no direct link between the CRISPR- and applied identical selective pressures onto four families of di- RNAi-based systems, in both defenses the host acquires genetic verse viruses. Using an RNAi-like defense, we found that the material from the virus and uses it to provide specificity to an capacity of positive-stranded RNA viruses to switch genomic otherwise nonspecific . templates during replication conferred an adaptability that In contrast to the small RNA-based defenses of , exceeded that of negative-stranded RNA viruses that were less plants, and invertebrates, vertebrates combat viruses using a able to perform this biology. Together, this work suggests that protein-based strategy referred to as the type I and type III IFN in the absence of an antiviral antagonist, fundamental aspects systems (6, 7). Also relying on unique aspects of virus replication of virus biology can provide an inherent advantage to evade to achieve specificity, the IFN response is initiated following the host defenses. engagement of so-called pattern recognition receptors and the Author contributions: L.C.A., T.X.J., E.H., and B.R.t. designed research; L.C.A., T.X.J., E.H., transcriptional induction of a family of IFN cytokines (5). IFN is J.H., and M.P. performed research; D.B.-M. contributed new reagents/analytic tools; secreted into the extracellular milieu and signals in both an L.C.A., T.X.J., D.B.-M., and B.R.t. analyzed data; and L.C.A., T.X.J., M.V., and B.R.t. wrote autocrine and paracrine manner to induce hundreds of host the paper. MICROBIOLOGY genes that slow virus replication by inhibiting aspects of tran- The authors declare no conflict of interest. scription, translation, metabolism, cell transport, and apoptosis This article is a PNAS Direct Submission. (5). While some sequencing data have suggested an RNAi-like Published under the PNAS license. – defense may be present at low levels in vertebrates (8 10), Data deposition: The data reported in this paper have been deposited in the Gene Ex- functionally RNAi and IFN have been found to be incompatible pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE108995). with each other, suggesting RNAi plays a minor physiological 1To whom correspondence should be addressed. Email: [email protected]. role in reducing viral replication (11–15). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Even with this notable absence, the biology of small RNA- 1073/pnas.1810229115/-/DCSupplemental. mediated control remains ubiquitous in almost all eukaryotic Published online September 12, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1810229115 PNAS | vol. 115 | no. 39 | E9211–E9219 Downloaded by guest on October 2, 2021 of viruses (19). While a direct phylogeny of viruses may never be needed to induce silencing (24, 25). Of note, no IFN signature known, it is clear that the prokaryotic cellular environment fa- genes were implicated in this screen, suggesting that this selective vored the expansion of DNA viruses, whereas in , pressure was based entirely on RNAi activity. RNA viruses flourished (20). These general trends suggest that the defenses of the host may inherently bias the diversity of the RNAi Can Effectively Neutralize Negative-Stranded Virus Replication. viruses that impact them. To explore this idea, we wanted to Given that our RNAi system appeared both potent and in- design a selective pressure that could be imposed equally onto dependent of the canonical IFN response, we next decided to different viruses to ascertain whether there are inherent repli- impose this selective pressure onto an essential component of cation attributes that enable more efficient evasion mechanisms. the virus. To this end, we moved the 5T and 5R cassettes from For this reason, we chose to exploit host miRNA expression in EGFP to the 3′ UTR of the (N) transcript of mammalian cells to recreate an RNAi-like response against Sendai virus (Fig. 2A). Infection of SeV-5T and -5R demon- viruses that would lack antagonists to this defense. We chose this strated no discernible difference in their capacity to replicate in selective pressure because use of small in antiviral de- silencing-deficient cells as measured by SeV-N protein levels fenses is a shared attribute in all but vertebrate life and because (Fig. 2B). In contrast, infection of SeV-5T and -5R in silencing- miRNAs do not possess the significant complementarity with any enabled cells demonstrated a complete loss of NP protein se- known virus necessary to induce silencing (15). lectively from the SeV-5T strain (Fig. 2C). Consistent with these To this end, we first verified the ubiquitous expression of five data, passaging experiments demonstrated that within 24 h (passage miRNAs in mammalian cells, notably miR-21–5p, miR-31–5p, one, P1), SeV titers were reduced by more than 5 logs and were miR-192–5p, miR-93–5p, and miR-29b-3p (SI Appendix, Fig. undetectable by P4 (Fig. 2D). In contrast, SeV-5R titers demon- S1A). We next engineered a cassette containing target sites with strated robust replication throughout the duration of the passages. perfect complementarity for each miRNA, and cloned this into These data could be further characterized by deep sequencing (Fig. the 3′ untranslated region (UTR) of enhanced green fluorescent 2 E–H). RNA sequencing of SeV-5R in both silencing-deficient protein (herein referred to as pEGFP-5T) (SI Appendix, Fig. and -enabled cells revealed no differences in coverage of the viral S1B). To test targeting of this cassette, we transfected pEGFP- genome or abundance of viral reads with variants 5T into RNaseIII-deficient fibroblasts (21) (herein referred to as appearing only in the 3′ UTR of each transcript at the site of silencing-deficient cells) and introduced miRNA mimetics in- polyadenylation (Fig. 2 E and F). Similarly, silencing-deficient dividually or as a pool. Analysis of EGFP expression by flow cells infected with SeV-5T demonstrated the same genomic cov- cytometry demonstrated that, compared with miR-1, which was erage and alignment profiles to SeV-5R (Fig. 2G). In striking not represented in the 5T cassette, each individual mimic was contrast, silencing-enabled cells were able to completely suppress sufficient to induce miRNA-mediated silencing (SI Appendix, all replication of SeV-5T, demonstrating only two reads that Fig. S1B). Moreover, this construct could additionally be si- aligned to the GFP ORF, corroborating the inability of SeV-5T to lenced in the presence of endogenous miRNAs in wild-type escape our RNAi-like pressure (Fig. 2 D and H). (WT) fibroblasts (herein referred to as silencing-enabled cells) To determine whether this phenotype was specific to para- (SI Appendix, Fig. S1C). myxoviruses, we also inserted the 5T and 5R cassettes into the 3′ UTR of segment five (encoding NP) of A virus (IAV) (SI RNAi-Mediated Selective Pressure Is IFN Independent. Having dem- Appendix,Fig.S2A). In silencing-deficient cells, TCID50 (tissue cul- onstrated that the inclusion of the 5T cassette mimicked an ture infectious dose to kill 50% of infected cells) titers of IAV-5T RNAi-like response, we next sought to determine whether it and -5R showed no significant difference (SI Appendix,Fig.S2B). In could be used to impose positive selection independent from the contrast, IAV replication in silencing-enabled cells demonstrated canonical IFN-I system and free from viral antagonism. To this robust titers of IAV-5R in contrast to IAV-5T, where infectious units end, we subcloned EGFP-5T into Sendai virus as an independent could only be observed in the initial 24 h and at levels 5 logs lower transcript (SeV-GFP-5T). As a control, we additionally made a than -5R (SI Appendix,Fig.S2B). These data could be further cor- construct in which the 3′ UTR of EGFP was composed of the roborated at the protein level in which IAV-5T NP production was same sequence but in the reverse orientation, rendering EGFP completely inhibited in silencing-enabled cells, with no indication of immune from miRNA-mediated repression (herein denoted as the cytopathic effects observed at 48 h postinfection (hpi), in stark SeV-GFP-5R) (Fig. 1A). To ascertain whether EGFP expression contrast to IAV-5R (SI Appendix,Fig.S2C and D). Overall effects was sensitive to the miRNA machinery in the context of virus of targeting on IAV-5T replication could also be observed by total infection, we infected wild-type murine fibroblasts as well as read count from RNA-Seq data with no indication that the tar- − − − − knockout cells lacking Dicer (Dcr / ), Ago2 (Ago2 / ), and geting cassette was excised to yield an escape mutant (SI Appendix, − − Ago1/3/4 (Ago1/3/4 / ). These data revealed fluorescence and Fig. S2E). Together, these data demonstrate the potency of RNAi hemagglutinin-neuraminidase (HN) expression in all genotypes in its capacity to silence RNA viruses of negative polarity. in response to SeV-GFP-5R, whereas EGFP expression was lim- − − − − ited to Dcr / and Ago2 / for SeV-GFP-5T (Fig. 1B). These data RNAi-Based Selective Pressure Induced Rapid Escape of Positive- suggest that a functional Ago2-based miRNA system is required Stranded RNA Viruses. To determine whether the difficulty in for efficient silencing which could be further confirmed by flow evading an RNAi response was equally evident in viruses of cytometry in both murine (Fig. 1C) and human fibroblasts (Fig. 1D). positive polarity we next imposed the same selective pressure on We next sought to define the central components required for Sindbis virus (SINV), an and a member of the targeting. To this end, we performed a whole-genome CRISPR Togaviridae family (26). To this end, we inserted the silencing screen on SeV-GFP-5T by selecting for cells in which silencing of cassette into an intergenic region between the nonstructural and the SeV-derived EGFP-5T had been disrupted (Fig. 1D). Cells structural transcripts in both forward (5T) and reverse (5R) expressing EGFP following infection of a previously established orientations (Fig. 3A). As this insertion site was in the proximity CRISPR cell library were selected and their gRNAs sequenced of the subgenomic of SINV (27, 28), we recreated the (22). Model-based analysis of the genome-wide CRISPR/ promoter downstream of the cassette to ensure normal pro- knockout (MAGeCK) successfully identified the canonical duction of the structural transcripts. Infection of silencing- members of the miRNA machinery including: Drosha, Dicer, deficient cells with either SINV-5T or -5R revealed compara- Ago2, and DGCR8 (Fig. 1E and SI Appendix, Table S1) (23). In ble replication kinetics between the two viruses as determined addition, the screen implicated p53 (TP53), miR-21, and XPO5, by Western blot (Fig. 3B). When silencing was enabled, we which would all impact the available cytoplasmic pool of miRNAs observed an initial delay in expression of the 5T virus,

E9212 | www.pnas.org/cgi/doi/10.1073/pnas.1810229115 Aguado et al. Downloaded by guest on October 2, 2021 Fig. 1. The 5T cassette only restricts through miRNA machinery. (A) Diagrams of recombinant SeV-eGFP-5T and SeV-eGFP-5R . The 5T (depicted with miRNA binding) and 5R (resistant to miRNA binding) cassettes are downstream of the GFP ORF, which is inserted between nucleoprotein and phosphoprotein ORFs. (B) Immunofluorescence microscopy of SeV-GFP-5T and -5R–infected MEFs. WT MEFS or Dicer (Dcr), Argonaute 2, or Argonaute 1, 3, and 4 knockout MEFs were infected at a MOI of 1 for 24 h. Cells were fixed and stained for SeV HN protein and DAPI. (C) Flow cytometry analysis of MEFs infected as stated in B.(D) FACS analysis of CRISPR KO library infected with SeV-GFP-5R or SeV-GFP-5T. Cells gated on FITC expression. (E) MAGeCK analysis of GFP+ CRISPR KO A549 cells infected with SeV-GFP-5T (D).

although in contrast to both SeV and IAV, the targeted SINV replication using deep sequencing (Fig. 3 D and E). Initial reached similar levels as early as 6 hpi (Fig. 3C). characterization of SINV-5R in silencing-enabled cells demon- To ensure that our selective pressure was functional in the strated the insertion of the genetic cassette was not overtly context of SINV infection, we next generated a SINV recombi- detrimental to the virus and appeared stable over 96 h of con- nant virus that expressed the EGFP-5T cassette under the con- tinuous replication (Fig. 3D). Moreover, we did not observe any trol of an extrasubgenomic promoter (SI Appendix, Fig. S3A). dramatic changes to the relative abundance of viral reads or the MICROBIOLOGY This virus construct, when used to infect silencing-deficient cells, appearance of minority variants. In contrast to -5R, the same demonstrated robust fluorescence as expected (SI Appendix, Fig. passaging experiment with SINV-5T showed the emergence of a S3B). In contrast, infection of silencing-enabled cells showed a single dominant species that had excised the 5T cassette but complete absence of EGFP fluorescence allowing us to conclude retained a fully functional subgenomic promoter by P4 compared that RNAi was functioning in the context of SINV infection de- with P0 (Fig. 3E). Interestingly, the selective pressure of RNAi spite the replication levels of SINV-5T in silencing-enabled cells. also resulted in a higher frequency of three residues To better understand how the 5T cassette could effectively (K232T, N370K, and G595V) that arose in three independent silence EGFP and the early production of capsid expression, we experiments (depicted as orange peaks in the nonstructural pro- serially passaged the SINV-5R and -5T variants and analyzed teins nsP1 and nsP2).

Aguado et al. PNAS | vol. 115 | no. 39 | E9213 Downloaded by guest on October 2, 2021 Fig. 2. SeV-5T lacks the capacity to adapt to a silencing-enabled environment. (A) Diagram of recombinant SeV-5T where the 5T cassette is downstream of the nucleoprotein (N) ORF and the ORF encoding GFP is inserted between N and the phosphoprotein (P). All other viral proteins and miRNA targets are also depicted. (B) Western blot of whole-cell extracts from silencing-deficient cells infected with either SeV-5T or SeV (MOI = 0.01) at 12, 24, 36, and 48 hpi. Immunoblots depict the levels of N and GAPDH. (C) Western blot of whole-cell extracts as in B from silencing-enabled cells infected with either SeV-5T or SeV- (MOI = 0.01) at 12, 24, 36, and 48 hpi. (D) Infectious virus released by silencing-deficient and silencing-enabled cells infected with SeV-GFP-5T or 5R (MOI 1) after passage 1 (black) and passage 4 (gray). (E and F) Relative read numbers (red) and minor variants (orange) plotted along the SeV-5R genome from silencing-deficient cells (E) or silencing-enabled (F) cells at 48 hpi. (G and H) Relative read numbers (red) and minor variants (orange) plotted along the SeV-5T genome from silencing-deficient (G) or silencing-enabled (H) cells at 48 hpi.

E9214 | www.pnas.org/cgi/doi/10.1073/pnas.1810229115 Aguado et al. Downloaded by guest on October 2, 2021 Fig. 3. SINV-5T rapidly adapts to RNAi-mediated silencing. (A) Diagrams of wild-type SINV (Top) and recombinant SINV-5T/-5R (Bottom), where the 5T cassette is downstream of the nonstructural ORF. (B) Western blot of whole-cell extracts from silencing-deficient cells infected with either SINV-5T or SINV-5R (MOI = 0.1) at 6, 12, 24, and 36 hpi. Immunoblots depict protein levels for SINV capsid (C or CP) and GAPDH. (C) Western blot as in B from silencing-enabled cells infected with either SINV-5T or SINV-5R (MOI = 0.1) at 6, 12, 24, and 36 hpi. (D) Relative read numbers (red) and minor variants (orange) from SINV-5R at 24 hpi (P0) and (P4) at 96 hpi in silencing-enabled cells. x axis denotes genomic alignment position. (E) Same as D from SINV-5T–infected silencing-enabled MICROBIOLOGY cells.

To better understand the dynamics of SINV-5T infection suggest that the 90% of nonmodified full-length genome during P0, we searched for gapped alignments (SI Appendix, Fig. would still contribute wild-type levels of the structural mRNA. S3C). While ∼90% of the virus remained intact, three distinct In contrast, the 10% of excision events observed, while dis- excision events accounted for the remaining 10% of viral rupting the capacity to generate the structural transcripts, RNA. As the 5T cassette was engineered to selectively would generate untargeted nonstructural mRNA (SI Appen- silence the nonstructural polyprotein, this RNA profile would dix,TableS2).

Aguado et al. PNAS | vol. 115 | no. 39 | E9215 Downloaded by guest on October 2, 2021 Next, we sought to ascertain whether the behavior of SINV-5T viruses of positive polarity that have been found to enable ex- could be applied to other positive-stranded RNA viruses. To this change of genetic material through polymerase-mediated template end, we applied the 5T cassette to a variant of Semliki Forest switching (31, 32). This has been particularly well characterized with virus (SFV) in which the endogenous structural proteins had respect to poliovirus (33–36). In an effort to phenocopy a virus of been replaced with the glycoprotein (G) of vesicular stomatitis negative polarity onto a positive-stranded virus such as polio, we virus (VSV) (29) (SI Appendix,Fig.S4A). We chose this recombi- utilized a well-characterized RdRp mutant incapable of homolo- nant chimeric virus (herein referred to as SFVG) because its repli- gous recombination (36). This poliovirus variant harbors a D79H cation rate was slower than SINV and allowed us to ascertain mutation in the 3D RdRp gene that has been shown to be incapable whether this attribute contributed to the inability of SeV- and IAV- of mediating a rescue of two nonreplicative genome segments. We 5T to escape. As such, we inserted the 5T or 5R cassettes into the 3′ engineered this mutation into the PV-GFP-5T backbone to gauge UTR of the nonstructural polyprotein transcript and initially com- the necessity for this activity in escaping RNAi. pared virus replication in silencing-deficient cells (SI Appendix,Fig. To this end, RNA for PV-GFP-5T or PV-GFP-5TD79H (herein S4B). Using VSV G protein expression as a readout for viral levels, referred to as PV-5T and PV-5TD79H) was transfected into we found no discernible difference between 5T and 5R variants, silencing-enabled or -deficient cells and supernatants were pas- although the slow kinetics of replication were apparent, as glyco- saged. We titered the supernatants from passages 0 and 4 and protein expression was not visible until 36 hpi. In contrast, infection performed RNA-Seq to monitor the virus populations. In silencing- of the 5T and 5R SFVG variants in silencing-enabled cells dem- deficient cells, PV-5T and PV-5TD79H showed comparable titers at onstrated a dramatic difference in glycoprotein levels during the first both P0 and P4 (Fig. 4B). Sequencing these viruses at the end of 36 h of infection with no detectable evidence of 5T replication (SI passaging demonstrated that the D79H mutant was stable and also Appendix,Fig.S4C). However, by 48 hpi, the 5T virus became de- indistinguishable from WT with regards to minority variants in the tectable, suggesting that, similar to SINV, an escape variant population (Fig. 4C). The only consistent minority variant observed had arisen. under these conditions were three nucleotide substitutions within To further discern the molecular dynamics of SFVG escape, the 2A cleavage sites that flank the GFP ORF. we serially passaged both the SFVG-5T and -5R variants in In contrast to the relatively homogenous population of PV-5T silencing-enabled cells. Similar to SINV, passaging of SFVG-5R and PV-5TD79H in silencing-deficient cells, when this same ex- demonstrated no excision event throughout the genome after a periment was performed in wild-type cells, PV-5TD79H titers × – combined 192 h of replication (4 48 h passages; P0 P4) (SI were undetectable by P4 in contrast to PV-5T (Fig. 4 B–D). In Appendix, Fig. S4D). However, P4 did show multiple variants agreement with the SINV and SFVG data, PV-5T sequencing throughout the genome even in the absence of positive selection, revealed an excision of the 5T cassette explaining how replica- likely a product of its relative lack of adaptation, a phenomenon tion persisted beyond P4 (Fig. 4D). However, sequencing of PV- that has been previously documented (30). 5TD79H resulted in very poor genomic coverage, consistent with When this same methodology was applied to SFVG-5T, we the inability to detect infectious virus. Interestingly, not only was found that initial replication yielded a heterogeneous population D79H ∼ PV-5T unable to excise the 5T cassette, extensive sequence of transcripts containing 65% wild-type genome (which would changes were apparent in the population. Examining the region generate VSV-G) and 45% 5T excision events (which would only that contains the D79H mutant under selective pressure, we generate nonstructural proteins), similar to the selection process observed that approximately half of the reads for this particular of SINV-5T (SI Appendix,Fig.S4E). By P4, a dominant genome codon display a reversion back to the WT nucleotide sequence arose which, like SINV-5T, showed a precise excision event where D79H WT (CAT → GAC )(SI Appendix, Fig. S5B). Together these the 5T element was removed but the downstream VSV-G sub- data illustrate the importance of homologous recombination in genomic promoter remained (SI Appendix,Fig.S4F). Collectively, escaping RNAi in the absence of an encoded antagonist. these results suggest that positive-stranded RNA viruses, in contrast to negative-stranded RNA viruses, can quickly adapt to a small Discussion RNA-mediated response regardless of their replication kinetics. Collectively, these results demonstrate the propensity of viruses RNAi-Induced Escape of Positive-Stranded Viruses Is Independent of of different polarities to evade RNAi by imposing a uniform Gene Expression Strategy. We next asked whether escape by ge- selective pressure onto each of them independently. Under nome excision is a universal strategy for viruses of positive po- similar intracellular viral RNA loads, viruses of positive-stranded larity in response to an RNAi-like system, regardless of genome polarity could rapidly excise genomic material to evade RNAi- organization. SINV and SFV represent members of the alpha- mediated repression, whereas negative-stranded viruses appear virus family of positive-stranded RNA viruses, and both produce to lack this plasticity. While the excision of genomic material tar- nonstructural and structural proteins from distinct mRNA geted by naturally occurring RNAi would not be as straightforward transcripts. Given that the initial escape of SINV and SFV occurs in a physiological setting, this system allowed us to monitor virus through complementation of a heterogenous population of viruses, evolution under comparable constraints and on a more rapid scale. we next explored whether escape would be evident in a mono- Previous studies utilizing miRNA targeting of positive-stranded cistronic RNA virus of positive polarity. To examine this, we utilized RNA viruses have shown disparate results as it relates to their poliovirus, a member of the family, that produces all of propensity to evade silencing. While some experimental designs its proteins from a single RNA molecule. To achieve this, we inserted have shown rapid escape or even an inability to be targeted (37, – the 5T cassette into the 3′ UTR of an EGFP-expressing poliovirus 38), others have achieved complete silencing (39 42). These dis- (PV-GFP-5T) and asked whether PV-GFP-5T could escape when crepancies likely reflect differences both in experimental systems placed under selective pressure (Fig. 4A). We transfected PV-GFP- and in the efforts by which virus populations and excision events 5T RNA into silencing-enabled and -deficient cells and measured were measured. Variables that would contribute to targeting di- release of infectious poliovirus at 48 hpi by TCID50 (SI Appendix,Fig. rectly include: the expression levels of the miRNA used, place- S5A). These data demonstrated a 2-log change in virus levels when ment of the target sites, the presence of other antiviral defense PV-GFP-5T was under selective pressure which was further reflected systems, and the capacity of the virus to inherently shield itself in the overall deep sequencing read counts (Fig. 4A). from targeting. The culmination of these dynamics would there- fore define the time necessary to generate an escape variant and RNAi-Induced Escape Demands Homologous Recombination. Ho- thus contribute to the disparate phenotypes that have been mologous recombination is an attribute largely associated with reported in the literature.

E9216 | www.pnas.org/cgi/doi/10.1073/pnas.1810229115 Aguado et al. Downloaded by guest on October 2, 2021 Fig. 4. Positive-stranded RNA virus escape from RNAi-like pressure requires genome recombination. (A) Graph depicts the total number of reads mapping to MICROBIOLOGY each position along the viral genome for PV-5T after passage 0 in silencing-deficient (blue) and silencing-enabled (red) cells. Diagram of recombinant PV-5T where the 5T cassette is downstream of the PV ORF. All viral proteins and miRNA targets are depicted. (B) Infectious virus release of PV-5T WT or D79H after

passage 0 (black) and passage 4 (gray) measured by TCID50 per milliliter. (C and D) Relative read numbers (red) and minor variants (orange) of PV5T-WT or PV- 5T-D79H plotted along the respective viral genome after passage 4 in silencing-deficient cells (C) and silencing-enabled cells (D).

While all RNA viruses generate truncated genomes during documented (30–32, 43). This phenomenon would have no replication, the idea that there is an inherent bias toward the doubt contributed to the rapid expansion of positive-stranded capacity of positive-stranded viruses to recombine has been well RNA viruses and is likely a product of the accessibility of their

Aguado et al. PNAS | vol. 115 | no. 39 | E9217 Downloaded by guest on October 2, 2021 genomic material and the biology of their RdRps (44). However, Cell Culture and Reagents. Dicer1- and Ago2-deficient murine embryonic cells while positive-stranded RNA viruses may have a greater pro- were a kind gift of A. Tarakhovsky, The Rockefeller University, New York, NY. pensity to recombine, this attribute has also been observed, albeit Ago1/3/4-deficient murine fibroblasts were a kind gift from M. Kay, Stanford − − significantly less frequently, in negative-stranded RNA viruses University, Palo Alto, CA. RNAi-deficient cells all refer to RNaseIII / cells (33, 45, 46). This stark dichotomy in RNA biology is undoubtedly which are described elsewhere (21, 49). Additional details regarding cells a result of the fact that, in contrast to positive-stranded RNA and culturing can be found in SI Appendix, SI Materials and Methods. viruses, viruses of negative polarity typically have encapsidated genomes (47). This added genomic protection makes them far CRISPR Screen. A549 cells were transduced with the whole-genome GeCKO less amenable to recombination and, for this reason, segmenta- library encoded in the lentiGuide-Puro, two-vector system to express Cas9 and a gene-specific sgRNA as previously described (22). For the SeV-GFP-5T screen, tion may be one of the more efficient means of creating genetic × 8 diversity. It should also be noted that negative-stranded viruses 2 10 cells were infected in triplicate at a multiplicity of infection (MOI) of do readily generate defective interfering particles (30). However, 1 and were maintained in culture for 5 d before fax sorting. gRNA pop- ulations were determined by amplicon-based Illumina sequencing. in these examples, the polymerase copies back on itself or maintains association with its ribonucleoprotein, making this Deep Sequencing Analyses. Purified RNA was fragmented and reverse tran- type of event distinct from template jumping. scribed, followed by second-strand synthesis, end repair, A-tailing, adapter Given the inherent inefficiency of negative-stranded viruses to ligation, and PCR amplification. Libraries were quantified using the universal escape RNAi-mediated targeting, one wonders what the evolu- complete KAPA library quantification kit (KAPA Biosystems) and sequenced tionary advantages of such a replicative strategy might be. While on an Illumina NextSeq with the NextSeq 500/550 Mid Output v2 kit. Raw purely speculative, one unique feature is that their biology is sequencing reads were aligned to the corresponding reference viral genome completely under their own control. In comparison with positive- using Bowtie2 (50) with default parameters. The resulting aligned reads stranded RNA viruses, whose genomes are directly translated, were visualized, and the read counts for each residue per site obtained, the initial mRNA of negative-stranded RNA viruses is controlled using the Integrative Genomics Viewer (51). Relative read counts and vari- exclusively by their RdRp. Such an attribute may prove benefi- ation analyses were performed using in-house Perl scripts. Major variant for cial in controlling the types of RNA structures formed during each site was defined as the residue (nucleotide) with higher read counts replication and thereby minimize both detection and genome compared with all other possible residues for the same site. For the analysis targeting by the host. of junction sites, raw sequencing reads were aligned to the corresponding Regardless of the evolutionary opportunity that enabled the reference viral genome using HISAT2 (52); resulting alignments were filtered invention of negative-stranded RNA viruses, their inability to for low mapping quality reads using samtools (53) (-q 10), and junction efficiently undergo homologous recombination does constrain counts were obtained using regtools (https://github.com/griffithlab/regtools) their ability to rapidly diversify in the absence of segmentation. (junctions extract -a 4). Further consolidation of junction sites and their In contrast, the capacity of positive-stranded RNA viruses to corresponding counts was performed using in-house Perl scripts. Relative rapidly recombine provides them a unique feature that would read number (counts) depicted throughout the paper is defined as the ratio significantly enable diversity in the context of RNAi. Given that of the total read counts per site over the total read count of the highest these dynamics have also been demonstrated in the context of covered residue over the entire genome/segment sequence (read count of the IFN system (36), it is tempting to speculate that the inability the highest peak). Percentage of reads corresponding to minor variants is of negative-stranded RNA viruses to efficiently undergo ho- defined as the cumulative read counts of minor variants (residues) over the mologous recombination is directly responsible for their lower total read count for each site. All sequencing data can be found at the GEO representation within the tree of life. In all, the findings here accession no. GSE108995. suggest that in the absence of an antiviral antagonist, virus bi- ology itself can provide an inherent advantage to evade ACKNOWLEDGMENTS. We thank Drs. J. Rose (Yale University), B. Lee [Icahn School of Medicine at Mount Sinai (ISMMS)], and P. Palese (ISMMS) for their host defenses. genetic rescue systems and corresponding reagents pertaining to SFV, SeV, and IAV, respectively. E.H. is supported by a National Science Foundation Materials and Methods Graduate Research Fellowship (1443116/20151986). T.X.J. is supported by the Virus Design, Rescue, and Quantification. The miRNA-silencing cassette (5T) New York University–ISMMS Mechanisms of Virus–Host Interactions National was designed as previously described (48). Further information regarding Institutes of Health T32 Training Grant (AI007647-09). B.R.t., M.V., and as- , rescue, and amplification of viruses can be found in SI Appendix, SI pects of this work are supported by the INTERCEPT program of the Defense Materials and Methods. Advanced Research Projects Agency.

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