Homologous recombination is an intrinsic defenseagainst antiviral RNA interferenceLauren 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, InstitutePasteur, 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 mechanismof plants and invertebrates, rendering the capacity to evade it adefining factor in shaping the viral landscape. Here we sought todetermine whether different virus replication strategies providedany inherent capacity to evade RNAi in the absence of an antagonist.Through the exploitation of host microRNAs, we recreated an RNAi-like environment in vertebrates and directly compared the capacityof positive- and negative-stranded RNA viruses to cope with thisselective pressure. Applying this defense against four distinctviral families revealed that the capacity to undergo homologousrecombination was the defining attribute that enabled evasion ofthis defense. Independent of gene expression strategy, positive-stranded RNA viruses that could undergo strand switching rapidlyexcised genomic material, while negative-stranded viruses wereeffectively targeted and cleared upon RNAi-based selection.These data suggest a dynamic relationship between host an-tiviral defenses and the biology of virus replication in shapingpathogen prevalence.

RNAi | virus polarity | virus adaptation | homologous recombination |miRNA

The capacity to elicit a defense to foreign material is an es-sential attribute to maintain life. With respect to antiviral

defenses, numerous strategies have evolved to inhibit virus rep-lication. In bacteria and archaea, viruses are subjected to acombination of restriction enzymes and clustered regularly inter-spaced short palindromic repeat (CRISPR) systems, in addition toother, less defined defense platforms (1, 2). These defenses pre-dominantly target foreign DNA reflecting the composition ofthe majority of prokaryotic viruses (3, 4). In plants and inverte-brates, the most dominant antiviral defense comes in the form ofRNA interference (RNAi) although additional strategies are alsoemployed (5). Despite no direct link between the CRISPR- andRNAi-based systems, in both defenses the host acquires geneticmaterial from the virus and uses it to provide specificity to anotherwise nonspecific nuclease.In contrast to the small RNA-based defenses of prokaryotes,

plants, and invertebrates, vertebrates combat viruses using aprotein-based strategy referred to as the type I and type III IFNsystems (6, 7). Also relying on unique aspects of virus replicationto achieve specificity, the IFN response is initiated following theengagement of so-called pattern recognition receptors and thetranscriptional induction of a family of IFN cytokines (5). IFN issecreted into the extracellular milieu and signals in both anautocrine and paracrine manner to induce hundreds of hostgenes that slow virus replication by inhibiting aspects of tran-scription, translation, metabolism, cell transport, and apoptosis(5). While some sequencing data have suggested an RNAi-likedefense may be present at low levels in vertebrates (8–10),functionally RNAi and IFN have been found to be incompatiblewith each other, suggesting RNAi plays a minor physiologicalrole in reducing viral replication (11–15).Even with this notable absence, the biology of small RNA-

mediated control remains ubiquitous in almost all eukaryotic

species. The RNAi machinery was evolutionarily repurposedseparately in both the plant and animal lineages to generatemicroRNAs (miRNAs) (16). A central difference between thesetwo systems, apart from their general function in the cell, is thesource of the small RNA. In contrast to being of viral origin, as isthe case for antiviral RNAi, miRNAs derive from genome-encoded hairpins and are generally used to control cell biologyand lineage fate (17). As miRNAs do not engage their targetwith perfect complementarity, cleavage does not occur and thustranscriptional regulation is subtle. While this dynamic is notpotent enough to be utilized during an acute infection, the long-term consequences that miRNA-based regulation can imposemake miRNAs an ideal platform for coordinating developmentalprocesses. A reflection of this biology can be inferred from thefact that of the four Argonaut (Ago) effector nucleases involvedin vertebrate miRNA biology, only Ago2 has retained the capacity fordouble-stranded RNA cleavage (17). Ago2 activity and the expressionof miRNAs, however, can be exploited by introducing a perfectbinding site into a target transcript to enable cleavage (18). Using thissystem, which mimics the RNAi response of invertebrates and plants,we set out to determine how different viruses would adapt to evadethe same selective pressure in the absence of an antagonist.

ResultsEngineering an Artificial Antiviral System. The combination of sheerdiversity, rapid mutation, and the evolutionary time scale of lifeon the planet severely limits the capacity to deduce the evolution

Significance

In an effort to determine whether host defenses can signifi-cantly influence the prevalence of different virus groups, weapplied identical selective pressures onto four families of di-verse viruses. Using an RNAi-like defense, we found that thecapacity of positive-stranded RNA viruses to switch genomictemplates during replication conferred an adaptability thatexceeded that of negative-stranded RNA viruses that were lessable to perform this biology. Together, this work suggests thatin the absence of an antiviral antagonist, fundamental aspectsof virus biology can provide an inherent advantage to evadehost defenses.

Author contributions: L.C.A., T.X.J., E.H., and B.R.t. designed research; L.C.A., T.X.J., E.H.,J.H., and M.P. performed research; D.B.-M. contributed new reagents/analytic tools;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. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE108995).1To whom correspondence should be addressed. Email: Benjamin.tenOever@mssm.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1810229115/-/DCSupplemental.

Published online September 12, 2018.

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of viruses (19). While a direct phylogeny of viruses may never beknown, it is clear that the prokaryotic cellular environment fa-vored the expansion of DNA viruses, whereas in eukaryotes,RNA viruses flourished (20). These general trends suggest thatthe defenses of the host may inherently bias the diversity of theviruses that impact them. To explore this idea, we wanted todesign a selective pressure that could be imposed equally ontodifferent viruses to ascertain whether there are inherent repli-cation attributes that enable more efficient evasion mechanisms.For this reason, we chose to exploit host miRNA expression inmammalian cells to recreate an RNAi-like response againstviruses that would lack antagonists to this defense. We chose thisselective pressure because use of small RNAs in antiviral de-fenses is a shared attribute in all but vertebrate life and becausemiRNAs do not possess the significant complementarity with anyknown virus necessary to induce silencing (15).To this end, we first verified the ubiquitous expression of five

miRNAs in mammalian cells, notably miR-21–5p, miR-31–5p,miR-192–5p, miR-93–5p, and miR-29b-3p (SI Appendix, Fig.S1A). We next engineered a cassette containing target sites withperfect complementarity for each miRNA, and cloned this intothe 3′ untranslated region (UTR) of enhanced green fluorescentprotein (herein referred to as pEGFP-5T) (SI Appendix, Fig.S1B). To test targeting of this cassette, we transfected pEGFP-5T into RNaseIII-deficient fibroblasts (21) (herein referred to assilencing-deficient cells) and introduced miRNA mimetics in-dividually or as a pool. Analysis of EGFP expression by flowcytometry demonstrated that, compared with miR-1, which wasnot represented in the 5T cassette, each individual mimic wassufficient to induce miRNA-mediated silencing (SI Appendix,Fig. S1B). Moreover, this construct could additionally be si-lenced in the presence of endogenous miRNAs in wild-type(WT) fibroblasts (herein referred to as silencing-enabled cells)(SI Appendix, Fig. S1C).

RNAi-Mediated Selective Pressure Is IFN Independent. Having dem-onstrated that the inclusion of the 5T cassette mimicked anRNAi-like response, we next sought to determine whether itcould be used to impose positive selection independent from thecanonical IFN-I system and free from viral antagonism. To thisend, we subcloned EGFP-5T into Sendai virus as an independenttranscript (SeV-GFP-5T). As a control, we additionally made aconstruct in which the 3′ UTR of EGFP was composed of thesame sequence but in the reverse orientation, rendering EGFPimmune from miRNA-mediated repression (herein denoted asSeV-GFP-5R) (Fig. 1A). To ascertain whether EGFP expressionwas sensitive to the miRNA machinery in the context of virusinfection, we infected wild-type murine fibroblasts as well asknockout cells lacking Dicer (Dcr−/−), Ago2 (Ago2−/−), andAgo1/3/4 (Ago1/3/4−/−). These data revealed fluorescence andhemagglutinin-neuraminidase (HN) expression in all genotypesin response to SeV-GFP-5R, whereas EGFP expression was lim-ited to Dcr −/− and Ago2−/− for SeV-GFP-5T (Fig. 1B). These datasuggest that a functional Ago2-based miRNA system is requiredfor efficient silencing which could be further confirmed by flowcytometry in both murine (Fig. 1C) and human fibroblasts (Fig. 1D).We next sought to define the central components required for

targeting. To this end, we performed a whole-genome CRISPRscreen on SeV-GFP-5T by selecting for cells in which silencing ofthe SeV-derived EGFP-5T had been disrupted (Fig. 1D). Cellsexpressing EGFP following infection of a previously establishedCRISPR cell library were selected and their gRNAs sequenced(22). Model-based analysis of the genome-wide CRISPR/Cas9 knockout (MAGeCK) successfully identified the canonicalmembers of the miRNA machinery including: Drosha, Dicer,Ago2, and DGCR8 (Fig. 1E and SI Appendix, Table S1) (23). Inaddition, the screen implicated p53 (TP53), miR-21, and XPO5,which would all impact the available cytoplasmic pool of miRNAs

needed to induce silencing (24, 25). Of note, no IFN signaturegenes were implicated in this screen, suggesting that this selectivepressure was based entirely on RNAi activity.

RNAi Can Effectively Neutralize Negative-Stranded Virus Replication.Given that our RNAi system appeared both potent and in-dependent of the canonical IFN response, we next decided toimpose this selective pressure onto an essential component ofthe virus. To this end, we moved the 5T and 5R cassettes fromEGFP to the 3′ UTR of the nucleoprotein (N) transcript ofSendai virus (Fig. 2A). Infection of SeV-5T and -5R demon-strated no discernible difference in their capacity to replicate insilencing-deficient cells as measured by SeV-N protein levels(Fig. 2B). In contrast, infection of SeV-5T and -5R in silencing-enabled cells demonstrated a complete loss of NP protein se-lectively from the SeV-5T strain (Fig. 2C). Consistent with thesedata, passaging experiments demonstrated that within 24 h (passageone, P1), SeV titers were reduced by more than 5 logs and wereundetectable by P4 (Fig. 2D). In contrast, SeV-5R titers demon-strated robust replication throughout the duration of the passages.These data could be further characterized by deep sequencing (Fig.2 E–H). RNA sequencing of SeV-5R in both silencing-deficientand -enabled cells revealed no differences in coverage of the viralgenome or abundance of viral reads with nucleotide variantsappearing only in the 3′ UTR of each transcript at the site ofpolyadenylation (Fig. 2 E and F). Similarly, silencing-deficientcells infected with SeV-5T demonstrated the same genomic cov-erage and alignment profiles to SeV-5R (Fig. 2G). In strikingcontrast, silencing-enabled cells were able to completely suppressall replication of SeV-5T, demonstrating only two reads thataligned to the GFP ORF, corroborating the inability of SeV-5T toescape our RNAi-like pressure (Fig. 2 D and H).To determine whether this phenotype was specific to para-

myxoviruses, we also inserted the 5T and 5R cassettes into the 3′UTR of segment five (encoding NP) of influenza A virus (IAV) (SIAppendix, Fig. S2A). In silencing-deficient cells, TCID50 (tissue cul-ture infectious dose to kill 50% of infected cells) titers of IAV-5Tand -5R showed no significant difference (SI Appendix, Fig. S2B). Incontrast, IAV replication in silencing-enabled cells demonstratedrobust titers of IAV-5R in contrast to IAV-5T, where infectious unitscould only be observed in the initial 24 h and at levels 5 logs lowerthan -5R (SI Appendix, Fig. S2B). These data could be further cor-roborated at the protein level in which IAV-5T NP production wascompletely inhibited in silencing-enabled cells, with no indication ofthe cytopathic effects observed at 48 h postinfection (hpi), in starkcontrast to IAV-5R (SI Appendix, Fig. S2 C and D). Overall effectsof targeting on IAV-5T replication could also be observed by totalread count from RNA-Seq data with no indication that the tar-geting cassette was excised to yield an escape mutant (SI Appendix,Fig. S2E). Together, these data demonstrate the potency of RNAiin its capacity to silence RNA viruses of negative polarity.

RNAi-Based Selective Pressure Induced Rapid Escape of Positive-Stranded RNA Viruses. To determine whether the difficulty inevading an RNAi response was equally evident in viruses ofpositive polarity we next imposed the same selective pressure onSindbis virus (SINV), an alphavirus and a member of theTogaviridae family (26). To this end, we inserted the silencingcassette into an intergenic region between the nonstructural andstructural transcripts in both forward (5T) and reverse (5R)orientations (Fig. 3A). As this insertion site was in the proximityof the subgenomic promoter of SINV (27, 28), we recreated thepromoter downstream of the cassette to ensure normal pro-duction of the structural transcripts. Infection of silencing-deficient cells with either SINV-5T or -5R revealed compara-ble replication kinetics between the two viruses as determinedby Western blot (Fig. 3B). When silencing was enabled, weobserved an initial delay in capsid expression of the 5T virus,

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although in contrast to both SeV and IAV, the targeted SINVreached similar levels as early as 6 hpi (Fig. 3C).To ensure that our selective pressure was functional in the

context of SINV infection, we next generated a SINV recombi-nant virus that expressed the EGFP-5T cassette under the con-trol of an extrasubgenomic promoter (SI Appendix, Fig. S3A).This virus construct, when used to infect silencing-deficient cells,demonstrated robust fluorescence as expected (SI Appendix, Fig.S3B). In contrast, infection of silencing-enabled cells showed acomplete absence of EGFP fluorescence allowing us to concludethat RNAi was functioning in the context of SINV infection de-spite the replication levels of SINV-5T in silencing-enabled cells.To better understand how the 5T cassette could effectively

silence EGFP and the early production of capsid expression, weserially passaged the SINV-5R and -5T variants and analyzed

replication using deep sequencing (Fig. 3 D and E). Initialcharacterization of SINV-5R in silencing-enabled cells demon-strated the insertion of the genetic cassette was not overtlydetrimental to the virus and appeared stable over 96 h of con-tinuous replication (Fig. 3D). Moreover, we did not observe anydramatic changes to the relative abundance of viral reads or theappearance of minority variants. In contrast to -5R, the samepassaging experiment with SINV-5T showed the emergence of asingle dominant species that had excised the 5T cassette butretained a fully functional subgenomic promoter by P4 comparedwith P0 (Fig. 3E). Interestingly, the selective pressure of RNAialso resulted in a higher frequency of three amino acid residues(K232T, N370K, and G595V) that arose in three independentexperiments (depicted as orange peaks in the nonstructural pro-teins nsP1 and nsP2).

Fig. 1. The 5T cassette only restricts through miRNA machinery. (A) Diagrams of recombinant SeV-eGFP-5T and SeV-eGFP-5R genomes. The 5T (depicted withmiRNA binding) and 5R (resistant to miRNA binding) cassettes are downstream of the GFP ORF, which is inserted between nucleoprotein and phosphoproteinORFs. (B) Immunofluorescence microscopy of SeV-GFP-5T and -5R–infected MEFs. WT MEFS or Dicer (Dcr), Argonaute 2, or Argonaute 1, 3, and 4 knockoutMEFs 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 inB. (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 KOA549 cells infected with SeV-GFP-5T (D).

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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 ofthe nucleoprotein (N) ORF and the ORF encoding GFP is inserted between N and the phosphoprotein (P). All other viral proteins and miRNA targets are alsodepicted. (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 fromsilencing-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-5Tgenome from silencing-deficient (G) or silencing-enabled (H) cells at 48 hpi.

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To better understand the dynamics of SINV-5T infectionduring P0, we searched for gapped alignments (SI Appendix, Fig.S3C). While ∼90% of the virus remained intact, three distinctexcision events accounted for the remaining 10% of viralRNA. As the 5T cassette was engineered to selectivelysilence the nonstructural polyprotein, this RNA profile would

suggest that the 90% of nonmodified full-length genomewould still contribute wild-type levels of the structural mRNA.In contrast, the 10% of excision events observed, while dis-rupting the capacity to generate the structural transcripts,would generate untargeted nonstructural mRNA (SI Appen-dix, Table S2).

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 5Tcassette 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-enabledcells 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 at24 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-enabledcells.

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Next, we sought to ascertain whether the behavior of SINV-5Tcould be applied to other positive-stranded RNA viruses. To thisend, we applied the 5T cassette to a variant of Semliki Forestvirus (SFV) in which the endogenous structural proteins hadbeen replaced with the glycoprotein (G) of vesicular stomatitisvirus (VSV) (29) (SI Appendix, Fig. S4A). We chose this recombi-nant chimeric virus (herein referred to as SFVG) because its repli-cation rate was slower than SINV and allowed us to ascertainwhether this attribute contributed to the inability of SeV- and IAV-5T to escape. As such, we inserted the 5T or 5R cassettes into the 3′UTR of the nonstructural polyprotein transcript and initially com-pared virus replication in silencing-deficient cells (SI Appendix, Fig.S4B). Using VSV G protein expression as a readout for viral levels,we found no discernible difference between 5T and 5R variants,although the slow kinetics of replication were apparent, as glyco-protein expression was not visible until 36 hpi. In contrast, infectionof the 5T and 5R SFVG variants in silencing-enabled cells dem-onstrated a dramatic difference in glycoprotein levels during the first36 h of infection with no detectable evidence of 5T replication (SIAppendix, Fig. S4C). However, by 48 hpi, the 5T virus became de-tectable, suggesting that, similar to SINV, an escape varianthad arisen.To further discern the molecular dynamics of SFVG escape,

we serially passaged both the SFVG-5T and -5R variants insilencing-enabled cells. Similar to SINV, passaging of SFVG-5Rdemonstrated no excision event throughout the genome after acombined 192 h of replication (4 × 48 h passages; P0–P4) (SIAppendix, Fig. S4D). However, P4 did show multiple variantsthroughout the genome even in the absence of positive selection,likely a product of its relative lack of adaptation, a phenomenonthat has been previously documented (30).When this same methodology was applied to SFVG-5T, we

found that initial replication yielded a heterogeneous populationof transcripts containing ∼65% wild-type genome (which wouldgenerate VSV-G) and 45% 5T excision events (which would onlygenerate nonstructural proteins), similar to the selection processof SINV-5T (SI Appendix, Fig. S4E). By P4, a dominant genomearose which, like SINV-5T, showed a precise excision event wherethe 5T element was removed but the downstream VSV-G sub-genomic promoter remained (SI Appendix, Fig. S4F). Collectively,these results suggest that positive-stranded RNA viruses, in contrastto negative-stranded RNA viruses, can quickly adapt to a smallRNA-mediated response regardless of their replication kinetics.

RNAi-Induced Escape of Positive-Stranded Viruses Is Independent ofGene Expression Strategy. We next asked whether escape by ge-nome excision is a universal strategy for viruses of positive po-larity in response to an RNAi-like system, regardless of genomeorganization. SINV and SFV represent members of the alpha-virus family of positive-stranded RNA viruses, and both producenonstructural and structural proteins from distinct mRNAtranscripts. Given that the initial escape of SINV and SFV occursthrough complementation of a heterogenous population of viruses,we next explored whether escape would be evident in a mono-cistronic RNA virus of positive polarity. To examine this, we utilizedpoliovirus, a member of the picornavirus family, that produces all ofits proteins from a single RNAmolecule. To achieve this, we insertedthe 5T cassette into the 3′ UTR of an EGFP-expressing poliovirus(PV-GFP-5T) and asked whether PV-GFP-5T could escape whenplaced under selective pressure (Fig. 4A). We transfected PV-GFP-5T RNA into silencing-enabled and -deficient cells and measuredrelease of infectious poliovirus at 48 hpi by TCID50 (SI Appendix, Fig.S5A). These data demonstrated a 2-log change in virus levels whenPV-GFP-5T was under selective pressure which was further reflectedin the overall deep sequencing read counts (Fig. 4A).

RNAi-Induced Escape Demands Homologous Recombination. Ho-mologous recombination is an attribute largely associated with

viruses of positive polarity that have been found to enable ex-change of genetic material through polymerase-mediated templateswitching (31, 32). This has been particularly well characterized withrespect to poliovirus (33–36). In an effort to phenocopy a virus ofnegative polarity onto a positive-stranded virus such as polio, weutilized a well-characterized RdRp mutant incapable of homolo-gous recombination (36). This poliovirus variant harbors a D79Hmutation in the 3D RdRp gene that has been shown to be incapableof mediating a rescue of two nonreplicative genome segments. Weengineered this mutation into the PV-GFP-5T backbone to gaugethe necessity for this activity in escaping RNAi.To this end, RNA for PV-GFP-5T or PV-GFP-5TD79H (herein

referred to as PV-5T and PV-5TD79H) was transfected intosilencing-enabled or -deficient cells and supernatants were pas-saged. We titered the supernatants from passages 0 and 4 andperformed RNA-Seq to monitor the virus populations. In silencing-deficient cells, PV-5T and PV-5TD79H showed comparable titers atboth P0 and P4 (Fig. 4B). Sequencing these viruses at the end ofpassaging demonstrated that the D79H mutant was stable and alsoindistinguishable from WT with regards to minority variants in thepopulation (Fig. 4C). The only consistent minority variant observedunder these conditions were three nucleotide substitutions withinthe 2A cleavage sites that flank the GFP ORF.In contrast to the relatively homogenous population of PV-5T

and PV-5TD79H in silencing-deficient cells, when this same ex-periment was performed in wild-type cells, PV-5TD79H titerswere undetectable by P4 in contrast to PV-5T (Fig. 4 B–D). Inagreement with the SINV and SFVG data, PV-5T sequencingrevealed an excision of the 5T cassette explaining how replica-tion persisted beyond P4 (Fig. 4D). However, sequencing of PV-5TD79H resulted in very poor genomic coverage, consistent withthe inability to detect infectious virus. Interestingly, not only wasPV-5TD79H unable to excise the 5T cassette, extensive sequencechanges were apparent in the population. Examining the regionthat contains the D79H mutant under selective pressure, weobserved that approximately half of the reads for this particularcodon display a reversion back to the WT nucleotide sequence(CATD79H → GACWT) (SI Appendix, Fig. S5B). Together thesedata illustrate the importance of homologous recombination inescaping RNAi in the absence of an encoded antagonist.

DiscussionCollectively, these results demonstrate the propensity of virusesof different polarities to evade RNAi by imposing a uniformselective pressure onto each of them independently. Undersimilar intracellular viral RNA loads, viruses of positive-strandedpolarity could rapidly excise genomic material to evade RNAi-mediated repression, whereas negative-stranded viruses appearto lack this plasticity. While the excision of genomic material tar-geted by naturally occurring RNAi would not be as straightforwardin a physiological setting, this system allowed us to monitor virusevolution under comparable constraints and on a more rapid scale.Previous studies utilizing miRNA targeting of positive-stranded

RNA viruses have shown disparate results as it relates to theirpropensity to evade silencing. While some experimental designshave shown rapid escape or even an inability to be targeted (37,38), others have achieved complete silencing (39–42). These dis-crepancies likely reflect differences both in experimental systemsand in the efforts by which virus populations and excision eventswere measured. Variables that would contribute to targeting di-rectly include: the expression levels of the miRNA used, place-ment of the target sites, the presence of other antiviral defensesystems, and the capacity of the virus to inherently shield itselffrom targeting. The culmination of these dynamics would there-fore define the time necessary to generate an escape variant andthus contribute to the disparate phenotypes that have beenreported in the literature.

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While all RNA viruses generate truncated genomes duringreplication, the idea that there is an inherent bias toward thecapacity of positive-stranded viruses to recombine has been well

documented (30–32, 43). This phenomenon would have nodoubt contributed to the rapid expansion of positive-strandedRNA viruses and is likely a product of the accessibility of their

Fig. 4. Positive-stranded RNA virus escape from RNAi-like pressure requires genome recombination. (A) Graph depicts the total number of reads mapping toeach position along the viral genome for PV-5T after passage 0 in silencing-deficient (blue) and silencing-enabled (red) cells. Diagram of recombinant PV-5Twhere 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 afterpassage 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).

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genomic material and the biology of their RdRps (44). However,while positive-stranded RNA viruses may have a greater pro-pensity to recombine, this attribute has also been observed, albeitsignificantly less frequently, in negative-stranded RNA viruses(33, 45, 46). This stark dichotomy in RNA biology is undoubtedlya result of the fact that, in contrast to positive-stranded RNAviruses, viruses of negative polarity typically have encapsidatedgenomes (47). This added genomic protection makes them farless amenable to recombination and, for this reason, segmenta-tion may be one of the more efficient means of creating geneticdiversity. It should also be noted that negative-stranded virusesdo readily generate defective interfering particles (30). However,in these examples, the polymerase copies back on itself ormaintains association with its ribonucleoprotein, making thistype of event distinct from template jumping.Given the inherent inefficiency of negative-stranded viruses to

escape RNAi-mediated targeting, one wonders what the evolu-tionary advantages of such a replicative strategy might be. Whilepurely speculative, one unique feature is that their biology iscompletely under their own control. In comparison with positive-stranded RNA viruses, whose genomes are directly translated,the initial mRNA of negative-stranded RNA viruses is controlledexclusively by their RdRp. Such an attribute may prove benefi-cial in controlling the types of RNA structures formed duringreplication and thereby minimize both detection and genometargeting by the host.Regardless of the evolutionary opportunity that enabled the

invention of negative-stranded RNA viruses, their inability toefficiently undergo homologous recombination does constraintheir ability to rapidly diversify in the absence of segmentation.In contrast, the capacity of positive-stranded RNA viruses torapidly recombine provides them a unique feature that wouldsignificantly enable diversity in the context of RNAi. Given thatthese dynamics have also been demonstrated in the context ofthe IFN system (36), it is tempting to speculate that the inabilityof negative-stranded RNA viruses to efficiently undergo ho-mologous recombination is directly responsible for their lowerrepresentation within the tree of life. In all, the findings heresuggest that in the absence of an antiviral antagonist, virus bi-ology itself can provide an inherent advantage to evadehost defenses.

Materials and MethodsVirus Design, Rescue, and Quantification. The miRNA-silencing cassette (5T)was designed as previously described (48). Further information regardingcloning, rescue, and amplification of viruses can be found in SI Appendix, SIMaterials and Methods.

Cell Culture and Reagents. Dicer1- and Ago2-deficient murine embryonic cellswere a kind gift of A. Tarakhovsky, The Rockefeller University, New York, NY.Ago1/3/4-deficient murine fibroblasts were a kind gift from M. Kay, StanfordUniversity, Palo Alto, CA. RNAi-deficient cells all refer to RNaseIII−/− cellswhich are described elsewhere (21, 49). Additional details regarding cellsand culturing can be found in SI Appendix, SI Materials and Methods.

CRISPR Screen. A549 cells were transduced with the whole-genome GeCKOlibrary encoded in the lentiGuide-Puro, two-vector system to express Cas9 anda gene-specific sgRNA as previously described (22). For the SeV-GFP-5T screen,2 × 108 cells were infected in triplicate at a multiplicity of infection (MOI) of1 and were maintained in culture for 5 d before fax sorting. gRNA pop-ulations were determined by amplicon-based Illumina sequencing.

Deep Sequencing Analyses. Purified RNA was fragmented and reverse tran-scribed, followed by second-strand synthesis, end repair, A-tailing, adapterligation, and PCR amplification. Libraries were quantified using the universalcomplete KAPA library quantification kit (KAPA Biosystems) and sequencedon an Illumina NextSeq with the NextSeq 500/550 Mid Output v2 kit. Rawsequencing reads were aligned to the corresponding reference viral genomeusing Bowtie2 (50) with default parameters. The resulting aligned readswere visualized, and the read counts for each residue per site obtained,using the Integrative Genomics Viewer (51). Relative read counts and vari-ation analyses were performed using in-house Perl scripts. Major variant foreach site was defined as the residue (nucleotide) with higher read countscompared with all other possible residues for the same site. For the analysisof junction sites, raw sequencing reads were aligned to the correspondingreference viral genome using HISAT2 (52); resulting alignments were filteredfor low mapping quality reads using samtools (53) (-q 10), and junctioncounts were obtained using regtools (https://github.com/griffithlab/regtools)(junctions extract -a 4). Further consolidation of junction sites and theircorresponding counts was performed using in-house Perl scripts. Relativeread number (counts) depicted throughout the paper is defined as the ratioof the total read counts per site over the total read count of the highestcovered residue over the entire genome/segment sequence (read count ofthe highest peak). Percentage of reads corresponding to minor variants isdefined as the cumulative read counts of minor variants (residues) over thetotal read count for each site. All sequencing data can be found at the GEOaccession no. GSE108995.

ACKNOWLEDGMENTS. We thank Drs. J. Rose (Yale University), B. Lee [IcahnSchool of Medicine at Mount Sinai (ISMMS)], and P. Palese (ISMMS) for theirgenetic rescue systems and corresponding reagents pertaining to SFV, SeV,and IAV, respectively. E.H. is supported by a National Science FoundationGraduate Research Fellowship (1443116/20151986). T.X.J. is supported by theNew York University–ISMMS Mechanisms of Virus–Host Interactions NationalInstitutes of Health T32 Training Grant (AI007647-09). B.R.t., M.V., and as-pects of this work are supported by the INTERCEPT program of the DefenseAdvanced Research Projects Agency.

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