w.nsogcidi1.03pa.74814PNAS www.pnas.org/cgi/doi/10.1073/pnas.1714385114 encoding What the possibilities? given alternative sequence of synonymous number as its astronomical is replicates conserved How PV of arise: rate Since tions error (10). interaction an CD155 and at quasispecies receptor (10), identity cellular serotype the (9), with structure virion ing 1 (Fig. cleavage tion and encoding (2A into region ORF itself 3’-heteropolymeric cleaves single 1 that complex (Fig. a a tail by by poly(A) followed followed nontrans- PP, 5’-terminal (5’-NTR), the its region in structures lated complex highly (2). children revealed healthy Mahoney three three iso- of was 1 in feces study, pooled type this from occurs in 1941 neurovirulent analyzed in PV species lated most viral poliomyelitis. main the the of [PV1(M)], which cause of the serotypes, sequences possible PV, these of fraction defines tiniest The code. genetic ate 1 (Fig. T virion poliovirus in role a plays precur- structures hairpin so genome/capsid of viruses array of formation. an +ssRNA cocondensation which (most in II, protein(s) sors step capsid studied); with far (PS) signal (poliovirus), ing proteins replication ( and acqui- or protein(s) I, ( step capsid either viruses: specificity, between +ssRNA packaging for pack- of strategy sition propose poliovirus we assembly (2017) in evidence, two-step al. role available a et critical all N, Considering the specificity. [Patel play aging genome” 2:17098] viral Microbiol entire numerousNat the that signals along unlikely packaging it mapping RNA makes dispersed This sequence-degenerate, sur- not recoding. “cryptic, do pair specificity packaging codon that determining vive structures in role secondary a local play might and/or con- that sites showed synonymous We recoding. served PV after the preserved were of packag- signal(s) cis-acting structures ing conserved evolutionary and if performed determine sequence to We genome of genotype. analysis single comprehensive a a of PV haploid infectivity of Si cellular broadly. discussed presents be will that phenotype P2 a vs. ticles), particles PFU/118 P2 1 (WT, PV1, (si) infectivity WT with for except pared nonviable are pairs) P2 under- (randomly codon of mutations synonymous (excess “SD” represented variants 2,104 “Min” to codons). synonymous up scrambled or syn- overrepresented pairs) of codon (excess mutations onymous car- “Max” nucleotides) 1,297 (6,189 to vari- ORF Kuhn) up whose viable J. ried (PV1), Richard 1 generated and type Gorbalenya poliovirus synthesis Alexander of by chemical ants reviewed 2017; and 17, design May review Computer for (sent 2017 17, August Wimmer, Eckard by Contributed 10305; NY Island, e Staten Hospital, University a Asare Emmanuel genomes Song poliovirus Yutong recoded massively genome of and packaging infectivity, specific variation, of Limits eateto optrSine tn ro nvriy tn ro,N,174 and 11794; NY, Brook, Stony University, Brook Stony Science, 11794; NY, Computer Brook, of Stony Department University, Brook Stony Microbiology, and Genetics Molecular of Department Min asddmi 1cnrl h dniyo Vb determin- by PV of identity the controls P1 domain Capsid PV1(M) of (3) organization gene and 4) (3, sequence Genome b P) apn oteNtriu fteplpoen(PP) polyprotein the of terminus N the poliovirus 1 to type of mapping acids) amino (PV), (881 P1 precursor capsid he pro ytehg fnt neato fasnl N packag- RNA single a of interaction affinity high the by ) ain eprtr-estv t3 n 39.5 and 33 at temperature-sensitive variant a , A n 3C and ,cnb noe n10 in encoded be can ), | eoerecoding genome a,1,2 pro /3CD lkad Gorbatsevych Oleksandr , a igJiang Ping , A A 5 ) h P()i natv molecule active an is (7) PP The 6). (5, ) 5 ,8). 6, (5, ) pro ∼ 9plppie ytovrlproteinases viral two by polypeptides 29 Min n nezm-needn matura- enzyme-independent an and ) | akgn signal packaging ipae atyrdcdspecific reduced vastly a displayed 442 a ∼ nk .Paul V. Aniko , 10 as()det h degener- the to due (1) ways − 4 c eateto hmsr,Uiest fIw,Iw iy A52242; IA City, Iowa Iowa, of University Chemistry, of Department a 1) h olwn ques- following the (11), yseicrecognition specific by ) | Min pcfi infectivity specific F/500par- PFU/35,000 1 , a,1 igLiu Ying , a tfe Mueller Steffen , ◦ .Com- C. a,b on Mugavero JoAnn , cdsqec ihwl-ye(T V(15). amino PV identical (WT) an wild-type sharing with results despite sequence codons acid construct synonymous with viral (human) nonviable domain a used” P1 in “rarely PV1 unequal of the the excess Encoding (14), an codons. bias” synonymous “codon of is use ORFs of C-CAVs, in restriction of well-known variability architecture second sequence A basic (13). was the parents sequences to evolutionary viruses its nucleotide adhered Coxsackie PV it of as C-cluster constrained evolution of the descendant (12), (C-CAVs) evolutionary an is capsid 881 specify to optimal residues? be would that coevolved have could 1073/pnas.1714385114/-/DCSupplemental at online information supporting contains article This 2 option. access open 1 PNAS the through Codagenix. online to A.G., available connection Y.S., Freely no unemployed. with currently members is laboratory laboratory no P.J. are the Codagenix. with E.A. left to retired J.M., have unrelated has A.V.P. C.B.W. are in Codagenix. and employments of pairs S.C., their manuscript focus Y.L., codon this the Codagenix. in synonymous with to described of interest co- work connection of The changes are Codagenix. conflict of They of no employee effects vaccines. presents an the of is S.M describing development genomes. patent and viral small a design a Inc., the of Codagenix, on holders of co-founders focusing are E.W. company and biotech S.M. statement: interest of Conflict Inflam- of Disease. Institute Purdue Infectious R.J.K., and Immunology, and mation, Center; Medical University the Leiden wrote A.G., E.W. Reviewers: and A.V.P., O.G., Y.S., and data; analyzed paper. O.G., E.W. Y.S., and tools; S.M., reagents/analytic A.V.P., P.J., new contributed E.A., C.B.W. and O.G. research; performed pairs codon underrepresented and (good), favorable see are details, that (for Methods ORF (16–18) pairs and codons individual codon Materials of SI synonymous use the of fit not frequency does the describes This uhrcnrbtos .. ... n ..dsge eerh ...... n S.H.S. and J.M., Y.L., Y.S., research; designed E.W. and A.V.P., Y.S., contributions: Author a,f [email protected]. or owo orsodnemyb drse.Eal [email protected] Email: addressed. be may correspondence whom To work. this to equally contributed O.G. and Y.S. ope n utpecgaecpisaddb multiple by aided capsids precursor cognate hairpins. capsid multiple genome-specific genome/single and single complex ( between precursor. capsid occurring cognate and ( signal or ing precursor capsid and follows protein assembly cation virus pro- +ssRNA ( We steps. of hypothesis. two progression this the support not that does pose analy- PVs Our recoded viruses. of +ssRNA genome, sis of of viral dozens specificity the single assembly that within determine a formed proposes initiate hairpins, hypothesis conserved to recent loosely virion A single infection. a cell of probability ( the infectivity specific defines prolifer- the studied particularly We phenotypes, ORF. to ation the up in carrying mutations variants synonymous (PV) 2,104 poliovirus viable constructed We Significance hr are osqec aito scdnpi is(CPB). bias pair codon is variation sequence to barrier third A genus the of member a PV, If n cadWimmer Eckard and , f oaei n. tn ro,N 11794 NY Brook, Stony Inc., Codagenix i a pcfiiy eonto ewe ihr( either between recognition Specificity: ) a .Shen H. Sam , | b ahlg n aoaoyMdcn,Sae Island Staten Medicine, Laboratory and Pathology ulse nieSpebr2,2017 25, September online Published .Oerpeetdcdnpisi an in pairs codon Overrepresented ). a,2 d oge n. onanVe,C 94043; CA View, Mountain Inc., Google, a,c . hre .Ward B. Charles , Enterovirus www.pnas.org/lookup/suppl/doi:10. b igeRApackag- RNA single ) ii Cocondensation, ) of Picornaviridae d,e | , > E8731–E8740 a er ago; years 2 si repli- ) that ) ,

MICROBIOLOGY PNAS PLUS A

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nts changed/ no. of nts of recoded segment

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Fig. 1. The PV genome and details of recoding by Max, Min, and SD designs. (A) The PV genome showing the 5’-terminal VPg-linked cloverleaf and IRES, the PP (open box) (divided into the P1 structural and P2, -3 nonstructural domains, and the polyadenylylated 3’NTR (1). The loci of three replication-essential hairpin structures, Cre (81), and α and β (23, 24), as well as the conserved but not-essential RNase L ci element (25, 26), are shown above the PP. The SnaB I and Bgl II restriction sites, used for subcloning the P2 and P3 domains, are indicated. (B) Growth phenotypes of PV1(M) and single-domain Max (green), Min (red), and SD (yellow) variants on HeLa monolayer R19 cells (Materials and Methods). For connotations of the constructs, see Table S1. Passage 0 means that full CPE emerged after transfection. The sequences of the essential Cre, α and β structures were restored after recoding in P2 and P3 variants (indicated as Maxcre, Mincre, or MaxWt, etc.). The total number of nucleotide changes in each recoded segment and the CPS of the recoded fragment relative to the WT in a segment are indicated in column 3 (please note the 12 unchanged nucleotides at the beginning of P1 SD are shown as a small white box). CPS, CPS of the recoded segments (compared with the same WT segment). Growth phenotypes of recoded variants were determined by the time of full CPE, together with virus titer and plaque size after transfection (Materials and Methods). (C) Same as B except more than one domain was recoded.

E8732 | www.pnas.org/cgi/doi/10.1073/pnas.1714385114 Song et al. ovnetrsrcinst,BlI,isd h 3dmi,was domain, al. et P3 Song the inside II, Bgl site, restriction convenient a the that proved P2 which (16), P1 variants P1 of viable segments produced subcloning PV1 and WT 16). analyses (15, Sequence signals 16). replication P1 P1 essential variant the harbor Unexpectedly, that confirms not result does This domain conditions. identical under WT PV from different phenotypes numerous replication Despite expressed individually 16). P1 (15, neither domain mutations, P1 silent published the of results in apparent is domains 1 PP Fig. different of sequences the changing Variants. of PV Recoded of Properties Growth 1 in summarized (Fig. is domains variants three 1 all (Fig. PV1(M) or domains the PP individual of ing ORF Methods Entire and the of Genome. Recoding Computer-Designed Results virion II and CPs step with in genome conserved the role loosely maturation. of a multiple cocondensation to the playing tributing that are suggest structures We hairpin 4). proteins capsid Fig. cognate its (CPs; formation with genome I, virion the II, step of step cocondensation viruses: by and +ssRNA assembly, of of propose specificity assembly we establishing for Here, morphogenesis. mechanism PV two-step explain a model to this serve that not show We does (22). genome” the as in throughout mapping described signals reside packaging been RNA viruses have dispersed sequence-degenerate, that +ssRNA “cryptic, hairpins of genome-specific of specificity deter- dozens important references assembly most and proposed for the 19–21 that recently minants implies (refs. a model assembly new se- to virus This large-scale therein). reference RNA to of in model tolerance also new genome’s but PV changes, the quence test to designed virology. the in discuss undervalued innate will the we with Below, example, cells response. for when immune interfere, or that lines drugs cell with different treated in are assayed is virus a when change related in conditions. comparable determined under when lines only is cell signature virions viral a to is (PFU) which values, unit plaque-forming a ∼ of For proliferation. ratio for the cells culture PV1(M) tissue favorable most the cells, (16) sequence aver- viral the a interpret of to CPS used then age have are Thus, pairs values use. dispreferred These codon scores. and negative account scores, into positive takes have pairs so ln preferred and = codon, occur- CPS the actual [i.e., of the pair rences the on the based of of is frequency frequency “Expected” observed expected (Observed/Expected)]. the the to of pair ratio codon the of logarithm natural (15). random at synony- codons language, shuffling mous involves third which a (“SD”), design” also “scrambling used the We consequences. for language biological (CPD)] testing over- deoptimization underrepre- pair the and [codon in (“Min”) optimization) sequences sented pair PV virus codon rewritten the have (“Max,” we kills represented a study, pairs rewriting this codon In small, bad (16). is excessive pairs with ORF. sequence bad) the overrepresented genome or of between (good function function underrepresented in and biological differences the the in Although (bad) unfavorable are /2.Orvrat ecie eeepesvsl different vastly express here described variants Our 1/120. h eoigeprmnsdsrbdhr eentonly not were here described experiments recoding The ( infectivity specific low a have PVs WT All the is which (CPS), score codon-pair a has pair codon Every Min Min B Fg 1 (Fig. lsi i o abrftlflw.I otatt P1 to contrast In flaws. fatal harbor not did plasmid and sn h a,Mn n Dsrtge ( strategies SD and Min, Max, the Using C B ,w aercddteP1M P y rt recod- first, by, PP, PV1(M) the recoded have we ), agtn igedmis ecnre recoding confirmed we domains, single Targeting . ie6 and 6, line , TbeS1 Table Min TbeS1 Table Max C a oval Fg 1 (Fig. nonviable was .Teiett fidvda viral individual of identity The ). 51260 o P1 nor (581/2,640) . B . ie6 a ibe(oethat (note viable was 6) line , ,floe yrcdn two recoding by followed ), si auscndramatically can values h eea outcome general The si hntp htis that phenotype si nHL R19 HeLa in ) , SD B osbycon- possibly IMaterials SI ie3 ref. 3; line , (930/2,628) Min into Min si , bevrsrpiain(5 6.Ti euti upre your by supported is result This dis- 26). not experiments. (25, did recoding it replication mutagenesis but virus L, Site-directed RNase 26). able for (25, activity L inhibitory entero- the RNase inactivated C-cluster for all 1 inhibitor of (Fig. an P3 viruses) as A to C-Coxsackie mapping (PV, RNA) viruses ci L (RNase ture 1 (Fig. P3 plaques with compared small (330) very and P3 titers 10). lower yielded 10) P3 and important cloning the ( in of 6,995–7,369) used (nucleotides WT site to restriction II reconstruction Bgl the the to due mutations) P3 1 (Fig. element recoding Cre from essential Max as the spared indicated variants, was P2 4,444–4,504) all in (nucleotides that note We sizes. 1 (Fig. PV P2 WT whereas to P2 identical passages. properties did blind growth the however, revealed during Sequencing, mutations adaptive below. indicate not described studies inoculum sufficient further accumulate for to P2 passages with blind transfection three required However, cloning). for used eue prxmtl wfl nP2 in twofold and ( approximately (qRT-PCR) (p.i.) reduced RT-PCR postinfection quantitative h 4 by Methods at 2) analysis of Northern (MOI cells 2 (Fig. reduced replication., was RNA RNA PV incoming inhibits of translation completely Therefore, mM) 2 immune (GnHCl; by 2 shown ride (Fig. as extracts infection), cellular of PV precipitation WT revealed similar however, with 2C P2 2], compared with of of cells production HeLa (MOI) of reduced infection Infection of cells. HeLa [multiplicity of form- virus layer plaque and a spread on viral for ing cell per virions of number large P2 ( PFUs by assayed P2 when of duction reduction significant a revealed WT however, cells, with 2 signif- compared CTP (Fig. disclose processing of transcripts not or PV absence did synthesis the in synthesis) in differences RNA (27) icant minimize extract (to cell-free UTP HeLa on and a focused biological in we the scripts Here, understood. influences poorly recoding P2 are SD PV or of Min, properties Max, which P2 by of Replication and Translation of Analysis P(1+2+3) the P(2+3) WT variant between P(1+2) in difference P(1+3) as phenotypes detectable replication yielded growth a ever, one-step reveal P(1+2+3) a and by not PV1 analyzed did as replication, curve, virus of synonymous kinetics 1,297 P(1+2+3) despite in are the replication, mutations variants that efficient these suggesting for in observation phe- pairs acceptable an codon growth 2–5), synonymous WT lines overrepresented produced 2, and (Fig. combinations notypes Cre Max WT All destroyed. with constructed β were P(1+2+3)] and 1 (Fig. ete etdtesnhsso P2 of synthesis the tested then We atnadcwreshv ecie ope N struc- RNA complex a described have coworkers and Barton P3 in domain P3 the of regions recoded The ete tde ainswt w rtrercdddomains recoded three or two with variants studied then We Max SD Min Min lmns hra h Ns iRAsrcue were structures RNA ci L RNase the whereas elements, h nyval i osrc.TasaigP2 Translating construct. Min viable only the , ainsaerltvl ml s stenme fsilent of number the is (so small relatively are variants iu a eylws au,wihidctstene fa of need the indicates which value, si low very a has virus elctdjs sW V,weesP3 whereas PV1, WT as just replicated C Min SD .A hw nFg 2 Fig. in shown As ). .AlMxadS ains[(+) (+) P(1+3), P(2+3), [P(1+2), variants SD and Max All ). n P(1+2+3) and , a oval,dsiecryn ee ietmutations silent fewer carrying despite nonviable, was SD Max Fg 1 (Fig. SD cre hthsfwrsln uain 114n)than nt) (1,174 mutations silent fewer has that ain 127nt). (1,297 variant PNAS Min , Max B A ie7 rdcdsihl mle plaque smaller slightly produced 7) line , .Aayi fgot fP2 of growth of Analysis ). SD ( cre | Max i.S1 Fig. SD ulse nieSpebr2,2017 25, September online Published n SD and , (497). al S1 Table ATPase Fg 1 (Fig. Fg 1 (Fig. C .Etne Drcdn,how- recoding, SD Extended ). i.S1 Fig. ( n t rcros(to precursors its and − cre C ie –0 es 3ad24). and 23 refs. 8–10; lines , C nC) N eeswere levels RNA GnHCl), ie5.Ipraty the Importantly, 5). line , ). Min ie –) hsincludes This 6–9). lines , B .A ewl hwbelow, show will we As ). Min .Gaiiehydrochlo- Guanidine ). seicRAi HeLa in RNA -specific ifce eacells HeLa -infected Min A α htcnfunction can that ) . SD and IMtrasand Materials SI h mechanisms The Max ( Min al S1 Table SD Min β Min P3 , P(2+3) , transcripts sequences B iu pro- virus Min nHeLa in ie5), line , Min B | α ∼ line , and , line , tran- E8733 30% and B Max Min SD B ). , ,

MICROBIOLOGY PNAS PLUS AB DE

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Fig. 2. Protein expression, and RNA levels in cells infected in vitro and in vivo with WT and recoded PVes. (A) In vitro translation of WT and P2Min recoded PV RNAs. Transcript RNAs of WT and P2Min constructs were translated in vitro in HeLa cell free extracts at 34 ◦C for 12 h in the presence of 35S-methionine. Samples were analyzed by SDS/polyacrylamide gel electrophoresis. (B) Western analysis of 2CATPase related proteins produced in cells infected with WT and P2Min viruses (Materials and Methods). Two sets of HeLa cells were infected with WT or recoded viruses at a MOI of 2, and the incubation continued either in the absence or presence of 2 mM GnHCl. At 4 h p.i., lysates were prepared, and the lysates were probed with antibody to PV 2CATPase and its precursors. (C) The synthesis of PV RNA was measured by qRT-PCR 4 h p.i. of HeLa cells that were infected with WT or P2Min viruses (MOI of 2) either in the absence or presence of GnHCl. Total RNA was isolated from cell lysates and subjected to qRT-PCR analyses. (D) PV RNA in infected HeLa cells was measured by Northern analyses. The cells were infected at an MOI of 2 with WT and P2Min viruses, at 0 and 4 h p.i. RNAs in lysates were quantitated by Northern analysis. Antisense RNA probes were used for the detection of PV RNA (3’-NTR) and for mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (E) Stability of newly made WT or recoded RNAs in infected cells. Two sets of HeLa cells were infected with WT and P2Min viruses at a MOI of 2 and incubated for 6 h at 37 ◦C. At 4 h p.i., 2 mM GnHCl was added to one batch. Lysates of infected cells were made at 4, 5, and 6 h p.i., and the total RNA was isolated. The amount of PV-specific RNA was measured by qRT-PCR. compared with the WT. If 2 mM GnHCl was added at the It is known that plating an average of ∼120 WT PV1(M) viri- time of infection, no RNA synthesis could be observed (Fig. ons onto a monolayer of 106 HeLa R19 cells produces one plaque 2C, +GnHCl). Northern analyses using a probe complementary (15). The si value, therefore, is 1/120. Note that in this experi- to the 3’-NTR of the PV genome were similar to the results ment, the multiplicity of virion per cell is 0.00012 (120 virions per Min obtained with qRT-PCR (Fig. 2D). To test the stability of P2 106 monolayer cells on the experimental plate), which excludes RNA, RNA replication was aborted after 4 h p.i. by the addi- the possibility that more than one virion is needed to produce tion of GnHCl. The fate of viral RNA was then monitored for a plaque. Therefore, the si of WT PV1(M) denotes that 1 of Min the next 1 and 2 h. We observed a slight degradation of P2 120 virions produced the plaque, whereas 119 virions failed. The RNA compared with WT RNA (Fig. 2E). These results are diffi- selection of the virion or of the single cell that leads to a success- cult to interpret because PV translation in vivo is linked in cis to ful infection is a stochastic process (Discussion). Importantly, si genome replication (28): A reduction of translation would inter- is the signature of the virus only when determined in related cell fere with genome synthesis. Defective genome synthesis, in turn, lines under comparable conditions. would interfere with translation. Whatever the mechanism, com- Max The si values that we observed with WT PV1(M), P(1+2+3) , pared with WT PV, there is a small defect in the production of and P2Min are presented in Table 1. For comparison, the P2Min RNA. observed si value of WT PV (1/118) was set at 1.0. In this context, Effect of Temperature on Plaque Assays of Recoded Viruses. An Max analysis of two of three recoded viruses revealed temperature- the si of P(1+2+3) was almost identical to that of WT virus, sensitive phenotypes (ts), as measured by plaque formation which was surprising considering the large sequence differences SD Min in the PP coding region. More surprising was the minute si value (Table S2). Specifically, P(1+2+3) and P2 did not pro- Min 6 duce measurable yields of virus at 33 and 39.5 ◦C, whereas of P2 (1/35, 285), as it took 35,285 virions plated onto 10 R19 HeLa monolayer cells to produce a single plaque. Even in the P(1+2+3)Max variant yielded WT PV titers at 33, 37, and this experiment, the multiplicity of variant/cell is low (0.035285). 39.5 ◦C. Hence, the probability that more than one virion interacted with SI PV and of Recoded PV Variants. The probability of a single one cell is low. It has been speculated that small si values may polio virion to initiate an infection of a favored host cell (HeLa reflect defective virions or clumping of the inoculum. However, R19) under tissue culture conditions is low. Owing to numerous Discussion hostile conditions presented by the cell, most attempts by the neither hypothesis is likely to apply for PV ( ). virus to invade, establish proliferation, and possibly kill the cell The reason for low to minute si values of WT PV1 and recoded are aborted. This phenomenon of restricted virus/cell interaction variants (15, 16) is not yet understood, but might be manifold is referred to as specific infectivity (si), a phenotype reproducible (ref. 15; Discussion). As mentioned, si values can change dras- under identical conditions of experimentation. si defines a known tically when the conditions of the experiments are modified. To prove this point, we have determined si values of WT PV1, minimum number n of virions that, when plated on a huge excess Max Min of a host cell layer, will allow one virion of “n” to successfully P(1+2+3) , and P2 on A549 cells that are known to express initiate an infection. Commonly, the successful infection will be a robust innate immune response (29). We speculated that using visible as a plaque. The term PFU, however, hides the number A549 cells may reduce the chance of any of the lonely virions to n: Depending on the virus or the cell, a PFU value may corre- produce a plaque. This was indeed the case: for PV1(M), the n spond to 100 virions of virus X in cell Y, whereas a PFU of the value changed from 1/118 (R19 HeLa cells) to 1/442 (A549 cells), same virus X tested on a different cell Z may correspond to 1,000 for P(1+2+3)Max from 1/141 (R19 HeLa) to 1/438 (A549), and virions. for P2Min from ∼1/105 (R19 HeLa) to ∼1/106 (A549) (Table 1).

E8734 | www.pnas.org/cgi/doi/10.1073/pnas.1714385114 Song et al. oge al. et Song added been has however, domain, P1 (Assembly morphogenesis The in in PV Poliovirion). sequence in role the the a because of play P1 not omitted does region We this P1. domain capsid the and RNA, ci L varia- RNase synonymous Cre, of conserved β rate the high of “limit” naturally outside natural the tion a to estimate due to variation difficult of however, Coxsackie is, from been It diverged likely (12). strain other viruses ancestral have no PV changes the and synonymous since 35), saturated that (12, guess encapsidation We ( PV variation. reduced in significantly role have elements a regions structural play these not Cre, However, do elements (32). cis-acting genus and the 3 C-Enterovirus Fig. ci, of in L location shown RNase the As (34). predicts clas- below) synplot2 a (“PS”; contain signal ele- that packaging structural serotypes sical and alphavirus pre- sequence different was among conserved Synplot2 ments identify 33). to (32, used we RNAs viously virus C), similar and regions between functional B conserved shared predicting (lines of pro- capable 3 synplot2 is which the Fig. to gram, using variability in leads site shown synonymous strategies analyzed for As have specific SD strategy. each or recoding sequences, Min, the nucleotide Max, in differences the profound by PVs. genome Synthetic PV1(M) of attempt any Variation and Genetic minute, P2 not of if spread the small, to plaques are the variants infection, virus an these in WT succeed of to of necessary that virus of to amount comparable intra- is ( its cells PV cell, HeLa a on replication of cellular infection P2 once productive that suggests established calculation successfully This 1). Table and 1, line ( was experiment 2.34 is, (6.7 is harvest (6.7 of time the at titer the If then plaque. a by produced signifi- the 1/71. a to reducing 1/118 cells, had from R19 stochastic BX795 PV HeLa that the on Note also in drug. effect the HeLa cant effects by with other reversed values not indicating the events observation reach an not did cells, of cells A549 inhibitor BX795-treated potent a IFN- and BX795, kinases with this should immunity confirmed innate with the revert interferes partially that inhibitor an brand, 10 comprising the monolayer If (cell cells host of excess vast a 6.4 with incubation after PFU single a yield to necessary are the that particles of number the 2.4 as 9.4 PFU/mL Titer, A549 PFU/mL Titer, BX795 inhibitor, TBK1/IKK of presence R19 and absence the in cells A549 and R19 HeLa in variants PVes recoded and PV WT of SI 1. Table Fg S2 Fig. regions. i.3i nopee si ak rsnaino uain in mutations of presentation lacks it as incomplete, is 3 Fig. the that note We formula the by calculated was virion) 1 = genome (1 equivalents genome or particles of number the and gradients, CsCl on purified were stocks virus All × si × Discussion 10 auswr omlzdt the to normalized were values si 10 12 × sdpneto h 59hs eladnto h virus the on not and cell host A549 the on dependent is 6 atce/L=1OD 1 = particles/mL 10 rsnstettro h ain;Fg ,ln ) that 6), line 1, Fig. variant; the of titer the presents ) Relative si 2.83 Relative si nM) (100 inhibitor TBK particles Viral sepce,nnyoyosmttosi h P1 the in mutations nonsynonymous expected, As . atce/F 442 118 particles/PFU , particles/PFU , 11 iin.Tettro Tvrs(o1)i h same the in (log10) virus WT of titer The virions. .Nvrhls,bcueo h nrdbylarge incredibly the of because Nevertheless, ). β > si si α 10 Min rdcin(0 1.Tealtered The 31). (30, production si and aust hs fR9HL el.W have We cells. HeLa R19 of those to values si 9 ) ntecl ae sgetyreduced. greatly is layer cell the on × au ie esr fvrsparticles virus of measure a gives value β 260 si 2;ta is, that 120; ngnmso 0 ebr fthe of members 200 of genomes in h netosttr(F/L fec iu a eemndb tnadpau sa ( assay plaque standard by determined was virus each of (PFU/mL) titer infectious The . is ∼ si /500(P2 1/35,000 au fW PV1. WT of value P eoigteOFo the of ORF the Recoding > < VW P(1+2+3) WT PV 1.2 × × .0)snnmu site synonymous 0.001) × × − .510 .5000 0.0023 0.006 0.0001 0.0033 1.15 1.26 1.01 0.84 1.25 1.66 1 1 ± ± 10 10 × 10 10 8354 18 71 9 10 Min 9 12 10 6 ) 11 × nHL cells), HeLa on n iin Fg 1, (Fig. virions 5 0 [where 000 35, au o WT for value si IKK 4 8 auswith values × × + ± ± -related 10 10 Min α 2438 141 32 3 10 9 and has B , 1.86 2.63 unmesntrlvrain tsol entd oee,that however, noted, is be CAV20 should It and num- P(1+2+3) variation. PVs the in natural Whereas the acquired outnumbers changes (36). in the P3 changes large, and synonymous P2, natural P1, of domain ber nucleotides total coding of the percentage a in as PV1(M) in from data changes the mous of interpretation the improve similar 3 To is Fig. P3. cur- and mutations at P2 synonymous but of that of domains, to density P3 and the P2 inspection, the sory in those outnumber domain ae nteosrainta ia eoe aetepropen- the 2013, [Dykeman, have hairpins genome-specific genomes of viral dozens form that to is observation sity It the proposed. recently on been based has morphogenesis virus +ssRNA steps 4 delicate (Fig. of genome progression cognate the the publi- and indicates CP other it between from as cocondensation 51), (50, term ( cations the uncertain borrowed remains steps have role these the We in but genome (35), PV detail great the in of assembly studies CP Previous PV (8). elucidated maturation have virion the by around followed (“pentamers”) (49), precursors genome capsid of condensation by A 4 (Fig. genus assembly of the specificity of viruses all of C-cluster possibly that and suggest We enteroviruses (45–48). 2C precursors PV CP related the mechanism closely (2C as protein This specific, key complex. highly a is replication by membrane-bound precursors the CP of of used recognition mechanism the The is interaction. protein/protein by precursors while genome, PV synthesized nascendi procap- the of viruses, ( role unknown +ssRNA precise still for is the morphogenesis but PV (8), in are structurally virions sids are remarkably, PV procapsids to that, These related RNAs. formed closely cellular are or viral 44) of (35, void capsids”) “empty VLP-associated as selection the in role critical in a (39–43). RNAs RNAs play RNA of can any abundance with cell the space whereby the internal cellular), by their or expressed called fill (viral VLPs cells available now host Indeed, (37), in (38)]. viruses virions [VLPs; many cognate particles” form like resemble to “virus information that the cellular structures carry CPs heterologous of 3D absence viral the abundant most in even genomes, However, the cognate cell. infected the packaging in acids avoid nucleic to Min. or cific Max Poliovirion. global the of no of identities is other Assembly variable there constructs, (with because Min exist part or in can mutants) Min Max and our Max for for calculated sequences CPS given a at 6 rgesmrhgnsso Va eitdi i.4 Fig. in depicted as PV of morphogenesis triggers ) × ifrn ehns edn oasml pcfiiyin specificity assembly to leading mechanism different A 4 (Fig. specificity to leading framework the that propose We known (also “procapsids” icosahedral cells, PV-infected In Picornaviridae × × − ± 10 ± 10 10 F 0386 10 10 94 6 Max eadded we , 11 13 sdlvrddrcl n pcfial ocgaecapsid cognate to specifically and directly delivered is , 6.8 2.8 TbeS3 Table s rti/rti eonto o achieving for recognition protein/protein use × × PNAS + ± ± 10 10 94.1 39 3.5 7 10 11 | ulse nieSpebr2,2017 25, September online Published hc eit h muto synony- of amount the depicts which , ia sebyms ehgl spe- highly be must assembly Viral I , ATPase A 4.9 1.2 1.4 ± ± aeil n Methods and Materials ). P2 0.2 0.2 × × × − Min 10 iciiae gis even against discriminates 10 10 × × 12 6 8 10 10 Max 6 4 Discussion II ( ) rPV(1+2+3) or Discussion 1.98 1.9 Discussion 2.6 2.5 ). ± Enterovirus 6 II ± .Unusual ). si el) Here, cells). perhaps : 0.2 0.3 × × sdefined is + nstatu in | ATPase 10 10 × ). × E8735 7 8 10 10 SD ). I 5 ) 4 ,

MICROBIOLOGY PNAS PLUS A

B

C

D

E

F

G

Fig. 3. An analysis of conserved RNA sequences and local secondary structures for viruses capable of assembling with a PV Mahoney capsid to produce infectious virus. (A) Cis-acting functional elements conserved in the RNA of enterovirus C cluster viruses are marked on the PVM sequence. The sequence regions mapping to these elements show a statistically significant reduction in variation at synonymous sites with respect to a neutral model of evolution among 200 enterovirus C viruses which share >75% amino acid identity with PVM (B and C). The dotted line in B signifies that the points above it are statistically significant (P < 0.005), and the dotted line in C represents the level at which variation at synonymous sites is observed as often as it is expected in a neutral model. D and E depict local secondary RNA structures conserved between PVM and natural viruses: Coxsackie A20, PV type 2, and PV type 3 (D), and synthetic viruses: Max and SD which were recoded over the P2 and P3 regions (E). A mathematically complete ensemble of structures was generated in 30-nt windows for each sequence with the crumple program (SI Materials and Methods). The resulting structures were filtered and compared between the viruses labeled at equivalent nucleotide positions. Each conserved structure is represented by a rectangle that spans the range of the structure’s position. (F and G) Synonymous (F) and nonsynonymous (G) in the same alignment of virus RNAs as used in D and E are compared by pairwise alignment to PV1(M); single nucleotide mutations are depicted by dark bars. no. 1019; no. 1103 (19); Rolfsson, 2016, no. 964 (21)], described morphogenesis based on properties of PV “defective interfer- as “cryptic, sequence-degenerate, dispersed RNA packaging signals” ing particles” (DI particles) (53, 54). PV DI particles naturally (22). These hairpins, which are loosely conserved and carry com- lack the P1 coding region. However, in competition experiments, mon “assembly motifs,” have been shown in in vitro experiments DI particles outcompete WT virus in genome replication sim- to promote RNA-dependent assembly of viral capsids, including ply because their genome is shorter. Moreover, DI particles elaborate structural rearrangements of capsid subunits (refs. 19 “interfere” with WT PV proliferation by stealing (in trans) the and 21 and references therein). We were curious as to whether WT PV CPs. This observation not only supports the mecha- the PV genome does also display an array of loosely conserved nism of protein/protein interaction, it also indicates that the large assembly structures along its genome with essential functions in genome segment encoding the capsid precursor is not important PV morphogenesis. We hypothesized that the ability of forming for assembly. these structures would be disturbed by extensive recoding of the Our analyses took into consideration that the choice of syn- PV genome. onymous codons for some amino acid residues, and the degen- Our analysis of the PV1 genome was confined to domains 2 eracy of nucleotide base pairing allows for the formation of and 3 (Fig. 3). The 5’- and 3’-terminal RNA segments do not conformationally identical RNA structures that do not share sig- determine assembly specificity, as was shown (35, 52). Domain nificant sequence similarity. We wanted to determine whether P1 has also been omitted because it does not play any role in any RNA structures could be present that are encoded by

E8736 | www.pnas.org/cgi/doi/10.1073/pnas.1714385114 Song et al. oge al. et Song S1 Fig. P(1+2+3) characteristics PV1 [for WT with proliferate changes) nt (1,297/6,189 synonymous base-pairing. possible of absence any or presence of reduced the selection had conserve would that that codon acids such amino choice, encoding codon regions synony- structures prevailing over after of occurred majority conserved the that structures revealed “multiple recoding of mous of inspection analyzed Manual hypothesis the (22). signals” the of packaging RNA regions accommodate dispersed P3 sequence-degenerate, cryptic, could and P2 that the viruses in exist structures 3 RNA (Figs. introduced 3 were 4 Fig. mutations in synonymous reduced only cooccurring distinctly that were those SD, SD with and and MAX compared of Max segments viruses, recoded con- the synthetic of pro- our profile to 3 to the capsids (Figs. structures 3 compared 2, RNA then CAV20, PV1, We served and into viruses. -3, encapsidated infectious -2, be duce can PV1, which occurring sequences of of naturally all windows the sliding to 30-nt all belonging generated in and with structures (55) detection RNA program possible Crumple escaped the have used We could Synplot2. that sequences degenerate with (used (83) Hagan and condensation. Perlmutter by permission). genome/CP published aiding figure a (22) of genome modification possible viral dots, entire orange the mapping (text); along signals”, genomes packaging ( viral RNA dispersed of complex. sequence-degenerate, absence “cryptic, with replication the or genome in RC, RNA RNAs cognate PS; cellular the with classical precursors (RNA/protein capsid genome PS, of RNA Cocondensation an membrane; genome. to viral unique M, the unit of structural interaction). PS RNA classical cognate a a to is CP PS precursor a of binding 2C High-affinity the complex, RC precur- assembly the this of capsid In protein the the cognate 2C where to (47) the the tailored assembly is PV to been in has process (RC) recognition interaction complex known This replication interaction). (protein/protein viral sor the of ber ( 4. Fig. I w ahaslaigt pcfiiyrcgiin ( specificity/recognition. to leading pathways Two ) G ainsP(2+3) Variants .Ti eutdmntae htfw fay ml conserved small any, if few, that demonstrates result This ). ,ee huhapoiaeyeeyfit uloiewas nucleotide fifth every approximately though even ], D ATPase yohssdsrbn h w-tpasml f+sN viruses. +ssRNA of assembly two-step the describing Hypothesis n 4 and hthd eonzn asdpnae (P,V3 VP1) VP3, [(VP0, pentamer capsid a recognizing (hatched) Max E .Tesrcue,i hywr osre within conserved were they if structures, The ). e h n-tpgot uv rsne in presented curve growth one-step the see , Max 73359n hne)adP(1+2+3) and changes) nt (723/3,549 B A ATPase cspsil sa lgmr(2.( (82). oligomer an as possibly acts I D , A ept h fact the despite , idn famem- a of Binding ) F II and I Max sa is , 5 B II ]. ) ) Mncntut) eg,a cuuaino underrepresented of accumulation an [e.g., constructs), either In (Min (15). retained. were PV sequences WT protein of the that and use to codon close method, CPS average the leaving and (16); CPSs (Min) ( with negative either average generated or is ( (Max) pairs positive codon average strategies: pro- of excess an macromolecular two Avoiding that such of codons followed proliferate. regulation to we the variants cesses, in the involved of sequences ability the nucleotide the preserving which by of extent the sequence test to us motivated Curiosity Discussion packaging PV in to role a us play lead not specificity. do PV, (22) WT signals” packaging dispersed over RNA sequence-degenerate, domain) cryptic, “multiple P1 that the conclude (lacking particles ( DI and cycle; packaging pack- and replication allowing ( yet aging; variants, P3 and P2 com- of The domains ( genomes. of their data encapsidated bined astounding. still is they were changed, but variants was debilitated nucleotide these third every of approximately which cycle in life the P(1+2+3) distinct Variants in phenotype PV replication WT known from any of Absence changed. niio B75 fteint muersos 3,31) (30, response immune innate the of (BX795) inhibitor (see PV WT with changes to similar also importantly, and, cells den- host PV a WT with of ( presented sity; result they where the centrifugation not gradient ( are ( genomes; (16), because: mutated al. virions et in Coleman defect by also observed VSV low or surprisingly (63) (PR8) cycle. virus influenza infectious for ( the [e.g., viruses of other abor- stage to Generally, mon any cells). at target occur single of The can of excess infection state huge of physiological the tion the (in or cells virions culture host of tissue genotype under or low cells underlie state host could reasons suitable Multiple us a conditions). to infecting known in viruses succeeds RNA low all principle, have In appreciated. widely Infectivity. Specific ( and (62); (16). ( protein virions viral (57); recoded cells a infected of ing the ( in (57); synthesis mRNA viral protein of yield the (56, of cell dation?) infected the in ( response 61); immune innate ( content enhanced CpG an chang- in to without increase increased The therefore, use. are, codon ing and pairs positions codon NNUpANN new additional or of two NNCpGNN in are occur which that UpA, dinucleotides and CpG specifically sequences, ( of proliferation viral related be in can deficiencies that constructs. CPD observed Min of to effect concluded certainty specific have single with we no animals, is experimental there in that or culture tissue in 1 (Fig. nonviability with associated C repli- often most viral is with and interfered cation inevitably pairs] codon (unfavorable) ii ∼ i nices ntecneto pcfi iuloie nCPD in dinucleotides specific of content the in increase an ) .B etn P fvrosmmainvrss(6 56–60) (16, viruses mammalian various of CPD testing By ). cabig(oig admysnnmu oos(SD), codons synonymous randomly (moving) scrambling ) hra a n Drcdnsddntdsryvaiiy CPD viability, destroy not did recodings SD and Max Whereas h xeiet ihA4 el eervaigbcuean because revealing were cells A549 with experiments The include: work this by part in supported mechanisms Possible /05 o hs eaiesrne N iue)(4] The (64)]. viruses) RNA negative-stranded these for 1/10–50 Note si ii iii outt hntp 5,6) ( 60); (59, phenotype ts robust a ) au fP1M 110 eaR9 si ag com- range a in is R19) HeLa (1/120, PV1(M) of value the ) ii h nw oeua tp fteP elcto and replication PV the of steps molecular known the ) ). si aus(.. o rbblte htasnl virion single a that probabilities low (e.g., values si > hntp fvrat a aibewt different with variable was variants of phenotype 0 fteP eoecnb oie while modified be can genome PV the of 80% i h xesv est fmttosi recoded in mutations of density excessive the ) si auso oo-ardotmzdP variants, PV deoptimized codon-pair of values PNAS h motneo h phenotype the of importance The SD ii l ainshv enprfidb CsCl by purified been have variants all ) 213617 n P(2+3) and (2,103/6,177) iii | h ueirasml fcec of efficiency assembly superior the ) ulse nieSpebr2,2017 25, September online Published i euneaaye i o reveal not did analyses sequence ) i hnigsynonymous changing ) vi iii atyrdcds of si reduced vastly a ) iv euto (degra- reduction a ) euto fviral of reduction a ) al S4 Table v rti misfold- protein ) SD si (1,174/3,549), h physical the : a lead may ) si | snot is i E8737 and

MICROBIOLOGY PNAS PLUS reversed to some extent the low si values in these cells of the viral genomes lack VPg and are never encapsidated, an obser- three viruses tested. Whereas the innate immune response is vation suggesting that putative RNA structures within the PV likely to play a role in reducing the replication of recoded viruses genome per se do not trigger assembly. The question then arises: (57, 61), attempts to identify any specific step related to innate How are mature PV virions formed at the replication complex immunity have failed (61). past the recognition process (Fig. 4 I, A), even though the PV In determining si values, the MOI (virion/cell) never exceeded genome lacks dPSs? the value of one. We suggest that the success of one virion over Currently, we cannot answer this question. However, we are all other virions incubated on a huge excess of host cells is a entertaining the thrilling hypothesis that the empty PV procap- stochastic process. This conclusion was also reached recently sids might serve as direct precursors to virions by first docking to by two other groups (65, 66). Since PV is haploid, studies of the replication complex via protein/protein interaction followed si present the infection of a single cell with a single genotype, by being filled with emerging genomic RNA, the “stuffing” cat- notwithstanding the fact that PV is a quasispecies that will form alyzed by the viral ATPase 2CATPase. As has been noted before genetic variants later during its replicative cycle. (79), this strategy would be analogous to the morphogenesis of numerous DNA viruses. Assembly. The key in virus encapsidation is specificity. It requires It is likely that the pathway of +ssRNA virus assembly shown a mechanism by which viral components (genome and CPs) rec- in Fig. 4 may have to be modified in the future as more details of ognize each other and assemble to near total exclusion of cellu- +ssRNA virus morphogenesis emerge. It may be prudent, there- lar or other viral components. We have discussed that the assem- fore, not to follow just one “Yellow Brick Road” (80) because bly specificity of C-cluster enteroviruses (PV, several Coxsackie this may lead to the wrong wizard. A viruses), and perhaps of all enteroviruses, is achieved through viral protein/protein interaction (Fig. 4 I, A; ref. 47). A mechanism Materials and Methods of protein/protein interaction to promote assembly specificity Recoding and Plasmids Design. These are described in SI Materials and is involved also in mechanisms of other viral systems (67–69). Methods. A common, alternative mechanism in +ssRNA virus assembly RNA Structure Alignment. This is described in SI Materials and Methods. is the specific, high-affinity binding of a genomic RNA element Cell Culture. Both HeLa R19 cells and A549 (human lung epithelial carci- to the cognate CP (Fig. 4 I, B). Available evidence suggests that noma) cells were maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and this highly specific selection between genome and CP will trigger ◦ 100 µg/mL streptomycin. All cells were grown at 37 C in a 5% CO2 incubator. assembly (Fig. 4 I, B). For two decades, this RNA element has Virus Growth and Virus Titration by Plaque Assay. Viruses were amplified been referred to as PS (70). The classical PS (i) is a single RNA by infection of HeLa R19 cell monolayers with ≥2 PFUs per cell. Infected hairpin (70, 71) or a closely mapping cluster of structures (34), cells were incubated in DMEM (2% FBS) at 37 ◦C until a complete cytopathic which bind strongly to a specific site in a CP; (ii) maps to a single effect (CPE) was detected. After three rounds of freezing and thawing, the genome locus that is distinct for related viruses (34); (iii) presents lysate was clarified of cell debris by a brief high-speed centrifugation, and with a sequence that is conserved for a given virus species. Con- the supernatant containing the virus was used for further passaging or rein- servation of the classical PS allows the direct discovery of the fection. For temperature sensitivity test, SD-, Max-, and Min-containing con- PS by synplot2 computer analyses (32, 33); (iv) triggers assembly structs were transfected into HeLa R19 cells. If there was no viable virus, in vitro or in vivo that can be prevented by site-directed muta- the cell lysate was harvested for blind passages up to six times. Viral titers were determined by a standard plaque assay on HeLa R19 cell monolayers genesis of even single nucleotides (71); (v) can be transplanted using a semisolid overlay of 0.6% tragacanth gum (Sigma-Aldrich) in min- into a different RNA bestowing to this heterologous RNA the imal Eagle’s medium. Plaques were visualized after 2 d of incubation (for ability to be encapsidated into the PS-cognate VLP (42); or normal plaque size) or 3- to 4-d incubation (for pinpoint plaque size), by (vi) can be transplanted into a different genome, thereby gen- staining cells with crystal violet. erating a chimeric virus (70). General Procedures. Reverse transcription-PCR (RT-PCR); sequencing of virus The known mechanisms triggering specificity of assembly (pro- variants; in vitro transcription and RNA transfection; labeling of PV pro- tein/protein or RNA/protein recognition) are portrayed in Fig. teins in vitro; virus purification and determination of viral particles via OD260 4I. We propose that the “cryptic, sequence-degenerate, dis- absorbance; Northern blot analysis; measurement of viral protein levels by persed RNA packaging signals” (22) provide a function in step II Western blot analysis; and assay for specific infectivity are described in SI Materials and Methods. of morphogenesis. The cocondensation of genome with cognate Note. During review of this paper, Aguilera et al., published a study CP, which have been characterized in genomes of RNA phage [mBio, March/April 2017 (84)] concluding that in infections with purified PV MS2 (19, 21), of plant virus satellite tobacco necrosis virus (72), (expressing no phenotype) at low MOI (0.000001 PFU/cell) approximately 5% and of two human viruses (22, 73, 74), is to facilitate “coconden- of the cells were infected with >1 virion. Dual infections increased (to 8%) sation” (step II). Cocondensation includes highly complex rear- when infections with two different PV mutants were analyzed and assayed rangements of precursor CPs, ultimately leading to mature viri- phenotypically. ons (19, 22, 50, 51, 75). To avoid confusion, we strongly suggest ACKNOWLEDGMENTS. We thank numerous colleagues with whom we dis- that, in distinction of the highly conserved entities called PSs, cussed aspects of RNA virus assembly, amongst them H. G. Krausslich,¨ the recently discovered “cryptic, sequence-degenerate, dispersed J. M. Hogle, M. F. Hagan, R. J. Kuhn, S. B. Larson, B. D. Lindenbach, RNA packaging signals” be abbreviated as dPS. E. I. Frolova, I. Frolov, A. L. N. Rao, D. J. Rowlands, P. G. Stockley, PV assembly depends on ongoing genome replication (76). P. E. Prevelidge, and S. P. J. Whelan. We thank D. Peabody for introduc- ing us into the genetics and assembly of phage MS2 and M. F. Hagan for Newly synthesized genomes carry a 5’-terminal VPg that is the permission to use a modified figure published previously. This work was cleaved off (77, 78) by a cellular enzyme once the RNA is supported in part by National Institute of Health Grants R01AI07521901 and released from the replication complex. The resulting infectious 5R37AI15122.

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E8740 | www.pnas.org/cgi/doi/10.1073/pnas.1714385114 Song et al.