Chemical Synthesis of PolioviruscDNA: Generation of InfectiousVirus in the Absence of NaturalTemplateJeronimo Cello, Aniko V. Paul, Eckard Wimmer*Full-length poliovirus complementary DNA (cDNA) was synthesized by assem-bling oligonucleotides of plus and minus strand polarity. The synthetic polio-virus cDNA was transcribed by RNA polymerase into viral RNA, which translatedand replicated in a cell-free extract, resulting in the de novo synthesis ofinfectious poliovirus. Experiments in tissue culture using neutralizing antibodiesand CD155 receptor–specific antibodies and neurovirulence tests in CD155transgenic mice confirmed that the synthetic virus had biochemical and patho-genic characteristics of poliovirus. Our results show that it is possible tosynthesize an infectious agent by in vitro chemical-biochemical means solelyby following instructions from a written sequence.Research on viruses is driven not only by anurgent need to understand, prevent, and cureviral disease. It is also fueled by a strong curi-osity about the minute particles that we canview both as chemicals and as “living” entities.Poliovirus can be crystallized (1) and its empir-ical formula can be calculated (2), yet this“chemical” replicates naturally in humans withhigh efficiency, occasionally causing the para-lyzing and lethal poliomyelitis.Poliovirus, an enterovirus of the Picorna-viridae, is a small, nonenveloped, icosahedralvirus consisting of five different macromole-cules: 60 copies each of capsid polypeptidesVP1, -2, -3, and -4 and one copy of thepositive-sense, single-stranded RNA genome(⬃7.5 kilobases in length) (Fig. 1A) (3). Thechemical sequence (4, 5), the genetic map ofthe genome (4 ), and the three-dimensionalcrystal structure of the virion (6 ) were deter-mined 2 decades ago. Poliovirus employs oneof the simplest genetic systems known forproliferation (3, 7). The virus enters the cellafter attaching to the cellular receptor CD155(8, 9). Immediately after the virus particleuncoats inside the cell, the genomic RNA istranslated under the control of the internalribosomal entry site (IRES) into a singlepolypeptide, the polyprotein (10, 11). Thepolyprotein is then processed into functionalproteins by two viral proteinases (3, 7 ). Withthe aid of viral proteins, most notably theRNA-dependent RNA polymerase 3Dpolandthe genome-linked protein VPg, along withcellular components, the viral RNA is tran-scribed into minus-strand copies that serve astemplates for the synthesis of new viral ge-nomes ( plus-strand RNA). Newly synthe-sized plus-strand RNA can serve as messen-ger RNA for more protein synthesis, engagefurther in RNA replication, or be encapsi-dated by an increasing pool of capsid proteins(7, 12). In suitable tissue culture cells (forexample, HeLa cells), the entire replicationcycle is complete in only 6 to 8 hours andyields 104to 105progeny virions per cell.Here we describe the de novo chemical-biochemical synthesis of infectious poliovi-rus from basic chemical building blocks, in-dependent of viral components previouslyformed in vivo and with the use of the knownsequence as the only instruction for engineer-ing the genome. The succession of macromo-lecular events in an infected cell was repro-duced in a test tube containing a cell-freeextract devoid of nuclei, mitochondria, andother cellular organelles and seeded with vi-ral RNA. This result confirms that the ge-nome sequence originally deciphered fromvirion RNA is correct (4, 5) and demonstratesthe feasibility of chemical-biochemical syn-thesis of an infectious agent in the absence ofa natural template.The strategy of synthesizing the genomeof poliovirus type 1 (Mahoney) [PV1(M)]began with the assembly of a full-lengthcDNA carrying a phage T7 RNA polymerasepromoter at the (left) 5⬘ end (Fig. 1) fromthree large, overlapping DNA fragments (F1,-2, and -3). Each DNA fragment was ob-tained by combining overlapping segments of400 to 600 base pairs (bp). The segmentswere synthesized by assembling purified oli-gonucleotides [average length, 69 nucleotides(nt)] of plus and minus polarity with overlap-ping complementary sequences at their termi-ni, and the segments were then ligated into aplasmid vector (13). Five to 15 clones weresequenced to identify either the correct DNAsegments or the segments containing smallnumbers of errors that could be eliminated,either by combining the error-free portions ofsegments by an internal cleavage site or bystandard site-directed mutagenesis (13). Toascertain the authenticity of the synthesizedviral genome [sPV1(M)] and to distinguish itfrom the wild-type (wt) sequence of PV1(M)[wt PV1(M)] (4, 5), we engineered nucleotidesubstitutions into the sPV1(M) cDNA as ge-netic markers (13).We have shown previously that polioviruscDNA carrying a phage T7 promoter for thephage RNA polymerase can be transcribedwith T7 RNA polymerase into highly infec-tious RNA (14 ). Accordingly, the sPV1(M)cDNA and wt PV1(M) cDNA were tran-scribed (13) and were found to yield tran-script RNAs of the same length as virionRNA (15). De novo synthesis of poliovirusfrom transcript RNA of wt PV1(M) cDNA ina cell-free extract of uninfected HeLa cellshas been previously described by Molla et al.(2). Therefore, the incubation of transcriptRNA from sPV1(M) cDNA in cytoplasmicextracts of uninfected HeLa cells should re-sult in the generation of poliovirus. To exam-ine this possibility, transcript RNA derivedfrom sPV1(M) cDNA was incubated with acytoplasmic extract of HeLa S3 cells, and thesynthesis of virus-specific proteins and infec-tious viruses were monitored. The productsof sPV1(M) cDNA– derived RNA translationand proteolytic processing were the same asthose obtained with wt PV1(M) RNA (Fig.2), an observation suggesting that the openreading frame (ORF) of the sPV1(M)-specif-ic RNA is intact. We then tested for thepresence of infectious virus particles in thecell-free incubation mixture by adding ali-quots of the incubation mixture to monolay-ers of HeLa cells. After 48 hours, plaquesappeared [0.5 to 1 ⫻ 105plaque-formingunits (PFU) per ␮g of transcript RNA in 50␮l of reaction] whose heterogeneous mor-phology was characteristic of those producedby authentic poliovirus (Fig. 3). All together,these results indicate that the input syntheticRNA was translated and replicated in thecell-free extract and that newly synthesizedRNA was encapsidated into newly synthe-sized coat proteins, resulting in the de novosynthesis of infectious