![Products of in vitro translation and proteolytic processing of poliovirus RNAs in a HeLa cell-free extract. Transcript RNA derived from sPVM cDNA ( 13 ) and virion RNA derived from wt PV1(M) were translated and analyzed as described in ( 13 ). Lane 1, wt PV1(M) marker (M) displaying a lysate of [ 35 S] methio- nine-labeled poliovirus- infected HeLa extract; lane 2, virion RNA derived from wt PV1(M); lane 3, RNA derived from sPV1(M) cDNA. Bands correspond with the seg- ments in Fig. 1A. VPO, 2BC, 3AB, and 3CD are precursor polypeptides.](https://www.researchgate.net/profile/Jeronimo-Cello/publication/11263253/figure/fig2/AS:282149744857090@1444281149280/Products-of-in-vitro-translation-and-proteolytic-processing-of-poliovirus-RNAs-in-a-HeLa_Q320.jpg)
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DOI: 10.1126/science.1072266
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et al.Jeronimo Cello,
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Chemical Synthesis of Poliovirus cDNA: Generation
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Chemical Synthesis of Poliovirus
cDNA: Generation of Infectious
Virus in the Absence of Natural
Template
Jeronimo 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 translated
and replicated in a cell-free extract, resulting in the de novo synthesis of
infectious poliovirus. Experiments in tissue culture using neutralizing antibodies
and CD155 receptor–specific antibodies and neurovirulence tests in CD155
transgenic mice confirmed that the synthetic virus had biochemical and patho-
genic characteristics of poliovirus. Our results show that it is possible to
synthesize an infectious agent by in vitro chemical-biochemical means solely
by following instructions from a written sequence.
Research on viruses is driven not only by an
urgent need to understand, prevent, and cure
viral disease. It is also fueled by a strong curi-
osity about the minute particles that we can
view 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 with
high efficiency, occasionally causing the para-
lyzing and lethal poliomyelitis.
Poliovirus, an enterovirus of the Picorna-
viridae, is a small, nonenveloped, icosahedral
virus consisting of five different macromole-
cules: 60 copies each of capsid polypeptides
VP1, -2, -3, and -4 and one copy of the
positive-sense, single-stranded RNA genome
(⬃7.5 kilobases in length) (Fig. 1A) (3). The
chemical sequence (4, 5), the genetic map of
the genome (4 ), and the three-dimensional
crystal structure of the virion (6 ) were deter-
mined 2 decades ago. Poliovirus employs one
of the simplest genetic systems known for
proliferation (3, 7). The virus enters the cell
after attaching to the cellular receptor CD155
(8, 9). Immediately after the virus particle
uncoats inside the cell, the genomic RNA is
translated under the control of the internal
ribosomal entry site (IRES) into a single
polypeptide, the polyprotein (10, 11). The
polyprotein is then processed into functional
proteins by two viral proteinases (3, 7 ). With
the aid of viral proteins, most notably the
RNA-dependent RNA polymerase 3D
pol
and
the genome-linked protein VPg, along with
cellular components, the viral RNA is tran-
scribed into minus-strand copies that serve as
templates 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, engage
further in RNA replication, or be encapsi-
dated by an increasing pool of capsid proteins
(7, 12). In suitable tissue culture cells (for
example, HeLa cells), the entire replication
cycle is complete in only 6 to 8 hours and
yields 10
4
to 10
5
progeny 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 previously
formed in vivo and with the use of the known
sequence 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-free
extract devoid of nuclei, mitochondria, and
other cellular organelles and seeded with vi-
ral RNA. This result confirms that the ge-
nome sequence originally deciphered from
virion RNA is correct (4, 5) and demonstrates
the feasibility of chemical-biochemical syn-
thesis of an infectious agent in the absence of
a natural template.
The strategy of synthesizing the genome
of poliovirus type 1 (Mahoney) [PV1(M)]
began with the assembly of a full-length
cDNA carrying a phage T7 RNA polymerase
promoter at the (left) 5⬘ end (Fig. 1) from
three large, overlapping DNA fragments (F1,
-2, and -3). Each DNA fragment was ob-
tained by combining overlapping segments of
400 to 600 base pairs (bp). The segments
were 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 a
plasmid vector (13). Five to 15 clones were
sequenced to identify either the correct DNA
segments or the segments containing small
numbers of errors that could be eliminated,
either by combining the error-free portions of
segments by an internal cleavage site or by
standard site-directed mutagenesis (13). To
ascertain the authenticity of the synthesized
viral genome [sPV1(M)] and to distinguish it
from the wild-type (wt) sequence of PV1(M)
[wt PV1(M)] (4, 5), we engineered nucleotide
substitutions into the sPV1(M) cDNA as ge-
netic markers (13).
We have shown previously that poliovirus
cDNA carrying a phage T7 promoter for the
phage RNA polymerase can be transcribed
with 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 virion
RNA (15). De novo synthesis of poliovirus
from transcript RNA of wt PV1(M) cDNA in
a cell-free extract of uninfected HeLa cells
has been previously described by Molla et al.
(2). Therefore, the incubation of transcript
RNA from sPV1(M) cDNA in cytoplasmic
extracts of uninfected HeLa cells should re-
sult in the generation of poliovirus. To exam-
ine this possibility, transcript RNA derived
from sPV1(M) cDNA was incubated with a
cytoplasmic extract of HeLa S3 cells, and the
synthesis of virus-specific proteins and infec-
tious viruses were monitored. The products
of sPV1(M) cDNA– derived RNA translation
and proteolytic processing were the same as
those obtained with wt PV1(M) RNA (Fig.
2), an observation suggesting that the open
reading frame (ORF) of the sPV1(M)-specif-
ic RNA is intact. We then tested for the
presence of infectious virus particles in the
cell-free incubation mixture by adding ali-
quots of the incubation mixture to monolay-
ers of HeLa cells. After 48 hours, plaques
appeared [0.5 to 1 ⫻ 10
5
plaque-forming
units (PFU) per g of transcript RNA in 50
l of reaction] whose heterogeneous mor-
phology was characteristic of those produced
by authentic poliovirus (Fig. 3). All together,
these results indicate that the input synthetic
RNA was translated and replicated in the
cell-free extract and that newly synthesized
RNA was encapsidated into newly synthe-
sized coat proteins, resulting in the de novo
synthesis of infectious poliovirus.
Experiments were then carried out to con-
firm that the infectious material isolated from
the cell-free extract was indeed sPV1(M), as
designated by the oligonucleotide sequence.
Restriction enzyme digestion of the reverse
transcriptase–polymerase chain reaction (RT-
PCR) product of the viral RNA recovered
from sPV1(M)-infected HeLa cells revealed
the presence of all engineered markers (fig.
S1, lanes 1 and 2).
We also tested the effects of the poliovirus
Department of Molecular Genetics and Microbiology,
School of Medicine, State University of New York at
Stony Brook, Stony Brook, NY 11794–5222, USA.
*To whom correspondence should be addressed. E-
mail: ewimmer@ms.cc.sunysb.edu
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on May 10, 2010 www.sciencemag.orgDownloaded from
receptor–specific monoclonal antibody
(Mab) D171 and type-specific hyperimmune
sera on plaque formation by sPV1(M) (Table
1). Mab D171 has been shown to completely
block infection of all three serotypes by spe-
cifically binding to CD155, the cellular re-
ceptor of poliovirus (8, 9, 16 ). The treatment
of HeLa cells with Mab D171 before the
addition of sPV1(M) completely abolished
plaque formation (Table 1). Similarly, no
plaques were observed when sPV1(M) was
incubated with poliovirus type 1–specific
rabbit hyperimmune serum [anti-PV1(M)].
Neutralization of the synthetic virus was
type-specific because hyperimmune serum to
poliovirus type 2 (Lansing) [PV2(L)] did not
inhibit plaque formation (Table 1). These
results were in full agreement with those
obtained with wt PV1(M) ( Table 1). They
imply that the de novo poliovirus particles
synthesized in the cell-free extract were se-
rotype 1, requiring the authentic poliovirus
receptor for infection.
The sPV1(M) virus was assayed to deter-
mine whether it expresses a neurovirulent phe-
notype in mice transgenic for the human polio-
virus receptor [CD155 tg mice strain
ICR.PVR.tg I (17)]. When injected with wt
poliovirus strains, these animals develop a neu-
rological disease indistinguishable, clinically
and histologically, from primate poliomyelitis
(17–19). Intracerebral injection of sPV1(M)
caused flaccid paralysis or death in CD155 tg
Fig. 1. Genomic structure of PV1(M) and strategy for the synthesis of its full-length cDNA. (A) The
positive-stranded RNA of poliovirus is shown with VPg at the 5⬘ end of the NTR. In the cDNA, VPg
is replaced by the T7 RNA polymerase promoter. The polyprotein contains one structural (P1) and
two nonstructural (P2 and P3) domains. The 3⬘ NTR contains a heteropolymeric region and is
polyadenylated (shown as AAA
n
). (B) PV1(M) cDNA carrying a T7 RNA polymerase promoter at the
5⬘ NTR end was subdivided into three large fragments for the synthesis of full-length sPV1(M)
cDNA. The sizes of the fragments (in bp) are depicted above or below each rectangle that
represents the respective fragment. The genome sequence encoded by each fragment was
described in (13). (C) The three DNA fragments were synthesized as described in the text. The DNA
fragments were assembled stepwise via common unique restriction endonuclease cleavage sites to
yield full-length sPV(M) cDNA (F1-2-3 pBR322). The sequence of sPV(M) cDNA was confirmed by
automated sequence analyses.
Fig. 2. Products of in
vitro translation and
proteolytic processing
of poliovirus RNAs in a
HeLa cell-free extract.
Transcript RNA derived
from sPVM cDNA (13)
and virion RNA derived
from wt PV1(M) were
translated and analyzed
as described in (13).
Lane 1, wt PV1(M)
marker (M) displaying a
lysate of [
35
S] methio-
nine-labeled poliovirus-
infected HeLa extract; lane 2, virion RNA derived
from wt PV1(M); lane 3, RNA derived from
sPV1(M) cDNA. Bands correspond with the seg-
ments in Fig. 1A. VPO, 2BC, 3AB, and 3CD are
precursor polypeptides.
Fig. 3. Plaque phenotypes of polioviruses gen-
erated in the HeLa cell-free extract. De novo
synthesis of poliovirus from transcript RNA in
cell-free extract of uninfected HeLa cells was
done as described in (13). (A) Plaque phenotype
of virus derived from transcript sPV1(M) RNA.
(B) Plaque phenotype of virus derived from
virion wt PV1(M) RNA.
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mice, resembling the disease produced by wt
PV1(M) (13). However, a larger inoculum of
sPV1(M) than PV1(M) was necessary to para-
lyze or kill the animals (Table 1). The increase
in the magnitude of attenuation was unexpect-
ed, because all nucleotide substitutions intro-
duced into sPV1(M) resulted in silent mutations
in the ORF, except for the newly created Xma
I and Stu I sites in the 5⬘ nontranslated region
(NTR) and 2B region, respectively. These latter
changes had been shown previously to have no
influence on viral replication in tissue culture
(20, 21). However, the silent mutations that we
introduced into the poliovirus genome may ex-
ert a strong influence on pathogenesis by hith-
erto unknown mechanisms.
The presence or absence of genetic mark-
ers in the inoculated virus and the virus iso-
lated from the spinal cords of paralyzed mice
was confirmed by amplification of the viral
RNA by RT-PCR and restriction enzyme
analysis. Our results show that the viruses
isolated from the spinal cords of paralyzed
mice resembled the inoculated virus (fig. S1).
Our data also confirm that the synthetic virus
was the causative agent of the flaccid paral-
ysis observed in the sPV1(M)-infected mice.
The chemical synthesis of the viral genome,
combined with de novo cell-free synthesis, has
yielded a synthetic virus with biochemical and
pathogenic characteristics of poliovirus. In
1828, when Wo¨hler synthesized urea, the theo-
ry of vitalism was shattered (22). If the ability to
replicate is an attribute of life, then poliovirus is
a chemical
[C
332,652
H
492,388
N
98,245
O
131,196
-P
7501
S
2340
,
see (2)] with a life cycle.
As a result of the World Health Organi-
zation’s vaccination campaign to eradicate
poliovirus (23), the global population is bet-
ter protected against poliomyelitis than ever
before. Any threat from bioterrorism will
arise only if mass vaccination stops (23) and
herd immunity against poliomyelitis is lost.
There is no doubt that technical advances will
permit the rapid synthesis of the poliovirus
genome, given access to sophisticated re-
sources. The potential for virus synthesis is
an important additional factor for consider-
ation in designing the closing strategies of the
poliovirus eradication campaign.
References and Notes
1. F. L. Schaffer, C. E. Schwerdt, Proc. Natl. Acad. Sci.
U.S.A. 41, 1020 (1955).
2. A. Molla, A. Paul, E. Wimmer, Science 254, 1647
(1991).
3. T. Pfister, C. Mirzayan, E. Wimmer, in The Encyclope-
dia of Virology, R. G. Webster, A. Granoff, Eds. (Aca-
demic Press Ltd., London, ed. 2, 1999), pp. 1330–
1348.
4. N. Kitamura et al., Nature 291, 547 (1981).
5. V. R. Racaniello, D. Baltimore, Proc. Natl. Acad. Sci.
U.S.A. 78, 4887 (1981).
6. J. M. Hogle, M. Chow, D. J. Filman, Science 229, 1358
(1985).
7. E. Wimmer, C. U. T. Hellen, X. Cao, Annu. Rev. Genet.
27, 353 (1993).
8. C. L. Mendelsohn, E. Wimmer, V. R. Racaniello, Cell
56, 855 (1989).
9. S. Koike et al., EMBO J. 9, 3217 (1990).
10. S. K. Jang et al., J. Virol. 62, 2636 (1988).
11. J. Pelletier, N. Sonenberg, Nature 334, 320 (1988).
12. W. K. Xiang, A.V. Paul, E. Wimmer, Semin. Virol. 8,
256 (1987).
13. Materials and methods are available as supporting
material on Science Online.
14. S. van der Werf, J. Bradley, E. Wimmer, F. W. Studier,
J. J. Dunn, Proc. Natl. Acad. Sci. U.S.A. 82, 2330
(1986).
15. J. Cello, A. V. Paul, E. Wimmer, unpublished data.
16. P. Nobis et al., J. Gen. Virol. 66, 2563 (1985).
17. S. Koike et al., Proc. Natl. Acad. Sci. U.S.A. 88, 951
(1991).
18. H. Horie et al., J. Virol. 68, 681 (1994).
19. M. Gromeier, H.-H. Lu, E. Wimmer, Microb. Pathog.
18, 253 (1995).
20. C. Mirzayan, E. Wimmer, Virology 189, 547 (1992).
21. W. Xiang, K. S. Harris, L. Alexander, E. Wimmer,
J. Virol. 69, 3658 (1995).
22. F. Wo¨hler, Ann. Phys. Chem. 88, 253 (1828).
23. A. Nomoto, I. Arita, Nature Immunol. 3, 205 (2002).
24. We thank A. Wimmer and J. Benach for valuable
comments on the manuscript. We are indebted to
B. L. Semler for a sample of cell-free HeLa cell extract.
Supported by Contracts N65236-99-C-5835 and
N65236-00-M-3707 from the Defense Advanced Re-
search Project Agency.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1072266/DC1
Materials and Methods
Fig. S1
References and Notes
26 March 2002; accepted 25 June 2002
Published online 11 July 2002;
10.1126/science.1072266
Include this information when citing this paper.
MAP Kinase Phosphatase As a
Locus of Flexibility in a
Mitogen-Activated Protein
Kinase Signaling Network
Upinder S. Bhalla,
1
*† Prahlad T. Ram,
2
*† Ravi Iyengar
2
Intracellular signaling networks receive and process information to control cellular
machines. The mitogen-activated protein kinase (MAPK) 1,2/protein kinase C (PKC)
system is one such network that regulates many cellular machines, including the
cell cycle machinery and autocrine/paracrine factor synthesizing machinery. We
used a combination of computational analysis and experiments in mouse NIH-3T3
fibroblasts to understand the design principles of this controller network. We find
that the growth factor–stimulated signaling network containing MAPK 1, 2/PKC can
operate with one (monostable) or two (bistable) stable states. At low concentra-
tions of MAPK phosphatase, the system exhibits bistable behavior, such that brief
stimulus results in sustained MAPK activation. The MAPK-induced increase in the
amounts of MAPK phosphatase eliminates the prolonged response capability and
moves the network to a monostable state, in which it behaves as a proportional
response system responding acutely to stimulus. Thus, the MAPK 1, 2/PKC con-
troller network is flexibly designed, and MAPK phosphatase may be critical for this
flexible response.
Intracellular signaling pathways communi-
cate extracellular information to modulate
cellular functions in response to external
stimuli. Signaling pathways function not only
to transmit information but also to process the
information as it is being transmitted. Such
processing occurs because signaling path-
ways interact with one another to form net-
works (1–3). The processing occurs both
through summation of inputs and through the
temporal characteristics of pathways. For in-
stance, the MAPK cascade communicates
signals from growth factors that bind to re-
ceptor tyrosine kinases to the transcriptional
Table 1. Biological characterization of sPV1(M). Plaque reduction assay in the presence (⫹) and absence
(⫺) of antibodies as described in (13). Anti-PV1(M) and anti-PV2(L) are neutralizing polyclonal antibodies
specific for types 1 and 2 poliovirus, respectively. Neuropathogenicity of sPV1(M) and wt PV1(M) was
assayed in hPVR-tg mice as described in (13). PLD
50
is defined as the amount of virus that caused paralysis
or death in 50% of the inoculated mice.
Virus
PFU
PLD
50
(log
10
PFU)
Mab D171 Anti-PV1(M) Anti-PV2(L)
– ⫹ – ⫹ – ⫹
sPV1(M) 83 0 91 0 88 92 6.2
wt PV1(M) 89 0 86 0 90 87 2.0
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