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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

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Materials and Methods

Synthesis of poliovirus cDNA fragments
Two different approaches were followed for the synthesis of the 3 gene fragments. F1 and F3 were synthesized by Integrated DNA Technologies, Inc. Briefly, gel-purified oligonucleotides of approximately 60 nt in length were designed to provide complete coverage of each segment with unique complementary overlaps of 15-30 nt. The oligonucleotides were annealed in a TE buffer [10 mM tris-HCl (pH 8.0), 1 mMEDTA, 50 mM NaCl] and ligated together with T4 DNA ligase to form the desired segment (size: 400-600 bp). These segments were gel purified, digested with the appropriate restriction enzymes and ligated into similarly cleaved pUC 18 plasmid vector. Several clones were sequenced to find a completely correct clone or a clone that could be corrected via standard site directed mutagenesis. The segments were ligated together to produce a given fragment which was cloned into pUC 18 plasmid vector, via designed 5� overhang ends compatible with Sal I and EcoR I restrictions sites. The sequence of each cDNA fragment was verified by sequencing of both DNA strands.

We synthesized the segments (size: 400-600 bp) of F2 by assembling in an asymmetric PCR assay 8 to 12 gel-purified oligonucleotides (S1). The length of the oligonucleotides varied between 40 to 93 nt (size average: 77) and the overlapping region between two oligonucleotides was on average 20 nt long. A mixture of Taq and Pwo DNA polymerases was used to reduce error frequency and increase of the yield of the PCR products (Expand High Fidelity PCR System, Roche). The PCR products were purified and ligated directly into a plasmid containing single 3�-T overhangs at the ends (pGEM®-T Easy Vector Systems, Promega) that complement those of the amplified segment. 5-15 clones were sequenced to identify either error-free DNA segments or segments containing small numbers of errors that could be eliminated by combining the error-free portions of segments via an internal cleavage site. All segments were assembled into pGEM®-T Easy Vector via their compatible unique cleavage sites. After completion, the sequence of F2 gene was verified on an ABI Prism 310 DNA Genetic Analyzer (Foster City, Ca), using a set of sequencing primers.

F1 encode the T7 RNA polymerase promoter, the 5�NTR and almost the entire structural region (VP4, VP2, VP3 and VP1 from position 2480 to position 2967). F1 overlaps F2 by 24 nts. F2 encodes part of VP1 (from position 2944), and the majority of the nonstructural protein P2 precursors (2A, 2B and 2C from position 4124 to position 4839). F2 overlaps F3 by 21 nt. F3 encodes part of 2C (from position 4819), the entire P3 region, 3�NTR and the poly (A) tail.

Designation of genotypic markers in sPV1 (M) cDNA
We engineered 13 new restriction sites into the sPV1 (M) cDNA by changing 20 nts of the wt PV1(M) sequence [S2 (GenBank accession number V01149)]. The positions assigned to the nts refer to the sequence of PV1 (M) published previously (S2). The nt changes and new restrictions sites created (in parentheses) are the following: nts 102 and 103 (T to G, A to G; Xma I); nt 778 (A to G; BssH II); nts 1399 and 1402 (T to C, C to G; Sma I); nts 2473 and 2476 (A to T, A to G; Fsp I); nts 2725 and 2728 (G to C, G to C; Sac II); nt 3836 (A to C; Stu I); nt 4435 (A to C; Xho I); nts 4831 and 4834 (T to C, A to G; Mlu I); nt 5242 (C to T; Hpa I); nts 5821 and 5824 (T to C, G to C; Not I); nt 6241 (C to G; Pvu II); nt 6706 (T to C; BbvC I); nts 7201 and 7204 (A to G, T to C; PpuM I). We also introduced 2 nt changes (T to G, A to C) at positions 1810 and 1813, which resulted in the elimination of a Pst I restriction site. In addition, the following substitutions were engineered: nt 3184 (T to C); nt 3262 (A to G); nt 3454 (T to C); nt 4084 (A to G); nt 4246 (A to G). All but three nts substitutions resulted in silent mutations in the ORF. One substitution engineered into the 2B coding region (creating a Stu I site) changed an amino acid from Ile to Leu . The other mapped to a sequence in the 5�NTR separating the cloverleaf from the IRES element. Both of these changes have previously been shown to have no influence on viral replication in tissue culture (S3, S4).

In vitro transcription
sPV1(M) cDNA and an infectious wt poliovirus cDNA, pT7PVM (S5) were linearized with EcoR I and 1Greek Letter Mug of linearized template DNA was transcribed in a 50 Greek Letter Mul reaction mixture which contained the following components: 40 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 10 mM dithiothreitol (DTT), 2 mM spermidine, 1 mM each NTP, 40 units of RNase inhibitor, and 40 units of T7 RNA polymerase. After 2 hours of incubation at 37°C, yields were about 30 Greek Letter Mug with > 80% full-length RNA, as estimated by gel electrophoresis.

In vitro translation
Transcript RNA derived from sPVM cDNA and virion RNA derived from wt PV1(M) were purified by phenol-chloroform extraction and ethanol precipitation, translated in the presence of [35S] Translabel (ICN Biochemicals) and processed at 34°C in HeLa cell-free extracts for 15 hours, as described previously (S6). Samples of the translation reactions were analyzed on SDS-12.5% polyacrylamide gels. The gel was fixed and treated with En3Hance and exposed to x-ray film.

De novo synthesis of poliovirus in a HeLa cell-free system and plaque assay
RNAs were either derived from sPV1(M) cDNA or from purified wt PV1(M). The translations were identical to those described in Fig. 2, except that they were supplemented with unlabeled methionine . One Greek Letter Mug of sPV1(M) RNA and 0.3 Greek Letter Mug of wt PV1(M) RNA were incubated with HeLa cell-free extracts in a total incubation volume of 50 Greek Letter Mul. Incubation was for 15 hours at 34 °C. The samples were then treated with RNase A (20 Greek Letter Mug/ml) and RNase T1 (100 U/ml) for 30 min at room temperature, diluted to 300 Greek Letter Mul in HBSS, and incubated with HeLa cell monolayers for 1 hour at room temperature. The monolayers were washed with HBSS and overlaid with 0.6% (wt/vol) gum tragacanth. After 48 hours at 37 °C and 5% CO2, the cells were stained with 1% crystal violet.

Detection of engineered genetic markers
Confluent HeLa cell monolayers on 35-mm-diameter plates were infected with sPV1(M) or wt PV1(M) at a multiplicity of infection of 10 PFU per cell. The cells were incubated at 37°C until they showed signs of cytopathic effect (CPE). The same procedure was applied to the viruses reisolated from the spinal cord of paralyzed mice. RNA was isolated from infected cells according to the TRIzol protocol (Life Technologies, Inc.). The region containing a given genetic marker was amplified by the Titan One Tube RT-PCR system (Roche Molecular Biochemicals) using downstream and upstream primers specifically designed for amplification of this region. To exclude the possibility that the signals detected in the RT-PCR assays were due to residual template DNA used in the transcriptions reactions, we tested all of the RNA samples by PCR without reverse transcription. No PCR bands were observed in the absence of cDNA synthesis, indicating that the signals detected were due to poliovirus RNA (data not shown). The specific products were analyzed by digestion with the respective restriction enzymes.

Plaque reduction assay
HeLa cells were grown as monolayers in six-well plates and incubated with MAb D171 at room temperature for 2 hours. Approximately 100 PFU of either sPV1(M) or wt PV1(M) was added to the cells and incubated for 1 hour at room temperature. Then the cells were washed and the number of plaques was determined after 48 hours, as described above. A control plate with unrelated Mab was similarly treated. The unrelated Mab had no effect on the virus infection (data not shown).

For the neutralization test, specific anti-poliovirus serum was mixed with approximately 100 PFU of either sPV1(M) or wt PV1(M) at room temperature for 2 hours. The antibody-virus mixture was added to the HeLa monolayers. Following incubation for 1 hour at room temperature, the cells were washed and the plaques stained after 2 days as described in Fig 3.

Neurovirulence assay
Groups of 4 CD155 tg mice (equal number of male and females) were inoculated with any given amount of virus ranging from 102 to 108 PFU (30 Greek Letter Mul/mouse) intracerebrally for sPV1(M) and wt PV(M). Mice were examined daily for 21 days post-inoculation for paralysis and/or death. Homogenized spinal cord materials were prepared from three representative mice for each virus tested, and viruses were reisolated from the spinal cord and tested for the presence or absence of the genetic markers as described above. The virus titer that induced paralysis or death in 50% of the mice (PLD50) was calculated by the method of Reed and Muench (S7). All procedures involving experimental mice were conducted according to protocols approved by the institutional committees on animal welfare.

References and Notes
S1. W. Pan et al., Nucleic Acids Res. 27, 1094 (1999).
S2. V. R Racaniello, D. Baltimore, Proc. Natl. Acad. Sci. U.S.A.78, 4887 (1981).
S3. C. Mirzayan, E. Wimmer, Virology 189, 547 (1992).
S4. W. Xiang, K. S. Harris, L. Alexander, E. Wimmer, J. Virol. 69, 3658 (1995).
S5. X. Cao, R. J. Kuhn, E. Wimmer, J. Virol. 67, 3149 (1993).
S6. A. Molla, A. Paul, E. Wimmer, Science 254, 1647 (1991).
S7. L. J. Reed, H. Muench, Am. J. Hyg. 27, 493 (1938).

Supplemental Figure 1. Test for the presence of engineered Not I genetic marker in sPV1(M) and wt PV1(M). An RT-PCR product, encompassing the region between nt 5260 and nt 6016, was generated from the inoculated viruses (A) and from the viruses isolated from spinal cord of paralyzed mice (B) as described above. The 757 bp RT-PCR product was treated with Not I in order to identify the restriction site introduced at position 5821 in sPV1 (M). The RT-PCR product was either untreated (-) or treated (+) with Not I. The positions of the 757 bp RT-PCR product and its 561 bp and 196 bp Not I cleavage products are indicated. M, DNA molecular weight marker VI (Boehringer Mannheim). Sizes of the bands are indicated in bp. Products were run on a 1.2% agarose gel. Electrophoretic analysis of the RT-PCR products of the inoculated sPV1(M), and of the virus recovered from sPV1(M)-infected mice showed the presence of 2 bands (561nt and 196 nt) after digestion with Not I, an observation indicating that the virus contained the engineered marker (lane 2 and 4). As expected, only a single band of 757 nt was observed when RT-PCR products of wt PV1(M) were digested with Not I, due the absence of the genetic marker (lane 6 and 8). We also confirmed the presence or absence of the additional markers in sPV1(M) and wt PV1(M), respectively (data not shown).

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