View source for Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template

(Veronika Jarc)

Jeronimo Cello et al. Chemical Synthesis of Poliovirus cDNA: Generation of Infectious virus in the Absence of Natural Template. Science 297, 1016-1018 (2002). URL: 


== Introduction ==

In this article they wanted to synthesis a poliovirus, so that they could see if it interacts with host just like the natural poliovirus. They also wanted to synthesis it by in vitro chemical-biochemical means just by following instructions from a written sequence of the virus itself.  Cello and his team have translated and replicated the poliovirus in a cell-free extract and they have done many experiments afterwards. Some experiments were done just so that they could describe biochemical and pathogenic characteristics of the virus. They included experiments in tissue culture with neutralizing antibodies, CD155 receptor-specific antibodies and neurovirulence tests in CD155 transgenic mice.


== Poliovirus ==

Poliovirus is an enterovirus and it belongs to family of Picornaviridae. It is a RNA 7500 nucleotides long non-enveloped virus composed of single-stranded positive-sense RNA genome. The virus is just 30 nm in diameter and has an icosahedral symmetry. It is so small in size and genome length so that it is one of the most characterized viruses. Because of its simplicity it is a model system for understanding the biology of the RNA viruses. (1)
We have 3 different serotypes of poliovirus PV1, PV2 and PV3. The difference between them is a slightly different capsid protein. The capsid proteins are very important for different things, one of them is cellular receptor specificity and virus antigenicity. All of the serotypes are highly infectious, you can get them from fecal-oral contact (2). This means that you can get them from sharing food or drink with a contaminated person. PV1 is the most common poliovirus seen in nature. Today it is most common found in regions of Pakistan, Afghanistan, Nigeria, Niger and Chad. PV2 it is instinct in nature and PV3 was found in parts of Nigeria and Pakistan. (3)

Poliovirus is a virus that infects human cells by binding to CD155, which is an immunoglobulin-like receptor (poliovirus receptor) on the cell surface. When it is attached to the receptor the viral particles enter the cell. Till now it is still not well understood, the whole mechanism, how polioviruses enter the cell.
As said before poliovirus is a positive stranded RNA virus and can be used as a messenger RNA (mRNA) and be immediately translated by the host cell. The poliovirus mRNA has a long 5´ end, which encodes for an internal ribosome entry side (IRES) and is necessary for the translation of the viral RNA. After the translation of the viral RNA it is transcribed into a single polypeptide, polyprotein. It is well processed by two internal proteinases into 10 individual viral proteins. RNA dependent RNA polymerase (3Dpol) is a viral polymerase whose function is to copy and transcribe the whole viral genome. 2Apro and 3Cpro/3CDpro are proteases that use the poliovirus-encoded polyprotein as a substrate. The possibility that these proteinases could degraded cellular proteins is still not explored enough. VPg is a small protein, which is coded as 3B between 3A and 3C. It binds to viral RNA and has a big role in the synthesis of viral positive and negative strand of RNA. 2BC, 2B, 2C, 3AB, 3A, 3B are proteins which are part of a protein complex needed for viral replication. All these elements of the virus above are prat of a non-structural region. The protein VP0 is the only protein, which is part of a structural region and is further cleaved into VP2, VP3, VP1 and VP4 proteins. The role of these proteins is to ensemble the viral capsid. With all this elements that compose a poliovirus we can newly synthesis plus-stranded RNA that represent mRNA for protein synthesis or to encapsidate the virus with the virus proteins. In different mammal tissue culture cells (HeLa) one replication cycle can be complete in 6-8 hours. In each dying cell we can release up from 104 to 105 polio virions per cell. (2)


== Sequence analysis of polioviral RNA ==

The poliovirus RNA was sequenced in three stages. The first was identification of the 3´- terminal poly(A) and the second was the identification of the internal sequences. At the end they characterized the 5´- terminal protein-linked fragment. Kitamura and colleagues used the Sanger´s chain termination method to sequence the poliovirus cDNA. First the poliovirus cDNA was synthesized and chains of 7000-7400 deoxyribonucleotides were selected by centrifugation. Then the poliovirion RNA was digested with RNase A and large oligonucleotides were separated on 2D gel electrophoresis. The large oligonucleotides were eluated from the gel, dephosphorylated on 3´ ends and labelled on 5´ ends with phosphorus-32. Then they prepared four different reaction mixtures with cDNA and 5’-32P-labelled primer was annealed and incubated with E.coli polymerase I in the presence of unlabeled dNTPs and one of the four 2’,3’-dideoxynucleotide triphosphates (ddNTPs). After that the synthesized DNA fragments were separated by gel electrophoresis. The sequence was then determined by reading the four different ladders of bands. The result was a nucleotide sequence of the genome of poliovirus type 1 (Mahoney). The base composition of the genome RNA is 30,2 % of A, 22,8 % of G, 23,1 % of C and 23.9 % of U and with the molecular weight of 2,411 X 106. The chemical structure of poliovirus is C332,652H492,388N98,245O131,196-P7501S2340. This empirical formula was the base for Cello and colleagues de novo chemical-biochemical synthesis of infectious poliovirus. (4)


== Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence ==


Racaniello and Baltimore have synthesized and purified polioviruses RNA and cloned the double-stranded molecules in a plasmid. They inserted the construct into the Pst I site on plasmid pBR322. After that they had made a screening of the tetracycline-resistant clones by colony hybridization using a calf thymus DNA-primed poliovirus cDNA probe. They prepared a restriction fragment form clone pVR103 form bases 149-200 with 5’-end labeled at BamHI site. The fragment was then hybridized to poliovirus RNA and extended with RNA-dependent DNA polymerase (revese transcriptase). Before cloning they chemically determined the sequence of the extension products. Then the extended fragment was given a tail with oligo (dC) and double-stranded with DNA polymerase I in the presence of (dG)12-18. After that it was tailed again with oligo(dC) and inserted into the Pst I site of pBR322. pVR105 contained the sequences from the BamHI site up to the first base of the poliovirus genome. The two plasmids were sequenced by Maxam—Gilbert method. They got the complete sequence of the virus. The nucleotide sequence is 7410 nucleotides long. An open reading frame has a beginning at base 671 and is followed by a methionine codon at 743 and continuous unitil a termination codon 71 bases from the 3’ end. (5)


== Chemical synthesis of poliovirus cDNA ==


As seen above, it is possible to synthesis the poliovirus RNA in a way that it is still functional. In this article Cello and colleagues want to assembly a poliovirus cDNA from scratch. Only with the knowledge of basic chemical building blocks independent of viral components previously formed in vivo and the use of the know sequence which was determined in the articles described above.
 
They synthesized the genome of poliovirus type 1 (Mahoney) [PV1(M)]. It began with the assembly of a cDNA carrying a phage T7 RNA polymerase promotor on the very end of the 5’ end. This promotor was in the beginning of three large DNA fragments F1, F2 and F3. On the right side of the promotor there was F1 with 3026 bp, then there was F2 with 1895 bp and on the end F3 2682 bp. All of those fragments were overlapping each other (400-600 bp) and they all covered one range of the genome of poliovirus. All of the fragments were synthesized by assembling purified oligonucleotides of plus and minus polarity with overlapping complementary sequences at their termini. The segments were then ligated in different plasmid vectors. (6)

To synthesis the poliovirus cDNA fragments two different approaches were taken. F1 and F3 were synthesized by Integrated DNA Technologies, Inc. they gel-purified oligonucleotides which were 60 nt long and provided complete coverage of each segment with complementary overlaps of 15-30 nt. The oligonucleotides were put in a TE buffer and ligated together with T4 DNA ligase to the size of the segment. The segments were then purified and cut with restriction enzymes (different for F1 and F3 fragment) and ligated into a pUC 18 plasmid vector.  Seven clones were sequenced so that they could find the completely correct clone or a clone that could be corrected by standard site mutagenesis. The correct fragments were then ligated and cloned into a pUC 18 vector. This vector was made with 5’ overhanging ends compatible with Sal I and EcoR I restriction sites. These vectors were then sequenced so that the cDNA of the fragment, which was in the plasmid vector, was the correct one. F1 pUC18 had two restricition sites SnaB I and EcoRI and F3 pUC18 had Mlu I and EcoR I. F2 fragment was assembled in a sdifferent way with an asymmetric PCr assay with 8 to 12 gel-purified oligonucleotides. The oligonucleotides were 40 to 93 nt long ans had an overlapping region between two oligonucleotides that was 20 nt long. For the PCR they used Taq and Pwo DNA polymerases which reduce error frequency in PCR products. The PCR products were then ligated into a pGEM-T plasmid vector, which has a 3’T overhangs on the end. Then they checked with sequencing 5 to 10 clones for errors. The right clones were then sequenced for the complete sequence of the F2 gene.  In F2 pGEM-T there are two restricition sites SnaB I and EcoR I.  (6)

The DNA fragments were assembled step by step with restriction cleavage sites. F1 pUC18 and F2 pGEM-T vectors mixed together so that F2 was inserted into F1 pUC18 trough the restricition sites SnaB I and EcoR I to assembly F 1-2 pUC18. To E. coli cloning vector pBR322, which has restricition sites EcoR I and Sal I, the F 1-2 pUC18 was given.  F1 and F2 fragments were inserted through these restricition sites to make a new vector F 1-2 pBR322. This vector has restricition sites EcoR I and Mlu I through which the fragment F3 from vector F3 pUC18 will insert. Now we have the full-length sPV(M) cDNA (F1-2-3 pBR322) as seen in figure 1 in the article. (6)


== sPV1(M) genetic marker ==


For the purpose of distinguishing the synthesized viral genome [sPV1(M)] form the wild type sequence of the PV1(M) [wt PV1(M)] we made nucleotide substitution into the sPV1(M) cDNA in a form of genetic markers. We designed 13 new recognition sites into the sPV1(M) cDNA by changing 20 nt of the wt PV1(M) sequence. They created nt changes in form of creating new restricition sites. The sPV1 (M) cDNA has now new restricition sites for Xma I, BssH II, Sma I, Fsp I, Sac II, Stu I, Xho I, Mlu I, Hpa I, Not I, Pvu II, BbvC I and also PpuM I. They did not just create new restriction sites, but also eliminated one site for the restricition enzyme Pst I. Silent mutations were also made in the ORF by substitution of three nucleotides. One substitution was also created into 2B coding region (creating Stu I site) changed an amino acid IIe to Leu. Another substitution made in the sequence in the 5’NTR separated the cloverleaf from IRES element. (6)


== In vitro transcription and translation ==


sPV1 (M) cDNA and wt poliovirus cDNA were put in a pT7PVM and linearized with EcoR I. after the transcription the results were put on a gel electrophoresis. The RNA transcript from the synthetic and wild type poliovirus were purified by phenol-chloroform extraction and ethanol precipitation and translated in the presence of [35S] Translabel. This mixture was then processed in HeLa cell-free extracts for 15 hours. The samples were then analyzed on SDS polyacrylamide gels. The gel was treated with En3Hamce and exposed to x-ray. With this experiment they wanted to see if the results will be the same for the synthetic as for the wild type poliovirus. The length of the transcripts from the wild type and the synthetic were the same as the virion RNA. (6)

De novo synthesis of poliovirus in a HeLa cell-free system and plaque assay

De novo synthesis is an experiment capable of translating RNA of poliovirus type 1 (Mahoney) with good accuracy. The viral proteins in poliovirus are visible except VP2 in VP4. VP2 can be detected by imunnoprecipitation with antibodies to VP2. The most difficult to detect is VP4 even in [35S] methionine-labeled extracts of infected cells, because it contains only one methionine. RNA was taken from the synthetic or wild type poliovirus and was incubated with HeLa cell-free extracts. After 15 hours the plagues appeared and the morphology of them was characteristic to poliovirus. To avoid problems such as transfection of monolayer cells with mRNA used for translation they added a mixture of ribonuclease A and RNase T1 before added to the cells. After 30 min they incubated the mixture with HeLa cell monolayers for 1 hour, so that they could test the presence of infectious virus particles in the cell-free incubating mixture. The monolayers were then washed and after 48 hours the cells were stained with 1% crystal violet. (6)

In the results for the proteolytic processing of poliovirus RNA in HeLa cell-free extract (seen in figure 2 in the article) we see that the transcript RNA from the synthetic poliovirus and the virion RNA from the wild type poliovirus were the same.  As seen in figure 3 in article the synthetic RNA was translated and replicated in the cell-free extract and that newly synthesized RNA was encapsidated into coat proteins which lead to de novo synthesis of the infectious poliovirus. The expression of the proteins in the synthetic poliovirus and the wild type poliovirus are the same. (6)

Detection of engineered genetic markers

Then they carried out experiments to confirm, that the infectious material from the cell-free extract was sPV1(M). They isolated viruses from the spinal cord of paralyzed mice. RNA was isolated from the infected cells with TRIZOL. Then they carried out RT-PCR using downstream and upstream primers for the amplification of the region. They tested all of the RNA samples by PCR without reverse transcription to exclude the possibility that some signals were residual DNA template. There was no band seen in the absence of cDNA synthesis, which indicates that the signals that were seen were poliovirus RNA. The products were then analyzed with restriction enzymes. (6)

Biological characterization of sPV1(M)

They tested the effects of the poliovirus receptor-specific monoclonal antibody (Mab) D171 and type-specific hyperimmune sera on plaque formation by sPV1(M). With this test they wanted to see if de novo poliovirus particles synthesized in the cell-free extract were serotype 1. They grown HeLa cells in monolayers and incubated them with MAb D171. After 1 hour they added 100 PFU of sPV1(M) or wt PV1(M) to the cells and incubated. After that they mixed anti-poliovirus serum with 100 PFU of sPV1(M) or wt PV1(M). They added the antibody-virus mixture to the HeLa monolayers. And then they washed and stained the plates. Mab D171 is shown to block infection of all three serotypes by binding to the CD155 (as seen in table 1 in the article). No plaques were observed when sPV1(M) was treated with poliovirus type 1 serum. Nevertheless synthetic virus was type-specific because hyperimmune serum to poliovirus type 2 did not inhibit plaque formation. The results were very similar also in the wild type poliovirus. (6)

Neurovirulence assay

Tis assay was made to determine whether synthetic poliovirus expresses a neurovirulent phenotype in mice transgenic for the human poliovirus receptor (CD155). Four tg CD155 mice were given poliovirus the synthetic one and the wild type one. Mice were then examined for 21 days for paralysis or death. After the 21 days the mice were taken the spinal cord and tested for the presence of the two viruses. The virus that induced paralysis or death in 50 % of the mice (PLD50) was calculated. The animals developed paralysis or death in animals injected with the wild type or synthetic poliovirus. The difference was in the inoculum between the polioviruses (as seen in table 1 in the article). There was an increase in the magnitude of attenuation in the mice treated with sPV1(M) which was the cause of silent mutations in the ORF. The mechanism of how silent mutations effect pathogenesis is not known. (6)

The region that had the engineered marker Not I was located in the region between 5260 and 6016 nt. The fragment (757 bp long) was treated with Not I in order to identify the restriction site in sPV1(M). The position of the RT-PCR product is in 561 bp and 196 bp.  After the RT-PCR they put the products on 1,2 % agarose gel. The results showed that synthetic poliovirus treated with Not I had presence of 2 bands (561 and 196 nt). When it was not treated with Not I it was no band observed at all, indicating that the virus contained the engineered marker. In wild type poliovirus we see only a single band at 757 nt when digested with Not I or without. (6)

Conclusion

The synthesis of the synthetic poliovirus is plausible with the right knowledge and experimental tools. In the article they showed that you can make a synthetic poliovirus from scratch and that it is functional. This article will be the basis of lots of new researches in developing new vaccination and treatments. It can be also used as an example how to synthesis other RNA viruses which are more complex.
 
References

1. Hogle J. Poliovirus cell entry: common structural themes in viral cell entry pathways. Annu Rev Microbiol 56: 677–702 (2002).

2. Racaniello and Baltimore, D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc. Natl. Acad. Sci. U.S.A. 78 (8): 4887–91 (1981)

3. Transmission of wild poliovirus type 2—apparent global interruption. Wkly. Epidemiol. Rec. 76 (13): 95–7 (2001).

4. Kitamura N. et al. Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature 291, 547 (1981).

5. V. R. Racaniello, D. Baltimore. Molecular cloning of poliovirus cDNA and the determination of the complete nucleotide sequence of the viral genome. Proc. Natl. Acad. Sci. U.S.A. 78, 4887 (1981).

6. Jeronimo Cello et al. Chemical Synthesis of Poliovirus cDNA: Generation of Infectious virus in the Absence of Natural Template. Science 297, 1016-1018 (2002).