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Chapter 36 : Cell-Free Genetics of Poliovirus

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Cell-Free Genetics of Poliovirus, Page 1 of 2

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Abstract:

This chapter discusses two processes of the polioviral life cycle that were successfully reproduced in the cell-free system, recombination and complementation. First, recombination, the components of the cell-free recombination system, and the alteration of crossover sites by temperature are discussed, and then the inadvertently discovered phenomenon of complementation are presented. The cell-free system has a limitation in that it works best when either viral RNA (extracted from purified poliovirus particles) or efficiently replicating in vitro-synthesized transcripts are used in conjunction with the HeLa extract. To prove that recombination had indeed occurred in the cell-free system, several controls were used. These experiments showed that only when the two parental RNAs were coreplicated, recombinants were generated. To analyze the recombinants, recombinant plaques were amplified once under the selection conditions and the reverse transcriptase PCR (RT-PCR) product sequenced to confirm the presence of the g mutation and subjected to restriction analysis. To determine the frequency of recombination, it was important to determine the yield of the parental viruses in extracts programmed by the parental RNAs. When guanidine hydrochloride (Gua-HCl) was added to the cell-free recombination reactions, it resulted in a significant reduction of binants (only one out of six reactions gave a single recombinant plaque). This observation is significant because it may provide insights into the mechanism of recombination. Studies involving intertypic poliovirus recombination have suggested a role for RNA secondary structure and a preference for crossover to occur in loop regions of stemloop structures.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36

Key Concept Ranking

Tobacco mosaic virus
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Foot-and-mouth disease virus
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Brome mosaic virus
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Figures

Image of FIGURE 1
FIGURE 1

Schematic representation of the parental molecules used in the recombination study and the resulting recombinant. PV1(M) viral RNA (A) is the wild-type poliovirus RNA and PV1(RIPO) (В) is the RNA of the chimeric poliovirus PV1(RIPO) that contains the mutation for guanidine resistance ( ) starting at nucleotide 4658. The sphere at the end represents the genome-linked nonstructural protein (VPg) at the 5′ terminus. 3′ to the VPg is the cloverleaf-like structure that contains the promoter for plus-strand synthesis. The stem-loop structures 3′ to the cloverleaf-like structure represent the IRES for poliovirus (solid lines) or HRV2 (broken lines). For both RNAs 3′ to the IRESs are the open reading frames of poliovirus followed by the 3′ nontranslated region (line) and the poly(A) tail. The restriction sites that are present on the cDNA of PV1(RIPO) only are shown. (C) The recombinant molecule that originated through a crossover event between the I and I restriction sites. (D) The scheme for studying polioviral recombination in the cell-free system. SK-N-MC cells are a line of neuroblastoma cells, and Gua-HCl is guanidine hydrochloride.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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Image of FIGURE 2
FIGURE 2

Mapping of crossover sites for five representative recombinants that were generated in the poliovirus cell-free system. The genome representation for wild-type poliovirus, PV1(M) with the four new silent restriction sites, and that of PV1(RIPO) with previous restriction sites, and the marker for Gua-HCl resistance ( ) are shown. Below the genome representation are agarose gels showing RT-PCR products of the parents [PV1(M) and PV1(RIPO) ] and five recombinants after being digested with restriction enzymes I and II (A and B), with restriction enzyme I (C), and with restriction enzyme I (D). All the gels shown are 0.8% agarose gels. Lane 5 in A and B, C, and D is DNA molecular weight marker. Sizes of the bands are indicated on the side. Reprinted from reference with permission.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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Image of FIGURE 3
FIGURE 3

Pattern of crossover of the recombinants obtained in the cell-free system at 34°C and after recombination in vivo at 37°C. The six different regions of crossover as defined by the new restriction sites on PV1(M) and the previously existing restriction sites on PV1(RIPO) are shown by six differently shaded columns between the two genomes. The columns in the bar graphs represent the percentage of the total recombinants generated in the cell-free system at 34°C (A) and in vivo at 37°C (B). Reprinted from reference with permission.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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Image of FIGURE 4
FIGURE 4

Pattern of crossover of the recombinants obtained in vivo at three different temperatures. The six different regions of crossover as defined by the new restriction sites on PV1(M) and the previously existing restriction sites on PV1(RIPO) are shown by six differently shaded columns. The columns in the bar graphs represent the percentage of the total recombinants generated in vivo at (A) 34°C, (B) 37°C, and (C) 40°C. Reprinted from reference with permission.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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Image of FIGURE 5
FIGURE 5

Secondary structure of the I-I region at (A) 30°C, (В) 34°C, (C) 37°C, and (D) 40°C.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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Image of FIGURE 6
FIGURE 6

Possible models for recombination based on the presence of the secondary structure in the 2BC region and the inclusion of Gua-HCl in the cell-free recombination system. (A) The established mechanism of strand switching during minus-strand synthesis that is facilitated by the secondary structure. (B) A possible mechanism of strand switching during plus-strand synthesis if the secondary structure also exists in the minus orientation. This mechanism draws support from the observation that very few recombinants were recovered during cell-free recombination in the presence of Gua-HCl.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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Image of FIGURE 7
FIGURE 7

A comparison of replication of two different in vitro-synthesized polioviral transcripts with that of virion-purified RNA in the cell-free system. The polioviral cDNA is depicted on top of the bar chart. The polioviral open reading frame (ORF) is shown as an open box and the lines on either side represent the 5′ and 3′ nontranslated regions. The two restriction sites used for linearizing the cDNA for carrying out runoff transcription, the RI site (adds 3 extra nonviral bases) and I (adds 626 nonviral bases), are shown. In the bar graph the three columns represent the RNAs and the polioviral titers (log PFU/ml). Reprinted from reference with permission.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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Image of FIGURE 8
FIGURE 8

The ability of a polioviral RNA to complement the replication of PV1(RIPO) RNA depends on its capacity to replicate in the cell-free system. The polioviral cDNA is depicted on top of the bar chart. The polioviral open reading frame (ORF) is shown as an open box, and the lines on either side represent the 5′ and 3′ nontranslated regions. The two restriction sites used for linearizing the cDNA for carrying out runoff transcription, the RI site (adds 3 extra nonviral bases) and I (adds 626 nonviral bases), are shown. In the bar graph the columns represent the vatious amounts of polioviral transcript RNAs and viral RNA (along the axis) that complement the replication of PV1(RIPO) , shown in terms of virus titer (log PFU/ml) along the axis. The yield of PV1(RIPO) in the absence of coreplicating wild-type polioviral RNA is 10 PFU/ml ( ). Reprinted from reference with permission.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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Image of FIGURE 9
FIGURE 9

A model for complementation in the cell-free system. This model tries to explain a requirement for replication for complementation to occur. Since virion-purified polioviral RNA can replicate efficiently and RIPO cannot, it is possible that the translational products (that form the replicase) from PV1(M) RNA during or soon after replication could be the molecules responsible for complementing the poor replication of PV1(RIPO) RNA.

Citation: Duggal R. 2002. Cell-Free Genetics of Poliovirus, p 451-460. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch36
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References

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1. Blumenthal, T.,, and G. G. Carmichael. 1979. RNA replication: function and structure of Qbeta-replicase. Annu. Rev. Biochem. 48:525548.
2. Cascone, P. J.,, T. F. Haydar,, and A. E. Simon. 1993. Sequences and structures required for recombination between virus-associated RNAs. Science 260:801805.
3. Duggal, R.,, A. Cuconati,, M. Gromeier,, and E. Wimmer. 1997. Genetic recombination of poliovirus in a cell-free system. Proc. Natl. Acad. Sci. USA 94:1378613791.
4. Duggal, R.,, and E. Wimmer. 1999. Genetic recombination of poliovirus in vitro and in vivo: temperature-dependent alteration of crossover sites. Virology 258:3041.
5. Goodfellow, I.,, Y. Chaudhry,, A. Richardson,, J. Meredith,, J. W. Almond,, W. Barclay,, and D. J. Evans. 2000. Identification of a cis-acting replication element within the poliovirus coding region. J. Virol. 74:45904600.
6. Gromeier, M.,, L. Alexander,, and E. Wimmer. 1996. Internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants. Proc. Natl. Acad. Sci. USA 93:23702375.
7. Hardy, S. F.,, T. L. German,, L. S. Loesch-Fries,, and T. C. Hall. 1979. Highly active template-specific RNA-dependent RNA polymerase from barley leaves infected with brome mosaic virus. Proc. Natl. Acad. Sci. USA 76:49564960.
8. Hiebert, E.,, J. B. Bancroft,, and C. E. Bracker. 1968. The assembly in vitro of some small spherical viruses, hybrid viruses, and other nucleoproteins. Virology 34:492508.
9. Kirkegaard, K.,, and D. Baltimore. 1986. The mechanism of RNA recombination in poliovirus. Cell 47:433443.
10. Lobert, P. E.,, N. Escriou,, J. Ruelle,, and T. Michiels. 1999. A coding RNA sequence acts as a replication signal in cardioviruses. Proc. Natl. Acad. Sci. USA 96:1156011565.
11. McKnight, K. L.,, and S. M. Lemon. 1998. The rhino-virus type 14 genome contains an internally located RNA structure that is required for viral replication. RNA 4: 15691584.
12. Molla, A.,, A. V. Paul,, and E. Wimmer. 1991. Cell-free, de novo synthesis of poliovirus. Science 254:16471651.
13. Romanova, L. I.,, V. M. Blinov,, E. A. Tolskaya,, E. G. Viktorova,, M. S. Kolesnikova,, E. A. Guseva,, and V. I. Agol. 1986. The primary structure of crossover regions of intertypic poliovirus recombinants: a model of recombination between RNA genomes. Virology 155:202213.
14. Tang, R. S.,, D. J. Barton,, J. B. Flanegan,, and K. Kirkegaard. 1997. Poliovirus RNA recombination in cell-free extracts. RNA 3:624633.
15. Tolskaya, E. A.,, L. I. Romanova,, V. M. Blinov,, E. G. Viktorova,, A. N. Sinyakov,, M. S. Kolesnikova,, and V. I. Agol. 1987. Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants. Virology 161:5461.
16. Wilson, V.,, P. Taylor,, and U. Desselberger. 1988. Crossover regions in foot-and-mouth disease virus (FMDV) recombinants correspond to regions of high local secondary structure. Arch. Virol. 102:131139.

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