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Chapter 37 : Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions

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Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, Page 1 of 2

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Abstract:

Poliovirus RNA replicates in membrane-associated replication complexes in the cytoplasm of infected cells. By using a reversible inhibitor of poliovirus RNA replication, it is possible to synchronize viral RNA replication. The processing of the viral polyprotein results in the formation of the individual viral proteins along with stable intermediates in the processing pathway. To expand the utility of the in vitro complementation assay, experiments were designed to determine if all of the viral replication proteins could be provided in trans to support the replication of mutant RNA templates. The authors engineered two transcript RNAs (DJB2 and DJB15) that contained large out-of-frame deletions in the polyprotein coding sequence. The results to date using the in vitro complementation assay indicate that the 5’ cloverleaf, the 3’ nontranslated region (NTR), and the poly(A) tail are the minimum sequences required for negative-strand synthesis. Previous studies have shown that the 5’ cloverleaf plays an important role in viral RNA replication. To investigate the role of the 5’ cloverleaf in negative-strand synthesis, the authors determined how cloverleaf mutations affected negative-strand synthesis in preinitiation RNA replication complexes. Results of these experiments showed that 5’ cloverleaf mutations dramatically diminished RNA stability and negative-strand RNA synthesis. The results of recent studies indicate that the 5’ cloverleaf is required for the initiation of negative-strand synthesis. Once the viral mRNA is cleared of translating ribosomes, it could then serve as a template for negative-strand synthesis.

Citation: Morasco B, Smerage L, Flanegan J, Barton D. 2002. Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, p 461-469. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch37

Key Concept Ranking

Protein Synthesis RNAs
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Viral Replication Proteins
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Agarose Gel Electrophoresis
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Figures

Image of FIGURE 1
FIGURE 1

Diagram of poliovirus RN As. (A) T7PV1(A) RNA. (В) 5′ Cloverleaf structure. Modified from reference by permission of Oxford University Press.

Citation: Morasco B, Smerage L, Flanegan J, Barton D. 2002. Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, p 461-469. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch37
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Image of FIGURE 2
FIGURE 2

Diagrams of negative- and positive-strand RNA synthesis using poliovirus RNA transcripts. (Left panel) Rz-T7PV1(A) transcript RNA with 5′ hammerhead ribozyme (Rz-RNA) undergoes autocatalytic cleavage to form ( + ) strand viral RNA with correct 5′-terminal sequence. This RNA is copied to form ( − ) strand RNA in replicative form (RF) RNA intermediate. The negative-strand RNA serves as a template for multiple rounds of VPg-UU primed ( + ) strand RNA synthesis. This results in the synthesis of replicative-intermediate RNA and ss ( + ) strand RNA. (Right panel) T7PV1(A) transcript RNA containing two nonviral 5′ guanine nucleotides (GG-RNA) is copied to form ( − ) strand RNA in RF RNA intermediate. The synthesis of ( + ) strand RNA, however, is inhibited below detectable levels.

Citation: Morasco B, Smerage L, Flanegan J, Barton D. 2002. Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, p 461-469. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch37
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Image of FIGURE 3
FIGURE 3

Two nonviral 5′ Gs in T7PV1(A) transcript RNA inhibit positive-strand RNA synthesis. Nondenaturing agarose gel electrophoresis of P-labeled viral RNAs synthesized in 60-min reactions containing preinitiation replication complexes formed with either poliovirion RNA or the transcript RNAs indicated. Positions of ( + ) strand RNA and RI/RF RNAs in gel are shown above. See diagrams in Fig. 2 for additional information.

Citation: Morasco B, Smerage L, Flanegan J, Barton D. 2002. Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, p 461-469. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch37
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Image of FIGURE 4
FIGURE 4

Complementation of 3D-M394T mutation requires cotranslation with helper RNA. RNA synthesis was assayed at 34 and 39°C in preinitiation RNA replication complexes isolated from HeLa S10 translation-replication reactions containing 3D-M394T RNA and/or RNA2 (a subgenomic replicon helper RNA) as indicated. The total translation time for each reaction was 4 h. For complementation, the reactions containing 3D-M394T RNA and RNA2 were mixed and cotranslated for the times indicated. For cotranslation times of less than 4 h, reactions containing each RNA were incubated separately for the appropriate amount of time before mixing. Labeled RNA was characterized by electrophoresis on a denaturing agarose gel and detected by autoradiography. The positions of 3D-M394T RNA and RNA2 are indicated.

Citation: Morasco B, Smerage L, Flanegan J, Barton D. 2002. Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, p 461-469. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch37
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Image of FIGURE 5
FIGURE 5

Complementation analysis of DJB2 and DJB15 RNA replication in vitro. (A) Diagram of poliovirus RNA, DJB2, and DJB15 transcript RNAs. DJB2 and DJB15 RNAs have large out-of-frame deletions in the polyprotein coding sequence, and therefore do not express any of the viral replication proreins. (B) Preinitiation replication complexes were isolated from translation-replication reactions containing the indicated RNAs. The helper RNA [RNA2(A)AGUA in reference ], contained a large in-frame deletion in the P1 coding region and encoded all of the viral replication proteins. Negative-strand RNA was synthesized by preinitiation complexes incubated at 37°C for 30 min. Radiolabeled negative-strand product RNAs were analyzed on denaturing agarose gels as previously described.

Citation: Morasco B, Smerage L, Flanegan J, Barton D. 2002. Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, p 461-469. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch37
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Image of FIGURE 6
FIGURE 6

Effect of 5′ cloverleaf mutation on RNA stability and negative-strand synthesis. DJB19 transcript RNA contains a four-nucleotide insertion in the 5′ cloverleaf structure following nucleotide 66 (see Fig. 1 ). PVl(A) RNA (wild type) and DJB19 RNA were transcribed in vitro with and without a 5′ cap. The stability of these RNAs and their ability to support negative-strand synthesis were measured in reactions containing preinitiation complexes. The reactions were incubated at 37°C for 30 min, and the total RNA and the labeled product RNAs were analyzed by denaturing agarose gel electrophoresis as described ( ). (A) The total RNA within the gel was stained with ethidium bromide and visualized with UV light. The position of the input viral RNA in the gel is indicated. (B) Labeled negative-strand product RNAs were detected in the gels by autoradiography. The position of negative-strand RNA in the gel is indicated. The results shown here were derived from results originally published in the ( ).

Citation: Morasco B, Smerage L, Flanegan J, Barton D. 2002. Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, p 461-469. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch37
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Image of FIGURE 7
FIGURE 7

Model of circular RNP complex used for the initiation of negative-strand RNA synthesis. PABP, PCBP, and viral proteins 3CD and VPg are proposed to interact with each other and the 5′ and 3′ ends of the viral RNA to form a circular RNP complex. Negative-strand synthesis initiates by the elongation of a VPg primer by the viral polymerase ЗD to form a nascent negative strand. Additional viral and cellular proteins, proteolytic processing of viral protein precursors, and cellular membranes are also required for negative-strand initiation but for clarity are not depicted in this model. See text for additional details. This is a modified version of a model originally published in the ( ).

Citation: Morasco B, Smerage L, Flanegan J, Barton D. 2002. Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions, p 461-469. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch37
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