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. 1992 Aug;66(8):5040–5046. doi: 10.1128/jvi.66.8.5040-5046.1992

A poliovirus replicon containing the chloramphenicol acetyltransferase gene can be used to study the replication and encapsidation of poliovirus RNA.

N Percy 1, W S Barclay 1, M Sullivan 1, J W Almond 1
PMCID: PMC241362  PMID: 1321286

Abstract

A poliovirus replicon, FLC/REP, which incorporates the reporter gene chloramphenicol acetyltransferase (CAT) in place of the region encoding the capsid proteins VP4, VP2, and part of VP3 in the genome of poliovirus type 3, has been constructed. Transfection of cells indicates that the FLC/REP replicon replicates efficiently and that active CAT enzyme is produced as a CAT-VP3 fusion protein. The level of CAT activity in transfected cells broadly reflects the level of FLC/REP RNA. A series of mutations in the 5' noncoding region of poliovirus type 3 were introduced into FLC/REP, and their effects were monitored by a simple CAT assay. These experiments helped to define further the stem-loop structures in the 5' noncoding region which are essential for RNA replication. The CAT-containing poliovirus replicon could also be packaged into poliovirus capsids provided by helper virus and was stable as a subpopulation of virus particles over at least four passages. The location of the CAT gene in FLC/REP excluded the presence of an encapsidation signal in the region of the poliovirus genome comprising nucleotides 756 to 1805.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andino R., Rieckhof G. E., Baltimore D. A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA. Cell. 1990 Oct 19;63(2):369–380. doi: 10.1016/0092-8674(90)90170-j. [DOI] [PubMed] [Google Scholar]
  2. Blair W. S., Semler B. L. Role for the P4 amino acid residue in substrate utilization by the poliovirus 3CD proteinase. J Virol. 1991 Nov;65(11):6111–6123. doi: 10.1128/jvi.65.11.6111-6123.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Choi W. S., Pal-Ghosh R., Morrow C. D. Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons. J Virol. 1991 Jun;65(6):2875–2883. doi: 10.1128/jvi.65.6.2875-2883.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dildine S. L., Semler B. L. The deletion of 41 proximal nucleotides reverts a poliovirus mutant containing a temperature-sensitive lesion in the 5' noncoding region of genomic RNA. J Virol. 1989 Feb;63(2):847–862. doi: 10.1128/jvi.63.2.847-862.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hagino-Yamagishi K., Nomoto A. In vitro construction of poliovirus defective interfering particles. J Virol. 1989 Dec;63(12):5386–5392. doi: 10.1128/jvi.63.12.5386-5392.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kajigaya S., Arakawa H., Kuge S., Koi T., Imura N., Nomoto A. Isolation and characterization of defective-interfering particles of poliovirus Sabin 1 strain. Virology. 1985 Apr 30;142(2):307–316. doi: 10.1016/0042-6822(85)90339-3. [DOI] [PubMed] [Google Scholar]
  8. Kaplan G., Racaniello V. R. Construction and characterization of poliovirus subgenomic replicons. J Virol. 1988 May;62(5):1687–1696. doi: 10.1128/jvi.62.5.1687-1696.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kitamura N., Semler B. L., Rothberg P. G., Larsen G. R., Adler C. J., Dorner A. J., Emini E. A., Hanecak R., Lee J. J., van der Werf S. Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature. 1981 Jun 18;291(5816):547–553. doi: 10.1038/291547a0. [DOI] [PubMed] [Google Scholar]
  10. Kräusslich H. G., Hölscher C., Reuer Q., Harber J., Wimmer E. Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity. J Virol. 1990 May;64(5):2433–2436. doi: 10.1128/jvi.64.5.2433-2436.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kuge S., Saito I., Nomoto A. Primary structure of poliovirus defective-interfering particle genomes and possible generation mechanisms of the particles. J Mol Biol. 1986 Dec 5;192(3):473–487. doi: 10.1016/0022-2836(86)90270-6. [DOI] [PubMed] [Google Scholar]
  12. Levis R., Weiss B. G., Tsiang M., Huang H., Schlesinger S. Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging. Cell. 1986 Jan 17;44(1):137–145. doi: 10.1016/0092-8674(86)90492-7. [DOI] [PubMed] [Google Scholar]
  13. Luytjes W., Krystal M., Enami M., Parvin J. D., Palese P. Amplification, expression, and packaging of foreign gene by influenza virus. Cell. 1989 Dec 22;59(6):1107–1113. doi: 10.1016/0092-8674(89)90766-6. [DOI] [PubMed] [Google Scholar]
  14. Minor P. D., Pipkin P. A., Hockley D., Schild G. C., Almond J. W. Monoclonal antibodies which block cellular receptors of poliovirus. Virus Res. 1984;1(3):203–212. doi: 10.1016/0168-1702(84)90039-x. [DOI] [PubMed] [Google Scholar]
  15. Moscufo N., Simons J., Chow M. Myristoylation is important at multiple stages in poliovirus assembly. J Virol. 1991 May;65(5):2372–2380. doi: 10.1128/jvi.65.5.2372-2380.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Park K. H., Huang T., Correia F. F., Krystal M. Rescue of a foreign gene by Sendai virus. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5537–5541. doi: 10.1073/pnas.88.13.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pelletier J., Sonenberg N. Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts. J Virol. 1989 Jan;63(1):441–444. doi: 10.1128/jvi.63.1.441-444.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Percy N., Belsham G. J., Brangwyn J. K., Sullivan M., Stone D. M., Almond J. W. Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis. J Virol. 1992 Mar;66(3):1695–1701. doi: 10.1128/jvi.66.3.1695-1701.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pilipenko E. V., Blinov V. M., Romanova L. I., Sinyakov A. N., Maslova S. V., Agol V. I. Conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence. Virology. 1989 Feb;168(2):201–209. doi: 10.1016/0042-6822(89)90259-6. [DOI] [PubMed] [Google Scholar]
  20. Racaniello V. R., 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. 1981 Aug;78(8):4887–4891. doi: 10.1073/pnas.78.8.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rivera V. M., Welsh J. D., Maizel J. V., Jr Comparative sequence analysis of the 5' noncoding region of the enteroviruses and rhinoviruses. Virology. 1988 Jul;165(1):42–50. doi: 10.1016/0042-6822(88)90656-3. [DOI] [PubMed] [Google Scholar]
  22. Skinner M. A., Racaniello V. R., Dunn G., Cooper J., Minor P. D., Almond J. W. New model for the secondary structure of the 5' non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence. J Mol Biol. 1989 May 20;207(2):379–392. doi: 10.1016/0022-2836(89)90261-1. [DOI] [PubMed] [Google Scholar]
  23. Stanway G., Cann A. J., Hauptmann R., Hughes P., Clarke L. D., Mountford R. C., Minor P. D., Schild G. C., Almond J. W. The nucleotide sequence of poliovirus type 3 leon 12 a1b: comparison with poliovirus type 1. Nucleic Acids Res. 1983 Aug 25;11(16):5629–5643. doi: 10.1093/nar/11.16.5629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stanway G., Hughes P. J., Mountford R. C., Reeve P., Minor P. D., Schild G. C., Almond J. W. Comparison of the complete nucleotide sequences of the genomes of the neurovirulent poliovirus P3/Leon/37 and its attenuated Sabin vaccine derivative P3/Leon 12a1b. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1539–1543. doi: 10.1073/pnas.81.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Toyoda H., Kohara M., Kataoka Y., Suganuma T., Omata T., Imura N., Nomoto A. Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants. J Mol Biol. 1984 Apr 25;174(4):561–585. doi: 10.1016/0022-2836(84)90084-6. [DOI] [PubMed] [Google Scholar]
  26. Westrop G. D., Wareham K. A., Evans D. M., Dunn G., Minor P. D., Magrath D. I., Taffs F., Marsden S., Skinner M. A., Schild G. C. Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine. J Virol. 1989 Mar;63(3):1338–1344. doi: 10.1128/jvi.63.3.1338-1344.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Xiong C., Levis R., Shen P., Schlesinger S., Rice C. M., Huang H. V. Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science. 1989 Mar 3;243(4895):1188–1191. doi: 10.1126/science.2922607. [DOI] [PubMed] [Google Scholar]
  28. Ypma-Wong M. F., Dewalt P. G., Johnson V. H., Lamb J. G., Semler B. L. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology. 1988 Sep;166(1):265–270. doi: 10.1016/0042-6822(88)90172-9. [DOI] [PubMed] [Google Scholar]
  29. van der Werf S., Bradley J., Wimmer E., Studier F. W., Dunn J. J. Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2330–2334. doi: 10.1073/pnas.83.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]

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