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. 1991 Jun;65(6):2875–2883. doi: 10.1128/jvi.65.6.2875-2883.1991

Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons.

W S Choi 1, R Pal-Ghosh 1, C D Morrow 1
PMCID: PMC240915  PMID: 1851859

Abstract

Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-P1 fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.

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Selected References

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

  1. Beckett D., Wu H. N., Uhlenbeck O. C. Roles of operator and non-operator RNA sequences in bacteriophage R17 capsid assembly. J Mol Biol. 1988 Dec 20;204(4):939–947. doi: 10.1016/0022-2836(88)90053-8. [DOI] [PubMed] [Google Scholar]
  2. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  3. Cole C. N. Defective interfering (di) particles of poliovirus. Prog Med Virol. 1975;20:180–207. [PubMed] [Google Scholar]
  4. Ehrenfeld E. Poliovirus-induced inhibition of host-cell protein synthesis. Cell. 1982 Mar;28(3):435–436. doi: 10.1016/0092-8674(82)90195-7. [DOI] [PubMed] [Google Scholar]
  5. French R., Janda M., Ahlquist P. Bacterial gene inserted in an engineered RNA virus: efficient expression in monocotyledonous plant cells. Science. 1986 Mar 14;231(4743):1294–1297. doi: 10.1126/science.231.4743.1294. [DOI] [PubMed] [Google Scholar]
  6. Fujimura T., Esteban R., Esteban L. M., Wickner R. B. Portable encapsidation signal of the L-A double-stranded RNA virus of S. cerevisiae. Cell. 1990 Aug 24;62(4):819–828. doi: 10.1016/0092-8674(90)90125-x. [DOI] [PubMed] [Google Scholar]
  7. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  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., Huang H., Schlesinger S. Engineered defective interfering RNAs of Sindbis virus express bacterial chloramphenicol acetyltransferase in avian cells. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4811–4815. doi: 10.1073/pnas.84.14.4811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. McClure M. A., Perrault J. Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs. Nucleic Acids Res. 1985 Oct 11;13(19):6797–6816. doi: 10.1093/nar/13.19.6797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McKeating J. A., Willey R. L. Structure and function of the HIV envelope. AIDS. 1989;3 (Suppl 1):S35–S41. doi: 10.1097/00002030-198901001-00005. [DOI] [PubMed] [Google Scholar]
  15. Olden K., Pratt R. M., Yamada K. M. Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts. Cell. 1978 Mar;13(3):461–473. doi: 10.1016/0092-8674(78)90320-3. [DOI] [PubMed] [Google Scholar]
  16. Owens R. J., Compans R. W. Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells. J Virol. 1989 Feb;63(2):978–982. doi: 10.1128/jvi.63.2.978-982.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pelletier J., Kaplan G., Racaniello V. R., Sonenberg N. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region. Mol Cell Biol. 1988 Mar;8(3):1103–1112. doi: 10.1128/mcb.8.3.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  19. Racaniello V. R., Baltimore D. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science. 1981 Nov 20;214(4523):916–919. doi: 10.1126/science.6272391. [DOI] [PubMed] [Google Scholar]
  20. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  21. Richards O. C., Ivanoff L. A., Bienkowska-Szewczyk K., Butt B., Petteway S. R., Jr, Rothstein M. A., Ehrenfeld E. Formation of poliovirus RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA. Virology. 1987 Dec;161(2):348–356. doi: 10.1016/0042-6822(87)90127-9. [DOI] [PubMed] [Google Scholar]
  22. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Semler B. L., Dorner A. J., Wimmer E. Production of infectious poliovirus from cloned cDNA is dramatically increased by SV40 transcription and replication signals. Nucleic Acids Res. 1984 Jun 25;12(12):5123–5141. doi: 10.1093/nar/12.12.5123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shaw G. M., Hahn B. H., Arya S. K., Groopman J. E., Gallo R. C., Wong-Staal F. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome. Science. 1984 Dec 7;226(4679):1165–1171. doi: 10.1126/science.6095449. [DOI] [PubMed] [Google Scholar]
  26. Sonenberg N. Regulation of translation by poliovirus. Adv Virus Res. 1987;33:175–204. doi: 10.1016/s0065-3527(08)60318-8. [DOI] [PubMed] [Google Scholar]
  27. Toyoda H., Nicklin M. J., Murray M. G., Anderson C. W., Dunn J. J., Studier F. W., Wimmer E. A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. Cell. 1986 Jun 6;45(5):761–770. doi: 10.1016/0092-8674(86)90790-7. [DOI] [PubMed] [Google Scholar]
  28. Trono D., Pelletier J., Sonenberg N., Baltimore D. Translation in mammalian cells of a gene linked to the poliovirus 5' noncoding region. Science. 1988 Jul 22;241(4864):445–448. doi: 10.1126/science.2839901. [DOI] [PubMed] [Google Scholar]
  29. Vaheri A., Pagano J. S. Infectious poliovirus RNA: a sensitive method of assay. Virology. 1965 Nov;27(3):434–436. doi: 10.1016/0042-6822(65)90126-1. [DOI] [PubMed] [Google Scholar]
  30. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Ypma-Wong M. F., Semler B. L. Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins. J Virol. 1987 Oct;61(10):3181–3189. doi: 10.1128/jvi.61.10.3181-3189.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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