Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 Aug;69(8):4797–4806. doi: 10.1128/jvi.69.8.4797-4806.1995

Construction and genetic analysis of dicistronic polioviruses containing open reading frames for epitopes of human immunodeficiency virus type 1 gp120.

H H Lu 1, L Alexander 1, E Wimmer 1
PMCID: PMC189292  PMID: 7541843

Abstract

On the basis of previous studies of dicistronic (dc) polioviruses that carried two internal ribosomal entry sites (L. Alexander, H.-H. Lu, and E. Wimmer, Proc. Natl. Acad. Sci. USA 91:1406-1410, 1994; A. Molla, S. K. Jang, A. V. Paul, Q. Reuer, and E. Wimmer, Nature [London] 356:255-257, 1992), we have constructed a variety of dc polioviruses which express foreign genetic elements that were inserted either between two internal ribosomal entry site elements upstream of the poliovirus open reading frame (pPNENPO derivatives) or upstream of the open reading frame for the poliovirus proteinase 2Apro (pDI-E2A derivatives). Surprisingly, the addition of an N-terminal secretory pathway signal sequence to the open reading frame of the inserted foreign sequences (specifying either truncated versions of human immunodeficiency virus type 1 [HIV-1] gp120 or chloramphenicol acetyltransferase) resulted in a null phenotype, whereas removal of the signal sequence led to the production of viable viruses. Constructs that carried a foreign gene with a signal sequence were negative in RNA synthesis, an observation that suggested a very early block in viral replication. The insertion of transmembrane sequences downstream of the leader sequence did not reverse the replication block. Studies of dc polioviruses that encoded the truncated versions of HIV-1 gp120 showed an increase in genetic stability that correlated with a decrease in the size of the insert. A dc construct that contained a minigene encoding the principal neutralization determinant of HIV-1 produced a stable virus that retained the foreign sequence through multiple passages in cultured cells. These data indicate that dc polioviruses have potential as vaccines for the expression of small foreign epitopes.

Full Text

The Full Text of this article is available as a PDF (809.2 KB).

Selected References

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

  1. Alexander L., Lu H. H., Wimmer E. Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1406–1410. doi: 10.1073/pnas.91.4.1406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andino R., Silvera D., Suggett S. D., Achacoso P. L., Miller C. J., Baltimore D., Feinberg M. B. Engineering poliovirus as a vaccine vector for the expression of diverse antigens. Science. 1994 Sep 2;265(5177):1448–1451. doi: 10.1126/science.8073288. [DOI] [PubMed] [Google Scholar]
  3. Dorner A. J., Semler B. L., Jackson R. J., Hanecak R., Duprey E., Wimmer E. In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate. J Virol. 1984 May;50(2):507–514. doi: 10.1128/jvi.50.2.507-514.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dougherty W. G., Semler B. L. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol Rev. 1993 Dec;57(4):781–822. doi: 10.1128/mr.57.4.781-822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hahn C. S., Hahn Y. S., Braciale T. J., Rice C. M. Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2679–2683. doi: 10.1073/pnas.89.7.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hellen C. U., Lee C. K., Wimmer E. Determinants of substrate recognition by poliovirus 2A proteinase. J Virol. 1992 Jun;66(6):3330–3338. doi: 10.1128/jvi.66.6.3330-3338.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hogle J. M., Chow M., Filman D. J. Three-dimensional structure of poliovirus at 2.9 A resolution. Science. 1985 Sep 27;229(4720):1358–1365. doi: 10.1126/science.2994218. [DOI] [PubMed] [Google Scholar]
  8. Huang H. V., Rice C. M., Xiong C., Schlesinger S. RNA viruses as gene expression vectors. Virus Genes. 1989 Sep;3(1):85–91. doi: 10.1007/BF00301989. [DOI] [PubMed] [Google Scholar]
  9. Jang S. K., Davies M. V., Kaufman R. J., Wimmer E. Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol. 1989 Apr;63(4):1651–1660. doi: 10.1128/jvi.63.4.1651-1660.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jang S. K., Kräusslich H. G., Nicklin M. J., Duke G. M., Palmenberg A. C., Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. doi: 10.1128/jvi.62.8.2636-2643.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Kräusslich H. G., Wimmer E. Viral proteinases. Annu Rev Biochem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Maynell L. A., Kirkegaard K., Klymkowsky M. W. Inhibition of poliovirus RNA synthesis by brefeldin A. J Virol. 1992 Apr;66(4):1985–1994. doi: 10.1128/jvi.66.4.1985-1994.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Molla A., Jang S. K., Paul A. V., Reuer Q., Wimmer E. Cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus. Nature. 1992 Mar 19;356(6366):255–257. doi: 10.1038/356255a0. [DOI] [PubMed] [Google Scholar]
  16. Molla A., Paul A. V., Wimmer E. Cell-free, de novo synthesis of poliovirus. Science. 1991 Dec 13;254(5038):1647–1651. doi: 10.1126/science.1661029. [DOI] [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. 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. Percy N., Barclay W. S., Sullivan M., Almond J. W. A poliovirus replicon containing the chloramphenicol acetyltransferase gene can be used to study the replication and encapsidation of poliovirus RNA. J Virol. 1992 Aug;66(8):5040–5046. doi: 10.1128/jvi.66.8.5040-5046.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pollard S. R., Rosa M. D., Rosa J. J., Wiley D. C. Truncated variants of gp120 bind CD4 with high affinity and suggest a minimum CD4 binding region. EMBO J. 1992 Feb;11(2):585–591. doi: 10.1002/j.1460-2075.1992.tb05090.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Porter D. C., Ansardi D. C., Choi W. S., Morrow C. D. Encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type 1 Gag and Pol proteins upon infection. J Virol. 1993 Jul;67(7):3712–3719. doi: 10.1128/jvi.67.7.3712-3719.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Raju R., Huang H. V. Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two subgenomic mRNA promoters. J Virol. 1991 May;65(5):2501–2510. doi: 10.1128/jvi.65.5.2501-2510.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. SALK J. E. Persistence of immunity after administration of formalin-treated poliovirus vaccine. Lancet. 1960 Oct 1;2(7153):715–723. doi: 10.1016/s0140-6736(60)91791-8. [DOI] [PubMed] [Google Scholar]
  24. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wimmer E., Hellen C. U., Cao X. Genetics of poliovirus. Annu Rev Genet. 1993;27:353–436. doi: 10.1146/annurev.ge.27.120193.002033. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES