Skip to main content
NCBI home page
RNA logoLink to RNA
. 2000 Mar;6(3):369–380. doi: 10.1017/s1355838200991337

A novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped RNA template.

K A Hudak 1, P Wang 1, N E Tumer 1
PMCID: PMC1369919  PMID: 10744021

Abstract

Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thereby inhibiting translation. We demonstrate here that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA. Recent results indicate that the antiviral activity of PAP may not be due to depurination of host ribosomes. Using PAP mutants that do not depurinate either tobacco or reticulocyte lysate rRNA, we show that PAP inhibits translation of brome mosaic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosomes. Furthermore, translation of only capped, but not uncapped, luciferase transcripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translation inhibition of BMV RNAs is overcome by treatment with PAP in the presence of increasing concentrations of the cap analog m7GpppG, but not GpppG or GTP, indicating that PAP recognizes the cap structure. Incubation of BMV RNAs or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinated after incubation with identical concentrations of PAP. These results demonstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Arias F. J., Rojo M. A., Muñoz R., Iglesias R., Ferreras J. M., Girbés T. Messenger-dependent action of the pokeweed antiviral protein and fusidic acid on in vitro Vicia sativa L. translation. Cell Mol Biol (Noisy-le-grand) 1993 May;39(3):333–337. [PubMed] [Google Scholar]
  2. Aron G. M., Irvin J. D. Inhibition of herpes simplex virus multiplication by the pokeweed antiviral protein. Antimicrob Agents Chemother. 1980 Jun;17(6):1032–1033. doi: 10.1128/aac.17.6.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbieri L., Valbonesi P., Bonora E., Gorini P., Bolognesi A., Stirpe F. Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A). Nucleic Acids Res. 1997 Feb 1;25(3):518–522. doi: 10.1093/nar/25.3.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barbieri L., Valbonesi P., Gorini P., Pession A., Stirpe F. Polynucleotide: adenosine glycosidase activity of saporin-L1: effect on DNA, RNA and poly(A). Biochem J. 1996 Oct 15;319(Pt 2):507–513. doi: 10.1042/bj3190507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dasgupta R., Harada F., Kaesberg P. Blocked 5' termini in brome mosaic virus RNA. J Virol. 1976 Apr;18(1):260–267. doi: 10.1128/jvi.18.1.260-267.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Endo Y., Mitsui K., Motizuki M., Tsurugi K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem. 1987 Apr 25;262(12):5908–5912. [PubMed] [Google Scholar]
  7. Endo Y., Tsurugi K., Lambert J. M. The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes: the RNA N-glycosidase activity of the proteins. Biochem Biophys Res Commun. 1988 Feb 15;150(3):1032–1036. doi: 10.1016/0006-291x(88)90733-4. [DOI] [PubMed] [Google Scholar]
  8. Endo Y., Tsurugi K. Mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. Nucleic Acids Symp Ser. 1986;(17):187–190. [PubMed] [Google Scholar]
  9. Endo Y., Tsurugi K. The RNA N-glycosidase activity of ricin A-chain. The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA. J Biol Chem. 1988 Jun 25;263(18):8735–8739. [PubMed] [Google Scholar]
  10. Hartley M. R., Legname G., Osborn R., Chen Z., Lord J. M. Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA. FEBS Lett. 1991 Sep 23;290(1-2):65–68. doi: 10.1016/0014-5793(91)81227-y. [DOI] [PubMed] [Google Scholar]
  11. Huang P. L., Chen H. C., Kung H. F., Huang P. L., Huang P., Huang H. I., Lee-Huang S. Anti-HIV plant proteins catalyze topological changes of DNA into inactive forms. Biofactors. 1992 Dec;4(1):37–41. [PubMed] [Google Scholar]
  12. Hudak K. A., Dinman J. D., Tumer N. E. Pokeweed antiviral protein accesses ribosomes by binding to L3. J Biol Chem. 1999 Feb 5;274(6):3859–3864. doi: 10.1074/jbc.274.6.3859. [DOI] [PubMed] [Google Scholar]
  13. Hur Y., Hwang D. J., Zoubenko O., Coetzer C., Uckun F. M., Tumer N. E. Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8448–8452. doi: 10.1073/pnas.92.18.8448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iordanov M. S., Pribnow D., Magun J. L., Dinh T. H., Pearson J. A., Chen S. L., Magun B. E. Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA. Mol Cell Biol. 1997 Jun;17(6):3373–3381. doi: 10.1128/mcb.17.6.3373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Irvin J. D., Uckun F. M. Pokeweed antiviral protein: ribosome inactivation and therapeutic applications. Pharmacol Ther. 1992;55(3):279–302. doi: 10.1016/0163-7258(92)90053-3. [DOI] [PubMed] [Google Scholar]
  16. Jacobson A., Peltz S. W. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem. 1996;65:693–739. doi: 10.1146/annurev.bi.65.070196.003401. [DOI] [PubMed] [Google Scholar]
  17. Li M. X., Yeung H. W., Pan L. P., Chan S. I. Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro. Nucleic Acids Res. 1991 Nov 25;19(22):6309–6312. doi: 10.1093/nar/19.22.6309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ling J., Liu W. Y., Wang T. P. Cleavage of supercoiled double-stranded DNA by several ribosome-inactivating proteins in vitro. FEBS Lett. 1994 May 30;345(2-3):143–146. doi: 10.1016/0014-5793(94)00421-8. [DOI] [PubMed] [Google Scholar]
  19. Lodge J. K., Kaniewski W. K., Tumer N. E. Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7089–7093. doi: 10.1073/pnas.90.15.7089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Montanaro L., Sperti S., Mattioli A., Testoni G., Stirpe F. Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes. Biochem J. 1975 Jan;146(1):127–131. doi: 10.1042/bj1460127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nicolas E., Beggs J. M., Haltiwanger B. M., Taraschi T. F. Direct evidence for the deoxyribonuclease activity of the plant ribosome inactivating protein gelonin. FEBS Lett. 1997 Apr 7;406(1-2):162–164. doi: 10.1016/s0014-5793(97)00267-6. [DOI] [PubMed] [Google Scholar]
  22. Osborn R. W., Hartley M. R. Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate. Inhibition of initiation and translocation. Eur J Biochem. 1990 Oct 24;193(2):401–407. doi: 10.1111/j.1432-1033.1990.tb19353.x. [DOI] [PubMed] [Google Scholar]
  23. Prestle J., Schönfelder M., Adam G., Mundry K. W. Type 1 ribosome-inactivating proteins depurinate plant 25S rRNA without species specificity. Nucleic Acids Res. 1992 Jun 25;20(12):3179–3182. doi: 10.1093/nar/20.12.3179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rajamohan F., Kurinov I. V., Venkatachalam T. K., Uckun F. M. Deguanylation of human immunodeficiency virus (HIV-1) RNA by recombinant pokeweed antiviral protein. Biochem Biophys Res Commun. 1999 Sep 24;263(2):419–424. doi: 10.1006/bbrc.1999.1335. [DOI] [PubMed] [Google Scholar]
  25. Ready M. P., Brown D. T., Robertus J. D. Extracellular localization of pokeweed antiviral protein. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5053–5056. doi: 10.1073/pnas.83.14.5053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rodes T. L., 3rd, Irvin J. D. Reversal of the inhibitory effects of the pokeweed antiviral protein upon protein synthesis. Biochim Biophys Acta. 1981 Jan 29;652(1):160–167. doi: 10.1016/0005-2787(81)90219-7. [DOI] [PubMed] [Google Scholar]
  27. Sonenberg N., Gingras A. C. The mRNA 5' cap-binding protein eIF4E and control of cell growth. Curr Opin Cell Biol. 1998 Apr;10(2):268–275. doi: 10.1016/s0955-0674(98)80150-6. [DOI] [PubMed] [Google Scholar]
  28. Sonenberg N., Shatkin A. J., Ricciardi R. P., Rubin M., Goodman R. M. Analysis of terminal structures of RNA from potato virus X. Nucleic Acids Res. 1978 Jul;5(7):2501–2512. doi: 10.1093/nar/5.7.2501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Taylor S., Massiah A., Lomonossoff G., Roberts L. M., Lord J. M., Hartley M. Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection. Plant J. 1994 Jun;5(6):827–835. doi: 10.1046/j.1365-313x.1994.5060827.x. [DOI] [PubMed] [Google Scholar]
  30. Tomlinson J. A., Walker V. M., Flewett T. H., Barclay G. R. The inhibition of infection by cucumber mosaic virus and influenza virus by extracts from Phytolacca americana. J Gen Virol. 1974 Feb;22(2):225–232. doi: 10.1099/0022-1317-22-2-225. [DOI] [PubMed] [Google Scholar]
  31. Tumer N. E., Hudak K., Di R., Coetzer C., Wang P., Zoubenko O. Pokeweed antiviral protein and its applications. Curr Top Microbiol Immunol. 1999;240:139–158. doi: 10.1007/978-3-642-60234-4_7. [DOI] [PubMed] [Google Scholar]
  32. Tumer N. E., Hwang D. J., Bonness M. C-terminal deletion mutant of pokeweed antiviral protein inhibits viral infection but does not depurinate host ribosomes. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3866–3871. doi: 10.1073/pnas.94.8.3866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tumer N. E., Parikh B. A., Li P., Dinman J. D. The pokeweed antiviral protein specifically inhibits Ty1-directed +1 ribosomal frameshifting and retrotransposition in Saccharomyces cerevisiae. J Virol. 1998 Feb;72(2):1036–1042. doi: 10.1128/jvi.72.2.1036-1042.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ussery M. A., Irvin J. D., Hardesty B. Inhibition of poliovirus replication by a plant antiviral peptide. Ann N Y Acad Sci. 1977 Mar 4;284:431–440. doi: 10.1111/j.1749-6632.1977.tb21979.x. [DOI] [PubMed] [Google Scholar]
  35. Wang P., Tumer N. E. Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA. Nucleic Acids Res. 1999 Apr 15;27(8):1900–1905. doi: 10.1093/nar/27.8.1900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yen Y., Green P. J. Identification and Properties of the Major Ribonucleases of Arabidopsis thaliana. Plant Physiol. 1991 Dec;97(4):1487–1493. doi: 10.1104/pp.97.4.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zarling J. M., Moran P. A., Haffar O., Sias J., Richman D. D., Spina C. A., Myers D. E., Kuebelbeck V., Ledbetter J. A., Uckun F. M. Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies. Nature. 1990 Sep 6;347(6288):92–95. doi: 10.1038/347092a0. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES