Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Apr;82(7):1959–1963. doi: 10.1073/pnas.82.7.1959

Translational control by adenovirus: lack of virus-associated RNAI during adenovirus infection results in phosphorylation of initiation factor eIF-2 and inhibition of protein synthesis.

J Siekierka, T M Mariano, P A Reichel, M B Mathews
PMCID: PMC397459  PMID: 3856874

Abstract

The dl331 mutant of adenovirus serotype 5 fails to produce virus-associated (VA) RNAI, and cells infected with this mutant do not synthesize proteins efficiently at late times in infection. The translational defect occurs at the level of polypeptide chain initiation, and cell-free extracts prepared from dl331-infected cells exhibit the defect observed in vivo. Addition of either eukaryotic initiation factor 2 (eIF-2) or guanine nucleotide exchange factor (GEF) to these cell-free extracts restores translational activity, with GEF functioning more efficiently in this regard. These results suggest that cells infected with the dl331 mutant develop a translational block at the level of GEF-catalyzed guanine nucleotide exchange and that this block is most likely established through phosphorylation of the alpha subunit of eIF-2. In the present investigation we show that endogenous HeLa cell GEF activity is significantly reduced in cells infected with the dl331 mutant. Further, in contrast to cells infected with wild-type serotype 2 adenovirus, dl331-infected cells contain increased eIF-2 alpha kinase activity. These results indicate that VA RNAI plays a role in suppressing eIF-2 alpha kinase activity during adenovirus infection of HeLa cells.

Full text

PDF
1959

Images in this article

1962Image
on p.1962

Selected References

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

  1. Benne R., Wong C., Luedi M., Hershey J. W. Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes. J Biol Chem. 1976 Dec 10;251(23):7675–7681. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Clemens M. J., Pain V. M., Wong S. T., Henshaw E. C. Phosphorylation inhibits guanine nucleotide exchange on eukaryotic initiation factor 2. Nature. 1982 Mar 4;296(5852):93–95. doi: 10.1038/296093a0. [DOI] [PubMed] [Google Scholar]
  4. Goss D. J., Parkhurst L. J., Mehta H. B., Woodley C. L., Wahba A. J. Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation. J Biol Chem. 1984 Jun 25;259(12):7374–7377. [PubMed] [Google Scholar]
  5. Hunter T., Hunt T., Jackson R. J., Robertson H. D. The characteristics of inhibition of protein synthesis by double-stranded ribonucleic acid in reticulocyte lysates. J Biol Chem. 1975 Jan 25;250(2):409–417. [PubMed] [Google Scholar]
  6. Jagus R., Anderson W. F., Safer B. The regulation of initiation of mammalian protein synthesis. Prog Nucleic Acid Res Mol Biol. 1981;25:127–185. doi: 10.1016/s0079-6603(08)60484-5. [DOI] [PubMed] [Google Scholar]
  7. Konieczny A., Safer B. Purification of the eukaryotic initiation factor 2-eukaryotic initiation factor 2B complex and characterization of its guanine nucleotide exchange activity during protein synthesis initiation. J Biol Chem. 1983 Mar 10;258(5):3402–3408. [PubMed] [Google Scholar]
  8. Leroux A., London I. M. Regulation of protein synthesis by phosphorylation of eukaryotic initiation factor 2 alpha in intact reticulocytes and reticulocyte lysates. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2147–2151. doi: 10.1073/pnas.79.7.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mathews M. B. Genes for VA-RNA in adenovirus 2. Cell. 1975 Oct;6(2):223–229. doi: 10.1016/0092-8674(75)90013-6. [DOI] [PubMed] [Google Scholar]
  10. Matts R. L., Levin D. H., London I. M. Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis. Proc Natl Acad Sci U S A. 1983 May;80(9):2559–2563. doi: 10.1073/pnas.80.9.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ochoa S. Regulation of protein synthesis initiation in eucaryotes. Arch Biochem Biophys. 1983 Jun;223(2):325–349. doi: 10.1016/0003-9861(83)90598-2. [DOI] [PubMed] [Google Scholar]
  12. Panniers R., Henshaw E. C. A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation. J Biol Chem. 1983 Jul 10;258(13):7928–7934. [PubMed] [Google Scholar]
  13. Pettersson U., Philipson L. Location of sequences on the adenovirus genome coding for the 5.5S RNA. Cell. 1975 Sep;6(1):1–4. doi: 10.1016/0092-8674(75)90066-5. [DOI] [PubMed] [Google Scholar]
  14. Ranu R. S., London I. M. Regulation of protein synthesis in rabbit reticulocyte lysates: preparation of efficient protein synthesis lysates and the purification and characterization of the heme-regulated translational inhibitory protein kinase. Methods Enzymol. 1979;60:459–484. doi: 10.1016/s0076-6879(79)60045-9. [DOI] [PubMed] [Google Scholar]
  15. Reich P. R., Forget B. G., Weissman S. M. RNA of low molecular weight in KB cells infected with adenovirus type 2. J Mol Biol. 1966 Jun;17(2):428–439. doi: 10.1016/s0022-2836(66)80153-5. [DOI] [PubMed] [Google Scholar]
  16. Reichel P. A., Merrick W. C., Siekierka J., Mathews M. B. Regulation of a protein synthesis initiation factor by adenovirus virus-associated RNA. Nature. 1985 Jan 17;313(5999):196–200. doi: 10.1038/313196a0. [DOI] [PubMed] [Google Scholar]
  17. Schneider R. J., Weinberger C., Shenk T. Adenovirus VAI RNA facilitates the initiation of translation in virus-infected cells. Cell. 1984 May;37(1):291–298. doi: 10.1016/0092-8674(84)90325-8. [DOI] [PubMed] [Google Scholar]
  18. Siekierka J., Manne V., Ochoa S. Mechanism of translational control by partial phosphorylation of the alpha subunit of eukaryotic initiation factor 2. Proc Natl Acad Sci U S A. 1984 Jan;81(2):352–356. doi: 10.1073/pnas.81.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Siekierka J., Mauser L., Ochoa S. Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha subunit of initiation factor 2. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2537–2540. doi: 10.1073/pnas.79.8.2537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Siekierka J., Mitsui K. I., Ochoa S. Mode of action of the heme-controlled translational inhibitor: relationship of eukaryotic initiation factor 2-stimulating protein to translation restoring factor. Proc Natl Acad Sci U S A. 1981 Jan;78(1):220–223. doi: 10.1073/pnas.78.1.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Söderlund H., Pettersson U., Vennström B., Philipson L., Mathews M. B. A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2. Cell. 1976 Apr;7(4):585–593. doi: 10.1016/0092-8674(76)90209-9. [DOI] [PubMed] [Google Scholar]
  22. Thimmappaya B., Weinberger C., Schneider R. J., Shenk T. Adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection. Cell. 1982 Dec;31(3 Pt 2):543–551. doi: 10.1016/0092-8674(82)90310-5. [DOI] [PubMed] [Google Scholar]
  23. de Haro C., Ochoa S. Further studies on the mode of action of the heme-controlled translational inhibitor. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1741–1745. doi: 10.1073/pnas.76.4.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES