Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1990 Jun;64(6):2732–2742. doi: 10.1128/jvi.64.6.2732-2742.1990

The adenovirus L4 100-kilodalton protein is necessary for efficient translation of viral late mRNA species.

B W Hayes 1, G C Telling 1, M M Myat 1, J F Williams 1, S J Flint 1
PMCID: PMC249453  PMID: 2335816

Abstract

When screening a number of adenovirus type 5 (Ad5) temperature-sensitive mutants for defects in viral gene expression, we observed that H5ts1-infected 293 cells accumulated reduced levels of newly synthesized viral late proteins. Pulse-labeling and pulse-chase experiments were used to establish that the late proteins synthesized in H5ts1-infected cells under nonpermissive conditions were as stable as those made in Ad5-infected cells. H5ts1-infected cells contained normal levels of viral late mRNAs. Because these observations implied that translation of viral mRNA species was defective in mutant virus-infected cells, the association of viral late mRNAs with polyribosomes was examined during the late phase of infection at a nonpermissive temperature. In Ad5-infected cells, the majority of the viral L2, L3, L4, pIX, and IVa2 late mRNA species were polyribosome bound. By contrast, these same mRNA species were recovered from H5ts1-infected cells in fractions nearer the top of polyribosome gradients, suggesting that initiation of translation was impaired. During the late phase of infection, neither the polyribosome association nor the translation of most viral early mRNA species was affected by the H5ts1 mutation. This lesion, mapped by marker rescue to the L4 100-kilodalton (kDa) nonstructural protein, has been identified as a single base pair substitution that replaces Ser-466 of the Ad5 100-kDa protein with Pro. A set of temperature-independent revertants of H5ts1 was isolated and characterized. Either true reversion of the H5ts1 mutation or second-site mutation of Pro-466 of the H5ts1 100-kDa protein to Thre, Leu, or His restored both temperature-independent growth and the efficient synthesis of viral late proteins. We therefore conclude that the Ad5 L4 100-kDa protein is necessary for efficient initiation of translation of viral late mRNA species during the late phase of infection.

Full text

PDF
2733

Images in this article

2734Image
on p.2734
2735Image
on p.2735
2736Image
on p.2736
2736Image
on p.2736
2737Image
on p.2737
2738Image
on p.2738
2738Image
on p.2738
2739Image
on p.2739
2739Image
on p.2739

Selected References

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

  1. Adam S. A., Dreyfuss G. Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells. J Virol. 1987 Oct;61(10):3276–3283. doi: 10.1128/jvi.61.10.3276-3283.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adam S. A., Nakagawa T., Swanson M. S., Woodruff T. K., Dreyfuss G. mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol Cell Biol. 1986 Aug;6(8):2932–2943. doi: 10.1128/mcb.6.8.2932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aiello L., Guilfoyle R., Huebner K., Weinmann R. Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology. 1979 Apr 30;94(2):460–469. doi: 10.1016/0042-6822(79)90476-8. [DOI] [PubMed] [Google Scholar]
  4. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Babich A., Feldman L. T., Nevins J. R., Darnell J. E., Jr, Weinberger C. Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination. Mol Cell Biol. 1983 Jul;3(7):1212–1221. doi: 10.1128/mcb.3.7.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beltz G. A., Flint S. J. Inhibition of HeLa cell protein synthesis during adenovirus infection. Restriction of cellular messenger RNA sequences to the nucleus. J Mol Biol. 1979 Jun 25;131(2):353–373. doi: 10.1016/0022-2836(79)90081-0. [DOI] [PubMed] [Google Scholar]
  7. Berget S. M., Moore C., Sharp P. A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3171–3175. doi: 10.1073/pnas.74.8.3171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berkner K. L., Schaffhausen B. S., Roberts T. M., Sharp P. A. Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant. J Virol. 1987 Apr;61(4):1213–1220. doi: 10.1128/jvi.61.4.1213-1220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Berkner K. L., Sharp P. A. Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant. Nucleic Acids Res. 1985 Feb 11;13(3):841–857. doi: 10.1093/nar/13.3.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Berkner K. L., Sharp P. A. Expression of dihydrofolate reductase, and of the adjacent EIb region, in an Ad5-dihydrofolate reductase recombinant virus. Nucleic Acids Res. 1984 Feb 24;12(4):1925–1941. doi: 10.1093/nar/12.4.1925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Binger M. H., Flint S. J. Accumulation of early and intermediate mRNA species during subgroup C adenovirus productive infections. Virology. 1984 Jul 30;136(2):387–403. doi: 10.1016/0042-6822(84)90175-2. [DOI] [PubMed] [Google Scholar]
  12. Blobel G. Gene gating: a hypothesis. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8527–8529. doi: 10.1073/pnas.82.24.8527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Blobel G., Sabatini D. Dissociation of mammalian polyribosomes into subunits by puromycin. Proc Natl Acad Sci U S A. 1971 Feb;68(2):390–394. doi: 10.1073/pnas.68.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cepko C. L., Sharp P. A. Analysis of Ad5 hexon and 100K ts mutants using conformation-specific monoclonal antibodies. Virology. 1983 Aug;129(1):137–154. doi: 10.1016/0042-6822(83)90402-6. [DOI] [PubMed] [Google Scholar]
  15. Cepko C. L., Sharp P. A. Assembly of adenovirus major capsid protein is mediated by a nonvirion protein. Cell. 1982 Dec;31(2 Pt 1):407–415. doi: 10.1016/0092-8674(82)90134-9. [DOI] [PubMed] [Google Scholar]
  16. Chow L. T., Broker T. R. The spliced structures of adenovirus 2 fiber message and the other late mRNAs. Cell. 1978 Oct;15(2):497–510. doi: 10.1016/0092-8674(78)90019-3. [DOI] [PubMed] [Google Scholar]
  17. Chow L. T., Gelinas R. E., Broker T. R., Roberts R. J. An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA. Cell. 1977 Sep;12(1):1–8. doi: 10.1016/0092-8674(77)90180-5. [DOI] [PubMed] [Google Scholar]
  18. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Crossland L. D., Raskas H. J. Identification of adenovirus genes that require template replication for expression. J Virol. 1983 Jun;46(3):737–748. doi: 10.1128/jvi.46.3.737-748.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dolph P. J., Racaniello V., Villamarin A., Palladino F., Schneider R. J. The adenovirus tripartite leader may eliminate the requirement for cap-binding protein complex during translation initiation. J Virol. 1988 Jun;62(6):2059–2066. doi: 10.1128/jvi.62.6.2059-2066.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Eron L. Post-transcriptional restriction of human adenovirus expression in monkey cells. J Virol. 1975 May;15(5):1256–1261. doi: 10.1128/jvi.15.5.1256-1261.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  23. Flint S. J. Regulation of adenovirus mRNA formation. Adv Virus Res. 1986;31:169–228. doi: 10.1016/s0065-3527(08)60264-x. [DOI] [PubMed] [Google Scholar]
  24. Gambke C., Deppert W. Specific complex of the late nonstructural 100,000-dalton protein with newly synthesized hexon in adenovirus type 2-infected cells. Virology. 1983 Jan 15;124(1):1–12. doi: 10.1016/0042-6822(83)90285-4. [DOI] [PubMed] [Google Scholar]
  25. Giard D. J., Aaronson S. A., Todaro G. J., Arnstein P., Kersey J. H., Dosik H., Parks W. P. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst. 1973 Nov;51(5):1417–1423. doi: 10.1093/jnci/51.5.1417. [DOI] [PubMed] [Google Scholar]
  26. Gonzalez I. L., Gorski J. L., Campen T. J., Dorney D. J., Erickson J. M., Sylvester J. E., Schmickel R. D. Variation among human 28S ribosomal RNA genes. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7666–7670. doi: 10.1073/pnas.82.22.7666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Katze M. G., Persson H., Philipson L. Control of adenovirus early gene expression: posttranscriptional control mediated by both viral and cellular gene products. Mol Cell Biol. 1981 Sep;1(9):807–813. doi: 10.1128/mcb.1.9.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Khalili K., Weinmann R. Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells. J Mol Biol. 1984 Jun 5;175(4):453–468. doi: 10.1016/0022-2836(84)90179-7. [DOI] [PubMed] [Google Scholar]
  31. Kitajewski J., Schneider R. J., Safer B., Munemitsu S. M., Samuel C. E., Thimmappaya B., Shenk T. Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase. Cell. 1986 Apr 25;45(2):195–200. doi: 10.1016/0092-8674(86)90383-1. [DOI] [PubMed] [Google Scholar]
  32. Kitajewski J., Schneider R. J., Safer B., Shenk T. An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines. Mol Cell Biol. 1986 Dec;6(12):4493–4498. doi: 10.1128/mcb.6.12.4493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kruijer W., van Schaik F. M., Sussenbach J. S. Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5. Nucleic Acids Res. 1981 Sep 25;9(18):4439–4457. doi: 10.1093/nar/9.18.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lindberg U., Sundquist B. Isolation of messenger ribonucleoproteins from mammalian cells. J Mol Biol. 1974 Jun 25;86(2):451–468. doi: 10.1016/0022-2836(74)90030-8. [DOI] [PubMed] [Google Scholar]
  36. Logan J., Shenk T. Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3655–3659. doi: 10.1073/pnas.81.12.3655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mathews M. B. Binding of adenovirus VA RNA to mRNA: a possible role in splicing? Nature. 1980 Jun 19;285(5766):575–577. doi: 10.1038/285575a0. [DOI] [PubMed] [Google Scholar]
  38. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. NATHANS D. PUROMYCIN INHIBITION OF PROTEIN SYNTHESIS: INCORPORATION OF PUROMYCIN INTO PEPTIDE CHAINS. Proc Natl Acad Sci U S A. 1964 Apr;51:585–592. doi: 10.1073/pnas.51.4.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. O'Malley R. P., Duncan R. F., Hershey J. W., Mathews M. B. Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells. Virology. 1989 Jan;168(1):112–118. doi: 10.1016/0042-6822(89)90409-1. [DOI] [PubMed] [Google Scholar]
  41. Oosterom-Dragon E. A., Anderson C. W. Polypeptide structure and encoding location of the adenovirus serotype 2 late, nonstructural 33K protein. J Virol. 1983 Jan;45(1):251–263. doi: 10.1128/jvi.45.1.251-263.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Oosterom-Dragon E. A., Ginsberg H. S. Characterization of two temperature-sensitive mutants of type 5 adenovirus with mutations in the 100,000-dalton protein gene. J Virol. 1981 Nov;40(2):491–500. doi: 10.1128/jvi.40.2.491-500.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Oosterom-Dragon E. A., Ginsberg H. S. Purification and preliminary immunological characterization of the type 5 adenovirus, nonstructural 100,000-dalton protein. J Virol. 1980 Mar;33(3):1203–1207. doi: 10.1128/jvi.33.3.1203-1207.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pilder S., Moore M., Logan J., Shenk T. The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs. Mol Cell Biol. 1986 Feb;6(2):470–476. doi: 10.1128/mcb.6.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  46. Reich N. C., Sarnow P., Duprey E., Levine A. J. Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein. Virology. 1983 Jul 30;128(2):480–484. doi: 10.1016/0042-6822(83)90274-x. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Sambrook J., Williams J., Sharp P. A., Grodzicker T. Physical mapping of temperature-sensitive mutations of adenoviruses. J Mol Biol. 1975 Sep 25;97(3):369–390. doi: 10.1016/s0022-2836(75)80046-5. [DOI] [PubMed] [Google Scholar]
  49. Schneider R. J., Safer B., Munemitsu S. M., Samuel C. E., Shenk T. Adenovirus VAI RNA prevents phosphorylation of the eukaryotic initiation factor 2 alpha subunit subsequent to infection. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4321–4325. doi: 10.1073/pnas.82.13.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schneider R. J., Shenk T. Impact of virus infection on host cell protein synthesis. Annu Rev Biochem. 1987;56:317–332. doi: 10.1146/annurev.bi.56.070187.001533. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Scorsone K. A., Panniers R., Rowlands A. G., Henshaw E. C. Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis. J Biol Chem. 1987 Oct 25;262(30):14538–14543. [PubMed] [Google Scholar]
  53. Siekierka J., Mariano T. M., Reichel P. A., Mathews M. B. 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. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1959–1963. doi: 10.1073/pnas.82.7.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Spector D. J., McGrogan M., Raskas H. J. Regulation of the appearance of cytoplasmic RNAs from region 1 of the adenovirus 2 genome. J Mol Biol. 1978 Dec 15;126(3):395–414. doi: 10.1016/0022-2836(78)90048-7. [DOI] [PubMed] [Google Scholar]
  55. Sundquist B., Persson T., Lindberg U. Characterization of mRNA-protein complexes from mammalian cells. Nucleic Acids Res. 1977 Apr;4(4):899–915. doi: 10.1093/nar/4.4.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tasseron-De Jong J. G., Brouwer J., Rietveld K., Zoetemelk C. E., Bosch L. Messenger ribonucleoprotein complexes in human KB cells infected with adenovirus type 5 contain tightly bound viral-coded '100K' proteins. Eur J Biochem. 1979 Oct;100(1):271–283. doi: 10.1111/j.1432-1033.1979.tb02058.x. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Thomas G. P., Mathews M. B. Alterations of transcription and translation in HeLa cells exposed to amino acid analogs. Mol Cell Biol. 1984 Jun;4(6):1063–1072. doi: 10.1128/mcb.4.6.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Thomas G. P., Mathews M. B. DNA replication and the early to late transition in adenovirus infection. Cell. 1980 Nov;22(2 Pt 2):523–533. doi: 10.1016/0092-8674(80)90362-1. [DOI] [PubMed] [Google Scholar]
  60. Van der Marel P., Tasseron-de Jong J. G., Bosch L. The proteins associated with mRNA from uninfected and adenovirus type 5-infected KB cells. FEBS Lett. 1975 Mar 1;51(1):330–334. doi: 10.1016/0014-5793(75)80919-7. [DOI] [PubMed] [Google Scholar]
  61. White D. O., Scharff M. D., Maizel J. V., Jr The polypeptides of adenovirus. 3. Synthesis in infected cells. Virology. 1969 Jul;38(3):395–406. doi: 10.1016/0042-6822(69)90152-4. [DOI] [PubMed] [Google Scholar]
  62. Williams J. F. Enhancement of adenovirus plaque formation on HeLa cells by magnesium chloride. J Gen Virol. 1970 Dec;9(3):251–255. doi: 10.1099/0022-1317-9-3-251. [DOI] [PubMed] [Google Scholar]
  63. Williams J. F., Gharpure M., Ustacelebi S., McDonald S. Isolation of temperature-sensitive mutants of adenovirus type 5. J Gen Virol. 1971 May;11(2):95–101. doi: 10.1099/0022-1317-11-2-95. [DOI] [PubMed] [Google Scholar]
  64. Willians J. F., Young C. S., Austin P. E. Genetic analysis of human adenovirus type 5 in permissive and nonpermissive cells. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):427–437. doi: 10.1101/sqb.1974.039.01.055. [DOI] [PubMed] [Google Scholar]
  65. Wilson M. C., Darnell J. E., Jr Control of messenger RNA concentration by differential cytoplasmic half-life. Adenovirus messenger RNAs from transcription units 1A and 1B. J Mol Biol. 1981 May 25;148(3):231–251. doi: 10.1016/0022-2836(81)90537-4. [DOI] [PubMed] [Google Scholar]
  66. Wold W. S., Cladaras C., Deutscher S. L., Kapoor Q. S. The 19-kDa glycoprotein coded by region E3 of adenovirus. Purification, characterization, and structural analysis. J Biol Chem. 1985 Feb 25;260(4):2424–2431. [PubMed] [Google Scholar]
  67. Zhang H., Scholl R., Browse J., Somerville C. Double stranded DNA sequencing as a choice for DNA sequencing. Nucleic Acids Res. 1988 Feb 11;16(3):1220–1220. doi: 10.1093/nar/16.3.1220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. van Beveren C. P., Maat J., Dekker B. M., van Ormondt H. The nucleotide sequence of the gene for protein IVa2 and of the 5' leader segment of the major late mRNAs of adenovirus type 5. Gene. 1981 Dec;16(1-3):179–189. doi: 10.1016/0378-1119(81)90074-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES