Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1982 Jun;42(3):932–950. doi: 10.1128/jvi.42.3.932-950.1982

Characterization of a Temperature-Sensitive Fiber Mutant of Type 5 Adenovirus and Effect of the Mutation on Virion Assembly

Cecilia Cheng Chee-Sheung 1, Harold S Ginsberg 1
PMCID: PMC256927  PMID: 7097864

Abstract

A temperature-sensitive, fiber-minus mutant of type 5 adenovirus, H5ts142, was biochemically and genetically characterized. Genetic studies revealed that H5ts142 was a member of one of the three apparent fiber complementation groups which were detected owing to intracistronic complementation. Recombination analyses showed that it occupied a unique locus at the right end of the adenovirus genetic map. At the nonpermissive temperature, the mutant made stable polypeptides, but they were not glycosylated like wild-type fiber polypeptides. Sedimentation studies of extracts of H5ts142-infected cells cultured and labeled at 39.5°C indicated that a limited number of the fiber polypeptides made at the nonpermissive temperature could assemble into a form having a sedimentation value of 6S (i.e., similar to the trimeric wild-type fiber), but that this 6S structure was not immunologically reactive. When H5ts142-infected cells were shifted to the permissive temperature, 32°C, fiber polypeptides synthesized at 39.5°C were as capable of being assembled into virions as fibers synthesized in wild type-infected cells; de novo protein synthesis was not required to allow this virion assembly. In H5ts142-infected cells incubated at 39.5°C, viral proteins accumulated and aggregated into particles having physical characteristics of empty capsids. These particles did not contain DNA or its associated core proteins. However, when the infected culture was shifted to 32°C, DNA appeared to enter the empty particles and complete virions developed. The intermediate particles obtained had the morphology of adenoviruses, but they contained less than unit-length viral genomes as measured by their buoyant density in a CsCl density gradient and the size of their DNA as determined in both neutral and alkaline sucrose gradients. The reduced size of the intermediate particle DNA was demonstrated to be the result of incompletely packaged DNA molecules being fragmented during the preparative procedures. Hybridization of labeled DNA extracted from the intermediate particles to filters containing restriction fragments of the adenovirus genome indicated that the molecular left end of the viral genome preferentially entered these particles.

Full text

PDF
935

Images in this article

934Image
on p.934
943Image
on p.943

Selected References

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

  1. Bellett A. J., Younghusband H. B. Replication of the DNA of chick embryo lethal orphan virus. J Mol Biol. 1972 Dec 30;72(3):691–709. doi: 10.1016/0022-2836(72)90185-4. [DOI] [PubMed] [Google Scholar]
  2. Burlingham B. T., Brown D. T., Doerfler W. Incomplete particles of adenovirus. I. Characteristics of the DNA associated with incomplete adenovirions of types 2 and 12. Virology. 1974 Aug;60(2):419–430. doi: 10.1016/0042-6822(74)90336-5. [DOI] [PubMed] [Google Scholar]
  3. CRICK F. H., ORGEL L. E. THE THEORY OF INTER-ALLELIC COMPLEMENTATION. J Mol Biol. 1964 Jan;8:161–165. doi: 10.1016/s0022-2836(64)80156-x. [DOI] [PubMed] [Google Scholar]
  4. Chow L. T., Roberts J. M., Lewis J. B., Broker T. R. A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids. Cell. 1977 Aug;11(4):819–836. doi: 10.1016/0092-8674(77)90294-x. [DOI] [PubMed] [Google Scholar]
  5. D'Halluin J. C., Martin G. R., Torpier G., Boulanger P. A. Adenovirus type 2 assembly analyzed by reversible cross-linking of labile intermediates. J Virol. 1978 May;26(2):357–363. doi: 10.1128/jvi.26.2.357-363.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. D'Halluin J. C., Milleville M., Boulanger P. A., Martin G. R. Temperature-sensitive mutant of adenovirus type 2 blocked in virion assembly: accumulation of light intermediate particles. J Virol. 1978 May;26(2):344–356. doi: 10.1128/jvi.26.2.344-356.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. D'Halluin J. C., Milleville M., Martin G. R., Boulanger P. Morphogenesis of human adenovirus type 2 studied with fiber- and fiber and penton base-defective temperature-sensitive mutants. J Virol. 1980 Jan;33(1):88–99. doi: 10.1128/jvi.33.1.88-99.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daniell E. Genome structure of incomplete particles of adenovirus. J Virol. 1976 Aug;19(2):685–708. doi: 10.1128/jvi.19.2.685-708.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dorsch-Häsler K., Fisher P. B., Weinstein I. B., Ginsberg H. S. Patterns of viral DNA integration in cells transformed by wild type or DNA-binding protein mutants of adenovirus type 5 and effect of chemical carcinogens on integration. J Virol. 1980 May;34(2):305–314. doi: 10.1128/jvi.34.2.305-314.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dorsett P. H., G8nsberg H. S. Characterization of type 5 adenovirus fiber protein. J Virol. 1975 Jan;15(1):208–216. doi: 10.1128/jvi.15.1.208-216.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. EAGLE H. Amino acid metabolism in mammalian cell cultures. Science. 1959 Aug 21;130(3373):432–437. doi: 10.1126/science.130.3373.432. [DOI] [PubMed] [Google Scholar]
  12. EDGAR R. S., LIELAUSIS I. TEMPERATURE-SENSITIVE MUTANTS OF BACTERIOPHAGE T4D: THEIR ISOLATION AND GENETIC CHARACTERIZATION. Genetics. 1964 Apr;49:649–662. doi: 10.1093/genetics/49.4.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edvardsson B., Everitt E., Jörnvall H., Prage L., Philipson L. Intermediates in adenovirus assembly. J Virol. 1976 Aug;19(2):533–547. doi: 10.1128/jvi.19.2.533-547.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edvardsson B., Ustacelebi S., Williams J., Philipson L. Assembly intermediates among adenovirus type 5 temperature-sensitive mutants. J Virol. 1978 Feb;25(2):641–651. doi: 10.1128/jvi.25.2.641-651.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ensinger M. J., Ginsberg H. S. Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. J Virol. 1972 Sep;10(3):328–339. doi: 10.1128/jvi.10.3.328-339.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Frost E., Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. doi: 10.1016/0042-6822(78)90353-7. [DOI] [PubMed] [Google Scholar]
  17. GINSBERG H. S., GOLD E., JORDAN W. S., Jr Tryptose phosphate broth as supplementary factor for maintenance of HeLa cell tissue cultures. Proc Soc Exp Biol Med. 1955 May;89(1):66–71. doi: 10.3181/00379727-89-21718. [DOI] [PubMed] [Google Scholar]
  18. GREEN M. Studies on the biosynthesis of viral DNA. Cold Spring Harb Symp Quant Biol. 1962;27:219–235. doi: 10.1101/sqb.1962.027.001.022. [DOI] [PubMed] [Google Scholar]
  19. Garon C. F., Berry K. W., Rose J. A. A unique form of terminal redundancy in adenovirus DNA molecules. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2391–2395. doi: 10.1073/pnas.69.9.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hammarskjöld M. L., Winberg G. Encapsidation of adenovirus 16 DNA is directed by a small DNA sequence at the left end of the genome. Cell. 1980 Jul;20(3):787–795. doi: 10.1016/0092-8674(80)90325-6. [DOI] [PubMed] [Google Scholar]
  21. Hsiao C. L., Black L. W. DNA packaging and the pathway of bacteriophage T4 head assembly. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3652–3656. doi: 10.1073/pnas.74.9.3652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ishibashi M., Maizel J. V., Jr The polypeptides of adenovirus. V. Young virions, structural intermediate between top components and aged virions. Virology. 1974 Feb;57(2):409–424. doi: 10.1016/0042-6822(74)90181-0. [DOI] [PubMed] [Google Scholar]
  23. Ishibashi M., Maizel J. V., Jr The polypeptides of adenovirus. VI. Early and late glycopolypeptides. Virology. 1974 Apr;58(2):345–361. doi: 10.1016/0042-6822(74)90070-1. [DOI] [PubMed] [Google Scholar]
  24. Kauffman R. S., Ginsberg H. S. Characterization of a temperature-sensitive, hexon transport mutant of type 5 adenovirus. J Virol. 1976 Aug;19(2):643–658. doi: 10.1128/jvi.19.2.643-658.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. King J., Casjens S. Catalytic head assembling protein in virus morphogenesis. Nature. 1974 Sep 13;251(5471):112–119. doi: 10.1038/251112a0. [DOI] [PubMed] [Google Scholar]
  26. King J., Lenk E. V., Botstein D. Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway. J Mol Biol. 1973 Nov 15;80(4):697–731. doi: 10.1016/0022-2836(73)90205-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lawrence W. C., Ginsberg H. S. Intracellular uncoating of type 5 adenovirus deoxyribonucleic acid. J Virol. 1967 Oct;1(5):851–867. doi: 10.1128/jvi.1.5.851-867.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lewis J. B., Anderson C. W., Atkins J. F. Further mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments. Cell. 1977 Sep;12(1):37–44. doi: 10.1016/0092-8674(77)90183-0. [DOI] [PubMed] [Google Scholar]
  30. Lewis J. B., Atkins J. F., Anderson C. W., Baum P. R., Gesteland R. F. Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1344–1348. doi: 10.1073/pnas.72.4.1344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lucas J. J., Ginsberg H. S. Synthesis of virus-specific ribonucleic acid in KB cells infected with type 2 adenovirus. J Virol. 1971 Aug;8(2):203–214. doi: 10.1128/jvi.8.2.203-214.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Maizel J. V., Jr, White D. O., Scharff M. D. The polypeptides of adenovirus. II. Soluble proteins, cores, top components and the structure of the virion. Virology. 1968 Sep;36(1):126–136. doi: 10.1016/0042-6822(68)90122-0. [DOI] [PubMed] [Google Scholar]
  33. McLIMANS W. F., DAVIS E. V., GLOVER F. L., RAKE G. W. The submerged culture of mammalian cells; the spinner culture. J Immunol. 1957 Nov;79(5):428–433. [PubMed] [Google Scholar]
  34. PHILIPSON L. Adenovirus assay by the fluorescent cell-counting procedure. Virology. 1961 Nov;15:263–268. doi: 10.1016/0042-6822(61)90357-9. [DOI] [PubMed] [Google Scholar]
  35. Peacock A. C., Dingman C. W. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry. 1968 Feb;7(2):668–674. doi: 10.1021/bi00842a023. [DOI] [PubMed] [Google Scholar]
  36. Persson H., Mathisen B., Philipson L., Pettersson U. A maturation protein in adenovirus morphogenesis. Virology. 1979 Feb;93(1):198–208. doi: 10.1016/0042-6822(79)90287-3. [DOI] [PubMed] [Google Scholar]
  37. Prage L., Höglund S., Philipson L. Structural proteins of adenoviruses. 8. Characterization of incomplete particles of adenovirus type 3. Virology. 1972 Sep;49(3):745–757. doi: 10.1016/0042-6822(72)90531-4. [DOI] [PubMed] [Google Scholar]
  38. Rekosh D. M., Russell W. C., Bellet A. J., Robinson A. J. Identification of a protein linked to the ends of adenovirus DNA. Cell. 1977 Jun;11(2):283–295. doi: 10.1016/0092-8674(77)90045-9. [DOI] [PubMed] [Google Scholar]
  39. Rosenwirth B., Tjia S., Westphal M., Doerfler W. Incomplete particles of adenovirus. II. Kinetics of formation and polypeptide composition of adenovirus type 2. Virology. 1974 Aug;60(2):431–437. doi: 10.1016/0042-6822(74)90337-7. [DOI] [PubMed] [Google Scholar]
  40. Russel W. C., Newman C., Williams J. F. Characterization of temperature-sensitive mutants of adenovirus type 5--serology. J Gen Virol. 1972 Dec;17(3):265–279. doi: 10.1099/0022-1317-17-3-265. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Steenbergh P. H., Maat J., van Ormondt H., Sussenbach J. S. The nucleotide sequence at the termini of adenovirus type 5 DNA. Nucleic Acids Res. 1977 Dec;4(12):4371–4389. doi: 10.1093/nar/4.12.4371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  44. Sundquist B., Everitt E., Philipson L., Hoglund S. Assembly of adenoviruses. J Virol. 1973 Mar;11(3):449–459. doi: 10.1128/jvi.11.3.449-459.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thiel J. F., Smith K. O. Fluorescent focus assay of viruses on cell monolayers in plastic Petri plates. Proc Soc Exp Biol Med. 1967 Jul;125(3):892–895. doi: 10.3181/00379727-125-32232. [DOI] [PubMed] [Google Scholar]
  46. Tibbetts C., Giam C. Z. In vitro association of empty adenovirus capsids with double-stranded DNA. J Virol. 1979 Dec;32(3):995–1005. doi: 10.1128/jvi.32.3.995-1005.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tibbetts C. Viral DNA sequences from incomplete particles of human adenovirus type 7. Cell. 1977 Sep;12(1):243–249. doi: 10.1016/0092-8674(77)90202-1. [DOI] [PubMed] [Google Scholar]
  48. Velicer L. F., Ginsberg H. S. Cytoplasmic synthesis of type 5 adenovirus capsid proteins. Proc Natl Acad Sci U S A. 1968 Dec;61(4):1264–1271. doi: 10.1073/pnas.61.4.1264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Velicer L. F., Ginsberg H. S. Synthesis, transport, and morphogenesis of type adenovirus capsid proteins. J Virol. 1970 Mar;5(3):338–352. doi: 10.1128/jvi.5.3.338-352.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. WILCOX W. C., GINSBERG H. S. Purification and immunological characterization of types 4 and 5 adenovirus-soluble antigens. Proc Natl Acad Sci U S A. 1961 Apr 15;47:512–526. doi: 10.1073/pnas.47.4.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wadell G., Hammarskjöld M. L., Varsanyi T. Incomplete virus particles of adenovirus type 16. J Gen Virol. 1973 Sep;20(3):287–302. doi: 10.1099/0022-1317-20-3-287. [DOI] [PubMed] [Google Scholar]
  52. Weber J. Genetic analysis of adenovirus type 2 III. Temperature sensitivity of processing viral proteins. J Virol. 1976 Feb;17(2):462–471. doi: 10.1128/jvi.17.2.462-471.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. 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]
  56. Wolfson J., Dressler D. Adenovirus-2 DNA contains an inverted terminal repetition. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3054–3057. doi: 10.1073/pnas.69.10.3054. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES