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. 1980 Oct;36(1):62–78. doi: 10.1128/jvi.36.1.62-78.1980

Synthesis and processing of polymerase proteins of wild-type and mutant avian retroviruses.

R N Eisenman, W S Mason, M Linial
PMCID: PMC353616  PMID: 6160263

Abstract

We have studied the biosynthesis of avian retrovirus proteins related to reverse transcriptase in permissive avian embryonic cells. Analysis of immune precipitates from avian sarcoma virus (ASV)-infected cells demonstrated the presence of the 180,000-dalton gag-pol "read-through" protein (Pr180gag-pol) and a 130,000-dalton polypeptide (Pr130gag-pol). Pr130gag-pol was found, in serological and peptide mapping studies, to consist primarily of sequences related to reverse transcriptase and the gag-encoded protein p15. Pr180gag-pol was found to be phosphorylated, whereas Pr130gag-pol was not. In addition, only Pr180gag-pol but not Pr130gag-pol was susceptible to cleavage with the virion protease p15. Although the structure of Pr130gag-pol would suggest that it is generated by removal of a portion of the gag region from Pr180gag-pol, an analysis of labeling kinetics has failed to demonstrate unequivocally whether Pr130gag-pol is a cleavage product of Pr180gag-pol or a primary translation product. We were repeatedly unable to detect either Pr180gag-pol or Pr130gag-pol in virus particles released from the cell, whereas both beta and alpha subunits were readily observed. Several presumed intermediates between Pr130gag-pol and the beta subunit of reverse transcriptase were also observed in virions. These studies indicate cleavage of polyemrase precursors at the time of virus budding. On the basis of these data, we present a processing scheme for the generation of reverse transcriptase subunits. We have also examined reverse transcriptase biosynthesis in cells producing two mutants that fail to package the enzyme. Previous work showed that integrated proviruses of both mutants are missing DNA sequences in pol: one mutant, PH9 (Mason et al., J. Virol. 30:132-140, 1979), contains a deletion near the 3' end of pol, whereas the other, SE52d (linial et al., Virology 87:130-141, 1978), may have inserted a host cell sequence near the 5' end of pol. Neither mutant synthesized Pr180gag-pol or Pr130gag-pol, but instead produced novel proteins comprised of sequences shared with gag proteins plus a region antigenically related to reverse transcriptase. Both proteins were defective as precursors to reverse transcriptase. Whereas Pr180gag-pol and Pr130gag-pol were precipitated by an antiserum raised against p32 (a virion protein derived from the portion of the beta subunit removed during processing of beta to alpha [Schiff and Grandgenett, J. Virol. 28:279-291, 1978]), the novel protein synthesized by PH9 ws not precipitated. This suggets that the alpha subunit is generated by a COOH-terminal cleavage of the beta subunit.

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Selected References

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

  1. Astrin S. M., Crittenden L. B., Buss E. G. ev 3, a structural gene locus for endogenous virus, segregates with the gs+chf+ phenotype in matings of line 63 chickens. Virology. 1979 Nov;99(1):1–9. doi: 10.1016/0042-6822(79)90031-x. [DOI] [PubMed] [Google Scholar]
  2. Beemon K., Hunter T. In vitro translation yields a possible Rous sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3302–3306. doi: 10.1073/pnas.74.8.3302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen J. H., Hanafusa H. Detection of a protein of avian leukoviruses in uninfected chick cells by radioimmunoassay. J Virol. 1974 Feb;13(2):340–346. doi: 10.1128/jvi.13.2.340-346.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cooper G. M. Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase. J Virol. 1978 Mar;25(3):788–796. doi: 10.1128/jvi.25.3.788-796.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Copeland T. D., Grandgenett D. P., Oroszlan S. Amino acid sequence analysis of reverse transcriptase subunits from avian myeloblastosis virus. J Virol. 1980 Oct;36(1):115–119. doi: 10.1128/jvi.36.1.115-119.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crittenden L. B., Smith E. J., Weiss R. A., Sarma P. S. Host gene control of endogenous avian leukosis virus production. Virology. 1974 Jan;57(1):128–138. doi: 10.1016/0042-6822(74)90114-7. [DOI] [PubMed] [Google Scholar]
  7. Dittmar K. J., Moelling K. Biochemical properties of p15-associated protease in an avian RNA tumor virus. J Virol. 1978 Oct;28(1):106–118. doi: 10.1128/jvi.28.1.106-118.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eisenman R., Shaikh R., Mason W. S. Identification of an avian oncovirus polyprotein in uninfected chick cells. Cell. 1978 May;14(1):89–104. doi: 10.1016/0092-8674(78)90304-5. [DOI] [PubMed] [Google Scholar]
  9. Erikson E., Brugge J. S., Erikson R. L. Phosphorylated and nonphosphorylated forms of avian sarcoma virus polypeptide p19. Virology. 1977 Jul 1;80(1):177–185. doi: 10.1016/0042-6822(77)90390-7. [DOI] [PubMed] [Google Scholar]
  10. Gerwin B. I., Rein A., Levin J. G., Bassin R. H., Benjers B. M., Kashmiri S. V., Hopkins D., O'Neill B. J. Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule. J Virol. 1979 Sep;31(3):741–751. doi: 10.1128/jvi.31.3.741-751.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grandgenett D. P., Golomb M., Vora A. C. Activation of an Mg2+-dependent DNA endonuclease of avian myeloblastosis virus alpha beta DNA polymerase by in vitro proteolytic cleavage. J Virol. 1980 Jan;33(1):264–271. doi: 10.1128/jvi.33.1.264-271.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grandgenett D. P., Vora A. C., Schiff R. D. A 32,000-dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity. Virology. 1978 Aug;89(1):119–132. doi: 10.1016/0042-6822(78)90046-6. [DOI] [PubMed] [Google Scholar]
  13. Hanafusa H., Baltimore D., Smoler D., Watson K. F., Yaniv A., Spiegelman S. Absence of polymerase protein in virions of alpha-type rous sarcoma virus. Science. 1972 Sep 29;177(4055):1188–1191. doi: 10.1126/science.177.4055.1188. [DOI] [PubMed] [Google Scholar]
  14. Hayman M. J. Viral polyproteins in chick embryo fibroblasts infected with avian sarcoma leukosis viruses. Virology. 1978 Mar;85(1):241–252. doi: 10.1016/0042-6822(78)90428-2. [DOI] [PubMed] [Google Scholar]
  15. Hayward W. S. Size and genetic content of viral RNAs in avian oncovirus-infected cells. J Virol. 1977 Oct;24(1):47–63. doi: 10.1128/jvi.24.1.47-63.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hizi A., Joklik W. K. RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the alpha, beta2, and alphabeta forms of the enzyme. J Biol Chem. 1977 Apr 10;252(7):2281–2289. [PubMed] [Google Scholar]
  17. Hizi A., Joklik W. K. The beta subunit of the DNA polymerase of avian sarcoma virus strain B77 is a phosphoprotein. Virology. 1977 May 15;78(2):571–575. doi: 10.1016/0042-6822(77)90132-5. [DOI] [PubMed] [Google Scholar]
  18. Jamjoom G. A., Naso R. B., Arlinghaus R. B. Further characterization of intracellular precursor polyproteins of Rauscher leukemia virus. Virology. 1977 May 1;78(1):11–34. doi: 10.1016/0042-6822(77)90075-7. [DOI] [PubMed] [Google Scholar]
  19. Kacian D. L., Watson K. F., Burny A., Spiegelman S. Purification of the DNA polymerase of avian myeloblastosis virus. Biochim Biophys Acta. 1971 Sep 24;246(3):365–383. doi: 10.1016/0005-2787(71)90773-8. [DOI] [PubMed] [Google Scholar]
  20. Kawai S., Hanafusa H. Isolation of defective mutant of avian sarcoma virus. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3493–3497. doi: 10.1073/pnas.70.12.3493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kerr I. M., Olshevsky U., Lodish H. F., Baltimore D. Translation of murine leukemia virus RNA in cell-free systems from animal cells. J Virol. 1976 May;18(2):627–635. doi: 10.1128/jvi.18.2.627-635.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Lai M. M. Phosphoproteins of Rous sarcoma viruses. Virology. 1976 Oct 15;74(2):287–301. doi: 10.1016/0042-6822(76)90336-6. [DOI] [PubMed] [Google Scholar]
  24. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  25. Ledbetter J. A., Nowinski R. C., Eisenman R. N. Biosynthesis and metabolism of viral proteins expressed on the surface of murine leukemia virus-infected cells. Virology. 1978 Nov;91(1):116–129. doi: 10.1016/0042-6822(78)90360-4. [DOI] [PubMed] [Google Scholar]
  26. Linial M., Brown S., Neiman P. A nonconditional mutant of Rous sarcoma virus containing defective polymerase. Virology. 1978 Jun 1;87(1):130–141. doi: 10.1016/0042-6822(78)90165-4. [DOI] [PubMed] [Google Scholar]
  27. Linial M., Mason W. S. Characterization of two conditional early mutants of Rous sarcoma virus. Virology. 1973 May;53(1):258–273. doi: 10.1016/0042-6822(73)90484-4. [DOI] [PubMed] [Google Scholar]
  28. Linial M., Neiman P. E. Infection of chick cells by subgroup E viruses. Virology. 1976 Sep;73(2):508–520. doi: 10.1016/0042-6822(76)90412-8. [DOI] [PubMed] [Google Scholar]
  29. Mason W. S., Hsu T. W., Yeater C., Sabran J. L., Mark G. E., Kaji A., Taylor J. M. Avian sarcoma virus-transformed quail clones defective in the production of focus-forming virus. J Virol. 1979 Apr;30(1):132–140. doi: 10.1128/jvi.30.1.132-140.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mason W. S., Yeater C., Bosch J. V., Wyke J. A., Friis R. R. Fourteen temperature-sensitive replication mutants of Rous sarcoma virus. Virology. 1979 Dec;99(2):226–240. doi: 10.1016/0042-6822(79)90003-5. [DOI] [PubMed] [Google Scholar]
  31. Moelling K. Reverse transcriptase and RNase H: present in a murine virus and in both subunits of an avian virus. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 2):969–973. doi: 10.1101/sqb.1974.039.01.111. [DOI] [PubMed] [Google Scholar]
  32. Moelling K., Scott A., Dittmar K. E., Owada M. Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors. J Virol. 1980 Feb;33(2):680–688. doi: 10.1128/jvi.33.2.680-688.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Murphy H. M. A new replication-defective variant of the Bryan high-titer strain Rous sarcoma virus. Virology. 1977 Apr;77(2):705–721. doi: 10.1016/0042-6822(77)90493-7. [DOI] [PubMed] [Google Scholar]
  34. Ng V. L., Wood T. G., Lyons D. D., Arlinghaus R. B. Characterization of intracellular precursor polyproteins of Moloney murine leukemia virus. J Virol. 1979 Dec;32(3):1051–1056. doi: 10.1128/jvi.32.3.1051-1056.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oppermann H., Bishop J. M., Varmus H. E., Levintow L. A joint produce of the genes gag and pol of avian sarcoma virus: a possible precursor of reverse transcriptase. Cell. 1977 Dec;12(4):993–1005. doi: 10.1016/0092-8674(77)90164-7. [DOI] [PubMed] [Google Scholar]
  36. Panet A., Baltimore D., Hanafusa T. Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay. J Virol. 1975 Jul;16(1):146–152. doi: 10.1128/jvi.16.1.146-152.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Paterson B. M., Marciani D. J., Papas T. S. Cell-free synthesis of the precursor polypeptide for avian myeloblastosis virus DNA polymerase. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4951–4954. doi: 10.1073/pnas.74.11.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Purchio A. F., Erikson E., Erikson R. L. Translation of 35S and of subgenomic regions of avian sarcoma virus RNA. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4661–4665. doi: 10.1073/pnas.74.10.4661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rettenmier C. W., Karess R. E., Anderson S. M., Hanafusa H. Tryptic peptide analysis of avian oncovirus gag and pol gene products. J Virol. 1979 Oct;32(1):102–113. doi: 10.1128/jvi.32.1.102-113.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Saborio J. L., Pong S. S., Koch G. Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J Mol Biol. 1974 May 15;85(2):195–211. doi: 10.1016/0022-2836(74)90360-x. [DOI] [PubMed] [Google Scholar]
  41. Scheele C. M., Hanafusa H. Proteins of helper-dependent RSV. Virology. 1971 Aug;45(2):401–410. doi: 10.1016/0042-6822(71)90341-2. [DOI] [PubMed] [Google Scholar]
  42. Schiff R. D., Grandgenett D. P. Virus-coded origin of a 32,000-dalton protein from avian retrovirus cores: structural relatedness of p32 and the beta polypeptide of the avian retrovirus DNA polymerase. J Virol. 1978 Oct;28(1):279–291. doi: 10.1128/jvi.28.1.279-291.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shaikh R., Linial M., Coffin J., Eisenman R. Recombinant avian oncoviruses. I. Alterations in the precursor to the internal structural proteins. Virology. 1978 Jun 15;87(2):326–338. doi: 10.1016/0042-6822(78)90138-1. [DOI] [PubMed] [Google Scholar]
  44. Shealy D. J., Mosser A. G., Rueckert R. R. Novel p19-related protein in Rous-associated virus type 61: implications for avian gag gene order. J Virol. 1980 May;34(2):431–437. doi: 10.1128/jvi.34.2.431-437.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Temin H. M., Baltimore D. RNA-directed DNA synthesis and RNA tumor viruses. Adv Virus Res. 1972;17:129–186. doi: 10.1016/s0065-3527(08)60749-6. [DOI] [PubMed] [Google Scholar]
  46. Tronick S. R., Stephenson J. R., Verma I. M., Aaronson S. A. Thermolabile reverse transcriptase of a mammalian leukemia virus mutant temperature sensitive in its replication and sarcoma virus helper functions. J Virol. 1975 Dec;16(6):1476–1482. doi: 10.1128/jvi.16.6.1476-1482.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Verma I. M. The reverse transcriptase. Biochim Biophys Acta. 1977 Mar 21;473(1):1–38. doi: 10.1016/0304-419x(77)90005-1. [DOI] [PubMed] [Google Scholar]
  48. Vogt V. M., Eisenman R., Diggelmann H. Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide. J Mol Biol. 1975 Aug 15;96(3):471–493. doi: 10.1016/0022-2836(75)90174-6. [DOI] [PubMed] [Google Scholar]
  49. Vogt V. M., Wight A., Eisenman R. In vitro cleavage of avian retrovirus gag proteins by viral protease p15. Virology. 1979 Oct 15;98(1):154–167. doi: 10.1016/0042-6822(79)90534-8. [DOI] [PubMed] [Google Scholar]
  50. Weiss S. R., Varmus H. E., Bishop J. M. The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses. Cell. 1977 Dec;12(4):983–992. doi: 10.1016/0092-8674(77)90163-5. [DOI] [PubMed] [Google Scholar]
  51. Witte O. N., Baltimore D. Relationship of retrovirus polyprotein cleavages to virion maturation studied with temperature-sensitive murine leukemia virus mutants. J Virol. 1978 Jun;26(3):750–761. doi: 10.1128/jvi.26.3.750-761.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. von der Helm K. Cleavage of Rous sarcoma viral polypeptide precursor into internal structural proteins in vitro involves viral protein p15. Proc Natl Acad Sci U S A. 1977 Mar;74(3):911–915. doi: 10.1073/pnas.74.3.911. [DOI] [PMC free article] [PubMed] [Google Scholar]

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