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. 1986 Aug;59(2):328–340. doi: 10.1128/jvi.59.2.328-340.1986

Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli.

N Tanese, M J Roth, S P Goff
PMCID: PMC253082  PMID: 2426463

Abstract

Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion proteins in Escherichia coli, and the purified proteins were used to elicit antibodies in Escherichia coli, and the purified proteins were used to elicit antibodies in rabbits. The sera were used to examine the mature pol gene products contained in virion particles and identified the reverse transcriptase and a second protein, P46pol, encoded by the 3' portion of the gene. The P46 protein was not phosphorylated and was present at the same molar abundance as the reverse transcriptase. The sera were also used to detect the Pr200gag-pol intracellular precursor protein and to analyze its processing to the mature forms. The proteins formed by several Moloney MuLV mutants were analyzed. Further tests revealed cross-reactivity with Friend MuLV and feline leukemia virus proteins, but not with avian retrovirus proteins.

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Selected References

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  1. Arcement L. J., Karshin W. L., Naso R. B., Arlinghaus R. B. "gag" polyprotein precursors of Rauscher murine leukemia virus. Virology. 1977 Sep;81(2):284–297. doi: 10.1016/0042-6822(77)90145-3. [DOI] [PubMed] [Google Scholar]
  2. BRYAN W. R. Biological studies on the Rous sarcoma virus. I. General introduction. II. Review of sources of experimental variation and of methods for their control. J Natl Cancer Inst. 1955 Aug;16(1):285–315. [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  5. Copeland T. D., Gerard G. F., Hixson C. W., Oroszlan S. Amino- and carboxyl-terminal sequence of Moloney murine leukemia virus reverse transcriptase. Virology. 1985 Jun;143(2):676–679. doi: 10.1016/0042-6822(85)90411-8. [DOI] [PubMed] [Google Scholar]
  6. Crawford S., Goff S. P. A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins. J Virol. 1985 Mar;53(3):899–907. doi: 10.1128/jvi.53.3.899-907.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Donehower L. A., Varmus H. E. A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6461–6465. doi: 10.1073/pnas.81.20.6461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duyk G., Leis J., Longiaru M., Skalka A. M. Selective cleavage in the avian retroviral long terminal repeat sequence by the endonuclease associated with the alpha beta form of avian reverse transcriptase. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6745–6749. doi: 10.1073/pnas.80.22.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duyk G., Longiaru M., Cobrinik D., Kowal R., deHaseth P., Skalka A. M., Leis J. Circles with two tandem long terminal repeats are specifically cleaved by pol gene-associated endonuclease from avian sarcoma and leukosis viruses: nucleotide sequences required for site-specific cleavage. J Virol. 1985 Nov;56(2):589–599. doi: 10.1128/jvi.56.2.589-599.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Edwards S. A., Fan H. gag-Related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms. J Virol. 1979 May;30(2):551–563. doi: 10.1128/jvi.30.2.551-563.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eisenman R. N., Vogt V. M. The biosynthesis of oncovirus proteins. Biochim Biophys Acta. 1978 Apr 6;473(3-4):187–239. doi: 10.1016/0304-419x(78)90014-8. [DOI] [PubMed] [Google Scholar]
  12. FRIEND C. Cell-free transmission in adult Swiss mice of a disease having the character of a leukemia. J Exp Med. 1957 Apr 1;105(4):307–318. doi: 10.1084/jem.105.4.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gerard G. F., Grandgenett D. P. Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus. J Virol. 1975 Apr;15(4):785–797. doi: 10.1128/jvi.15.4.785-797.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Golomb M., Grandgenett D. P. Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus. J Biol Chem. 1979 Mar 10;254(5):1606–1613. [PubMed] [Google Scholar]
  15. Golomb M., Grandgenett D. P., Mason W. Virus-coded DNA endonuclease from avian retrovirus. J Virol. 1981 May;38(2):548–555. doi: 10.1128/jvi.38.2.548-555.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grandgenett D. P., Gerard G. F., Green M. A single subunit from avian myeloblastosis virus with both RNA-directed DNA polymerase and ribonuclease H activity. Proc Natl Acad Sci U S A. 1973 Jan;70(1):230–234. doi: 10.1073/pnas.70.1.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Grandgenett D. P., Knaus R. J., Hippenmeyer P. J. Antibodies against a synthetic peptide of the avian retrovirus pp32 protein and the beta DNA polymerase subunit. Virology. 1983 Oct 15;130(1):257–262. doi: 10.1016/0042-6822(83)90137-x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Grandgenett D., Quinn T., Hippenmeyer P. J., Oroszlan S. Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains. J Biol Chem. 1985 Jul 15;260(14):8243–8249. [PubMed] [Google Scholar]
  21. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  22. Hanafusa T., Wang L. H., Anderson S. M., Karess R. E., Hayward W. S., Hanafusa H. Characterization of the transforming gene of Fujinami sarcoma virus. Proc Natl Acad Sci U S A. 1980 May;77(5):3009–3013. doi: 10.1073/pnas.77.5.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. JARRETT W. F., CRAWFORD E. M., MARTIN W. B., DAVIE F. A VIRUS-LIKE PARTICLE ASSOCIATED WITH LEUKEMIA (LYMPHOSARCOMA). Nature. 1964 May 9;202:567–569. doi: 10.1038/202567a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Katz L., Kingsbury D. T., Helinski D. R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591. doi: 10.1128/jb.114.2.577-591.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kleid D. G., Yansura D., Small B., Dowbenko D., Moore D. M., Grubman M. J., McKercher P. D., Morgan D. O., Robertson B. H., Bachrach H. L. Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine. Science. 1981 Dec 4;214(4525):1125–1129. doi: 10.1126/science.6272395. [DOI] [PubMed] [Google Scholar]
  27. Kopchick J. J., Harless J., Geisser B. S., Killam R., Hewitt R. R., Arlinghaus R. B. Endodeoxyribonuclease activity associated with Rauscher murine leukemia virus. J Virol. 1981 Jan;37(1):274–283. doi: 10.1128/jvi.37.1.274-283.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kopchick J. J., Jamjoom G. A., Watson K. F., Arlinghaus R. B. Biosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a gag-pol read-through viral precursor polyprotein. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2016–2020. doi: 10.1073/pnas.75.4.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kopchick J. J., Karshin W. L., Arlinghaus R. B. Tryptic peptide analysis of gag and gag-pol gene products of Rauscher murine leukemia virus. J Virol. 1979 May;30(2):610–623. doi: 10.1128/jvi.30.2.610-623.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kurth R., Tanaka T., Moelling K. Isolation of monoclonal antibodies specific for Rous sarcoma virus structural, polymerase and transforming proteins and their use for the study of mutant virus-infected cells. J Gen Virol. 1985 Apr;66(Pt 4):827–837. doi: 10.1099/0022-1317-66-4-827. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Leis J., Duyk G., Johnson S., Longiaru M., Skalka A. Mechanism of action of the endonuclease associated with the alpha beta and beta beta forms of avian RNA tumor virus reverse transcriptase. J Virol. 1983 Feb;45(2):727–739. doi: 10.1128/jvi.45.2.727-739.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Levin J. G., Hu S. C., Rein A., Messer L. I., Gerwin B. I. Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure. J Virol. 1984 Aug;51(2):470–478. doi: 10.1128/jvi.51.2.470-478.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  35. Misra T. K., Grandgenett D. P., Parsons J. T. Avian retrovirus pp32 DNA-binding protein. I. Recognition of specific sequences on retrovirus DNA terminal repeats. J Virol. 1982 Oct;44(1):330–343. doi: 10.1128/jvi.44.1.330-343.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mizutani S., Temin H. M. Specific serological relationships among partially purified DNA polymerases of avian leukosis-sarcoma viruses, reticuloendotheliosis viruses, and avian cells. J Virol. 1974 May;13(5):1020–1029. doi: 10.1128/jvi.13.5.1020-1029.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nissen-Meyer J., Nes I. F. Purification and properties of DNA endonucleases associated with Friend leukemia virus. Nucleic Acids Res. 1980 Nov 11;8(21):5043–5055. doi: 10.1093/nar/8.21.5043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pal B. K., McAllister R. M., Gardner M. B., Roy-Burman P. Comparative studies on the structural phosphoproteins of mammalian type C viruses. J Virol. 1975 Jul;16(1):123–131. doi: 10.1128/jvi.16.1.123-131.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. RUBIN H., VOGT P. K. An avian leukosis virus associated with stocks of Rous sarcoma virus. Virology. 1962 May;17:184–194. doi: 10.1016/0042-6822(62)90096-x. [DOI] [PubMed] [Google Scholar]
  40. Rees-Jones R. W., Taylor S. I. An endogenous substrate for the insulin receptor-associated tyrosine kinase. J Biol Chem. 1985 Apr 10;260(7):4461–4467. [PubMed] [Google Scholar]
  41. Roth M. J., Tanese N., Goff S. P. Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J Biol Chem. 1985 Aug 5;260(16):9326–9335. [PubMed] [Google Scholar]
  42. SCHMIDT-RUPPIN K. H. HETEROTRANSPLANTATION OF ROUS SARCOMA AND ROUS SARCOMA VIRUS TO MAMMALS. Oncology. 1964;17:247–272. doi: 10.1159/000224235. [DOI] [PubMed] [Google Scholar]
  43. Saris C. J., van Eenbergen J., Liskamp R. M., Bloemers H. P. Structure of glycosylated and unglycosylated gag and gag-pol precursor proteins of Moloney murine leukemia virus. J Virol. 1983 Jun;46(3):841–859. doi: 10.1128/jvi.46.3.841-859.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schwartzberg P., Colicelli J., Goff S. P. Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection. Cell. 1984 Jul;37(3):1043–1052. doi: 10.1016/0092-8674(84)90439-2. [DOI] [PubMed] [Google Scholar]
  45. Schwartzberg P., Colicelli J., Goff S. P. Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent. J Virol. 1983 May;46(2):538–546. doi: 10.1128/jvi.46.2.538-546.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  47. Spindler K. R., Rosser D. S., Berk A. J. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol. 1984 Jan;49(1):132–141. doi: 10.1128/jvi.49.1.132-141.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tanese N., Roth M., Goff S. P. Expression of enzymatically active reverse transcriptase in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4944–4948. doi: 10.1073/pnas.82.15.4944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Van Zaane D., Dekker-Michielsen J. A., Bloemers H. P. Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus: synthesis, identification, and processing. Virology. 1976 Nov;75(1):113–129. doi: 10.1016/0042-6822(76)90011-8. [DOI] [PubMed] [Google Scholar]
  50. Verma I. M. Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. J Virol. 1975 Apr;15(4):843–854. doi: 10.1128/jvi.15.4.843-854.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Watson K. E., Nowinski R. C., Yaniv A., Spiegelman S. Serological analysis of the deoxyribonucleic acid polymerase of avian oncornaviruses. I. Preparation and characterization of monospecific antiserum with purified deoxyribonucleic acid polymerase. J Virol. 1972 Nov;10(5):951–958. doi: 10.1128/jvi.10.5.951-958.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Weis J., Smith D., Burns N., Furcht L., Faras A. Production and characterization of monoclonal antibodies against avian retrovirus reverse transcriptase. J Virol. 1983 Feb;45(2):859–863. doi: 10.1128/jvi.45.2.859-863.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Yoshinaka Y., Katoh I., Copeland T. D., Oroszlan S. Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1618–1622. doi: 10.1073/pnas.82.6.1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yoshinaka Y., Luftig R. B. Properties of a P70 proteolytic factor of murine leukemia viruses. Cell. 1977 Nov;12(3):709–719. doi: 10.1016/0092-8674(77)90271-9. [DOI] [PubMed] [Google Scholar]

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