Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1988 Jul;62(7):2358–2365. doi: 10.1128/jvi.62.7.2358-2365.1988

Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli.

R Terry 1, D A Soltis 1, M Katzman 1, D Cobrinik 1, J Leis 1, A M Skalka 1
PMCID: PMC253392  PMID: 2836618

Abstract

The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95- and 63-kilodalton (kDa) beta and alpha subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36pol) which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32pol and p36pol proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32pol and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.

Full text

PDF
2358

Images in this article

2361Image
on p.2361
2361Image
on p.2361
2362Image
on p.2362
2362Image
on p.2362
2363Image
on p.2363
2363Image
on p.2363

Selected References

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

  1. Alexander F., Leis J., Soltis D. A., Crowl R. M., Danho W., Poonian M. S., Pan Y. C., Skalka A. M. Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain. J Virol. 1987 Feb;61(2):534–542. doi: 10.1128/jvi.61.2.534-542.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boros I., Pósfai G., Venetianer P. High-copy-number derivatives of the plasmid cloning vector pBR322. Gene. 1984 Oct;30(1-3):257–260. doi: 10.1016/0378-1119(84)90130-6. [DOI] [PubMed] [Google Scholar]
  3. Cobrinik D., Katz R., Terry R., Skalka A. M., Leis J. Avian sarcoma and leukosis virus pol-endonuclease recognition of the tandem long terminal repeat junction: minimum site required for cleavage is also required for viral growth. J Virol. 1987 Jun;61(6):1999–2008. doi: 10.1128/jvi.61.6.1999-2008.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Crowl R., Seamans C., Lomedico P., McAndrew S. Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene. 1985;38(1-3):31–38. doi: 10.1016/0378-1119(85)90200-8. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Eisenman R. N., Mason W. S., Linial M. Synthesis and processing of polymerase proteins of wild-type and mutant avian retroviruses. J Virol. 1980 Oct;36(1):62–78. doi: 10.1128/jvi.36.1.62-78.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Grandgenett D. P., Vora A. C. Site-specific nicking at the avian retrovirus LTR circle junction by the viral pp32 DNA endonuclease. Nucleic Acids Res. 1985 Sep 11;13(17):6205–6221. doi: 10.1093/nar/13.17.6205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grandgenett D. P., Vora A. C., Swanstrom R., Olsen J. C. Nuclease mechanism of the avian retrovirus pp32 endonuclease. J Virol. 1986 Jun;58(3):970–974. doi: 10.1128/jvi.58.3.970-974.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Hishinuma F., DeBona P. J., Astrin S., Skalka A. M. Nucleotide sequence of acceptor site and termini of integrated avian endogenous provirus ev1: integration creates a 6 bp repeat of host DNA. Cell. 1981 Jan;23(1):155–164. doi: 10.1016/0092-8674(81)90280-4. [DOI] [PubMed] [Google Scholar]
  14. Houts G. E., Miyagi M., Ellis C., Beard D., Beard J. W. Reverse transcriptase from avian myeloblastosis virus. J Virol. 1979 Feb;29(2):517–522. doi: 10.1128/jvi.29.2.517-522.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Katz R. A., Fu X. D., Skalka A. M., Leis J. Avian retrovirus nucleocapsid protein, pp12, produced in Escherichia coli has biochemical properties identical to unphosphorylated viral protein. Gene. 1986;50(1-3):361–369. doi: 10.1016/0378-1119(86)90340-9. [DOI] [PubMed] [Google Scholar]
  16. Katz R. A., Skalka A. M. A C-terminal domain in the avian sarcoma-leukosis virus pol gene product is not essential for viral replication. J Virol. 1988 Feb;62(2):528–533. doi: 10.1128/jvi.62.2.528-533.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Miyamoto C., Chizzonite R., Crowl R., Rupprecht K., Kramer R., Schaber M., Kumar G., Poonian M., Ju G. Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7232–7236. doi: 10.1073/pnas.82.21.7232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Morris-Vasios C., Kochan J. P., Skalka A. M. Avian sarcoma-leukosis virus pol-endo proteins expressed independently in mammalian cells accumulate in the nucleus but can be directed to other cellular compartments. J Virol. 1988 Jan;62(1):349–353. doi: 10.1128/jvi.62.1.349-353.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Panganiban A. T., Temin H. M. The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locus. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7885–7889. doi: 10.1073/pnas.81.24.7885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schiff R. D., Grandgenett D. P. Partial phosphorylation in vivo of the avian retrovirus pp32 DNA endonuclease. J Virol. 1980 Dec;36(3):889–893. doi: 10.1128/jvi.36.3.889-893.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  28. 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]

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

RESOURCES