1887

Abstract

Human immunodeficiency virus type 1 Gag and Gag–Pol precursors are translated from an mRNA which is indistinguishable from the full-length genomic RNA. The ratio of Gag to Gag–Pol polyproteins is approximately 20:1 and is controlled by a frameshift of the reading frame, which takes place downstream of the p7 nucleocapsid (NC) in the N terminus of the p1 peptide. The viral precursors Gag and Gag–Pol are cleaved by the virus-encoded protease (PR) into the structural proteins, and into p6, PR, reverse transcriptase and integrase. Due to the frameshift event, the cleavage site at the C terminus of NC coded in the Gag frame (ERQAN-FLGKI) changes either to ERQANFLRED or ERQANFFRED. The results presented in this report demonstrate that the NC released from the Gag–Pol precursor is 8 amino acid residues longer than the NC cleaved from the Gag polyprotein. Our results also show that truncated Gag–Pol precursors bearing cleavage site mutation at the NC/p6, and/or p6/PR junctions, undergo autoprocessing in bacterial and eukaryotic cells, indicating that PR is active when part of the precursor.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-82-3-581
2001-03-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/82/3/0820581a.html?itemId=/content/journal/jgv/10.1099/0022-1317-82-3-581&mimeType=html&fmt=ahah

References

  1. Accola M., Hoglund S., Gottlinger H. 1998; A putative alpha-helical structure which overlaps the capsid–p2 boundary in the human immunodeficiency virus type 1 Gag precursor is crucial for viral particle assembly. Journal of Virology 72:2072–2078
    [Google Scholar]
  2. Almog N., Roller R., Arad G., Passi-Even L., Wainberg M., Kotler M. 1996; A p6Pol protease fusion protein is present in mature particles of immunodeficiency virus type 1. Journal of Virology 70:7228–7232
    [Google Scholar]
  3. Arad G., Bar-Meir R., Kotler M. 1995; Ribosomal frameshifting at the Gag Pol junction in avian leukemia sarcoma virus forms a novel cleavage site. FEBS Letters 364:1–4
    [Google Scholar]
  4. Baraz L., Friedler A., Blumenzweig I., Nussinov O., Chen N., Steinitz M., Gilon C., Kotler M. 1998; Human immunodeficiency virus type 1 Vif-derived peptides inhibit the viral protease and arrest virus production. FEBS Letters 441:419–426
    [Google Scholar]
  5. Beissinger M., Paulus C., Bayer P., Wolf H., Rosch P., Wagner R. 1996; Sequence-specific resonance assignments of the H-NMR spectra and structural characterization solution of the HIV-1 transfer protein p6*. European Journal of Biochemistry 237:383–392
    [Google Scholar]
  6. Cherry E., Liang C., Rong L., Quan Y., Inouye P., Li X., Kotler M., Wainberg M. 1998; Characterization of human immunodeficiency virus type 1 (HIV-1) particles that express protease–reverse transcriptase fusion proteins. Journal of Molecular Biology 284:43–56
    [Google Scholar]
  7. Demetrios A., Welkie N. 1984; Expression of exogenous DNA in mammalian cells. In Transcription and Translation pp 1–45 Edited by Hames B., Higgins S. Boca Raton, FL: IRL Press;
    [Google Scholar]
  8. Friedler A., Blumenzweig I., Baraz L., Steinitz M., Kotler M., Gilon C. 1999; Peptides derived from HIV-1 Vif: a non-substrate based novel type of HIV-1 protease inhibitors. Journal of Molecular Biology 287:93–101
    [Google Scholar]
  9. Fuerst T. R., Niles E. G., Studier F. W., Moss B. 1986; Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences, USA 83:8122–8126
    [Google Scholar]
  10. Gillespie P., Gillespie S. 1997; Improved electrophoresis and transfer picogram amounts of protein with hemoglobin. Analytical Biochemistry 246:239–245
    [Google Scholar]
  11. Henderson L., Copeland D., Sowder C., Schultz A., Oroszlan S. 1988; Analysis of proteins and peptides purified from sucrose gradient banded HTLV-III. In Human Retroviruses, Cancer, and AIDS: Approaches to Prevention and Therapy pp 135–147 Edited by Bolognesi D. New York: Alan M. Liss;
    [Google Scholar]
  12. Henderson L., Bowers M., Sowder R., Serabyn S., Johnson D., Bess J., Arthur L., Bryant D., Fenselau C. 1992; Gag proteins of the highly replicative MN strain of the human immunodeficiency virus type 1: post translational modifications, proteolytic processing and complete amino acid sequences. Journal of Virology 66:1856–1865
    [Google Scholar]
  13. Jacks T., Power M., Masiarz F., Luciw P., Barr P., Varmus H. 1988; Characterization of ribosomal frameshifting in HIV-1 gag–pol expression. Nature 331:280–283
    [Google Scholar]
  14. Karacostas V., Wolffe K., Nagashima K., Gonda M., Moss B. 1993; Overexpression of the HIV-1 Gag–Pol proteins results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles. Virology 193:661–671
    [Google Scholar]
  15. Kotler M., Arad G., Hughes S. 1992; Human immunodeficiency virus type 1 gag protease fusion proteins are enzymatically active. Journal of Virology 66:6781–6783
    [Google Scholar]
  16. Krausslich H.-G., Facke M., Heuser A., Konvalinka J., Zentgraft H. 1995; The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity. Journal of Virology 69:3407–3419
    [Google Scholar]
  17. Louis J. M., Nashed N. T., Parris K. D., Kimmel A. R., Jerina D. M. 1994; Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag–Pol polyprotein. Proceedings of the National Academy of Sciences, USA 91:7970–7974
    [Google Scholar]
  18. Louis J. M., Clore G. M., Gronenborn A. M. 1999; Autoprocessing of HIV-1 protease is tightly coupled to protein folding. Nature Structural Biology 6:868–875
    [Google Scholar]
  19. Norrander J., Vieira J., Rubenstein I., Messing J. 1985; Manipulation and expression of the maize zein storage proteins in Escherichia coli. Journal of Biotechnology 2:157–175
    [Google Scholar]
  20. Pettit S., Moody M., Wehbie R., Kaplan A., Nantermet P., Klein C., Swanstrom R. 1994; The P2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions. Journal of Virology 68:8017–8027
    [Google Scholar]
  21. Phylip L., Mills J., Parten B., Dunn B., Kay J. 1992; Intrinsic activity of precursor forms of HIV-1 proteinase. FEBS Letters 314:449–454
    [Google Scholar]
  22. Ratner L., Haseltine W., Patarca R., Livak K., Starcich B., Joseph S., Doran E., Rafalski J., Whitehorn K., Ivanoff L., Petteway S. J., Pearson M., Lautenberg J., Papas T., Ghrayeb J., Chang N., Gallo R., Wong-Staal F. 1985; Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313:277–284
    [Google Scholar]
  23. Roberts N., Martin J., Kinchington D., Broadharst A., Craig J., Duncan I., Galpin S., Handa B., Kay J., Krohn A., Lambert R., Merrett J., Mills J., Parks K., Redshaw S., Taylor D., Thomas G., Machin P. 1990; Rational design of peptide-based HIV proteinase inhibitors. Science 248:358–361
    [Google Scholar]
  24. Sedlacek J., Strop P., Kapralek F., Pecenka V., Kostka V., Travnicek M., Riman J. 1988; Processed enzymatically active protease (p15gag) of avian retrovirus obtained in an E. coli system expressing a recombinant precursor (Pr25lac-delta gag. FEBS Letters 237:187–190
    [Google Scholar]
  25. Smith H., Srinivasakuman N., Hammarskjold M.-L., Rekosh D. 1993; Requirements for incorporation of Pr160 gag–pol from human immunodeficiency virus type 1 into virus-like particles. Journal of Virology 67:2266–2275
    [Google Scholar]
  26. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. 1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185:60–89
    [Google Scholar]
  27. Swanstrom R., Wills J. W. 1997; Synthesis, assembly, and processing of viral proteins. In Retroviruses Edited by Coffin J. M., Hughes S. H., Varmus H. E. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  28. Tessmer U., Krausslich H.-G. 1998; Cleavage of human immunodeficiency virus type 1 proteinase from the N-terminally adjacent p6* protein is essential for efficient Gag polyprotein processing and viral infectivity. Journal of Virology 72:3459–3463
    [Google Scholar]
  29. Tritch R., Cheng Y.-S. E., Yin F., Erickson-Vitanen S. 1991; Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein. Journal of Virology 65:922–930
    [Google Scholar]
  30. Vieira J., Messing J. 1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
    [Google Scholar]
  31. Vogt V. 1996; Proteolytic processing and particle maturation. Current Topics in Microbiology and Immunology 214:95–131
    [Google Scholar]
  32. Wondrak E., Louis J., de Rocquigny H., Chermann J., Roques B. 1993; The gag precursor contains a specific HIV-1 protease cleavage site between the NC(P7) and p1 proteins. FEBS Letters 333:21–24
    [Google Scholar]
  33. Zybarth G., Carter C. 1995; Domains upstream of the protease (PR) in human immunodeficiency virus type 1 Gag–Pol influence PR autoprocessing. Journal of Virology 69:3878–3884
    [Google Scholar]
  34. Zybarth G., Krausslich H.-G., Partin K., Carter C. 1994; Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain. Journal of Virology 68:240–250
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-82-3-581
Loading
/content/journal/jgv/10.1099/0022-1317-82-3-581
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error