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
2019-10-22
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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [Google Scholar]
  7. Demetrios, A. & Welkie, N. ( 1984; ). Expression of exogenous DNA in mammalian cells. In Transcription and Translation , pp. 1-45. Edited by B. Hames & S. Higgins. Boca Raton, FL:IRL Press.
  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.[CrossRef]
    [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.[CrossRef]
    [Google Scholar]
  10. Gillespie, P. & Gillespie, S. ( 1997; ). Improved electrophoresis and transfer picogram amounts of protein with hemoglobin. Analytical Biochemistry 246, 239-245.[CrossRef]
    [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 D. Bolognesi. New York:Alan M. Liss.
  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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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 J. M. Coffin, S. H. Hughes & H. E. Varmus. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  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.[CrossRef]
    [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.[CrossRef]
    [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...

Most Cited This Month

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