Stabilization from Autoproteolysis and Kinetic Characterization of the Human T-cell Leukemia Virus Type 1 Proteinase*

We have developed a system for expression and purification of wild-type human T-cell leukemia virus type 1 (HTLV-1) proteinase to attain sufficient quantities for structural, kinetic, and biophysical investigations. However, similar to the human immunodeficiency virus type 1 (HIV-1) proteinase, HTLV-1 proteinase also undergoes autoproteolysis rapidly upon renaturation to produce two products. The site of this autoproteolytic cleavage was mapped, and a resistant HTLV-1 proteinase construct (L40I) as well as another construct, wherein the two cysteine residues were exchanged to alanines, were expressed and purified. Oligopeptide substrates representing the naturally occurring cleavage sites in HTLV-1 were good substrates of the HTLV-1 proteinase. The kinetic parametersk cat and K m were nearly identical for all the three enzymes. Although three of four peptides representing HTLV-1 proteinase cleavage sites were fairly good substrates of HIV-1 proteinase, only two of nine peptides representing HIV-1 proteinase cleavage sites were hydrolyzed by the HTLV-1 proteinase, suggesting substantial differences in the specificity of the two enzymes. The large difference in the specificity of the two enzymes was also demonstrated by inhibition studies. Of the several inhibitors of HIV-1 or other retroviral proteinases that were tested on HTLV-1 proteinase, only two inhibit the enzyme with aK i lower than 100 nm.

[1]  A. Kakinuma,et al.  Purification and characterization of human T‐cell leukemia virus type I protease produced in Escherichia coli , 1991, FEBS letters.

[2]  W. Sheremata,et al.  High‐dose zidovudine induction in HTLV‐I‐associated myelopathy , 1993, Neurology.

[3]  S. Pettit,et al.  HIV type 1 protease inhibitors fail to inhibit HTLV-I Gag processing in infected cells. , 1998, AIDS research and human retroviruses.

[4]  D. Gotte,et al.  Moloney murine leukemia virus protease: bacterial expression and characterization of the purified enzyme. , 1993, Virology.

[5]  A. Tomasselli,et al.  Specificity of retroviral proteases: an analysis of viral and nonviral protein substrates. , 1994, Methods in enzymology.

[6]  Hoffbrand Bi.  Beta-adrenergic blockage in congestive cardiomyopathy. , 1980 .

[7]  R. Lal,et al.  Efficient Expression and Rapid Purification of Human T-Cell Leukemia Virus Type 1 Protease , 1998, Journal of Virology.

[8]  K. Hui,et al.  A rational approach in the search for potent inhibitors against HIV proteinase , 1991, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  A. Kakinuma,et al.  Expression of human T-cell leukemia virus type I protease in Escherichia coli. , 1991, Biochemical and biophysical research communications.

[10]  I B Duncan,et al.  Rational design of peptide-based HIV proteinase inhibitors. , 1990, Science.

[11]  D. Decamp,et al.  Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2. , 1990, The Journal of biological chemistry.

[12]  J. Louis,et al.  Structural basis for specificity of retroviral proteases. , 1998, Biochemistry.

[13]  R. Khabbaz,et al.  The Epidemiology of Human T‐lymphotropic Virus Types I and II , 1993 .

[14]  A. Wlodawer,et al.  [14]Subsite preferences of retroviral proteinases , 1994 .

[15]  I. Weber,et al.  Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates. , 1993, Biochemistry.

[16]  John D. Minna,et al.  Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma , 1980, Proceedings of the National Academy of Sciences.

[17]  J. Louis,et al.  A Transient Precursor of the HIV-1 Protease , 1996, The Journal of Biological Chemistry.

[18]  H. Kräusslich,et al.  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 , 1998, Journal of Virology.

[19]  J. Louis,et al.  Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Craik,et al.  Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions. , 1993, The Journal of biological chemistry.

[21]  Mitsuhiro Osame,et al.  HTLV-I ASSOCIATED MYELOPATHY, A NEW CLINICAL ENTITY , 1986, The Lancet.

[22]  L. Menéndez-Arias,et al.  Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli. , 1992, The Journal of biological chemistry.

[23]  T. Yamazaki,et al.  Three‐dimensional solution structure of the HIV‐1 protease complexed with DMP323, a novel cyclic urea‐type inhibitor, determined by nuclear magnetic resonance spectroscopy , 1996, Protein science : a publication of the Protein Society.

[24]  J. Louis,et al.  Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli. , 1991, European journal of biochemistry.

[25]  D. Baltimore,et al.  Standardized and simplified nomenclature for proteins common to all retroviruses , 1988, Journal of virology.

[26]  T. Copeland,et al.  Comparison of the HIV‐1 and HIV‐2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag‐Pol polyproteins , 1991, FEBS letters.

[27]  PatrickY.-S. Lam,et al.  Rational design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors. , 1994, Science.

[28]  K. Nagata,et al.  Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[29]  P. Štrop,et al.  Cloning, Bacterial Expression, and Characterization of the Mason-Pfizer Monkey Virus Proteinase (*) , 1995, The Journal of Biological Chemistry.

[30]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[31]  A. Wlodawer,et al.  Expression, characterisation and mutagenesis of the aspartic proteinase from equine infectious anaemia virus. , 1996, European journal of biochemistry.

[32]  D. Hatfield,et al.  Translational Suppression in Retroviral GENE Expression , 1992, Advances in Virus Research.

[33]  A. Olson,et al.  Molecular analysis of the feline immunodeficiency virus protease: generation of a novel form of the protease by autoproteolysis and construction of cleavage-resistant proteases , 1997, Journal of virology.

[34]  E. Fenyö,et al.  Analysis of cross reactivity of retrovirus proteases using a vaccinia virus-T7 RNA polymerase-based expression system. , 1995, The Journal of general virology.

[35]  D. Grobelny,et al.  Selective phosphinate transition-state analogue inhibitors of the protease of human immunodeficiency virus. , 1990, Biochemical and biophysical research communications.

[36]  J. Louis,et al.  Kinetic and modeling studies of S3-S3' subsites of HIV proteinases. , 1992, Biochemistry.

[37]  J. Louis,et al.  Influence of flanking sequences on the dimer stability of human immunodeficiency virus type 1 protease. , 1996, Biochemistry.

[38]  W. Howe,et al.  The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties. , 1994, Biochemistry.

[39]  G. Pavlakis,et al.  Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I , 1992, Journal of virology.

[40]  C. Bangham,et al.  Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding. , 1994, The Journal of general virology.

[41]  A. Dalgleish,et al.  HIV-1 proteinase is required for synthesis of pro-viral DNA. , 1991, Biochemical and biophysical research communications.

[42]  M. Dixon.  The determination of enzyme inhibitor constants. , 1953, The Biochemical journal.

[43]  Alexander Wlodawer,et al.  Structure of an inhibitor complex of the proteinase from feline immunodeficiency virus , 1995, Nature Structural Biology.

[44]  V. Kostka,et al.  Specificity studies on retroviral proteinase from myeloblastosis-associated virus. , 1991, Biochemistry.

[45]  C. Flexner.  HIV-protease inhibitors. , 1998, The New England journal of medicine.

[46]  A. Wlodawer,et al.  Structure-based inhibitors of HIV-1 protease. , 1993, Annual review of biochemistry.