Human immunodeficiency virus type 1 protease inhibitors irreversibly block infectivity of purified virions from chronically infected cells

Synthetic peptide analog inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were used to study the effects of inhibition of polyprotein processing on the assembly, structure, and infectivity of virions released from a T-cell line chronically infected with HIV-1. Inhibition of proteolytic processing of both Pr55gag and Pr160gag-pol was observed in purified virions from infected T cells after treatment. Protease inhibition was evident by the accumulation of precursors and processing intermediates of Pr55gag and by corresponding decreases in mature protein products. Electron microscopy revealed that the majority of the virion particles released from inhibitor-treated cells after a 24-h treatment had an immature or aberrant capsid morphology. This morphological change correlated with the inhibition of polyprotein processing and a loss of infectivity. The infectivity of virion particles purified from these chronically infected cell cultures was assessed following treatment with the inhibitor for 1 to 3 days. Virions purified from cultures treated with inhibitor for 1 or 2 days demonstrated a 95- to 100-fold reduction in virus titers, and treatment for 3 days resulted in complete loss of detectable infectivity. The fact that virions from treated cultures were unable to establish infection over the 7- to 10-day incubation period in the titration experiments strongly suggests that particles produced by inhibitor-treated cells were unable to reactivate to an infectious form when they were purified away from exogenous protease inhibitor. Thus, a block of HIV-1 protease processing of viral polyproteins by specific inhibitors results in a potent antiviral effect characterized by the production of noninfectious virions with altered protein structures and immature morphologies.

[1]  R. Redfield,et al.  Genomic diversity of human T-lymphotropic virus type III (HTLV-III). , 1985, Science.

[2]  S. Devare,et al.  Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24 , 1985, Journal of virology.

[3]  J. Sodroski,et al.  Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[4]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. Copeland,et al.  Immunological and chemical analysis of P6, the carboxyl-terminal fragment of HIV P15. , 1987, AIDS research and human retroviruses.

[6]  B. Moss,et al.  An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[7]  L J Davis,et al.  Active human immunodeficiency virus protease is required for viral infectivity. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Hershfield,et al.  Detection of HIV-1 neutralizing antibodies by a simple, rapid, colorimetric assay. , 1988, AIDS research and human retroviruses.

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

[10]  Brian W. Metcalf,et al.  Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues , 1990, Nature.

[11]  D. Baltimore,et al.  Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase , 1981, Journal of virology.

[12]  F. Arenzana‐Seisdedos,et al.  HIV enhancer activity perpetuated by NF-κB induction on infection of monocytes , 1991, Nature.

[13]  C. Debouck,et al.  Inhibition of human immunodeficiency virus 1 protease in vitro: rational design of substrate analogue inhibitors. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[14]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[15]  H. Ellens,et al.  Morphometric analysis of recombinant soluble CD4-mediated release of the envelope glycoprotein gp120 from HIV-1. , 1990, AIDS research and human retroviruses.

[16]  A. Fauci,et al.  Interferon-alpha but not AZT suppresses HIV expression in chronically infected cell lines. , 1989, Science.

[17]  D. Norbeck,et al.  Design, activity, and 2.8 A crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease. , 1990, Science.

[18]  B. Ho,et al.  Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity , 1989, Journal of virology.

[19]  E. Lillehoj,et al.  The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors , 1988, Journal of virology.

[20]  L. Reed,et al.  A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS , 1938 .

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

[22]  T. Copeland,et al.  Biochemical and immunological analysis of human immunodeficiency virus gag gene products p17 and p24 , 1988, Journal of virology.

[23]  Tomi K. Sawyer,et al.  A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation. , 1990, Disease markers.

[24]  Arindam Banerjee,et al.  Submitted for publication , 1981 .

[25]  K. Steimer,et al.  Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). , 1985, Science.

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

[27]  S. Cha,et al.  Tight-binding inhibitors-I. Kinetic behavior. , 1975, Biochemical pharmacology.

[28]  G. Nabel Tampering with transcription , 1991, Nature.

[29]  P. Earl,et al.  In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity , 1988, Journal of virology.

[30]  M. Gonda,et al.  Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus. , 1985, Science.

[31]  A. Israël,et al.  Processing of the precursor of NF-κB by the HIV-1 protease during acute infection , 1991, Nature.

[32]  H. Lyerly,et al.  Interaction between the human T-cell lymphotropic virus type IIIB envelope glycoprotein gp120 and the surface antigen CD4: role of carbohydrate in binding and cell fusion. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[33]  V. Potter,et al.  Enzyme Inhibition in Relation to Chemotherapy.∗ , 1949, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[34]  Mark L. Pearson,et al.  Complete nucleotide sequence of the AIDS virus, HTLV-III , 1985, Nature.

[35]  C. Debouck,et al.  Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[36]  G. Moore,et al.  Rosette-forming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes. , 1972, Journal of the National Cancer Institute.