       Document 0383
 DOCN  M9620383
 TI    An active-site mutation in the human immunodeficiency virus type 1
       proteinase (PR) causes reduced PR activity and loss of PR-mediated
       cytotoxicity without apparent effect on virus maturation and
       infectivity.
 DT    9602
 AU    Konvalinka J; Litterst MA; Welker R; Kottler H; Rippmann F; Heuser AM;
       Krausslich HG; Angewandte Tumorvirologie, Deutsches
       Krebsforschungszentrum,; Heidelberg, Germany.
 SO    J Virol. 1995 Nov;69(11):7180-6. Unique Identifier : AIDSLINE
       MED/96013822
 AB    Infectious retrovirus particles are derived from structural polyproteins
       which are cleaved by the viral proteinase (PR) during virion
       morphogenesis. Besides cleaving viral polyproteins, which is essential
       for infectivity, PR of human immunodeficiency virus (HIV) also cleaves
       cellular proteins and PR expression causes a pronounced cytotoxic
       effect. Retroviral PRs are aspartic proteases and contain two copies of
       the triplet Asp-Thr-Gly in the active center with the threonine adjacent
       to the catalytic aspartic acid presumed to have an important structural
       role. We have changed this threonine in HIV type 1 PR to a serine. The
       purified mutant enzyme had an approximately 5- to 10-fold lower activity
       against HIV type 1 polyprotein and peptide substrates compared with the
       wild-type enzyme. It did not induce toxicity on bacterial expression and
       yielded significantly reduced cleavage of cytoskeletal proteins in
       vitro. Cleavage of vimentin in mutant-infected T-cell lines was also
       markedly reduced. Mutant virus did, however, elicit productive infection
       of several T-cell lines and of primary human lymphocytes with no
       significant difference in polyprotein cleavage and with similar
       infection kinetics and titer compared with wild-type virus. The
       discrepancy between reduced processing in vitro and normal virion
       maturation can be explained by the observation that reduced activity was
       due to an increase in Km which may not be relevant at the high substrate
       concentration in the virus particle. This mutation enables us therefore
       to dissociate the essential function of PR in viral maturation from its
       cytotoxic effect.
 DE    Amino Acid Sequence  Animal  Binding Sites  Blotting, Western  Cell Line
       *Cell Survival  Cercopithecus aethiops  Cytoskeletal Proteins/ISOLATION
       & PURIF/*METABOLISM  Genes, pol  Human  HIV
       Protease/BIOSYNTHESIS/*METABOLISM
       HIV-1/ENZYMOLOGY/*PHYSIOLOGY/*PATHOGENICITY  Kinetics  Molecular
       Sequence Data  Mutagenesis, Site-Directed  Peptide
       Fragments/CHEMISTRY/ISOLATION & PURIF  Recombinant
       Proteins/BIOSYNTHESIS/METABOLISM  Substrate Specificity  Support,
       Non-U.S. Gov't  Transfection  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

