Document 1141
 DOCN  M94A1141
 TI    Mechanism of ddC resistance of the K65R substitution in HIV-1 reverse
       transcriptase.
 DT    9412
 AU    Gu Z; Arts E; Li X; Parniak M; Wainberg MA; McGill AIDS Centre-Jewish
       General Hospital, Montreal, Qc, Canada.
 SO    Int Conf AIDS. 1994 Aug 7-12;10(2):20 (abstract no. 374A). Unique
       Identifier : AIDSLINE ICA10/94371434
 AB    Substitution mutations K65R and M184V in the HIV-1 RT coding region have
       been shown to be responsible for HIV-1 resistance to each of ddC, ddI
       and 3TC. We introduced these two mutations into both p66 and p51
       subunits of the HXB2 HIV RT gene by site-directed mutagenesis and used
       an E. coli expression system to generate recombinant p66/p51 heterodimer
       RT proteins that were purified to > 95% by FPLC. Steady-state kinetic
       parameters for each of Km and Kcat were determined for wild-type (wt)
       and mutant HIV-1 RTs under both processive and non-processive conditions
       using the template/primer poly(rA).(dT) 12-18 and poly(rI).(dC) 12-18. A
       2.2-fold increased Km value was observed for dCTP in the case of RT
       compared to wt RT. No significant changes were seen in Kcat for dCTP, or
       for either Km or Kcat for dTTP, between wt and mutant K65R RT.
       Inhibition assays showed that the Ki value of K65R was 8.5-fold
       increased for ddCTP and 3.3-fold increased for ddTTP, in comparison to
       wt enzyme. However, ddCTP did not exert competitive inhibition effects
       on poly(rA).(dt) template/primer and dTTP substrates. We also assayed
       for incorporation of and chain termination by ddCTP, 3TC-TP, ddATP and
       AZT-TP during the synthesis of (-) strong-stop DNA using in vitro
       assays. Recombinant HIV RTs containing only K65R or both the K65R and
       M184V mutations yielded significantly more (-_ strong-stop product in
       the presence of ddCTP, 3TC-TP and ddATP than did wt HIV-1 RT. A slight
       decrease in degree of chain termination was observed with each of AZT-TP
       and ddITP. Altered nucleoside-analog recognition and chain termination
       are likely involved in drug resistance mechanisms for K65R.
 DE    *Codon  Comparative Study  Cytosine/ANALOGS & DERIVATIVES/PHARMACOLOGY
       Didanosine/PHARMACOLOGY  Drug Resistance, Microbial/GENETICS
       HIV-1/*DRUG EFFECTS/ENZYMOLOGY/GENETICS  Kinetics  Mutagenesis,
       Site-Directed  *Point Mutation  Recombinant Fusion Proteins/ANTAGONISTS
       & INHIB  Reverse Transcriptase/ANTAGONISTS & INHIB/*GENETICS
       Zalcitabine/*PHARMACOLOGY  Zidovudine/PHARMACOLOGY  MEETING ABSTRACT

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