3rd International Workshop on HIV Drug Resistance


2-5 August 1994, Kauai, Hawaii, USA


MECHANISMS OF ddC RESISTANCE OF THE K65R SUBSTITUTION IN HIV-1 REVERSE TRANSCRIPTASE

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:36 (abstract no. 35)

Z. Gu, E. Arts, X. Li, M. Parniak and M.A. Wainberg
McGill AIDS Centre:Jewish General Hospital, Montreal, Qc, Canada

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)l2-18, poly(rl)•(dC)l2-18, poly(rC)•(dG)l2-18 and heteropolymeric template/primer. A 2- to 3-fold increased Km value was observed for dCTP and ddATP in the case of RT compared to wt RT. No significant changes were seen in Kcat for dCTP, ddATP or for either Km or Kcat for dTTP and ddGTP between wt and mutant K65R RT. Inhibition assays showed that the Ki value of K65R was about 10-fold increased for ddCTP, ddATP and 3TCTP and about 5-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 and AZTTP 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.

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1994-08-02
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