Second International Congress Drug Therapy in HIV Infection 18-22 November 1994 Glasgow, UK

MECHANISTIC STUDIES OF NUCLEOSIDE RESISTANCE INVOLVING RECOMBINANT PURIFIED HIV REVERSE TRANSCRIPTASE

Mark A. Wainberq, Zhengxian Gu, Eric Arts, Xuguang Li and Michael Parniak
McGill AIDS Centre-Jewish General Hospital, 3755 Cote-Ste-Catherine Road, Hontreal, Qc, Canada

Int Cong Drug Therapy HIV 1994 Nov 18-22;2:Abstract No. 5.4
AIDS 1994, Vol. 8 (Suppl. 4);S4

We have previously described substitution mutations Ml84V and K65R in the HIV-1 RT coding region and shown that these are responsible for varying degrees of resistance to each of 3TC, ddC' and ddI. We have used site-directed mutagenesis is to introduce these two mutations into both the p51 and p66 subunits of the HXB2 HIV RT gene. We then used an E.coli expression system to generate recombinant p66/p51 heterodimer RT proteins that were purified by FPLC to >95%. 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(rA). (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. Most importantly, we have employed endogenous RT assays to measure incorporation of and chain termination by each of ddCTP, 3TC-TP, ddATP and AZT-TP during the synthesis of (-) strong-stop DNA and subsequent products of reverse transcription. This method involves the use of genomic RNA as template and tRNAL^LYS3 as a primer of RT activity. We found that recombinant HIV RTs containing only K65R or both the K65R and M184V mutations yielded significantly more (-) strong-stop product in the presence of ddCTP, 3TCTP and ddATP than did wild-type HIV-1 RT.

1994-11-18
5.4

Originally published in AIDS Volume 8, Supplement 4 and hosted with permission of the publisher Lippincott Williams & Wilkins, 250 Waterloo Road, London, SE1 8RD, UK. Tel: +44 (0)20 7981 0700 Fax: +44 (0) 7981 0701

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