Nguyen Laura A, Domaoal Robert A, Kennedy Edward M, Kim Dong-Hyun, Schinazi Raymond F, Kim Baek
Department of Pathology, University of Rochester Medical Center, Rochester, NY, USA.
Center for Drug Discovery, Emory Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
Antiviral Res. 2015 Mar;115:75-82. doi: 10.1016/j.antiviral.2014.12.016. Epub 2014 Dec 31.
Non-dividing macrophages maintain extremely low cellular deoxyribonucleotide triphosphate (dNTP) levels, but high ribonucleotide triphosphate (rNTP) concentrations. The disparate nucleotide pools kinetically forces Human Immunodeficiency Virus 1 (HIV-1) reverse transcriptase (RT) to incorporate non-canonical rNTPs during reverse transcription. HIV-1 RT pauses near ribonucleoside monophosphates (rNMPs) embedded in the template DNA, which has previously been shown to enhance mismatch extension. Here, pre-steady state kinetic analysis shows rNTP binding affinity (Kd) of HIV-1 RT for non-canonical rNTPs was 1.4- to 43-fold lower, and the rNTP rate of incorporation (kpol) was 15- to 1551-fold slower than for dNTPs. This suggests that RT is more selective for incorporation of dNTPs rather than rNTPs. HIV-1 RT selectivity for dNTP versus rNTP is the lowest for ATP, implying that HIV-1 RT preferentially incorporates ATP when dATP concentration is limited. We observed that incorporation of a dNTP occurring one nucleotide before an embedded rNMP in the template had a 29-fold greater Kd and a 20-fold slower kpol as compared to the same template containing dNMP. This reduced the overall dNTP incorporation efficiency of HIV-1 RT by 581-fold. Finally, the RT mutant Y115F displayed lower discrimination against rNTPs due to its increase in binding affinity for non-canonical rNTPs. Overall, these kinetic results demonstrate that HIV-1 RT utilizes both substrate binding and a conformational change during: (1) enzymatic discrimination of non-canonical rNTPs from dNTPs and (2) during dNTP primer extension with DNA templates containing embedded rNMP.
非分裂巨噬细胞维持极低的细胞脱氧核糖核苷三磷酸(dNTP)水平,但核糖核苷三磷酸(rNTP)浓度较高。不同的核苷酸库在动力学上迫使人类免疫缺陷病毒1型(HIV-1)逆转录酶(RT)在逆转录过程中掺入非规范的rNTP。HIV-1 RT在嵌入模板DNA中的单磷酸核糖核苷(rNMP)附近停顿,此前已证明这会增强错配延伸。在此,稳态前动力学分析表明,HIV-1 RT对非规范rNTP的rNTP结合亲和力(Kd)低1.4至43倍,rNTP掺入速率(kpol)比dNTP慢15至1551倍。这表明RT对dNTP掺入的选择性高于rNTP。HIV-1 RT对dNTP与rNTP的选择性在ATP方面最低,这意味着当dATP浓度有限时,HIV-1 RT优先掺入ATP。我们观察到,与含有dNMP的相同模板相比,在模板中嵌入的rNMP前一个核苷酸处掺入dNTP时,Kd大29倍,kpol慢20倍。这使HIV-1 RT的总体dNTP掺入效率降低了581倍。最后,RT突变体Y115F由于对非规范rNTP的结合亲和力增加,对rNTP的辨别能力较低。总体而言,这些动力学结果表明,HIV-1 RT在以下过程中利用底物结合和构象变化:(1)对非规范rNTP与dNTP进行酶促辨别;(2)在用含有嵌入rNMP的DNA模板进行dNTP引物延伸过程中。