title: Pre-steady state kinetic analysis of HIV-1 reverse transcriptase for non-canonical ribonucleoside triphosphate incorporation and DNA synthesis from ribonucleoside-containing DNA template.
abstract: 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.
authors.affiliation: Department of Pathology, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Nguyen
authors.affiliation: Center for Drug Discovery, Emory Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Domaoal
authors.affiliation: Department of Pathology, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Kennedy
authors.affiliation: College of Pharmacy, Kyung-Hee University, Seoul, South Korea.
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Center for Drug Discovery, Emory Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA; Veterans Affairs Medical Center, Decatur, GA, USA.
authors.forename: Raymond F
authors.initials: RF
authors.lastname: Schinazi
authors.affiliation: Center for Drug Discovery, Emory Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA. Electronic address: baek.kim@emory.edu.
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
mesh_terms: D004261:DNA Replication / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis*; D003854:Deoxyribonucleotides / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D012263:Ribonucleosides / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D004279:DNA, Viral; D003854:Deoxyribonucleotides; D012263:Ribonucleosides; D012265:Ribonucleotides; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
keywords: HIV-1; Kinetics; Macrophages; Reverse transcriptase; dNTPs; rNTPs