Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

[Synthesis, Structure and Some Biochemical-properties of 3'-branched Thymidines and Their 5'-phosphate Derivatives]

A full scheme of synthesizing 3'-C-methyl-2'-deoxynucleosides and 3'-C-methylidene-28,3'-dideoxythymidine has been developed by using 2-deoxyribose. The stereoselectivity of the Grignard reagent's attachment to 2-deoxyfuranose 3-ulosides determined by the substitute configuration at Cl and the condensation stereoselectivity of 3-C-methyl-2-deoxyfuranosides with silylated thymine dependent on the configuration of the hydroxyl or-OBz group at C3 have been studied. The structure of the resultant compounds has been evidenced by H-1 and C-13 NMR, UV spectroscopies and C, H, and N analysis. The C2'-endo-C1-exo-conformation, the anti-conformation of the thymine base in relation to the glycoside bond and the gosh+-conformation in relation to the C4'-C5' bond are characteristic of the structure of 3'-C-methyl-2'-deoxythymidine in the crystal. 3'-C-Metyl-2'-deoxythymidine-5'-triphosphate exhibited the properties of the competitive inhibitor against 2'-deoxythimidine 5'-triphosphate in the synthesis of DNA catalyzed by various DNA-polymerases and reverse transcriptases. But none of these enzymes incorporated this compound into the growing DNA chain. At the same time 3'-C-methylidene-2',3'-dideoxythymidine-5'-triphosphate was incorporated into the 3'-end of the chain of DNA catalyzed by HIV reverse transcriptase, though the latter having a low efficacy. 3'-C-Methyl-2'-deoxythymidine failed to suppress HIV-1 production in the cultured MT-4 cells, its 5'-phosphite exhibiting a low activity under the same conditions