utilization of a deoxynucleoside diphosphate substrate by hiv reverse transcriptase利用二磷酸deoxynucleoside衬底的艾滋病毒逆转录酶.pdfVIP

Utilization of a Deoxynucleoside Diphosphate Substrate by HIV Reverse Transcriptase 1 2 1 Scott J. Garforth , Michael A. Parniak , Vinayaka R. Prasad * 1 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America Abstract Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA synthesis 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 synthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics for treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the c-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 dNDPs 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 K65 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 39-terminal nucleoside inhibitors such as AZT forms the basis for HIV-1