The pyrophosphate analogue foscarnet traps the pre-translocational state of HIV-1 reverse transcriptase in a Brownian ratchet model of polymerase translocation.
ABSTRACT The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified "hot spots" for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre- and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. In these cases, the enzyme is seen at multiple positions around the 3' end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation.
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ABSTRACT: DNA polymerases need to be engineered to achieve optimal performance for biotechnological applications, which often require high fidelity replication when using modified nucleotides and when replicating difficult DNA sequences. These tasks are achieved for the bacteriophage T4 DNA polymerase by replacing leucine with methionine in the highly conserved Motif A sequence (L412M). The costs are minimal. Although base substitution errors increase moderately, accuracy is maintained for templates with mono- and dinucleotide repeats while replication efficiency is enhanced. The L412M substitution increases intrinsic processivity and addition of phage T4 clamp and single-stranded DNA binding proteins further enhance the ability of the phage T4 L412M-DNA polymerase to replicate all types of difficult DNA sequences. Increased pyrophosphorolysis is a drawback of increased processivity, but pyrophosphorolysis is curbed by adding an inorganic pyrophosphatase or divalent metal cations, Mn(2+) or Ca(2+). In the absence of pyrophosphorolysis inhibitors, the T4 L412M-DNA polymerase catalyzed sequence-dependent pyrophosphorolysis under DNA sequencing conditions. The sequence specificity of the pyrophosphorolysis reaction provides insights into how the T4 DNA polymerase switches between nucleotide incorporation, pyrophosphorolysis and proofreading pathways. The L-to-M substitution was also tested in the yeast DNA polymerases delta and alpha. Because the mutant DNA polymerases displayed similar characteristics, we propose that amino acid substitutions in Motif A have the potential to increase processivity and to enhance performance in biotechnological applications. An underlying theme in this chapter is the use of genetic methods to identify mutant DNA polymerases with potential for use in current and future biotechnological applications.Frontiers in microbiology. 01/2014; 5:380.
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ABSTRACT: The introduction of potent combination therapies in the mid-90s had a tremendous effect on AIDS mortality. However, drug resistance has been a major factor contributing to antiretroviral therapy failure. Currently, there are 26 drugs approved for treating human immunodeficiency virus (HIV) infections, although some of them are no longer prescribed. Most of the available antiretroviral drugs target HIV genome replication (i.e. reverse transcriptase inhibitors) and viral maturation (i.e. viral protease inhibitors). Other drugs in clinical use include a viral coreceptor antagonist (maraviroc), a fusion inhibitor (enfuvirtide) and two viral integrase inhibitors (raltegravir and elvitegravir). Elvitegravir and the nonnucleoside reverse transcriptase inhibitor rilpivirine have been the most recent additions to the antiretroviral drug armamentarium. An overview of the molecular mechanisms involved in antiretroviral drug resistance and the role of drug resistance-associated mutations was previously presented (Menéndez-Arias, L., 2010. Molecular basis of human immunodeficiency virus drug resistance: an update. Antiviral Res. 85, 210-231). This article provides now an updated review that covers currently approved drugs, new experimental agents (e.g. neutralizing antibodies) and selected drugs in preclinical or early clinical development (e.g. experimental integrase inhibitors). Special attention is dedicated to recent research on resistance to reverse transcriptase and integrase inhibitors. In addition, recently discovered interactions between HIV and host proteins and novel strategies to block HIV assembly or viral entry emerge as promising alternatives for the development of effective antiretroviral treatments.Antiviral research 02/2013; · 3.61 Impact Factor
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ABSTRACT: Nucleotide-competing reverse transcriptase inhibitors (NcRTIs) were shown to bind reversibly to the nucleotide-binding site of the RT enzyme of human immunodeficiency virus type 1 (HIV-1). Here we show that the presence of ATP can enhance the inhibitory effects of the prototype compound INDOPY-1. We employed a combination of cell-free and cell-based assays to shed light on the underlying molecular mechanism. Binding studies and site-specific footprinting experiments demonstrate the existence of a stable quaternary complex with HIV-1 RT, its nucleic acid substrate, INDOPY-1, and ATP. The complex is frozen in the post-translocational state that usually accommodates the incoming nucleotide substrate. Structure-activity relationship (SAR) studies show that both the base and the phosphate moieties of ATP are elements that play important roles in enhancing the inhibitory effects of INDOPY-1. In vitro susceptibility measurements with mutant viruses containing amino acid substitutions K70G, V75T, L228R and K219R in the putative ATP binding pocket revealed unexpectedly a hypersusceptible phenotype with respect to INDOPY-1. The same mutational cluster was previously shown to reduce susceptibility to the pyrophosphate analogue phosphonoformic acid (PFA). However, in the absence of INDOPY-1, ATP can bind and act as a pyrophosphate donor under conditions that favor formation of the pre-translocated RT complex. We therefore conclude that the mutant enzyme facilitates simultaneous binding of INDOPY-1 and ATP to the post-translocated complex. Based on this data, we propose a model in which the bound ATP traps the inhibitor, which, in turn, compromises its dissociation.Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor