() DNA-dependent DNA polymerase activity of Ty3 DNA polymerase active site mutants Asp213→Glu, Asp214→Asn, Asp214→Glu and Asp214→Gln

<p><b>Copyright information:</b></p><p>Taken from "Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase"</p><p>Nucleic Acids Research 2005;33(1):171-181.</p><p>Published online 12 Jan 2005</p><p>PMCID:PMC546138.</p><p>© 2005, the authors © </p> Enzyme:substrate ratios and lane notations are as indicated in the legend to . ( and ) DNA-dependent DNA polymerase activity evaluated in the absence (B) and presence of heparin (C), respectively. Lane notations are as in the legend to

Mn-supported RNA-dependent DNA polymerase activity of wild-type (lane w) and mutant Ty3 derivatives (lanes 1–7)

<p><b>Copyright information:</b></p><p>Taken from "Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase"</p><p>Nucleic Acids Research 2005;33(1):171-181.</p><p>Published online 12 Jan 2005</p><p>PMCID:PMC546138.</p><p>© 2005, the authors © </p> Lane c, no enzyme. Enzymes were analyzed in the absence () and presence of heparin (). Substrate and lane notations are according to the legend of

Designed and SELEX selected duplexes tested for binding to RTs.

<p>Shown is the sequence of the duplex constructs tested for binding to the various RTs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#pone-0041712-t001" target="_blank">Table 1</a>). Each template strand was 45 nts in length while primer strands were 41 nts. All constructs had a four nt 5′ overhang. Capital letters are sequences that were derived from the PPT containing region of the genome for the particular designed duplex (other than the Random duplex) or those that were selected from the randomized region for the SELEX-derived duplexes. The sequences with small lettering are identical in all the constructs and were derived from the fixed primer region in the primer-template SELEX protocol.</p

Sequences recovered from SELEX experiments.

<p>The nucleotide sequence of material recovered with the indicated enzyme in the indicated round(s) of the primer-template SELEX protocol are shown. Only the sequence in the 25 nt randomized region of the primer strand is shown. The sequence of a DNA version of the PPT for each RT is shown at the top of each set of recovered sequences for reference. During selection, the primer strand was only 21 nts long, the last four nts at the 3′ end of each sequence are underlined since they were not present on the primer during selection. However, complementary nts were present on the template strand. Specific sequences recovered with MuLV and AMV and used to prepare constructs for K_d determinations are designated with names corresponding to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#pone-0041712-g001" target="_blank">Fig. 1</a>. Sequences recovered more than once are indicated by an “X” (times).</p

Example of assays and plots used to determine K_d values for the duplex shown in <b>Fig. 1</b>.

<p>(A) An autoradiogram of an experiment performed with HIV (top panel) or AMV (bottom panel) RT on their cognate designed DNA PPT constructs (HIV DNA PPT, and AMV DNA PPT duplex, respectively) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#pone-0041712-g001" target="_blank">Fig. 1</a>). Positions for the unextended and extend primer are indicated as is the amount of enzyme used in each assay. –E, no enzyme control; TC, trap control to test for trap efficiency in which the enzyme and trap were mixed prior to addition to the reactions; FE, full extension control contained the highest amount of enzyme used in the assay incubated with the substrate in the absence of trap for 10 min. Refer to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#s4" target="_blank">Methods</a> section for details. (B) A graph of extended primer <i>vs.</i> [HIV RT] for four different designed duplexes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#pone-0041712-g001" target="_blank">Fig. 1</a>). The line shown for each duplex was made by fitting the data points to the binding equation described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#s4" target="_blank">Methods</a> section. This line was used to determine the enzyme's K_d for the particular construct. The data shown is from a single experiment with each construct. Experiments were repeated 2–4 times and data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#pone-0041712-t001" target="_blank">Table 1</a> is an average of those experiments ± standard deviations.</p

K_d values for RTs on various duplex constructs.

1<p>Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#pone-0041712-g001" target="_blank">Fig. 1</a> for duplex sequences.</p>2<p>Experiments for determining K_d values are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#pone-0041712-g002" target="_blank">Fig. 2</a>. All values are an average of 2–4 independent experiments ± standard deviations. Values are in nM and were calculated based on protein mass as provided by the manufacturer (HIV, MuLV, and AMV RTs) or determined as described (Ty3) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041712#pone.0041712-Rausch2" target="_blank">[31]</a>. Active site concentrations were not determined.</p

Development of Small Molecules with a Noncanonical Binding Mode to HIV‑1 Trans Activation Response (TAR) RNA

Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has long been an important model system for the chemistry of targeting RNA. Here we report the synthesis, biochemical, and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue, <b>15</b>, retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC₅₀ of 40 μM. NMR characterization of a soluble analogue, <b>2</b>, revealed a noncanonical binding mode for this class of compounds. Finally, evaluation of <b>2</b> and <b>15</b> by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) indicates specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5′-untranslated region (UTR). Thus, these compounds exhibit a novel and specific mode of interaction with TAR, providing important suggestions for RNA ligand design

Exploiting Drug-Resistant Enzymes as Tools To Identify Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)­thieno­[2,3-<i>d</i>]­pyrimidin-4­(3<i>H</i>)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3′,4′-dihydroxyphenyl (catechol) substituted thienopyrimidinones with submicromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones <i>destabilize</i> the enzyme, in some instances reducing the <i>T</i>_m by 5 °C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthen the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy

Sesquiterpenoid Tropolone Glycosides from <i>Liriosma ovata</i>

Two new sesquiterpenoid tropolone glycosides, liriosmasides A (<b>1</b>) and B (<b>2</b>), along with two known compounds, secoxyloganin and oplopanpheside C, were isolated from a methanol extract of the roots of <i>Liriosma ovata</i>. The structures of <b>1</b> and <b>2</b> were elucidated by spectroscopic methods including 1D and 2D NMR and by high-resolution mass spectrometry involving an ultra-high-performance liquid chromatography–quadrupole-orbital ion trap mass spectrometric (UHPLC–Q-Orbitrap MS) method. Compound <b>1</b> showed weak inhibitory activity against HIV RNase H

SiRNA-Induced Mutation in HIV-1 Polypurine Tract Region and Its Influence on Viral Fitness

<div><p>Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3′ and central polypurine tracts (3′ and cPPTs). The 5′ and 3′ termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3′PPT sequence. Introducing either or both mutations into the 3′PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3′PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3′PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3′PPT is capable of replication, this nucleotide substitution shifts the 3′-terminal cleavage site in the 3′PPT by one nucleotide (nt) and significantly reduces viral fitness.</p></div