David G. Nickens

HIV-1 inactivation by nucleic acid aptamers.

Although developments in small-molecule therapeutics for HIV-1 have been dramatic in recent years, the rapid selection of drug-resistant viral strains and the adverse side effects associated with long-term exposure to current treatments propel continued exploration of alternative anti-HIV-1 agents. Non-coding nucleic acids have emerged as potent inhibitors that dramatically suppress viral function both in vitro and in cell culture. In particular, RNA and DNA aptamers inhibit HIV-1 function by directly interfering with essential proteins at critical stages in the viral replication cycle (Figure 1). Their antiviral efficacy is expected to be a function, in part, of the biochemical properties of the aptamer-target interaction. Accordingly, we present an overview of biochemical and cell culture analyses of the expanding list of aptamers targeting HIV-1. Our discussion focuses on the inhibition of viral enzymes (reverse transcription, proteolytic processing, and chromosomal integration), viral expression (Rev/RRE and Tat/TAR), viral packaging (p55Gag, matrix and nucleocapsid), and viral entry (gp120) (Table 1). Additional nucleic acid-based strategies for inactivation of HIV-1 function (including RNAi, antisense, and ribozymes) have also demonstrated their utility. These approaches are reviewed in other chapters of this volume and elsewhere.

Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice

Significance Hepatitis B virus (HBV) is a double-stranded DNA virus that packages a single-stranded RNA pregenome (pgRNA). The linear pgRNA is reverse transcribed to a gapped circular dsDNA within the confines of the virus capsid. We hypothesized that a specific capsid-RNA-reverse transcriptase structure would be required to accomplish this task. In this article, we report the structure of the authentic pgRNA-filled HBV core as determined by cryo-EM and asymmetric 3D reconstruction. The observed ordered structure suggests the assembly process and the first steps of reverse transcription follow a single, determinate pathway. Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well.

Biotype of the purple nonsulfur photosynthetic bacterium, Rhodospirillum centenum

Rhodospirillum centenum exhibited a number of general properties typically observed in nonsulfur purple bacteria, but also displayed a number of unusual characteristics that include: (1) conversion of the vibrioid/spiral cells to thick-walled cysts under certain growth conditions; (2) absence of O2 repression of photopigment synthesis; (3) synthesis of “R-bodies”; and (4) swarming motility on agar surfaces that allows macroscopic observation of colony phototaxis. The unusual characteristics indicate that Rsp.centenum will prove to be a valuable experimental system for investigating various basic problems, especially in connection with photosensory phenomena and the regulation of photopigment synthesis by dioxygen and light. The present comparative study of 13 strains was undertaken to further define the Rsp. centenum biotype.

Convergent donor and acceptor substrate utilization among kinase ribozymes

Accommodation of donor and acceptor substrates is critical to the catalysis of (thio)phosphoryl group transfer, but there has been no systematic study of donor nucleotide recognition by kinase ribozymes, and there is relatively little known about the structural requirements for phosphorylating internal 2′OH. To address these questions, new self-phosphorylating ribozymes were selected that utilize ATP(gammaS) or GTP(gammaS) for 2′OH (thio)phosphorylation. Eight independent sequence families were identified among 57 sequenced isolates. Kinetics, donor nucleotide recognition and secondary structures were analyzed for representatives from each family. Each ribozyme was highly specific for its cognate donor. Competition assays with nucleotide analogs showed a remarkable convergence of donor recognition requirements, with critical contributions to recognition provided by the Watson–Crick face of the nucleobase, lesser contributions from donor nucleotide ribose hydroxyls, and little or no contribution from the Hoogsteen face. Importantly, most ribozymes showed evidence of significant interaction with one or more donor phosphates, suggesting that—unlike most aptamers—these ribozymes use phosphate interactions to orient the gamma phosphate within the active site for in-line displacement. All but one of the mapped (thio)phosphorylation sites are on unpaired guanosines within internal bulges. Comparative structural analysis identified three loosely-defined consensus structural motifs for kinase ribozyme active sites.

Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

Background In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5′ or 2′ OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. Methodology/Principal Findings This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5′ phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A2>A1>A3>A4>A0>A6>A8>A10 and T2>T3>T4>T6≈T1>T8>T10. Bridging Ts generally gave higher yield of ligated product than bridging As. ATP concentrations above 33 µM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or T∶G wobble-paired, substrate on the 3′ side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The “universal” analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide. Conclusions/Significance Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.

A Mutation That Affects Isoprenoid Biosynthesis Results in Altered Expression of Photosynthesis Genes and Synthesis of the Photosynthetic Apparatus in Rhodobacter Capsulatus

Observations of the repressing effects of high light intensity on synthesis of the purple bacterial photosystem were first reported in 1957 by Cohen-Bazire et al. [1]. Their study demonstrated that shifting a growing culture from low to high light intensity resulted in an abrupt decrease in synthesis of the bacterial photosystem. Subsequent physiological studies demonstrated that photosynthetic bacteria respond to alterations in light intensity by adjusting the amounts of reaction centers, light harvesting-I, and light harvest-ing-II complexes located in an intracytoplasmic membrane2-4. Studies of the regulation of photopigment synthesis have supported earlier observations concerning light and oxygen control of biosynthesis of reaction centers, light harvesting-I, and light harvesting-II complexes5-7.

An organoleptic survey of meads made with lactic acid-producing yeasts

We previously reported the isolation a suite of wild lactic acid-producing yeasts (LAYs) that enable “primary souring” during beer fermentation without the use of lactic acid bacteria. With sour meads gaining popularity in modern mead making, we were interested in exploring the same primary souring approach to traditional semi-sweet meads. In this study, we utilized 13 LAY strains to produce semi-sweet meads using a standardized batch of honey must to ensure consistent starting conditions. Thirteen 11-L batches of mead were prepared, and each was inoculated with one of the LAY strains, along with two control batches inoculated with champagne yeast. The initial pH and specific gravity were measured for each batch before inoculation. Traditional organic staggered nutrient addition was utilized for the first 72 h of fermentation with specific gravities being taken throughout the mead making process. Meads were racked, tasted, stabilized, cold crashed, bottled, and transported to the American Mead Maker’s Association 2018 Conference in Broomfield, Colorado. There, organoleptic surveys were conducted on these meads utilizing an array of tasters with varying levels of mead sensory analysis experience. The results of the sensory analysis, focusing on aroma and flavor, are discussed.

The S. cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro

Telomere length homeostasis is vital to maintaining genomic stability and is regulated by multiple factors, including DNA helicases. The Saccharomyces cerevisiae Pif1 helicase was the first discovered catalytic inhibitor of telomerase, but recent experimental evidence suggests that Hrq1, the yeast homolog of the disease-linked human RECQL4 helicase, plays a similar role via an undefined mechanism. Using yeast extracts enriched for telomerase activity and an in vitro primer extension assay, we determined the effects of recombinant wild-type and inactive Hrq1 and Pif1 on total telomerase activity and telomerase processivity. We found that titrations of these helicases alone have equal-but-opposite biphasic effects on telomerase, with Hrq1 stimulating activity at high concentrations. When the helicases were combined in reactions, however, they synergistically inhibited or stimulated telomerase activity dependent on which helicase was catalytically active. These results suggest that Hrq1 and Pif1 interact and that their concerted activities ensure proper telomere length homeostasis in vivo.