A Branched SELEX Approach Identifies RNA Aptamers That Bind Distinct HIV-1 Capsid Structural Components.

The HIV-1 capsid protein (CA) assumes distinct structural forms during replication, each presenting unique, solvent-accessible surfaces that facilitate multifaceted functions and host factor interactions. However, functional contributions of individual CA structures remain unclear, as evaluation of CA presents several technical challenges. To address this knowledge gap, we identified CA-targeting aptamers with different structural specificities, which emerged through a branched SELEX approach using an aptamer library previously selected to bind the CA hexamer lattice. Subsets were either highly specific for the CA lattice or bound both the CA lattice and CA hexamer. We then evaluated four representatives to reveal aptamer regions required for binding, highlighting interesting structural features and challenges in aptamer structure determination. Further, we demonstrate binding to biologically relevant CA structural forms and aptamer-mediated affinity purification of CA from cell lysates without virus or host modification, supporting the development of structural form-specific aptamers as exciting new tools for the study of CA.

Retroviral PBS-segment sequence and structure: Orchestrating early and late replication events.

An essential regulatory hub for retroviral replication events, the 5' untranslated region (UTR) encodes an ensemble of cis-acting replication elements that overlap in a logical manner to carry out divergent RNA activities in cells and in virions. The primer binding site (PBS) and primer activation sequence initiate the reverse transcription process in virions, yet overlap with structural elements that regulate expression of the complex viral proteome. PBS-segment also encompasses the attachment site for Integrase to cut and paste the 3' long terminal repeat into the host chromosome to form the provirus and purine residues necessary to execute the precise stoichiometry of genome-length transcripts and spliced viral RNAs. Recent genetic mapping, cofactor affinity experiments, NMR and SAXS have elucidated that the HIV-1 PBS-segment folds into a three-way junction structure. The three-way junction structure is recognized by the host's nuclear RNA helicase A/DHX9 (RHA). RHA tethers host trimethyl guanosine synthase 1 to the Rev/Rev responsive element (RRE)-containing RNAs for m7-guanosine Cap hyper methylation that bolsters virion infectivity significantly. The HIV-1 trimethylated (TMG) Cap licenses specialized translation of virion proteins under conditions that repress translation of the regulatory proteins. Clearly host-adaption and RNA shapeshifting comprise the fundamental basis for PBS-segment orchestrating both reverse transcription of virion RNA and the nuclear modification of m7G-Cap for biphasic translation of the complex viral proteome. These recent observations, which have exposed even greater complexity of retroviral RNA biology than previously established, are the impetus for this article. Basic research to fully comprehend the marriage of PBS-segment structures and host RNA binding proteins that carry out retroviral early and late replication events is likely to expose an immutable virus-specific therapeutic target to attenuate retrovirus proliferation.

Kinetic pathway of HIV-1 TAR cotranscriptional folding.

The Trans-Activator Receptor (TAR) RNA, located at the 5'-end untranslated region (5' UTR) of the human immunodeficiency virus type 1 (HIV-1), is pivotal in the virus's life cycle. As the initial functional domain, it folds during the transcription of viral mRNA. Although TAR's role in recruiting the Tat protein for trans-activation is established, the detailed kinetic mechanisms at play during early transcription, especially at points of temporary transcriptional pausing, remain elusive. Moreover, the precise physical processes of transcriptional pause and subsequent escape are not fully elucidated. This study focuses on the folding kinetics of TAR and the biological implications by integrating computer simulations of RNA folding during transcription with nuclear magnetic resonance (NMR) spectroscopy data. The findings reveal insights into the folding mechanism of a non-native intermediate that triggers transcriptional pause, along with different folding pathways leading to transcriptional pause and readthrough. The profiling of the cotranscriptional folding pathway and identification of kinetic structural intermediates reveal a novel mechanism for viral transcriptional regulation, which could pave the way for new antiviral drug designs targeting kinetic cotranscriptional folding pathways in viral RNAs.

Effect of montmorillonite K10 clay on RNA structure and function.

One of the earliest living systems was likely based on RNA ("the RNA world"). Mineral surfaces have been postulated to be an important environment for the prebiotic chemistry of RNA. In addition to adsorbing RNA and thus potentially reducing the chance of parasitic takeover through limited diffusion, minerals have been shown to promote a range of processes related to the emergence of life, including RNA polymerization, peptide bond formation, and self-assembly of vesicles. In addition, self-cleaving ribozymes have been shown to retain activity when adsorbed to the clay mineral montmorillonite. However, simulation studies suggest that adsorption to minerals is likely to interfere with RNA folding and, thus, function. To further evaluate the plausibility of a mineral-adsorbed RNA world, here we studied the effect of the synthetic clay montmorillonite K10 on the malachite green RNA aptamer, including binding of the clay to malachite green and RNA, as well as on the formation of secondary structures in model RNA and DNA oligonucleotides. We evaluated the fluorescence of the aptamer complex, adsorption to the mineral, melting curves, Förster resonance energy transfer interactions, and 1H-NMR signals to study the folding and functionality of these nucleic acids. Our results indicate that while some base pairings are unperturbed, the overall folding and binding of the malachite green aptamer are substantially disrupted by montmorillonite. These findings suggest that minerals would constrain the structures, and possibly the functions, available to an adsorbed RNA world.

Differentiation SELEX approach identifies RNA aptamers with different specificities for HIV-1 capsid assembly forms.

The HIV-1 capsid protein (CA) assumes distinct assembly forms during replication, each presenting unique, solvent-accessible surfaces that facilitate multifaceted functions and host factor interactions. However, contributions of individual CA assemblies remain unclear, as the evaluation of CA in cells presents several technical challenges. To address this need, we sought to identify CA assembly form-specific aptamers. Aptamer subsets with different specificities emerged from within a highly converged, pre-enriched aptamer library previously selected to bind the CA hexamer lattice. Subsets were either highly specific for CA lattice or bound both CA lattice and CA hexamer. We further evaluated four representatives to reveal aptamer structural features required for binding, highlighting interesting features and challenges in aptamer structure determination. Importantly, our aptamers bind biologically relevant forms of CA and we demonstrate aptamer-mediated affinity purification of CA from cell lysates without virus or host modification. Thus, we have identified CA assembly form-specific aptamers that represent exciting new tools for the study of CA.

Embedding Biometric Information in Interpolated Medical Images with a Reversible and Adaptive Strategy.

How to hide messages in digital images so that messages cannot be discovered and tampered with is a compelling topic in the research area of cybersecurity. The interpolation-based reversible data hiding (RDH) scheme is especially useful for the application of medical image management. The biometric information of patients acquired by biosensors is embedded into an interpolated medical image for the purpose of authentication. The proposed scheme classifies pixel blocks into complex and smooth ones according to each block's dynamic range of pixel values. For a complex block, the minimum-neighbor (MN) interpolation followed by DIM embedding is applied, where DIM denotes the difference between the block's interpolated pixel values and the maximum pixel values. For a smooth block, the block mean (BM) interpolation is followed by a prediction error histogram (PEH) embedding and a difference expansion (DE) embedding is applied. Compared with previous methods, this adaptive strategy ensures low distortion due to embedding for smooth blocks while it provides a good payload for complex blocks. Our scheme is suitable for both medical and general images. Experimental results confirm the effectiveness of the proposed scheme. Performance comparisons with state-of-the-art schemes are also given. The peak signal to noise ratio (PSNR) of the proposed scheme is 10.32 dB higher than the relevant works in the best case.

An RNA aptamer that shifts the reduction potential of metabolic cofactors.

The discovery of ribozymes has inspired exploration of RNA's potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme's catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by -40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π-π and donor atom-π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms.

Anomalous HIV-1 RNA, How Cap-Methylation Segregates Viral Transcripts by Form and Function.

The acquisition of m7G-cap-binding proteins is now recognized as a major variable driving the form and function of host RNAs. This manuscript compares the 5'-cap-RNA binding proteins that engage HIV-1 precursor RNAs, host mRNAs, small nuclear (sn)- and small nucleolar (sno) RNAs and sort into disparate RNA-fate pathways. Before completion of the transcription cycle, the transcription start site of nascent class II RNAs is appended to a non-templated guanosine that is methylated (m7G-cap) and bound by hetero-dimeric CBP80-CBP20 cap binding complex (CBC). The CBC is a nexus for the co-transcriptional processing of precursor RNAs to mRNAs and the snRNA and snoRNA of spliceosomal and ribosomal ribonucleoproteins (RNPs). Just as sn/sno-RNAs experience hyper-methylation of m7G-cap to trimethylguanosine (TMG)-cap, so do select HIV RNAs and an emerging cohort of mRNAs. TMG-cap is blocked from Watson:Crick base pairing and disqualified from participating in secondary structure. The HIV TMG-cap has been shown to license select viral transcripts for specialized cap-dependent translation initiation without eIF4E that is dependent upon CBP80/NCBP3. The exceptional activity of HIV precursor RNAs secures their access to maturation pathways of sn/snoRNAs, canonical and non-canonical host mRNAs in proper stoichiometry to execute the retroviral replication cycle.

HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR.

Appended to the 5' end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1-reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5' untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.

Safety and efficacy profile of a phytase produced by fermentation and used as a feed additive.

Enzymes can aid in optimal feed stock utilization when used as feed additives. A range of toxicological studies were performed to evaluate the safety profile of a novel phytase (phytase HM) from Citrobacter b raakii produced in Aspergillus oryzae. Phytase HM was found to be non-mutagenic and non-clastogenic in in vitro tests. Further, the phytase HM preparation did not exhibit irritative potential to the eye and skin when applied in in vitro models. A 13-week subchronic toxicity study with oral administration of phytase HM to rats did not show any adverse effects. Efficacy studies showed that the dietary supplementation of this phytase significantly improved growth performance and bone mineralization in broiler chickens and piglets fed P-deficient diets, and increased retention of phosphorus (P) and calcium (Ca), and phytate-P degradation in excreta of broiler chickens in a dose-dependent manner. In conclusion, there are no safety concerns using phytase HM as a feed additive and the phytase is well tolerated by broiler chickens and pigs. Further, phytase HM improves with high efficacy the growth performance in both broiler chickens and pigs.

The three-way junction structure of the HIV-1 PBS-segment binds host enzyme important for viral infectivity.

HIV-1 reverse transcription initiates at the primer binding site (PBS) in the viral genomic RNA (gRNA). Although the structure of the PBS-segment undergoes substantial rearrangement upon tRNALys3 annealing, the proper folding of the PBS-segment during gRNA packaging is important as it ensures loading of beneficial host factors. DHX9/RNA helicase A (RHA) is recruited to gRNA to enhance the processivity of reverse transcriptase. Because the molecular details of the interactions have yet to be defined, we solved the solution structure of the PBS-segment preferentially bound by RHA. Evidence is provided that PBS-segment adopts a previously undefined adenosine-rich three-way junction structure encompassing the primer activation stem (PAS), tRNA-like element (TLE) and tRNA annealing arm. Disruption of the PBS-segment three-way junction structure diminished reverse transcription products and led to reduced viral infectivity. Because of the existence of the tRNA annealing arm, the TLE and PAS form a bent helical structure that undergoes shape-dependent recognition by RHA double-stranded RNA binding domain 1 (dsRBD1). Mutagenesis and phylogenetic analyses provide evidence for conservation of the PBS-segment three-way junction structure that is preferentially bound by RHA in support of efficient reverse transcription, the hallmark step of HIV-1 replication.

Research Note: The effect of sequential displacement of dietary dextrose with myo-inositol on broiler chicken growth performance, bone characteristics, ileal nutrient digestibility, and total tract nutrient retention.

A total of 480 male Cobb 500 broiler chicks were assigned to one of 6 dietary treatments to explore the energy equivalence of myo-inositol compared with dextrose. The 6 dietary treatments included a corn and soy-based control ration formulated with 5% anhydrous dextrose and 5 further diets that were generated by the sequential displacement of increments of 1% dextrose with myo-inositol. Each diet was fed to 8 replicate cages of 10 chicks per cage from day 8 to day 18 after hatch. The BW gain, feed intake, and feed conversion ratio (FCR) were measured, and on day 15 to day 17, excreta were collected to estimate the total tract nutrient retention. Ileal digestibility of nutrients and tibia mineral content was assessed on day 18. The displacement of dextrose with myo-inositol generated a significant linear reduction in the FCR that did not reach a plateau at 5% dietary inclusion of myo-inositol. There was no effect of the displacement of dextrose with myo-inositol on bone mineral concentration. However, supplemental myo-inositol linearly reduced ileal digestibility of DM, calcium, and ileal digestible energy. Myo-inositol addition resulted in a significant linear increase in the total tract retention of CP. It can be concluded that myo-inositol has an energy equivalence equal to approximately 78% of that of dextrose for young broiler chicks but exerts a range of extra caloric effects that improve feed efficiency and may influence nitrogen (N) retention and the uric acid cycle. Future work should focus on the role of phytase and myo-inositol on uric acid, creatine kinase, and other metabolites involved in renal function and biochemical flows of N in urine and feces in nonruminants.

Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity.

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3'-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Feasibility of Known RNA Polymerase Inhibitors as Anti-SARS-CoV-2 Drugs.

Coronaviruses (CoVs) are positive-stranded RNA viruses that infect humans and animals. Infection by CoVs such as HCoV-229E, -NL63, -OC43 and -HKU1 leads to the common cold, short lasting rhinitis, cough, sore throat and fever. However, CoVs such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the newest SARS-CoV-2 (the causative agent of COVID-19) lead to severe and deadly diseases with mortality rates ranging between ~1 to 35% depending on factors such as age and pre-existing conditions. Despite continuous global health threats to humans, there are no approved vaccines or drugs targeting human CoVs, and the recent outbreak of COVID-19 emphasizes an urgent need for therapeutic interventions. Using computational and bioinformatics tools, here we present the feasibility of reported broad-spectrum RNA polymerase inhibitors as anti- SARS-CoV-2 drugs targeting its main RNA polymerase, suggesting that investigational and approved nucleoside RNA polymerase inhibitors have potential as anti-SARS-CoV-2 drugs. However, we note that it is also possible for SARS-CoV-2 to evolve and acquire drug resistance mutations against these nucleoside inhibitors.

Tamarix chinensis Lour inhibits chronic ethanol-induced liver injury in mice.

BACKGROUND: Tamarix chinensis Lour (TCL) is a shrub that usually grows in arid or semiarid desert areas and saline-alkali fields. It is a traditional Chinese herbal medicine with hepatoprotective, antioxidant, antibacterial, and antitumor activities. AIM: To investigate the possible protective effects of TCL against liver injury induced by chronic ethanol intake. METHODS: C57BL/6J male mice were fed a Lieber-DeCarli lipid diet containing alcohol and received (by gavage) a water-alcohol extract (80%) of TCL (100 and 200 mg/kg BW) or distilled water for 4 wk. After euthanasia, liver tissues were observed histologically with hematoxylin and eosin staining and Oil red O staining, and the levels of alanine aminotransferase, aspartate transaminase, hepatic lipids, reactive oxygen species, malondialdehyde, and superoxide dismutase were measured. In addition, expression of the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and downstream proinflammatory cytokines were determined. RESULTS: Compared with the ethanol group, mice in the TCL-treated group (200 mg/kg) had significantly lower serum levels of alanine aminotransferase (mean, 34.1 IU/L vs 45.3 IU/L, P < 0.01) and aspartate transaminase (mean, 89.6 IU/L vs 115.7 IU/L, P < 0.01), as well as marked reduction of hepatic tissue reactive oxygen species (decreased by 27.5%, P < 0.01) and malondialdehyde (decreased by 76.6%, P < 0.01) levels, with a significant increase of superoxide dismutase (Increased by 73.2%, P < 0.01). Expression of the NLRP3 inflammasome and its downstream cytokines [interleukin (IL)-1ß, tumor necrosis factor-α, and IL-6], and recruitment of natural killer T cells to the liver, were reduced in the TCL-treated incubation with a Lieber-DeCaril ethanol lipid diet group. CONCLUSION: These findings suggest that a TCL extract (200 mg/kg) protects against chronic ethanol-induced liver injury, probably by inhibiting the NLRP3-caspase-1-IL-1ß signaling pathway and suppressing oxidative stress.

Binding interface and impact on protease cleavage for an RNA aptamer to HIV-1 reverse transcriptase.

RNA aptamers that bind HIV-1 reverse transcriptase (RT) inhibit RT in enzymatic and viral replication assays. Some aptamers inhibit RT from only a few viral clades, while others show broad-spectrum inhibition. Biophysical determinants of recognition specificity are poorly understood. We investigated the interface between HIV-1 RT and a broad-spectrum UCAA-family aptamer. SAR and hydroxyl radical probing identified aptamer structural elements critical for inhibition and established the role of signature UCAA bulge motif in RT-aptamer interaction. HDX footprinting on RT ± aptamer shows strong contacts with both subunits, especially near the C-terminus of p51. Alanine scanning revealed decreased inhibition by the aptamer for mutants P420A, L422A and K424A. 2D proton nuclear magnetic resonance and SAXS data provided constraints on the solution structure of the aptamer and enable computational modeling of the docked complex with RT. Surprisingly, the aptamer enhanced proteolytic cleavage of precursor p66/p66 by HIV-1 protease, suggesting that it stabilizes the productive conformation to allow maturation. These results illuminate features at the RT-aptamer interface that govern recognition specificity by a broad-spectrum antiviral aptamer, and they open new possibilities for accelerating RT maturation and interfering with viral replication.

Safety and efficacy evaluation of a novel dietary muramidase for swine.

The safety of a novel microbial muramidase (Muramidase 007) as a feed additive for swine was evaluated in a target animal safety study (Experiment 1). Forty weanling pigs were allotted to 4 dietary treatments: T1 control group, and 3 groups receiving Muramidase 007 in increasing doses: T2 65,000 (1X), T3 325,000 (5X) and T4 650,000 (10X) LSU(F)/kg feed. The efficacy of Muramidase 007 on growth performance was evaluated in a feeding experiment (Experiment 2). A total of 288 piglets were allotted to two groups: T1 control group and T2 receiving Muramidase 007 at 50,000 (LSU(F)/kg feed. In Experiment 1, no growth depression of pigs was observed. No adverse effects of Muramidase 007 were observed for any of the hematology and serum chemistry parameters measured or on pig health status. Post-mortem evaluation showed no adverse effects due to Muramidase 007 supplementation in the gross pathology or in the histological examination. In Experiment 2, Muramidase 007 significantly increased overall (d 0-42) average daily gain (ADG) and tended to improve overall average daily feed intake (ADFI) and day 42 body weight of nursery pigs and had no effect on feed conversion ratio (FCR). Overall, results of these studies show that there were no adverse effects of Muramidase 007 compared to the control group.

Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA.

DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNALys3-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.

Synthesis of distal and proximal fleximer base analogues and evaluation in the nucleocapsid protein of HIV-1.

Anti-HIV-1 drug design has been notably challenging due to the virus' ability to mutate and develop immunity against commercially available drugs. The aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of HIV (NC). The compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. The target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against NC and HIV-1. The results of those studies are described herein.