Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex.

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

The Repurposing of Cellular Proteins during Enterovirus A71 Infection.

Viruses pose a great threat to people's lives. Enterovirus A71 (EV-A71) infects children and infants all over the world with no FDA-approved treatment to date. Understanding the basic mechanisms of viral processes aids in selecting more efficient drug targets and designing more effective antivirals to thwart this virus. The 5'-untranslated region (5'-UTR) of the viral RNA genome is composed of a cloverleaf structure and an internal ribosome entry site (IRES). Cellular proteins that bind to the cloverleaf structure regulate viral RNA synthesis, while those that bind to the IRES also known as IRES trans-acting factors (ITAFs) regulate viral translation. In this review, we survey the cellular proteins currently known to bind the 5'-UTR and influence viral gene expression with emphasis on comparing proteins' functions and localizations pre- and post-(EV-A71) infection. A comprehensive understanding of how the host cell's machinery is hijacked and reprogrammed by the virus to facilitate its replication is crucial for developing effective antivirals.

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures.

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for antiviral therapeutic development. Here, we report on the identification of amiloride-based small molecules that potently inhibit OC43 and SARS-CoV-2 replication through targeting of conserved structured elements within the viral 5'-end. Nuclear magnetic resonance­based structural studies revealed specific amiloride interactions with stem loops containing bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Amilorides represent the first antiviral small molecules that target RNA structures within the 5' untranslated regions and proximal region of the CoV genomes. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA­targeted antivirals.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Integrated approaches to reveal mechanisms by which RNA viruses reprogram the cellular environment.

RNA viruses are major threats to global society and mass outbreaks can cause long-lasting damage to international economies. RNA and related retro viruses represent a large and diverse family that contribute to the onset of human diseases such as AIDS; certain cancers like T cell lymphoma; severe acute respiratory illnesses as seen with COVID-19; and others. The hallmark of this viral family is the storage of genetic material in the form of RNA, and upon infecting host cells, their RNA genomes reprogram the cellular environment to favor productive viral replication. RNA is a multifunctional biomolecule that not only stores and transmits heritable information, but it also has the capacity to catalyze complex biochemical reactions. It is therefore no surprise that RNA viruses use this functional diversity to their advantage to sustain chronic or lifelong infections. Efforts to subvert RNA viruses therefore requires a deep understanding of the mechanisms by which these pathogens usurp cellular machinery. Here, we briefly summarize several experimental techniques that individually inform on key physicochemical features of viral RNA genomes and their interactions with proteins. Each of these techniques provide important vantage points to understand the complexities of virus-host interactions, but we attempt to make the case that by integrating these and similar methods, more vivid descriptions of how viruses reprogram the cellular environment emerges. These vivid descriptions should expedite the identification of novel therapeutic targets.

Predictors of 30-Day Readmission After Inpatient Endoscopic Retrograde Cholangiopancreatography: A Single-Center Experience.

INTRODUCTION: The 30-day hospital readmission rate is a nationally recognized quality measure. Nearly one-fifth of medicare beneficiaries are hospitalized within 30 days of discharge, resulting in a cost of over $26 billion dollars annually. Endoscopic retrograde cholangiopancreatography (ERCP) remains the endoscopic procedure with the highest risk of morbidity and mortality. We set out to analyze the clinical characteristics predictive of 30-day readmission after an inpatient ERCP. METHODS: We performed a retrospective chart review of all inpatient ERCPs performed at our institution between 12/1/2014 and 9/30/2018. Clinical characteristics and outcomes of these patients were compared to determine predictors of 30-day readmission. RESULTS: A total of 497 inpatient ERCP procedures done for biliary or pancreatic indications, constituting 483 patients, were identified. There were 52 readmissions that occurred among 48 patients within 30 days of discharge. Basic demographic characteristics were similar between both groups. Comorbidities were significantly higher in those who were readmitted. Multivariate analysis revealed significantly greater odds of readmission with prior liver transplantation (OR = 4.15), cirrhosis (OR = 3.20), and pancreatic duct stent placement (OR = 2.56). Subgroup analysis for biliary indications revealed cholecystectomy before discharge and early ERCP to be protective against readmission. CONCLUSION: A history of liver transplantation and cirrhosis are predictive of increased 30-day readmission rates after an inpatient ERCP. Pancreatic duct stent placement is associated with readmission; however, this phenomenon is likely related to stenting for pancreatic endotherapy. Cholecystectomy before discharge and early ERCP are predictive of decreased need for readmission in procedures done for biliary indications.

Toxic emissions resulting from sucralose added to electronic cigarette liquids.

Electronic cigarettes (ECIGs) are appealing in part because of the many flavors of the liquids used in them. Concerns have been raised that some ECIG liquid flavors, especially those that are sweet, are attracting otherwise nicotine-naïve youth to ECIGs. Sucralose is an artificial, non-caloric sweetener that is added to some ECIG liquids. In this study, we evaluated the toxicants, namely isomers of chloropropanols that can be produced when sucralose-containing ECIG liquid is aerosolized. An analytical separation method relying on solid-phase extraction (SPE) to isolate chloropropanols from the propylene glycol/glycerol matrix was developed. Chloropropanols were then derivatized by silylation before they were analyzed on GC-MS. The influence of different ECIG operating conditions on the generation of chloropropanols was studied by varying ECIG device design and power output and also the sucralose concentration of the liquid. Heated sucralose-containing ECIG liquids produce two toxic compounds that can be found in the resulting aerosols. The two chloropropanols, 3-monochloro-1,2-propanediol (3-MCPD) and 1,3-dichloropropanol (1,3-DCP) that were detected under all conditions were found to be correlated significantly with liquid sucralose content. Effective regulation of ECIGs will minimize user and bystander exposure to these and other ECIG toxicants.

Free-Base and Total Nicotine, Reactive Oxygen Species, and Carbonyl Emissions From IQOS, a Heated Tobacco Product.

INTRODUCTION: IQOS is an emerging heated tobacco product marketed by Philip Morris International (PMI). Because the tobacco in IQOS is electrically heated and not combusted, PMI claims that it generates significantly lower toxicant levels than combustible cigarettes. To date, a few independent studies have addressed IQOS toxicant emissions, and none have reported reactive oxygen species (ROS), and the form of the nicotine emitted by the device. METHODS: In this study, IQOS aerosol was generated using a custom-made puffing machine. Two puffing regimens were used: Health Canada Intense and ISO. ROS, carbonyl compounds (CCs), and total nicotine and its partitioning between free-base and protonated forms were quantified in the IQOS aerosol by fluorescence, high-performance liquid chromatography, and gas chromatography, respectively. The same toxicants were also quantified in combustible cigarette aerosols for comparison. In addition, propylene glycol and vegetable glycerin were also measured in the IQOS tobacco and aerosol. RESULTS: IQOS and combustible cigarettes were found to emit similar quantities of total and free-base nicotine. IQOS total ROS (6.26 ± 2.72 nmol H2O2/session) and CC emissions (472 ± 19 µg/session) were significant, but 85% and 77% lower than levels emitted by combustible cigarettes. CONCLUSIONS: IQOS emits harmful constituents that are linked to cancer, pulmonary disease, and addiction in cigarette smokers. For a given nicotine intake, inhalation exposure to ROS and CCs from IQOS is likely to be significantly less than that for combustible cigarettes. IMPLICATIONS: IQOS is PMI's new heated tobacco product. PMI claims that because IQOS heats and does not burn tobacco it generates low toxicant yields. We found that one IQOS stick can emit similar free-base and total nicotine yields as a combustible cigarette. A pack-a-day equivalent user of IQOS may experience significant inhalation exposure of ROS and CCs compared to background air. However, substituting IQOS for combustible cigarettes will likely result in far lower ROS and carbonyl inhalation exposure for a given daily nicotine intake.

Reactive Oxygen Species Emissions from Supra- and Sub-Ohm Electronic Cigarettes.

Electronic cigarettes (ECIGs) are battery-powered devices that heat and vaporize solutions containing propylene glycol (PG) and/or vegetable glycerin (VG), nicotine and possible trace flavorants to produce an inhalable aerosol. The heating process can lead to the formation of reactive oxygen species (ROS), which are linked to various oxidative damage-initiated diseases. Several studies in the literature have addressed ROS emissions in ECIG aerosols, but the effects of power, ECIG device design and liquid composition on ROS are relatively unknown. In addition, ROS emissions have not been examined in the emerging high power, sub-Ohm device (SOD) category. In this study, an acellular 2',7'-dichlorofluorescin (DCFH) probe technique was optimized to measure ROS in ECIG aerosols. The technique was deployed to measure ROS emissions in SOD and supra-Ohm ECIGs while varying power, heater coil head design and liquid composition (PG/VG ratio and nicotine concentration). Liquids were made from analytical standards of PG, VG and nicotine and contained no flavorants. At high powers, ROS emissions in ECIGs and combustible cigarettes were similar. Across device designs, ROS emissions were uncorrelated with power (R2 = 0.261) but were highly correlated with power per unit area (R2 = 0.78). It was noticed that an increase in the VG percentage in the liquid yielded higher ROS flux, and nicotine did not affect ROS emissions. ROS emissions are a function of device design and liquid composition at a given power. For a given liquid composition, a promising metric for predicting ROS emissions across device designs and operating conditions is power per unit area of the heating coil. Importantly, ROS formation is significant even when the ECIG liquid consists of pure analytical solutions of PG and VG; it can therefore be viewed as intrinsic to ECIG operation and not solely a by-product of particular flavorants, contaminants or additives.

Acidianus sulfidivorans sp. nov., an extremely acidophilic, thermophilic archaeon isolated from a solfatara on Lihir Island, Papua New Guinea, and emendation of the genus description.

A novel, extremely thermoacidophilic, obligately chemolithotrophic archaeon (strain JP7(T)) was isolated from a solfatara on Lihir Island, Papua New Guinea. Cells of this organism were non-motile, Gram-negative staining, irregular-shaped cocci, 0.5-1.5 microm in size, that grew aerobically by oxidation of sulfur, Fe(2+) or mineral sulfides. Cells grew anaerobically using Fe(3+) as a terminal electron acceptor and H(2)S as an electron donor but did not oxidize hydrogen with elemental sulfur as electron acceptor. Strain JP7(T) grew optimally at 74 degrees C (temperature range 45-83 degrees C) and pH 0.8-1.4 (pH range 0.35-3.0). On the basis of 16S rRNA gene sequence similarity, strain JP7(T) was shown to belong to the Sulfolobaceae, being most closely related to the type strains of Acidianus ambivalens (93.7 %) and Acidianus infernus (93.6 %). Cell-membrane lipid structure, DNA base composition and 16S rRNA gene sequence similarity data support the placement of this strain in the genus Acidianus. Differences in aerobic and anaerobic metabolism, temperature and pH range for growth, and 16S rRNA gene sequence differentiate strain JP7(T) from recognized species of the genus Acidianus, and an emendation of the description of the genus is proposed. Strain JP7(T) is considered to represent a novel species of the genus Acidianus, for which the name Acidianus sulfidivorans sp. nov. is proposed. The type strain is JP7(T) (=DSM 18786(T)=JCM 13667(T)).

Characterization of a thermophilic sulfur oxidizing enrichment culture dominated by a Sulfolobus sp. obtained from an underground hot spring for use in extreme bioleaching conditions.

A thermoacidophilic elemental sulfur and chalcopyrite oxidizing enrichment culture VS2 was obtained from hot spring run-off sediments of an underground mine. It contained only archaeal species, namely a Sulfolobus metallicus-related organism (96% similarity in partial 16S rRNA gene) and Thermoplasma acidophilum (98% similarity in partial 16S rRNA gene). The VS2 culture grew in a temperature range of 35-76 degrees C. Sulfur oxidation by VS2 was optimal at 70 degrees C, with the highest oxidation rate being 99 mg S(0 )l(-1 )day(-1). At 50 degrees C, the highest sulfur oxidation rate was 89 mg l(-1 )day(-1 )(in the presence of 5 g Cl(-) l(-1)). Sulfur oxidation was not significantly affected by 0.02-0.1 g l(-1) yeast extract or saline water (total salinity of 0.6 M) that simulated mine water at field application sites with availability of only saline water. Chloride ions at a concentration above 10 g l(-1) inhibited sulfur oxidation. Both granular and powdered forms of sulfur were bioavailable, but the oxidation rate of granular sulfur was less than 50% of the powdered form. Chalcopyrite concentrate oxidation (1% w/v) by the VS2 resulted in a 90% Cu yield in 30 days.