Structure of saguaro cactus virus 3' translational enhancer mimics 5' cap for eIF4E binding.

The genomes of several plant viruses contain RNA structures at their 3' ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5' cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5' cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3' CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5' cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE-eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5' cap for eIF4E binding. Such 5' cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

Structural basis for a highly conserved RNA-mediated enteroviral genome replication.

Enteroviruses contain conserved RNA structures at the extreme 5' end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Šresolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3' spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3' spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform.

Advances in chaperone-assisted RNA crystallography using synthetic antibodies.

RNA molecules play essential roles in many biological functions, from gene expression regulation, cellular growth, and metabolism to catalysis. They frequently fold into three-dimensional structures to perform their functions. Therefore, determining RNA structure represents a key step for understanding the structure-function relationships and developing RNA-targeted therapeutics. X-ray crystallography remains a method of choice for determining high-resolution RNA structures, but it has been challenging due to difficulties associated with RNA crystallization and phasing. Several natural and synthetic RNA binding proteins have been used to facilitate RNA crystallography. Having unique properties to help crystal packing and phasing, synthetic antibody fragments, specifically the Fabs, have emerged as promising RNA crystallization chaperones, and so far, over a dozen of RNA structures have been solved using this strategy. Nevertheless, multiple steps in this approach need to be improved, including the recombinant expression of these anti-RNA Fabs, to warrant the full potential of these synthetic Fabs as RNA crystallization chaperones. This review highlights the nuts and bolts and recent advances in the chaperone-assisted RNA crystallography approach, specifically emphasizing the Fab antibody fragments as RNA crystallization chaperones.

Efficient aqueous molybdenum removal using commercial Douglas fir biochar and its iron oxide hybrids.

Molybdenum (Mo) is a naturally-occurring trace element in drinking water. Most commonly, molybdate anions (MoO42-) are in well water and breast milk. In addition, it is used in medical image testing. Recently, the EPA classified Mo as a potential contaminant, as exposure can lead to health effects such as gout, hyperuricemia, and even lung cancer. We have assessed the sorptive removal of aqueous molybdate using Douglas fir biochar (DFBC) and a hybrid DFBC/Fe3O4 composite containing chemically-coprecipitated iron oxide (Fe3O4). Adsorption was studied at various: pH values, equilibrium times (5 min-24 h), initial Mo concentrations (2.5-1000 mg/L), and temperatures (5, 25, and 40 °C) using batch sorption and fixed-bed column equilibrium methods. Langmuir capacities for DFBC and DFBC/Fe3O4 (at pH 3, 2 hrs equilibrium) were within 459.3-487.9 mg/g and 288-572 mg/g, respectively. These adsorbents and their Mo-laden counterparts were characterized by elemental analysis, BET, PZC, SEM, TEM, EDS, XRD, and XPS. MoO42- adsorption on DFBC is thought to be governed primarily via electrostatic attraction. Adsorption by DFBC/Fe3O4 is primarily governed by chemisorption onto magnetite surface hydroxyl groups, while electrostatics prevail in the DFBC-exposed phase. Stoichiometric precipitation of iron molybdates triggered by iron dissolution was also considered. The data suggest that DFBC and DFBC/Fe3O4 are promising candidates for molybdate sorption.