All coronaviruses (CoVs), including the causative agent of severe acute respiratory syndrome (SARS), encode a nucleocapsid (N) protein that harbors two independent RNA binding domains of known structure, but poorly characterized RNA binding properties. We show here that the N-terminal domain (NTD) of N protein from mouse hepatitis virus (MHV), a virus most closely related to SARSCoV, employs aromatic amino acid-nucleobase stacking interactions with a triple adenosine motif to mediate high-affinity binding to single-stranded RNAs containing the transcriptional regulatory sequence (TRS) or its complement (cTRS). Stoichiometric NTD fully unwinds a TRS-cTRS duplex that mimics a transiently formed transcription intermediate in viral subgenomic RNA synthesis. Mutation of the solvent-exposed Y127, positioned on the β-platform surface of our 1.75 Å structure, binds the TRS far less tightly and is severely crippled in its RNA unwinding activity. In contrast, the C-terminal domain (CTD) exhibits no RNA unwinding activity. Viruses harboring Y127A N mutation are strongly selected against and Y127A N does not support an accessory function in MHV replication. We propose that the helix melting activity of the coronavirus N protein NTD plays a critical accessory role in subgenomic RNA synthesis and other processes requiring RNA remodeling.

The 59 untranslated region (UTR) of the mouse hepatitis virus (MHV) genome contains cis-acting sequences necessary for transcription and replication. A consensus secondary structural model of the 59 140 nucleotides of the 59 UTRs of nine coronaviruses (CoVs) derived from all three major CoV groups is presented and characterized by three major stem-loops, SL1, SL2, and SL4. NMR spectroscopy provides structural support for SL1 and SL2 in three group 2 CoVs, including MHV, BCoV, and HCoV-OC43. SL2 is conserved in all CoVs, typically containing a pentaloop (C47-U48-U49-G50-U51 in MHV) stacked on a 5 base-pair stem, with some sequences containing an additional U 39 to U51; SL2 therefore possesses sequence features consistent with a U-turn-like conformation. The imino protons of U48 in the wild-type RNA, and G48 in the U48G SL2 mutant RNA, are significantly protected from exchange with solvent, consistent with a hydrogen bonding interaction critical to the hairpin loop architecture. SL2 is required for MHV replication; MHV genomes containing point substitutions predicted to perturb the SL2 structure (U48C, U48A) were not viable, while those that maintain the structure (U48G and U49A) were viable. The U48C MHV mutant supports both positive-and negative-sense genome-sized RNA synthesis, but fails to direct the synthesis of positive-or negative-sense subgenomic RNAs. These data support the existence of the SL2 in our models, and further suggest a critical role in coronavirus replication.

SummaryThe leader RNA of the 5' untranslated region (UTR) of coronaviral genomes contains two stem-loop structures denoted SL1 and SL2. Herein, we show that SL1 is functionally and structurally bipartite. While the upper region of SL1 is required to be paired, we observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a non-canonical base pair near A35. Viruses containing destabilizing SL1-ΔA35 mutations also contain one of two specific mutations in the 3' UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-ΔA35 substitutions are characterized by one or more features of the wild-type SL1. We propose a "dynamic SL1" model, in which the base of SL1 has an optimized lability required to mediate a physical interaction between the 5' and 3' UTRs that stimulates subgenomic RNA synthesis. Although not conserved at the nucleotide sequence level, these general structural characteristics of SL1 appear to be conserved in other coronaviral genomes.

We report characterization of Hepatitis B virus (HBV) capsids by resistive-pulse sensing through single track-etched conical nanopores formed in poly(ethylene terephthalate) membranes. The pores were ~40 nm in diameter at the tip, and the pore surface was covalently modified with triethylene glycol to reduce surface charge density, minimize adsorption of the virus capsids, and suppress electroosmotic flow in the pore. The HBV capsids were assembled in vitro from Cp149, the assembly domain of HBV capsid protein. Assembled T=3 (90 Cp149 dimer) and T=4 (120 dimer) capsids are 31 and 36 nm in diameter, respectively, and were easily discriminated by monitoring the change in current as capsids passed through an electrically biased pore. The ratio of the number of T=3 to T=4 capsids transiting a pore did not reflect actual concentrations, but favored transport of smaller T=3 capsids. These results combined with longer transit times for the T=4 capsids indicated that the capsids must overcome an entropic barrier to enter a pore.Nanopores and nanochannels exhibit unique transport properties1 and have a number of potential applications.2 Of particular interest is developing label-free, nondestructive techniques for rapid sensing, characterization, and sorting of particles with nanometer dimensions. The resistive-pulse technique3 measures changes in ion current resulting from transit of particles through an electrically biased nanopore filled with electrolyte. As sensing elements, protein pores,4 e.g., α-hemolysin, exhibit highly reproducible pore composition and dimensions, but lack robustness when suspended in lipid bilayers. Alternatively, microand nanofabrication techniques are used to fabricate solid-state and synthetic nanopores5 , 6 with a wide range of well-defined geometries and dimensions. Forming these pores parallel or perpendicular to the substrate surface permits straightforward integration with other device features. Solid-state and synthetic nanopores exhibit ion depletion/concentration,7 -9 ion permittivity,10 enhanced channel conductance,11 ion current rectification,12 , 13 and pressure-induced salt flux rectification.14 , 15 The ability to control pore dimensions over a range of length scales permits analysis of a variety of samples, including DNA,16 -18 proteins,19 viruses,20 immune complexes,21 nanoparticles,22 and small molecules,23 and similarly designed pores may be used to sequence DNA.24 In some cases, the molecule of interest, e.g., DNA, must overcome an entropic barrier to enter nanoscale slits25 and pores. 26 Related to this work is the characterization of viruses with track-etched pores20 and immune complexes with femtosecond laser-machined pores.21 In both examples, the studied protein complexes are ~100-150 nm in diameter. The reassembly process is inherently of interest, and this system offers a unique opportunity to characterize capsid transport, capsid properties, and nanopore properties. The T=3 and T=4 capsids are similar in diameter, 31 and 36 nm, respectively, and have identic...

Silica aerogels are sol-gel-derived materials consisting of interconnected nanoparticle building blocks that form an open and highly porous three-dimensional silica network. Flexible aerogel films could have wide applications in various thermal insulation systems. However, aerogel thin films produced with a pure sol-gel process have inherent disadvantages, such as high fragility and moisture sensitivity, that hinder wider applications of these materials. We have developed synthesis and manufacturing methods to incorporate electrospun polyurethane nanofibers into the cast sol film prior to gelation of the silica-based gel in order to reinforce the structure and overcome disadvantages such as high fragility and poor mechanical strength. In this method, a two-stage sol-gel process was employed: (1) acid-catalyzed tetraethyl orthosilicate hydrolysis and (2) base-catalyzed gelation. By precisely controlling the sol gelation kinetics with the amount of base present in the formulation, nanofibers were electrospun into the sol before the onset of the gelation process and uniformly embedded in the silica network. Nanofiber reinforcement did not alter the thermal conductivity and rendered the final composite film bendable and flexible.