Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus‐like particles

Virus‐like particles (VLPs) derived from nonenveloped viruses result from the self‐assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self‐assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N‐ and C‐terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant‐based production of nucleic acid‐free VLPs. Remarkably, expression of N‐ or C‐terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.     

The long-distance abscisic acid signal in the droughted plant: the fate of the hormone on its way from root to shoot

Abscisic acid (ABA) is a potent molecule that certainly modifies stomatal behaviour and plant water loss and probably acts to modify the growth of leaves. The hormone is synthesized both in the leaves and the roots of the plant and in the soil and may move freely from plant to soil and soil to plant. It can also move rapidly through the plant in both the xylem and the phloem and will partition between different compartments in different tissues largely as a function of pH. It is described here how perturbations in soil conditions around the roots and the water status of the air can modify the fluxes of ABA around the plant and its accumulation in different compartments and different tissues. These fluxes can be interpreted as signals of different stresses imposed on the plant and consideration is given to how different perturbations can exert subtle changes which are manifest as modified shoot growth rates and functioning. Most emphasis in the discussion is placed upon the plant's responses to the imposition of soil and atmospheric drought.     

A sulfate pocket formed by three GoU pairs in the 0.97 A resolution X-ray structure of a nonameric RNA

The crystal structure of the RNA duplex [r(CGUGAUCG)dC]2 has been solved at a resolution of 0.97 A. The model has been refined to R-work and R-free of 14.88% and 19.54% for 23,838 independent reflections. The base-pairing scheme forces the 5'-rC to be excluded from the helix and to be disordered. In the crystals, the sequence promotes the formation of two GoU wobble pairs that cluster around a crystallographic threefold axis in two different ways. In the first contact type, the GoU pairs are exclusively surrounded by water molecules, whereas in the other contact type, the three amino groups of the guanine residues of the symmetry-related GoU pairs trap a sulfate ion. This work provides the first example of the interaction of a GoU pair with a sulfate ion in a helical context. Despite the negative charge on the polynucleotide backbone, the guanine amino N2 is able to attract negatively charged groups that could, in the folding of complex RNA molecules, belong to a negative phosphodiester group from a neighboring strand and, in a RNA-protein complex, to a negative carboxyl group of an aspartate or glutamate side chain.     

Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA. Common features of RNA binding in archaea and eukarya

The Sm proteins are conserved in all three domains of life and are always associated with U-rich RNA sequences. Their proposed function is to mediate RNA-RNA interactions. We present here the crystal structures of Pyrococcus abyssi Sm protein (PA-Sm1) and its complex with a uridine heptamer. The overall structure of the protein complex, a heptameric ring with a central cavity, is similar to that proposed for the eukaryotic Sm core complex and found for other archaeal Sm proteins. RNA molecules bind to the protein at two different sites. They interact specifically inside the ring with three highly conserved residues, defining the uridine-binding pocket. In addition, nucleotides also interact on the surface formed by the N-terminal alpha-helix as well as a conserved aromatic residue in beta-strand 2 of the PA-Sm1 protein. The mutation of this conserved aromatic residue shows the importance of this second site for the discrimination between RNA sequences. Given the high structural homology between archaeal and eukaryotic Sm proteins, the PA-Sm1.RNA complex provides a model for how the small nuclear RNA contacts the Sm proteins in the Sm core. In addition, it suggests how Sm proteins might exert their function as modulators of RNA-RNA interactions.     

Radial transport of abscisic acid conjugates in maize roots: its implication for long distance stress signals

Conjugated, alkaline hydrolysable ABA (predominantly abscisic acid glucose ester, ABA-GE), which is transported in the xylem from roots to shoots of Zea mays L. plants, has its origin in the root symplast rather than from soil, although it was detectable in soil solution with concentrations up to 30 nM. External ABA glucose ester cannot be dragged with the water flow across the exodermis and the endodermis because of its hydrophobic properties. Experimental evidence is presented that enzymes in the cortical apoplast cleave ABA-GE thus releasing ABA from its conjugates. Liberated ABA can then be translocated apoplastically and symplastically to the xylem vessels. Endogenous ABA-GE can be released from isolated cortical and stelar tissues to the surrounding media, with rates that are up to 5-fold higher from stelar tissues than those from cortical tissues. Release of ABA-GE is highest under conditions of inhibited ABA-metabolism.     

A rev-like NES mediates cytoplasmic localization of HERV-K cORF

The human endogenous retrovirus K (HERV-K)-encoded protein cORF has recently been shown to be a functional homolog of the HIV Rev protein. Rev-mediated RNA export requires interaction between a leucine-rich nuclear export signal (NES) in Rev and the nuclear export receptor Crm1/exportin1. Like Rev, cORF binds to Crm1 and cORF-mediated RNA export depends on Crm1 activity. Here we document that mutation of the putative NES in cORF results in trapping of the protein in the nucleus, suggesting that the cORF NES functions in analogy to the Rev NES.