RNA interference against animal viruses: how morbilliviruses generate extended diversity to escape small interfering RNA control

Viruses are serious threats to human and animal health. Vaccines can prevent viral diseases, but few antiviral treatments are available to control evolving infections. Among new antiviral therapies, RNA interference (RNAi) has been the focus of intensive research. However, along with the development of efficient RNAi-based therapeutics comes the risk of emergence of resistant viruses. In this study, we challenged the in vitro propensity of a morbillivirus (peste des petits ruminants virus), a stable RNA virus, to escape the inhibition conferred by single or multiple small interfering RNAs (siRNAs) against conserved regions of the N gene. Except with the combination of three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The genetic modifications involved consisted of single or multiple point nucleotide mutations and a deletion of a stretch of six nucleotides, illustrating that this virus has an unusual genomic malleability.     

TRPM1 Is Mutated in Patients with Autosomal-Recessive Complete Congenital Stationary Night Blindness

Night vision requires signaling from rod photoreceptors to adjacent bipolar cells in the retina. Mutations in the genes NYX and GRM6, expressed in ON bipolar cells, lead to a disruption of the ON bipolar cell response. This dysfunction is present in patients with complete X-linked and autosomal-recessive congenital stationary night blindness (CSNB) and can be assessed by standard full-field electroretinography (ERG), showing severely reduced rod b-wave amplitude and slightly altered cone responses. Although many cases of complete CSNB (cCSNB) are caused by mutations in NYX and GRM6, in ∼60% of the patients the gene defect remains unknown. Animal models of human diseases are a good source for candidate genes, and we noted that a cCSNB phenotype present in homozygous Appaloosa horses is associated with downregulation of TRPM1. TRPM1, belonging to the family of transient receptor potential channels, is expressed in ON bipolar cells and therefore qualifies as an excellent candidate. Indeed, mutation analysis of 38 patients with CSNB identified ten unrelated cCSNB patients with 14 different mutations in this gene. The mutation spectrum comprises missense, splice-site, deletion, and nonsense mutations. We propose that the cCSNB phenotype in these patients is due to the absence of functional TRPM1 in retinal ON bipolar cells.     

Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2

Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.     

Proton magnetic resonance studies of the binding of oligopeptides containing tryptophan to polyribonucleotides poly A, poly U and poly C

The binding of oligopeptides Lys-Trp-Gly-Lys OtBu, Lys-Gly-Trp-Lys OtBu and Lys-Trp-Lys to Polyadenylic, Polycytidylic and Polyuridylic acid has been studied by Proton NMR at 90 MHz and 500 MHz at oligopeptide/Polynucleotide ratios ranging from 0.01 to 0.20 at 275-365 K. Downfield shift of 0.01-0.2 ppm at 296 K of the H2, H8 and H1' resonances of Poly A due to binding with oligopeptides is accompanied by a marked narrowing of resonance lines of Poly A. The ring protons of tryptophan shift upfield by 0.3-0.6 ppm at 296 K on binding to Poly A. Changes in chemical shift of both adenine and tryptophan protons on binding are much smaller at 355 K than that at 275 K. These observations are ascribed to intercalation of the tryptophan ring in the adenine bases resulting in partial destacking of adenine bases in Poly A. Using the magnetic anisotropy ring current shifts, an overlap geometry of tryptophan ring in the adenine has been proposed. Addition of oligopeptides to Poly C and Poly U, on the other hand, suggests that tryptophan ring does not stack in Poly U and Poly C.     

Solution structure of the parallel-stranded duplex oligonucleotide alpha-d(TCTAAAC)-beta-d(AGATTTG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations

The solution structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAAC) with its natural and parallel complementary sequence beta-d(AGATTTG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. The structure was refined on the basis of interproton distances determined by NOE measurements for a series of mixing times. The NOE values were converted to distances by using the complete 134 x 134 relaxation matrix including all proton dipole-dipole interactions and spin diffusion. The computation of the relaxation matrix requires the Cartesian coordinates of the oligonucleotide, which are not known, a priori. To avoid this ambiguity, we used an iterative procedure in which the new distance constraints are obtained by using the complete relaxation matrix calculated from the previous structure. After three iterations, the process converged. The unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG) adopts in solution a right-helical structure with Watson-Crick base pairing, an anti conformation on the glycosyl linkage on the beta-strand, a syn conformation on the alpha-strand, and a 3'-exo conformation of the deoxyriboses for both sugar anomers. The three-dimensional structure obtained allowed us to describe the local heterogeneity of the duplex.     

The role of iron in the bacterial degradation of organic matter derived from Phaeocystis antarctica

In high-nutrient low-chlorophyll areas, bacterial degradation of organic matter may be iron-limited. The response of heterotrophic bacteria to Fe addition may be directly controlled by Fe availability and/or indirectly controlled through the effect of enhanced phytoplankton productivity and the subsequent supply of organic matter suitable for bacteria. In the present study, the role of Fe on bacterial carbon degradation was investigated through regrowth experiments by monitoring bacterial response to organic substrates derived from Phaeocystis antarctica cultures set up in <1 nM Fe (LFe) and in Fe-amended (HFe) Antarctic seawater. Results showed an impact of Fe addition on the morphotype dominance (colonies vs. single cells) of P. antarctica and on the quality of Phaeocystis-derived organic matter. Fe addition leaded to a decrease of C/N ratio of Phaeocystis material. The bacterial community composition was modified as observed from denaturing gradient gel electrophoresis (DGGE) profiles in LFe as compared to HFe bioassays. The percentage of active bacteria as well as their specific metabolic activities (ectoenzymatic hydrolysis, growth rates and bacterial growth efficiency) were enhanced in HFe bioassays. As a consequence, the lability of Phaeocystis-derived organic matter was altered, i.e., after seven days more than 90% was degraded in HFe and only 9% (dissolved) and 55% (total) organic carbon were degraded in LFe bioassays. By inducing increased bacterial degradation and preventing the accumulation of dissolved organic carbon, the positive effect of Fe supply on the carbon biological pump may partly be counteracted.