Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphospborylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5' end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Thermodynamic and kinetic properties of interpolymer complexes assessed by isothermal titration calorimetry and surface plasmon resonance

Interpolymer complexes (IPCs) formed between complimentary polymers in solution have shown a wide range of applications from drug delivery to biosensors. This work describes the combined use of isothermal titration calorimetry and surface plasmon resonance to investigate the thermodynamic and kinetic processes during hydrogen-bonded interpolymer complexation. Varied polymers that are commonly used in layer-by-layer coatings and pharmaceutical preparations were selected to span a range of chemical functionalities including some known IPCs previously characterized by other techniques, and other polymer combinations with unknown outcomes. This work is the first to comprehensively detail the thermodynamic and kinetic data of hydrogen bonded IPCs, aiding understanding and detailed characterization of the complexes. The applicability of the two techniques in determining thermodynamic, gravimetric and kinetic properties of IPCs is considered.

On the distortion of turbulence by a progressive surface wave

A rapid-distortion model is developed to investigate the interaction of weak turbulence with a monochromatic irrotational surface water wave. The model is applicable when the orbital velocity of the wave is larger than the turbulence intensity, and when the slope of the wave is sufficiently high that the straining of the turbulence by the wave dominates over the straining of the turbulence by itself. The turbulence suffers two distortions. Firstly, vorticity in the turbulence is modulated by the wave orbital motions, which leads to the streamwise Reynolds stress attaining maxima at the wave crests and minima at the wave troughs; the Reynolds stress normal to the free surface develops minima at the wave crests and maxima at the troughs. Secondly, over several wave cycles the Stokes drift associated with the wave tilts vertical vorticity into the horizontal direction, subsequently stretching it into elongated streamwise vortices, which come to dominate the flow. These results are shown to be strikingly different from turbulence distorted by a mean shear flow, when `streaky structures' of high and low streamwise velocity fluctuations develop. It is shown that, in the case of distortion by a mean shear flow, the tendency for the mean shear to produce streamwise vortices by distortion of the turbulent vorticity is largely cancelled by a distortion of the mean vorticity by the turbulent fluctuations. This latter process is absent in distortion by Stokes drift, since there is then no mean vorticity. The components of the Reynolds stress and the integral length scales computed from turbulence distorted by Stokes drift show the same behaviour as in the simulations of Langmuir turbulence reported by McWilliams, Sullivan & Moeng (1997). Hence we suggest that turbulent vorticity in the upper ocean, such as produced by breaking waves, may help to provide the initial seeds for Langmuir circulations, thereby complementing the shear-flow instability mechanism developed by Craik & Leibovich (1976). The tilting of the vertical vorticity into the horizontal by the Stokes drift tends also to produce a shear stress that does work against the mean straining associated with the wave orbital motions. The turbulent kinetic energy then increases at the expense of energy in the wave. Hence the wave decays. An expression for the wave attenuation rate is obtained by scaling the equation for the wave energy, and is found to be broadly consistent with available laboratory data.

A global perspective on Langmuir turbulence in the ocean surface boundary layer.

The turbulent mixing in thin ocean surface boundary layers (OSBL), which occupy the upper 100 m or so of the ocean, control the exchange of heat and trace gases between the atmosphere and ocean. Here we show that current parameterizations of this turbulent mixing lead to systematic and substantial errors in the depth of the OSBL in global climate models, which then leads to biases in sea surface temperature. One reason, we argue, is that current parameterizations are missing key surface-wave processes that force Langmuir turbulence that deepens the OSBL more rapidly than steady wind forcing. Scaling arguments are presented to identify two dimensionless parameters that measure the importance of wave forcing against wind forcing, and against buoyancy forcing. A global perspective on the occurrence of waveforced turbulence is developed using re-analysis data to compute these parameters globally. The diagnostic study developed here suggests that turbulent energy available for mixing the OSBL is under-estimated without forcing by surface waves. Wave-forcing and hence Langmuir turbulence could be important over wide areas of the ocean and in all seasons in the Southern Ocean. We conclude that surfacewave- forced Langmuir turbulence is an important process in the OSBL that requires parameterization.

Hepatitis C virus NS5A protein binds the SH3 domain of the Fyn tyrosine kinase with high affinity: mutagenic analysis of residues within the SH3 domain that contribute to the interaction

Background: The hepatitis C virus (HCV) non-structural 5A protein (NS5A) contains a highly conserved C-terminal polyproline motif with the consensus sequence Pro-X-X- Pro-X-Arg that is able to interact with the Src-homology 3 (SH3) domains of a variety of cellular proteins. Results: To understand this interaction in more detail we have expressed two N-terminally truncated forms of NS5A in E. coli and examined their interactions with the SH3 domain of the Src-family tyrosine kinase, Fyn. Surface plasmon resonance analysis revealed that NS5A binds to the Fyn SH3 domain with what can be considered a high affinity SH3 domain-ligand interaction (629 nM), and this binding did not require the presence of domain I of NS5A (amino acid residues 32-250). Mutagenic analysis of the Fyn SH3 domain demonstrated the requirement for an acidic cluster at the C-terminus of the RT-Src loop of the SH3 domain, as well as several highly conserved residues previously shown to participate in SH3 domain peptide binding. Conclusion: We conclude that the NS5A: Fyn SH3 domain interaction occurs via a canonical SH3 domain binding site and the high affinity of the interaction suggests that NS5A would be able to compete with cognate Fyn ligands within the infected cell.

Glycosaminoglycans and protein disulfide isomerase-mediated reduction of HIV Env

Conformational changes within the human immunodeficiency virus-1 (HIV-1) surface glycoprotein gp120 result from binding to the lymphocyte surface receptors and trigger gp41-mediated virus/cell membrane fusion. The triggering of fusion requires cleavage of two of the nine disulfide bonds of gp120 by a cell-surface protein disulfide-isomerase (PDI). Soluble glycosaminoglycans such as heparin and heparan sulfate bind gp120 via V3 and, possibly, a CD4-induced domain. They exert anti-HIV activity by interfering with the HIV envelope glycoprotein ( Env)/cell-surface interaction. Env also binds cell-surface glycosaminoglycans. Here, using surface plasmon resonance, we observed an inverse relationship between heparin binding by gp120 and its thiol content. In vitro, and in conditions in which gp120 could bind CD4, heparin and heparan sulfate reduced PDI-mediated gp120 reduction by approximately 80%. Interaction of Env with the surface of lymphocytes treated using sodium chlorate, an inhibitor of glycosaminoglycan synthesis, led to gp120 reduction. We conclude that besides their capacity to block Env/cell interaction, soluble glycosaminoglycans can effect anti-HIV activity via interference with PDI- mediated gp120 reduction. In contrast, their presence at the cell surface is dispensable for Env reduction during the course of interaction with the lymphocyte surface. This work suggests that the reduction of exofacial proteins in various diseases can be inhibited by compounds targeting the substrates ( not by targeting PDI, as is usually done), and that glycosaminoglycans that primarily protect proteins by preserving them from proteolysis also have a role in preventing reduction.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC50 value of 5.521 mg/mL is high compared with 0.2811 mu g/mL (182.5 pM or 1.825 x 10(-10) M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E (R) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%. 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease. (C) 2009 Elsevier Ltd. All rights reserved

DC-SIGN increases the affinity of HIV-1 envelope glycoprotein interaction with CD4

Mannose-binding C-type lectin receptors, expressed on Langerhans cells and subepithelial dendritic cells (DCs) of cervico-vaginal tissues, play an important role in HIV-1 capture and subsequent dissemination to lymph nodes. DC-SIGN has been implicated in both productive infection of DCs and the DC-mediated trans infection of CD4(+) T cells that occurs in the absence of replication. However, the molecular events that underlie this efficient transmission have not been fully defined. In this study, we have examined the effect of the extracellular domains of DC-SIGN and Langerin on the stability of the interaction of the HIV-1 envelope glycoprotein with CD4 and also on replication in permissive cells. Surface plasmon resonance analysis showed that DC-SIGN increases the binding affinity of trimeric gp140 envelope glycoproteins to CD4. In contrast, Langerin had no effect on the stability of the gp140:CD4 complex. In vitro infection experiments to compare DC-SIGN enhancement of CD4-dependent and CD4-independent strains demonstrated significantly lower enhancement of the CD4-independent strain. In addition DC-SIGN increased the relative rate of infection of the CD4-dependent strain but had no effect on the CD4-independent strain. DC-SIGN binding to the HIV envelope protein effectively increases exposure of the CD4 binding site, which in turn contributes to enhancement of infection.

Optimizing layer-by-layer deposition of interpolymer complexes on solid substrates using Biacore

The layer-by-layer deposition of polymers onto surfaces allows the fabrication of multilayered materials for a wide range of applications, from drug delivery to biosensors. This work describes the analysis of complex formation between poly(acrylic acid) and methylcellulose in aqueous solutions using Biacore, a surface plasmon resonance analytical technique, traditionally used to examine biological interactions. This technique characterized the layer-by-layer deposition of these polymers on the surface of a Biacore sensor chip. The results were subsequently used to optimize the experimental conditions for sequential layer deposition on glass slides. The role of the solution pH and poly(acrylic acid) molecular weight on the formation of interpolymer multilayered coatings was researched, and showed that the optimal deposition of the polymer complexes was achieved at pHs ≤2.5 with a poly(acrylic acid) molecular weight of 450 kDa.

On the momentum fluxes associated with mountain waves in directionally sheared flows

The direct impact of mountain waves on the atmospheric circulation is due to the deposition of wave momentum at critical levels, or levels where the waves break. The first process is treated analytically in this study within the framework of linear theory. The variation of the momentum flux with height is investigated for relatively large shears, extending the authors’ previous calculations of the surface gravity wave drag to the whole atmosphere. A Wentzel–Kramers–Brillouin (WKB) approximation is used to treat inviscid, steady, nonrotating, hydrostatic flow with directional shear over a circular mesoscale mountain, for generic wind profiles. This approximation must be extended to third order to obtain momentum flux expressions that are accurate to second order. Since the momentum flux only varies because of wave filtering by critical levels, the application of contour integration techniques enables it to be expressed in terms of simple 1D integrals. On the other hand, the momentum flux divergence (which corresponds to the force on the atmosphere that must be represented in gravity wave drag parameterizations) is given in closed analytical form. The momentum flux expressions are tested for idealized wind profiles, where they become a function of the Richardson number (Ri). These expressions tend, for high Ri, to results by previous authors, where wind profile effects on the surface drag were neglected and critical levels acted as perfect absorbers. The linear results are compared with linear and nonlinear numerical simulations, showing a considerable improvement upon corresponding results derived for higher Ri.

Assessing wind profile effects on the global atmospheric torque

The impact of a new approach to the evaluation of surface gravity wave drag (GWD) is assessed. This approach uses linear theory, but incorporates the effects of wind profile shear and curvature, by means of a second-order WKB approximation. While the theory predicts the possibility of either drag enhancement or reduction, depending on the wind profile, results obtained with the ERA-40 reanalysis data clearly indicate the predominance of local drag enhancement. However, the global impact of shear on the atmospheric axial GWD torque comes mostly from regions with predominantly easterly flow, contributing to a slight reduction of the bias found in different studies of the global angular momentum budget. The relative correction due to shear on linear GWD is found not to depend too strongly on the levels chosen for the computation of the low-level wind derivatives.

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P,0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.