Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation

AbstractThe PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface

A Case Study of Stratospheric Ozone Affecting Ground-Level Oxidant Concentrations

During the predawn hours of 19 November 1972, the air pollution monitoring station at Santa Rosa, Calif., recorded five consecutive hours of oxidant concentrations in excess of the present National Ambient Air Quality Standard. The highest of the hourly averages was 0.23 ppm. From a detailed analysis of the meteorological conditions surrounding this incident, it is shown that the ozone responsible for the anomalous concentrations originated in the stratosphere and not from anthropogenic sources

Numerico-empirical analyses of atmospheric diffusion theories

Numerico-empirical expressions for the particle displacement probability density function from which the mean concentration of material in turbulent fluid may be obtained are derived from the numerical planetary boundary layer model of Deardorff. These expressions are then used to compute profiles of the mean, cross-wind-integrated concentration of an inert pollutant issuing from a continuous point source below a stable layer. Profiles are derived for each of two conditions of atmospheric stability: zi/L=0 and –4.5, where zi is the inversion base height and L the Monin-Obukhov length. The resulting concentration profiles [referred to as the numerico-empirical (NE) profiles] are then used in two separate experiments designed to assess the adequacy of conventional atmospheric diffusion formulations. First, the validity of the atmospheric diffusion equation is assessed by determining for each of the two stabilities cited above the profile of vertical eddy diffusivity that produces the closest fit of the mean concentration predicted by the atmospheric diffusion equation with the NE profiles. Second, comparisons are made between the NE profiles and the corresponding concentration distributions predicted by the Gaussian plume formula with Pasquill-Gifford dispersion parameters, and the Gaussian puff equation with McElroy-Pooler travel-time-dependent dispersion parameters

Fusion Protein of the Paramyxovirus SV5: Destabilizing and Stabilizing Mutants of Fusion Activation

AbstractThe fusion (F) protein of the paramyxovirus SV5 strain W3A causes syncytium formation without coexpression of the SV5 hemagglutinin-neuraminidase (HN) glycoprotein, whereas the F protein of the SV5 strain WR requires coexpression of HN for fusion activity. SV5 strains W3A and WR differ by three amino acid residues at positions 22, 443, and 516. The W3A F protein residues P22, S443, and V516 were changed to amino acids found in the WR F protein (L22, P443, and A516, respectively). Three single-mutants, three double-mutants, and the triple-mutant were constructed, expressed, and assayed for fusion using three different assays. Mutant P22L did not cause fusion under physiological conditions, but fusion was activated at elevated temperatures. Compared with the W3A F protein, mutant S443P enhanced the fusion kinetics with a faster rate and greater extent, and had a lower activation temperature. Mutant V516A had little effect on F protein-mediated fusion. The double-mutant P22L,S443P was capable of causing fusion, suggesting that the two mutations have opposing effects on fusion activation. The WR F protein requires coexpression of HN to cause fusion at 37°C, and does not cause fusion at 37°C when coexpressed with influenza virus hemagglutinin (HA); however, at elevated temperatures coexpression of WR F protein with HA resulted in fusion activation. In the crystal structure of the core trimer of the SV5 F protein (Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309–319), S443 is the last residue (with interpretable electron density) in an extended chain region and the temperature factor for S443 is high, suggesting conformational flexibility at this point. Thus, the presence of prolines at residues 22 and 443 may destabilize the F protein and thereby decrease the energy required to trigger the presumptive conformational change to the fusion-active state

Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures

AbstractParamyxoviruses enter cells by fusion of their lipid envelope with the target cell plasma membrane. Fusion of the viral membrane with the plasma membrane allows entry of the viral genome into the cytoplasm. For paramyxoviruses, membrane fusion occurs at neutral pH, but the trigger mechanism that controls the viral entry machinery such that it occurs at the right time and in the right place remains to be elucidated. Two viral glycoproteins are key to the infection process—an attachment protein that varies among different paramyxoviruses and the fusion (F) protein, which is found in all paramyxoviruses. For many of the paramyxoviruses (parainfluenza viruses 1–5, mumps virus, Newcastle disease virus and others), the attachment protein is the hemagglutinin/neuraminidase (HN) protein. In the last 5 years, atomic structures of paramyxovirus F and HN proteins have been reported. The knowledge gained from these structures towards understanding the mechanism of viral membrane fusion is described

A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity

This is the published version, also available here: http://dx.doi.org/10.1083/jcb.140.6.1463.The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members

Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

This is the published version, also available here: http://dx.doi.org/10.1091/mbc.9.12.3505.Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a DrosophilaProtein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor ofEgfr Elp, a hypermorphic form of theDrosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis ofcoracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphiccoracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults

Influenza B virus BM2 protein is an oligomeric integral membrane protein expressed at the cell surface

AbstractThe influenza B virus BM2 protein contains 109 amino acid residues and it is translated from a bicistronic mRNA in an open reading frame that is +2 nucleotides with respect to the matrix (M1) protein. The amino acid sequence of BM2 contains a hydrophobic region (residues 7–25) that could act as a transmembrane (TM) anchor. Analysis of properties of the BM2 protein, including detergent solubility, insolubility in alkali pH 11, flotation in membrane fractions, and epitope-tagging immunocytochemistry, indicates BM2 protein is the fourth integral membrane protein encoded by influenza B virus in addition to hemagglutinin (HA), neuraminidase (NA), and the NB glycoprotein. Biochemical analysis indicates that the BM2 protein adopts an NoutCin orientation in membranes and fluorescence microscopy indicates BM2 is expressed at the cell surface. As the BM2 protein possesses only a single hydrophobic domain and lacks a cleavable signal sequence, it is another example of a Type III integral membrane protein, in addition to M2, NB, and CM2 proteins of influenza A, B, and C viruses, respectively. Chemical cross-linking studies indicate that the BM2 protein is oligomeric, most likely a tetramer. Comparison of the amino acid sequence of the TM domain of the BM2 protein with the sequence of the TM domain of the proton-selective ion channel M2 protein of influenza A virus is intriguing as M2 protein residues critical for ion selectivity/activation and channel gating (H37 and W41, respectively) are found at the same relative position and spacing in the BM2 protein (H19 and W23)

A stochastic-Lagrangian particle system for the Navier-Stokes equations

This paper is based on a formulation of the Navier-Stokes equations developed by P. Constantin and the first author (\texttt{arxiv:math.PR/0511067}, to appear), where the velocity field of a viscous incompressible fluid is written as the expected value of a stochastic process. In this paper, we take $N$ copies of the above process (each based on independent Wiener processes), and replace the expected value with $\frac{1}{N}$ times the sum over these $N$ copies. (We remark that our formulation requires one to keep track of $N$ stochastic flows of diffeomorphisms, and not just the motion of $N$ particles.) We prove that in two dimensions, this system of interacting diffeomorphisms has (time) global solutions with initial data in the space \holderspace{1}{\alpha} which consists of differentiable functions whose first derivative is $\alpha$ H\"older continuous (see Section \ref{sGexist} for the precise definition). Further, we show that as $N \to \infty$ the system converges to the solution of Navier-Stokes equations on any finite interval $[0,T]$. However for fixed $N$, we prove that this system retains roughly $O(\frac{1}{N})$ times its original energy as $t \to \infty$. Hence the limit $N \to \infty$ and $T\to \infty$ do not commute. For general flows, we only provide a lower bound to this effect. In the special case of shear flows, we compute the behaviour as $t \to \infty$ explicitly.Comment: v3: Typo fixes, and a few stylistic changes. 17 pages, 2 figure