Packaging determinants in the UL11 tegument protein of herpes simplex virus type 1

The UL11 gene of herpes simplex virus type 1 encodes a 96-amino-acid tegument protein that is myristylated, palmitylated, and phosphorylated and is found on the cytoplasmic faces of nuclear, Golgi apparatus-derived, and plasma membranes of infected cells. Although this protein is thought to play a role in virus budding, its specific function is unknown. Purified virions were found to contain approximately 700 copies of the UL11 protein per particle, making it an abundant component of the tegument. Moreover, comparisons of cell-associated and virion-associated UL11 showed that packaging is selective for underphosphorylated forms, as has been reported for several other tegument proteins. Although the mechanism by which UL11 is packaged is unknown, previous studies have identified several sequence motifs in the protein that are important for membrane binding, intracellular trafficking, and interaction with UL16, another tegument protein. To ascertain whether any of these motifs are needed for packaging, a transfection/infection-based assay was used in which mutant forms of the protein must compete with the wild type. In this assay, the entire C-terminal half of UL11 was found to be dispensable. In the N-terminal half, the sites of myristylation and palmitylation, which enable membrane-binding and Golgi apparatus-specific targeting, were found to be essential for efficient packaging. The acidic cluster motif, which is not needed for Golgi apparatus-specific targeting but is involved in recycling the protein from the plasma membrane and for the interaction with UL16, was found to be essential, too. Thus, something other than mere localization of UL11 to Golgi apparatus-derived membranes is needed for packaging. The critical factor is unlikely to be the interaction with UL16 because other mutants that fail to bind this protein (due to removal of the dileucine-like motif or substitutions with foreign acidic clusters) were efficiently packaged. Collectively, these results suggest that UL11 packaging is not driven by a passive mechanism but instead requires trafficking through a specific pathway.     

Characterization of the N-deacetylase domain from the heparan sulfate N-deacetylase/N-sulfotransferase 2

Heparin and heparan sulfate are linear sulfated polysaccharides that exert a multitude of biological functions. Heparan sulfate glucosaminyl N-deacetylase/N-sulfotransferase isoform 2 (NDST-2), a key enzyme in the biosynthesis of heparin, contains two distinct activities. This bifunctional enzyme removes the acetyl group from N-acetylated glucosamine (N-deacetylase activity) and transfers a sulfuryl group to the unsubstituted amino position (N-sulfotransferase activity). The N-sulfotransferase activity of NDST has been unambiguously localized to the C-terminal domain of NDST. Here, we report that the N-terminal domain of NDST-2 retains N-deacetylase activity. The N-terminal domain (A66-P604) of human NDST-2, designated as N-deacetylase (NDase), was cloned as a (His)(6)-fusion protein, and protein expression was carried out in Escherichia coli. Heparosan treated with NDase contains N-unsubstituted glucosamine and is highly susceptible to N-sulfation by N-sulfotransferase. Our results conclude that the N-terminal domain of NDST-2 contains functional N-deacetylase activity. This finding helps further elucidate the mechanism of action of heparan sulfate N-deacetylase/N-sulfotransferases and the biosynthesis of heparan sulfate in general.     

Epithelial Adhesion Mediated by Pilin SpaC Is Required for Lactobacillus rhamnosus GG-Induced Cellular Responses

Lactobacillus rhamnosus GG is a widely used probiotic, and the strain''s salutary effects on the intestine have been extensively documented. We previously reported that strain GG can modulate inflammatory signaling, as well as epithelial migration and proliferation, by activating NADPH oxidase 1-catalyzed generation of reactive oxygen species (ROS). However, how strain GG induces these responses is unknown. Here, we report that strain GG''s probiotic benefits are dependent on the bacterial-epithelial interaction mediated by the SpaC pilin subunit. By comparing strain GG to an isogenic mutant that lacks SpaC (strain GGΩspaC), we establish that SpaC is necessary for strain GG to adhere to gut mucosa, that SpaC contributes to strain GG-induced epithelial generation of ROS, and that SpaC plays a role in strain GG''s capacity to stimulate extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in enterocytes. In addition, we show that SpaC is required for strain GG-mediated stimulation of cell proliferation and protection against radiologically inflicted intestinal injury. The identification of a critical surface protein required for strain GG to mediate its probiotic influence advances our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat intestinal diseases.     

Lrp4 Regulates Initiation of Ureteric Budding and Is Crucial for Kidney Formation – A Mouse Model for Cenani-Lenz Syndrome

Background

Development of the kidney is initiated when the ureteric bud (UB) branches from the Wolffian duct and invades the overlying metanephric mesenchyme (MM) triggering the mesenchymal/epithelial interactions that are the basis of organ formation. Multiple signaling pathways must be integrated to ensure proper timing and location of the ureteric bud formation.

Methods and Principal Findings

We have used gene targeting to create an Lrp4 null mouse line. The mutation results in early embryonic lethality with a subpenetrant phenotype of kidney agenesis. Ureteric budding is delayed with a failure to stimulate the metanephric mesenchyme in a timely manner, resulting in failure of cellular differentiation and resulting absence of kidney formation in the mouse as well as comparable malformations in humans with Cenani-Lenz syndrome.

Conclusion

Lrp4 is a multi-functional receptor implicated in the regulation of several molecular pathways, including Wnt and Bmp signaling. Lrp4^−/− mice show a delay in ureteric bud formation that results in unilateral or bilateral kidney agenesis. These data indicate that Lrp4 is a critical regulator of UB branching and lack of Lrp4 results in congenital kidney malformations in humans and mice.     

gA and gB glycoproteins of herpes simplex virus type 1: two forms of a single polypeptide.

Utilizing a combination of preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-hydroxylapatite column chromatography, we have separated and purified the gA and gB glycoproteins of the major virus-specific glycoprotein region from herpes simplex virus type 1-infected cells. By using purified antigen preparations, antisera specific to each of these glycoproteins were produced. Immunoprecipitation from detergent extracts of infected cells and radioimmune precipitation of the purified antigens have shown that the anti-gA and anti-gB sera each recognize both the gA and the gB glycoproteins. The anti-gA serum was also shown to neutralize virus despite the presence of only minute quantities of the gA glycoprotein in virions. Pulse-chase studies have indicated that the gA and gB glycoproteins are synthesized from a common precursor polypeptide. Together, these data demonstrate that the gA and gB glycoproteins of herpes simplex virus type 1 are antigenically similar but not identical and probably represent two different forms of the same polypeptide which differ in their degree of glycosylation.     

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.     

PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12

Most peroxisomal enzymes are targeted to peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1) at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Like its C-terminal fragment, full-length tetrameric PEX5 exhibits high intrinsic affinity for the PTS1, with a K(d) of 35 nm for the peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO(-). The specificity of this interaction was demonstrated by the fact that PEX5 had no detectable affinity for a peptide in which the Lys was replaced with Glu, a substitution that inactivates PTS1 signals in vivo. Hsp70 has been found to regulate the affinity of PEX5 for a PTS1-containing protein, but we found that the kinetics of PEX5-PTS1 binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In addition, we found that another protein known to interact with the PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no discernable effect on PEX5-PTS1 binding kinetics. Taken together, these results suggest that the initial step in peroxisomal protein import, the recognition of enzymes by PEX5, is a relatively simple process and that Hsp70 most probably stimulates this process by catalyzing the folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.     

The value of wetlands for water quality improvement: an example from the St. Johns River watershed,Florida

Wetlands provide many valuable ecosystem functions such as sediment and nutrient retention, high biological productivity and biodiversity, flood control, and opportunities to recreate. Despite their importance, estimating the value of wetlands is difficult as the worth of these functions and services is not easily quantified. The overall objective of this study was to estimate the value of freshwater wetlands in the Saint Johns River (SJR) watershed, Florida based on their ability to remove nutrients, namely nitrogen (N) and phosphorus (P). We used a combination of literature review, geospatial analysis of land cover, and regression analysis to determine the total wetland area in the SJR watershed and the rates of nitrogen and phosphorus burial in the wetlands. We then estimated the economic value of these wetlands based on the replacement cost of nutrient removal by wastewater treatment plants. Nitrogen burial rates ranged from 27 g/m²/year to a background rate of 6.56 g/m²/year, and phosphorus burial rates range from 1.31 g/m²/year to a background of 0.11 g/m²/year. Using these rates, we calculate wetlands of the SJR catchment remove 79,873 MT of nitrogen annually just from burial in the soil, with a replacement cost of between $240 million to $150 billion per year. The amount of phosphorus buried yearly is more than 2400 MT with an annual replacement cost of $17 to $497 million. Though they are based on limited data and include a variety of watershed-scale research limitations, these findings highlight the significant potential value of conserving functional wetlands based solely on their nutrient retention functions. If we were to consider the benefits associated with other wetland functions such as flood control, biological productivity, and biodiversity in addition to their ability to retain nutrients, the value of the SJR wetlands would be even greater.     

Mucosal allergic sensitization to cockroach allergens is dependent on proteinase activity and proteinase-activated receptor-2 activation

We have shown that proteinase-activated receptor-2 (PAR(2)) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR(2) in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR(2). To study the role of PAR(2) in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR(2) activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR(2), mice were exposed intranasally to a receptor-blocking anti-PAR(2) Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR(2) blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR(2) activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR(2)-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR(2) activation may be a general mechanism used by aeroallergens to induce allergic sensitization.