Budding of retroviruses utilizing divergent L domains requires nucleocapsid

We recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPX(n)L L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPX(n)L-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif.     

Multiple-line cross QTL mapping for biomass yield and plant height in triticale (× Triticosecale Wittmack)

Key message

QTL mapping in multiple families identifies trait-specific and pleiotropic QTL for biomass yield and plant height in triticale.

Abstract

Triticale shows a broad genetic variation for biomass yield which is of interest for a range of purposes, including bioenergy. Plant height is a major contributor to biomass yield and in this study, we investigated the genetic architecture underlying biomass yield and plant height by multiple-line cross QTL mapping. We employed 647 doubled haploid lines from four mapping populations that have been evaluated in four environments and genotyped with 1710 DArT markers. Twelve QTL were identified for plant height and nine for biomass yield which cross-validated explained 59.6 and 38.2 % of the genotypic variance, respectively. A major QTL for both traits was identified on chromosome 5R which likely corresponds to the dominant dwarfing gene Ddw1. In addition, we detected epistatic QTL for plant height and biomass yield which, however, contributed only little to the genetic architecture of the traits. In conclusion, our results demonstrate the potential of genomic approaches for a knowledge-based improvement of biomass yield in triticale.     

CELLULOSE SYNTHASE-LIKE A2, a Glucomannan Synthase,Is Involved in Maintaining Adherent Mucilage Structure in Arabidopsis Seed

Mannans are hemicellulosic polysaccharides that are considered to have both structural and storage functions in the plant cell wall. However, it is not yet known how mannans function in Arabidopsis (Arabidopsis thaliana) seed mucilage. In this study, CELLULOSE SYNTHASE-LIKE A2 (CSLA2; At5g22740) expression was observed in several seed tissues, including the epidermal cells of developing seed coats. Disruption of CSLA2 resulted in thinner adherent mucilage halos, although the total amount of the adherent mucilage did not change compared with the wild type. This suggested that the adherent mucilage in the mutant was more compact compared with that of the wild type. In accordance with the role of CSLA2 in glucomannan synthesis, csla2-1 mucilage contained 30% less mannosyl and glucosyl content than did the wild type. No appreciable changes in the composition, structure, or macromolecular properties were observed for nonmannan polysaccharides in mutant mucilage. Biochemical analysis revealed that cellulose crystallinity was substantially reduced in csla2-1 mucilage; this was supported by the removal of most mucilage cellulose through treatment of csla2-1 seeds with endo-β-glucanase. Mutation in CSLA2 also resulted in altered spatial distribution of cellulose and an absence of birefringent cellulose microfibrils within the adherent mucilage. As with the observed changes in crystalline cellulose, the spatial distribution of pectin was also modified in csla2-1 mucilage. Taken together, our results demonstrate that glucomannans synthesized by CSLA2 are involved in modulating the structure of adherent mucilage, potentially through altering cellulose organization and crystallization.Mannan polysaccharides are a complex set of hemicellulosic cell wall polymers that are considered to have both structural and storage functions. Based on the particular chemical composition of the backbone and the side chains, mannan polysaccharides are classified into four types: pure mannan, glucomannan, galactomannan, and galactoglucomannan (Moreira and Filho, 2008; Wang et al., 2012; Pauly et al., 2013). Each of these polysaccharides is composed of a β-1,4-linked backbone containing Man or a combination of Glc and Man residues. In addition, the mannan backbone can be substituted with side chains of α-1,6-linked Gal residues. Mannan polysaccharides have been proposed to cross link with cellulose and other hemicelluloses via hydrogen bonds (Fry, 1986; Iiyama et al., 1994; Obel et al., 2007; Scheller and Ulvskov, 2010). Furthermore, it has been reported that heteromannans with different levels of substitution can interact with cellulose in diverse ways (Whitney et al., 1998). Together, these observations indicate the complexity of mannan polysaccharides in the context of cell wall architecture.CELLULOSE SYNTHASE-LIKE A (CSLA) enzymes have been shown to have mannan synthase activity in vitro. These enzymes polymerize the β-1,4-linked backbone of mannans or glucomannans, depending on the substrates (GDP-Man and/or GDP-Glc) provided (Richmond and Somerville, 2000; Liepman et al., 2005, 2007; Pauly et al., 2013). In Arabidopsis (Arabidopsis thaliana), nine CSLA genes have been identified; different CSLAs are responsible for the synthesis of different mannan types (Liepman et al., 2005, 2007). CSLA7 has mannan synthase activity in vitro (Liepman et al., 2005) and has been shown to synthesize stem glucomannan in vivo (Goubet et al., 2009). Disrupting the CSLA7 gene results in defective pollen growth and embryo lethality phenotypes in Arabidopsis, indicating structural or signaling functions of mannan polysaccharides during plant embryo development (Goubet et al., 2003). A mutation in CSLA9 results in the inhibition of Agrobacterium tumefaciens-mediated root transformation in the rat4 mutant (Zhu et al., 2003). CSLA2, CSLA3, and CSLA9 are proposed to play nonredundant roles in the biosynthesis of stem glucomannans, although mutations in CSLA2, CSLA3, or CSLA9 have no effect on stem development or strength (Goubet et al., 2009). All of the Arabidopsis CSLA proteins have been shown to be involved in the biosynthesis of mannan polysaccharides in the plant cell wall (Liepman et al., 2005, 2007), although the precise physiological functions of only CSLA7 and CSLA9 have been conclusively demonstrated.In Arabidopsis, when mature dry seeds are hydrated, gel-like mucilage is extruded to envelop the entire seed. Ruthenium red staining of Arabidopsis seeds reveals two different mucilage layers, termed the nonadherent and the adherent mucilage layers (Western et al., 2000; Macquet et al., 2007a). The outer, nonadherent mucilage is loosely attached and can be easily extracted by shaking seeds in water. Compositional and linkage analyses suggest that this layer is almost exclusively composed of unbranched rhamnogalacturonan I (RG-I) (>80% to 90%), with small amounts of branched RG-I, arabinoxylan, and high methylesterified homogalacturonan (HG). By contrast, the inner, adherent mucilage layer is tightly attached to the seed and can only be removed by strong acid or base treatment, or by enzymatic digestion (Macquet et al., 2007a; Huang et al., 2011; Walker et al., 2011). As with the nonadherent layer, adherent mucilage is also mainly composed of unbranched RG-I, but with small numbers of arabinan and galactan ramifications (Penfield et al., 2001; Willats et al., 2001; Dean et al., 2007; Macquet et al., 2007a, 2007b; Arsovski et al., 2009; Haughn and Western, 2012). There are also minor amounts of pectic HG in the adherent mucilage, with high methylesterified HG in the external domain compared with the internal domain of the adherent layer (Willats et al., 2001; Macquet et al., 2007a; Rautengarten et al., 2008; Sullivan et al., 2011; Saez-Aguayo et al., 2013). In addition, the adherent mucilage contains cellulose (Blake et al., 2006; Macquet et al., 2007a), which is entangled with RG-I and is thought to anchor the pectin-rich mucilage onto seeds (Macquet et al., 2007a; Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011). As such, Arabidopsis seed mucilage is considered to be a useful model for investigating the biosynthesis of cell wall polysaccharides and how this process is regulated in vivo (Haughn and Western, 2012).Screening for altered seed coat mucilage has led to the identification of several genes encoding enzymes that are involved in the biosynthesis or modification of mucilage components. RHAMNOSE SYNTHASE2/MUCILAGE-MODIFIED4 (MUM4) is responsible for the synthesis of UDP-l-Rha (Usadel et al., 2004; Western et al., 2004; Oka et al., 2007). The putative GALACTURONSYLTRANSFERASE11 can potentially synthesize mucilage RG-I or HG pectin from UDP-d-GalUA (Caffall et al., 2009). GALACTURONSYLTRANSFERASE-LIKE5 appears to function in the regulation of the final size of the mucilage RG-I (Kong et al., 2011, 2013). Mutant seeds defective in these genes display reduced thickness of the extruded mucilage layer compared with wild-type Arabidopsis seeds.RG-I deposited in the apoplast of seed coat epidermal cells appears to be synthesized in a branched form that is subsequently modified by enzymes in the apoplast. MUM2 encodes a β-galactosidase that removes Gal residues from RG-I side chains (Dean et al., 2007; Macquet et al., 2007b). β-XYLOSIDASE1 encodes an α-l-arabinfuranosidase that removes Ara residues from RG-I side chains (Arsovski et al., 2009). Disruptions of these genes lead to defective hydration properties and affect the extrusion of mucilage. Furthermore, correct methylesterification of mucilage HG is also required for mucilage extrusion. HG is secreted into the wall in a high methylesterified form that can then be enzymatically demethylesterified by pectin methylesterases (PMEs; Bosch and Hepler, 2005). PECTIN METHYLESTERASE INHIBITOR6 (PMEI6) inhibits PME activities (Saez-Aguayo et al., 2013). The subtilisin-like Ser protease (SBT1.7) can activate other PME inhibitors, but not PMEI6 (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). Disruption of either PMEI6 or SBT1.7 results in the delay of mucilage release.Although cellulose is present at low levels in adherent mucilage, it plays an important adhesive role for the attachment of mucilage pectin to the seed coat epidermal cells. The orientation and amount of pectin associated with the cellulose network is largely determined by cellulose conformation properties (Macquet et al., 2007a; Haughn and Western, 2012). Previous studies have demonstrated that CELLULOSE SYNTHASE A5 (CESA5) is required for the production of seed mucilage cellulose and the adherent mucilage in the cesa5 mutant can be easily extracted with water (Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011).Despite all of these discoveries, large gaps remain in the current knowledge of the biosynthesis and functions of mucilage polysaccharides in seed coats. In this study, we show that CSLA2 is involved in the biosynthesis of mucilage glucomannan. Furthermore, we show that CSLA2 functions in the maintenance of the normal structure of the adherent mucilage layer through modifying the mucilage cellulose ultrastructure.     

Origins and evolution of AIDS viruses: estimating the time-scale

The primate lentiviruses comprise SIV strains from various host species, as well as two viruses, HIV-1 and HIV-2, that cause AIDS in humans. The origins of HIV-1 and HIV-2 have been traced to cross-species transmissions from chimpanzees and sooty mangabey monkeys respectively. Two approaches have been taken to estimate the time-scale of the evolution of these viruses. Certain groups of SIV strains appear to have evolved in a host-dependent manner, implying a time-scale of many thousands or even millions of years. In stark contrast, molecular clock calculations have previously been used to estimate a time-scale of only tens or hundreds of years. Those calculations largely ignored heterogeneity of evolutionary rates across different sites within sequences. In fact, the distribution of rates at different sites seems extremely skewed in HIV-1, and so the time-depth of the primate lentivirus evolutionary tree may have been underestimated by at least a factor of ten. However, these date estimates still seem to be far too recent to be consistent with host-dependent evolution.     

Identification of Cell-Binding Adhesins of Leptospira interrogans

Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and vaccines.     

Inhibition of recombinant human maltase glucoamylase by salacinol and derivatives

Inhibitors targeting pancreatic alpha-amylase and intestinal alpha-glucosidases delay glucose production following digestion and are currently used in the treatment of Type II diabetes. Maltase-glucoamylase (MGA), a family 31 glycoside hydrolase, is an alpha-glucosidase anchored in the membrane of small intestinal epithelial cells responsible for the final step of mammalian starch digestion leading to the release of glucose. This paper reports the production and purification of active human recombinant MGA amino terminal catalytic domain (MGAnt) from two different eukaryotic cell culture systems. MGAnt overexpressed in Drosophila cells was of quality and quantity suitable for kinetic and inhibition studies as well as future structural studies. Inhibition of MGAnt was tested with a group of prospective alpha-glucosidase inhibitors modeled after salacinol, a naturally occurring alpha-glucosidase inhibitor, and acarbose, a currently prescribed antidiabetic agent. Four synthetic inhibitors that bind and inhibit MGAnt activity better than acarbose, and at comparable levels to salacinol, were found. The inhibitors are derivatives of salacinol that contain either a selenium atom in place of sulfur in the five-membered ring, or a longer polyhydroxylated, sulfated chain than salacinol. Six-membered ring derivatives of salacinol and compounds modeled after miglitol were much less effective as MGAnt inhibitors. These results provide information on the inhibitory profile of MGAnt that will guide the development of new compounds having antidiabetic activity.     

Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation

High cell density fed-batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3-hydroxybutyrate) (PHB) in a 60-L fermentor. During the fermentation, pH was controlled with NH(4)OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0-20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed-batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L . h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28-32, 1997.