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PubMed2006

A heterologous system for assembly of retroviral gene vectors: intracellular budding in yeast?

Mammalian retroviral gene vectors are of particular importance in gene therapy because of their efficient chromosomal integration and resulting stable maintenance of transgenes. These vectors are currently produced in mammalian cells, with low yield and at substantial expense. Therefore, a more efficient heterologous production system for retroviral vectors would be desirable. With their impressive track record in biotechnology, various yeast species appear like ideal organisms to generate retroviral vectors. Typically, retroviral vector particles emerge from mammalian cells after budding at the plasma membrane. However, in yeast, viral budding at the plasma membrane is blocked by the cell wall. At the same time, mass production of enveloped viral vectors in yeast protoplasts is technically challenging. Recent reports indicated the generation of infectious virions via intracellular budding for some combinations of retroviruses and mammalian cells. Relying on these data I hypothesise that a successful assembly of the retroviral transducing particles can be accomplished intracellularly inside yeast cells with a normal cell wall. Firstly, it is possible that some of the intracellular yeast compartments have all the necessary host factors that are required for successful RNA packaging, budding and maturation of infectious retroviral vector particles. Secondly, it might be possible to improve intracellular viral vector production by artificially targeting viral cores to bud at specific intracellular vesicular structures using appropriate targeting or retention signals. A suitable envelope protein, conferring infectivity and specific cellular tropism to the vector particles, can be expressed in yeast or, alternatively, 'bald' viral particles without envelope protein can be produced in yeast and later complexed with a desired envelope protein in vitro. Retroviral budding on yeast intracellular membranes can rely on the same host factors that are used by yeast retrotransposons driving intracellular formation of non-infectious virus-like particles. It is likely that optimal vector packaging system can be found as a result of the dedicated screening of the various retroviral vectors against an array of yeast species. The central implication of the hypothesis is that budding of retroviral vector particles inside yeast cells, with no need for obtaining and maintaining yeast spheroplasts or protoplasts, can be an efficient and economical method of mass production of these valuable gene therapy vectors.

Biotechnology↗

PubMed1993

How does a virus bud?

How does a virus bud from the plasma membrane of its host? Here we investigate several possible rate-limiting processes, including thermal fluctuations of the plasma membrane, hydrodynamic interactions, and diffusion of the glycoprotein spikes. We find that for bending moduli greater than 3 x 10(-13) ergs, membrane thermal fluctuations are insufficient to wrap the viral capsid, and the mechanical force driving the budding process must arise from some other process. If budding is limited by the rate at which glycoprotein spikes can diffuse to the budding site, we compute that the budding time is 10-20 min, in accord with the experimentally determined upper limit of 20 min. In light of this, we suggest some alternative mechanisms for budding and provide a rationale for the observation that budding frequently occurs in regions of high membrane curvature.

Animals↗

PubMed2000

Peroral infection of nuclear polyhedrosis virus budded particles in the host, Bombyx mori l., enabled by an optical brightener, Tinopal UNPA-GX.

Perorally inoculated budded particles of a nuclear polyhedrosis virus was used to infect Bombyx mori (BmNPV) (Lepidoptera; Bombycidae), aided by an optical brightener, Tinopal UNPA-GX (Tinopal). BmNPV budded particles not occluded in the occlusion body do not infect successfully the host, B. mori, when administered perorally. It was found that feeding the host Tinopal enabled perorally delivered BmNPV budded particles to infect the host. B. mori larvae ingesting BmNPV budded particles (1.3 x 10(6) TCID(50) units per larva) after they consumed an artificial diet containing 0. 3% Tinopal died of the viral infection. Peroral administration of these particles to host larvae with 1% Tinopal also resulted in virus infection. Tinopal is a candidate for viral activity enhancing agent promoting viral insecticide infection in hosts. The results suggest that B. mori-BmNPV budded particles are convenient for detecting viral infection enhancement activity of a chemical of interest. Since recombinant baculovirus vectors are constructed by replacing the polyhedrin gene with the foreign gene of interest, they do not produce occlusion bodies, i.e. polyhedra. Budded particles of a baculovirus vector not occluded in polyhedra cannot infect their hosts when administered perorally. The peroral inoculation of BmNPV budded particles by Tinopal leads to industrial pharmaceutics production using a baculovirus vector for a huge number of insect hosts, i.e. an 'insect factory'.

Administration, Oral↗

PubMed1999

Ibd1p, a possible spindle pole body associated protein, regulates nuclear division and bud separation in Saccharomyces cerevisiae.

The proper spatial and temporal coordination of mitosis and cytokinesis is essential for maintaining genomic integrity. We describe the identification and characterization of the Saccharomyces cerevisiae IBD1 gene, which encodes a novel protein that regulates the proper nuclear division and bud separation. IBD1 was identified by the limited homology to byr4, a dosage-dependent regulator of cytokinesis in Schizosaccharomyces pombe. IBD1 is not an essential gene, and the knock-out cells show no growth defects except for the reduced mating efficiency [1]. However, upon ectopic expression from an inducible promoter, IBD1 is lethal to the cell and leads to abnormal nuclear division and bud separation. In detail, approximately 90% of the IBD1 overexpressing cells arrest at large bud stages with dividing or divided nuclei. In some IBD1 overexpressing cells, spindle elongation and chromosome separation occur within the mother cell, leading to anucleated and binucleate daughter cells. The anucleated cell can not bud, but the binucleate cell proceeds through another cell cycle(s) to produce a cell with multiple nuclei and multiple buds. Observations of the F-actin and chitin rings in the IBD1 overexpressing cells reveal that these cells lose the polarity for bud site selection and growth or attain the hyper-polarity for growth. Consistent with the phenotypes, the IBD1 overexpressing cells contain a broad range of DNA content, from 2 to 4 N or more. A functional Ibd1p-GFP fusion protein localizes to a single dot at the nuclear DNA boundary in the divided nuclei or to double dots in dividing nuclei, suggesting its localization on the spindle pole body (SPB). The cross-species expressions of IBD1 in S. pombe and byr4 in S. cerevisiae cause defects in shape, implicating the presence of a conserved mechanism for the control of cytokinesis in eukaryotes. We propose that Ibd1p is an SPB associated protein that links proper nuclear division to cytokinesis and bud separation.

Cell Cycle Proteins↗

PubMed2002

Selective inhibition of protein kinases blocks the formation of a new axis, the beginning of budding, in Hydra.

In Hydra, head regeneration and bud formation appear to be very similar processes. The fact that there are genes whose expression is specific for one of the two processes suggests that they do not have identical molecular bases. We analyzed the signal transduction pathways regulating bud development using inhibitors of protein kinase C, Src, PI3K and ERK. The four inhibitors reversibly blocked bud formation in Hydra when applied before stage 1. Once the bud reached stage 3, three of them had no effect and the bud developed normally. The inhibitors blocked the expression of Budhead, an early head marker, and of CnOtx which are specific for bud formation. The results are in agreement with the central role of a signaling pathway mediated by Src on bud development.

Animals↗

PubMed2003

Mapping of quantitative trait loci controlling timing of bud flush in Salix.

Dormancy release is an important phenological stage, which determines plant growth and survival in northern temperate regions. Spring bud flushing was studied in a Salix pedigree (n=82) derived from a cross between the male hybrid clone "Björn" (Salix viminalis x Salix schwerinii) and the female clone "78183" (Salix viminalis). The timing of bud flush was recorded outdoors in two consecutive years (1998, 1999) and indoor in the spring of 1998. Timing of bud flush was found to be under moderately strong genetic control (clonal mean heritabilities ranging from 0.43 to 0.72). Phenotypic correlations between height growth and bud flushing were negative but non-significant (r=0.1-0.3). Using a Salix linkage map composed of 325 AFLP and 38 RFLP markers, six quantitative trait loci (QTLs) and three unmapped marker loci associated with timing of bud flush were detected. Four QTLs were detected in the field experiment while two QTLs and three unmapped marker loci were identified in the indoor experiment. One QTL associated with indoor bud flushing coincided with one of the QTL detected from the field data. Individual QTL explained 6-16% of the phenotypic variance [corrected]. None of the bud flush QTLs coincided with QTLs controlling height growth identified previously in the same pedigree.

Adaptation, Physiological↗

PubMed1993

BUD2 encodes a GTPase-activating protein for Bud1/Rsr1 necessary for proper bud-site selection in yeast.

Cells of the budding yeast Saccharomyces cerevisiae bud at either axial or bipolar sites depending on their cell type. Bud-site selection directs both cell polarity and the cytoskeletal orientation during budding and is determined by at least five genes: BUD1/RSR1, BUD2, BUD3, BUD4 and BUD5. Mutants defective in BUD1, BUD2 or BUD5 choose bud sites randomly. Bud1 protein (Bud1p) has sequence similarity to Ras, a small GTP-binding protein, and Bud5p is similar to Cdc25p (refs 4, 5), a GDP-GTP exchange factor. Here we report that Bud2p is a GTPase-activating protein (GAP) for Bud1p with a sequence similar to the catalytic domain of rasGAPs, and that Bud2p purified from yeast stimulates GTP hydrolysis by Bud1p. Chromosomal deletion of BUD2 causes a random budding pattern but no obvious growth defect. Overexpression of BUD2 also causes a random budding pattern by wild-type cells.

Amino Acid Sequence↗

PubMed2005

MAX1, a regulator of the flavonoid pathway, controls vegetative axillary bud outgrowth in Arabidopsis.

We show that MAX1, a specific repressor of vegetative axillary bud outgrowth in Arabidopsis, acts a positive regulator of the flavonoid pathway, including 11 structural genes and the transcription factor An2. Repression of bud outgrowth requires MAX1-dependent flavonoid gene expression. As the flavonoidless state leads to lateral outgrowth in Arabidopsis, our data suggest that a flavonoid-based mechanism regulates axillary bud outgrowth and that this mechanism is under the control of MAX1. Flavonoid gene expression results in the diminished expression of auxin transporters in the bud and stem, and this, in turn, decreases the rate of polar auxin transport. We speculate that MAX1 could repress axillary bud outgrowth via regulating flavonoid-dependent auxin retention in the bud and underlying stem. Because MAX1 is implicated in synthesis of the carotenoid-derived branch regulator(s) from the root, it likely links long-distance signaling with local control of bud outgrowth.

Arabidopsis↗

PubMed2004

Specific residues of the GDP/GTP exchange factor Bud5p are involved in establishment of the cell type-specific budding pattern in yeast.

Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type. Haploid a and alpha cells bud in an axial pattern whereas diploid a/alpha cells bud in a bipolar pattern. The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types. Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding. These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/alpha cells without affecting Bud5p localization in haploid alpha cells. In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types. Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site. Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues. Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues.

Alanine↗

PubMed1985

Blocked coated pits in AtT20 cells result from endocytosis of budding retrovirions.

AtT20 cells support the replication of two endogenous retroviruses, a murine leukemia virus and a mouse mammary tumor virus. On glass or plastic substrates, AtT20 cells grow in clumps. In this situation, retroviruses budding from the plasma membrane of one cell can, on rare occasions, be invested by coated pits in the plasma membranes of contiguous cells. These pits can invaginate to depths of 2,000-4,000 A within the cytoplasm drawing with them the viral buds which remain connected to their parental cells by tubular stalks, some of which are only 225 +/- 15 A in diameter. These stalks run down the straight necks of the pits from the buds to the parental cell surfaces. Several lines of evidence indicate that these unique structures are blocked such that neither endocytosis nor budding can go to completion, and that they persist for several hours. The properties of these blocked coated pits are relevant to models of both endocytosis and viral budding. First, they indicate that the invagination of a coated pit is not absolutely dependent on its pinching off to form a coated vesicle, but that uncoating appears to be dependent upon the generation of a free vesicle. Secondly, they suggest that the final stages in the maturation of a retroviral core into a mature nucleoid are dependent on the detachment of the bud from its parental cell and that the driving force of budding is the association of viral transmembrane proteins with viral core proteins. An explanation is offered to account for the formation of these structures despite the phenomenon of viral interference.

Animals↗

PubMed1994

Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step.

Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl-galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO-permeabilized cells were treated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), the budding compartment accumulated a large number of beta-cop-containing buds and vesicular profiles. Complementary biochemical experiments were carried out to determine whether vesicular transport was required for the newly synthesized M protein, that contains only O-linked oligosaccharides, to acquire first, GalNAc and second, the Golgi modifications galactose and sialic acid. The results from both in vivo studies and from the use of SLO-permeabilized cells showed that, while GalNAc addition occurred under conditions which block vesicular transport, both cytosol and ATP were prerequisites for the M protein oligosaccharides to acquire Golgi modifications. Collectively, our data argue that transport from the rough ER to the Golgi complex requires only one vesicular transport step and that the intermediate compartment is a specialized domain of the endoplasmatic reticulum that extends to the first cisterna on the cis side of the Golgi stack.

Acetylgalactosamine↗

PubMed1997

Astral microtubule dynamics in yeast: a microtubule-based searching mechanism for spindle orientation and nuclear migration into the bud.

Localization of dynein-green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at approximately 0.3-1.5 micron/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.

Anaphase↗

PubMed2002

Sec16p potentiates the action of COPII proteins to bud transport vesicles.

SEC16 encodes a 240-kD hydrophilic protein that is required for transport vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes, hence it is not one of the cytosolic proteins required to reconstitute transport vesicle budding in a cell-free reaction. However, Sec16p is removed from the membrane by salt washes, and using such membranes we have reconstituted a vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII vesicle budding is promoted by GTP or a nonhydrolyzable analogue, guanylimide diphosphate (GMP-PNP), Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated vesicle budding were explored with synthetic liposomes composed of a mixture of lipids, including acidic phospholipids (major-minor mix), or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major-minor mix liposomes and facilitates the recruitment of COPII proteins and vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major-minor mix are required to recruit pure Sec16p to liposomes, PC/PE liposomes bind Sar1p-GTP, which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP-dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.

Biological Transport↗

PubMed2004

Stable and dynamic axes of polarity use distinct formin isoforms in budding yeast.

Bud growth in yeast is guided by myosin-driven delivery of secretory vesicles from the mother cell to the bud. We find transport occurs along two sets of actin cables assembled by two formin isoforms. The Bnr1p formin assembles cables that radiate from the bud neck into the mother, providing a stable mother-bud axis. These cables also depend on septins at the neck and are required for efficient transport from the mother to the bud. The Bni1p formin assembles cables that line the bud cortex and target vesicles to varying locations in the bud. Loss of these cables results in morphological defects as vesicles accumulate at the neck. Assembly of these cables depends on continued polarized secretion, suggesting vesicular transport provides a positive feedback signal for Bni1p activation, possibly by rho-proteins. By coupling different formin isoforms to unique cortical landmarks, yeast uses common cytoskeletal elements to maintain stable and dynamic axes in the same cell.

Actins↗

PubMed1998

Requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus.

The cytoplasmic domains of viral glycoproteins are often involved in specific interactions with internal viral components. These interactions can concentrate glycoproteins at virus budding sites and drive efficient virus budding, or can determine virion morphology. To investigate the role of the vesicular stomatitis virus (VSV) glycoprotein (G) cytoplasmic and transmembrane domains in budding, we recovered recombinant VSVs expressing chimeric G proteins with the transmembrane and cytoplasmic domains derived from the human CD4 protein. These unrelated foreign sequences were capable of supporting efficient VSV budding. Further analysis of G protein cytoplasmic domain deletion mutants showed that a cytoplasmic domain of only 1 amino acid did not drive efficient budding, whereas 9 amino acids did. Additional studies in agreement with the CD4-chimera experiments indicated the requirement for a short cytoplasmic domain on VSV G without the requirement for a specific sequence in that domain. We propose a model for VSV budding in which a relatively non-specific interaction of a cytoplasmic domain with a pocket or groove in the viral nucleocapsid or matrix proteins generates a glycoprotein array that promotes viral budding.

Amino Acid Sequence↗

PubMed2003

Lack of expression of EGF and TGF-alpha in the fetal mouse alters formation of prostatic epithelial buds and influences the response to TCDD.

In utero, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure causes abnormal ventral, dorsolateral, and anterior prostate development in C57BL/6J mice. Androgens, mesenchymal-epithelial interactions, and growth factor expression all have roles in initiating and regulating development and growth of the prostate. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), both of which bind the EGF receptor (EGFR), are expressed in human and rodent developing prostate. This study examines the influence of null expression of EGF and/or TGF-alpha on prostatic bud development and on the ability of TCDD to inhibit prostatic budding. Growth factor knockout (-/-) and wild-type (WT) mice were exposed either to vehicle or to TCDD (0, 0.2, 1, 5, 10, 50, 100, or 150 microg/kg) on gestation day (GD) 12. The number of anterior, dorsal, and lateral prostatic buds (ADLB) and ventral buds (VB) were counted on GD 17.5. Control WT and EGF (-/-) fetuses had similar numbers of ADLB and VB. In control TGF-alpha (-/-) fetuses, the number of ADLBs was higher relative to the C57BL/6J. Control EGF + TGF-alpha (-/-) had poor bud outgrowth, especially in the ADL region. TCDD induced a dose-related decrease in bud formation in all strains with the formation of VBs being more sensitive than ADLBs. The severity of the response depended on growth factor expression, with the most severe effects on VBs in the EGF (-/-) and on ADLBs in the EGF + TGF-alpha (-/-) fetuses. TGF-alpha (-/-) and C57BL/6J fetuses responded to TCDD similarly. In conclusion, EGF and TGF-alpha expression are important for the formation of ADLBs and VBs, and expression of EGF and TGF-alpha affects the ability of TCDD to inhibit prostatic bud formation in a region-specific manner.

Abnormalities, Drug-Induced↗

PubMed1986

An effect of gravity on bud-burst in balsam fir.

Bud-burst on first order lateral branches of Abies bafsamea L. (balsam fir) was delayed when the branches were rotated 180 degrees about their long axis. This was not a consequence of injury caused by the treatment because buds rotated 180 degrees on inverted plants flushed at the same time as the controls, whereas flushing of all other buds was delayed. Buds thus appear to be more vigorous when maintained in the same orientation to gravity in which they are formed and the site of gravitational stimulus perception appears to be the bud itself. Except on the leading shoot, leaves from inverted buds turned so that their adaxial surface faced upward, unless there was intense illumination from below. However, both anisophylly and positioning of leaves on lateral shoots were apparently predetermined because the shorter, more forward pointing leaves appeared below the longer distichous leaves on shoots from inverted buds. Shoots with normally oriented leaves appeared the next season.

Journal Article↗

PubMed1995

Effects of "near-lethal" stress on bud dormancy and stem cold hardiness in red-osier dogwood.

We studied the effects of "near-lethal" (NL, 47 degrees C for 1 h) heat stress, applied to intact shoots of red-osier dogwood (Cornus sericea L.) during early (October), deep (November) or late (December) dormancy, on bud dormancy release and development of stem tissue cold hardiness under natural conditions and at a constant temperature of 0 or 23 degrees C in the dark. The NL heat-stress treatment overcame bud dormancy when applied during the early and late stages of dormancy. During October and December, all plants in the 23 degrees C + dark post-stress environment broke bud within 35 and 12 days, respectively, whereas the corresponding values for days to bud break in the control plants were more than 150 and 110 days, respectively. Application of NL heat stress during deep dormancy caused only slightly earlier bud break compared to the control plants. In the 0 degrees C + dark post-stress environment, all NL heat-treated plants died within 9 weeks. Under natural post-stress conditions, bud break in plants receiving NL heat stress during early and deep dormancy occurred at the same time as in control plants, whereas bud break of plants receiving NL heat stress during late dormancy occurred 55 days earlier than in control plants. Under both natural and 23 degrees C + dark post-stress conditions, cold hardiness of plants receiving NL heat stress during early dormancy was similar to that of controls. Application of NL heat stress during deep dormancy hastened the rate of deacclimation under the 23 degrees C + dark post-stress conditions but had no effect on deacclimation under natural post-stress conditions. Application of NL heat stress during late dormancy enhanced deacclimation of plants in both the 23 degrees C + dark and natural post-stress environments.

Journal Article↗