Ecological factors rather than temporal factors dominate the evolution of vesicular stomatitis virus

Vesicular stomatitis New Jersey virus (VSV-NJ) is a rhabdovirus that causes economically important disease in cattle and other domestic animals in endemic areas from southeastern United States to northern South America. Its negatively stranded RNA genome is capable of undergoing rapid evolution, which allows phylogenetic analysis and molecular epidemiology studies to be performed. Previous epidemiological studies in Costa Rica showed the existence of at least two distinct ecological zones of high VSV-NJ activity, one located in the highlands (premontane tropical moist forest) and the other in the lowlands (tropical dry forest). We wanted to test the hypothesis that the viruses circulating in these ecological zones were genetically distinct. For this purpose, we sequenced the hypervariable region of the phosphoprotein gene for 50 VSV-NJ isolates from these areas. Phylogenetic analysis showed that viruses from each ecological zone had distinct genotypes. These genotypes were maintained in each area for periods of up to 8 years. This evolutionary pattern of VSV-NJ suggests an adaptation to ecological factors that could exert selective pressure on the virus. As previous data indicated an absence of virus adaptation to factors related to the bovine host (including immunological pressure), it appears that VSV genetic divergence represents positive selection to adapt to specific vectors and/or reservoirs at each ecological zone.

Reduced facilitation and vesicular uptake in crustacean and mammalian neuromuscular junction by T-588, a neuroprotective compound

Bath application of compound T-588, a neuroprotective agent, reduced paired-pulse and repetitive-pulse facilitation at mammalian and crustacean neuromuscular junctions. In addition, it reduced voltage-gated sodium and potassium currents in a use-dependent fashion, but had only a small effect on the presynaptic Ca2+ conductance. By contrast, it blocked FM 1–43 vesicular uptake but not its release, in both species. Postsynaptically, T-588 reduced acetylcholine currents at the mammalian junction in a voltage-independent manner, but had no effect on the crayfish glutamate junction. All of these effects were rapidly reversible and were observed at concentrations close to the compound’s acute protective level. We propose that this set of mechanisms, which reduces high-frequency synaptic transmission, is an important contributory factor in the neuroprotective action of T-588.

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.

Differential quantal release of histamine and 5-hydroxytryptamine from mast cells of vesicular monoamine transporter 2 knockout mice

The recent availability of mice lacking the neuronal form of the vesicular monoamine transporter 2 (VMAT2) affords the opportunity to study its roles in storage and release. Carbon fiber microelectrodes were used to measure individual secretory events of histamine and 5-hydroxytryptamine (5-HT) from VMAT2-expressing mast cells as a model system for quantal release. VMAT2 is indispensable for monoamine storage because mast cells from homozygous (VMAT2−/−) mice, while undergoing granule-cell fusion, do not release monoamines. Cells from heterozygous animals (VMAT2+/−) secrete lower amounts of monoamine per granule than cells from wild-type controls. Investigation of corelease of histamine and 5-HT from granules in VMAT2+/− cells revealed 5-HT quantal size was reduced more than that of histamine. Thus, although vesicular transport is the limiting factor determining quantal size of 5-HT and histamine release, intragranular association with the heparin matrix also plays a significant role.

Quantitative ER ↔ Golgi Transport Kinetics and Protein Separation upon Golgi Exit Revealed by Vesicular Integral Membrane Protein 36 Dynamics in Live CellsV⃞

To quantitatively investigate the trafficking of the transmembrane lectin VIP36 and its relation to cargo-containing transport carriers (TCs), we analyzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expressed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized with epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, including the closely related lectin ERGIC53. VIP36-SP-FP trafficking structures comprised tubules and globular elements, which translocated in a saltatory manner. Simultaneous visualization of anterograde secretory cargo and VIP36-SP-FP indicated that the globular structures were pre-Golgi carriers, and that VIP36-SP-FP segregated from cargo within the Golgi and was not included in post-Golgi TCs. Organelle-specific bleach experiments directly measured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulum (ER). Fitting a two-compartment model to the recovery data predicted first order rate constants of 1.22 ± 0.44%/min for ER → Golgi, and 7.68 ± 1.94%/min for Golgi → ER transport, revealing a half-time of 113 ± 70 min for leaving the ER and 1.67 ± 0.45 min for leaving the Golgi, and accounting for the measured steady-state distribution of VIP36-SP-FP (13% Golgi/87% ER). Perturbing transport with AlF4− treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model suggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secretory components.

Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles.

In a previous study we demonstrated that vesicular stomatitis virus (VSV) can be used as a vector to express a soluble protein in mammalian cells. Here we have generated VSV recombinants that express four different membrane proteins: the cellular CD4 protein, a CD4-G hybrid protein containing the ectodomain of CD4 and the transmembrane and cytoplasmic tail of the VSV glycoprotein (G), the measles virus hemagglutinin, or the measles virus fusion protein. The proteins were expressed at levels ranging from 23-62% that of VSV G protein and all were transported to the cell surface. In addition we found that all four proteins were incorporated into the membrane envelope of VSV along with the VSV G protein. The levels of incorporation of these proteins varied from 6-31% of that observed for VSV G. These results suggest that many different membrane proteins may be co-incorporated quite efficiently with VSV G protein into budding VSV virus particles and that specific signals are not required for this co-incorporation process. In fact, the CD4-G protein was incorporated with the same efficiency as wild type CD4. Electron microscopy of virions containing CD4 revealed that the CD4 molecules were dispersed throughout the virion envelope among the trimeric viral spike glycoproteins. The recombinant VSV-CD4 virus particles were about 18% longer than wild type virions, reflecting the additional length of the helical nucleocapsid containing the extra gene. Recombinant VSVs carrying foreign antigens on the surface of the virus particle may be useful for viral targeting, membrane protein purification, and for generation of immune responses.

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.

Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

We have previously shown that the G protein of vesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracycline-dependent and can be modulated by beta-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.

Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.

Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells.

14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae.

Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.