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sindbis-group alphavirus replication in periosteum and endosteum of long bones in adult mice.several alphaviruses, including the sindbis-group viruses, ross river virus, o'nyong-nyong virus, and chikungunya virus, are associated with outbreaks of acute and persistent arthralgia and arthritis in humans. mechanisms underlying alphavirus-induced arthralgia and arthritis are not clearly understood, though direct viral replication within or around the affected joints is thought to contribute to disease. s.a.ar86 is a sindbis-group alphavirus closely related to the arthralgia-associated ockel ...200010982376
sindbis virus glycoprotein e1 is divided into two discrete domains at amino acid 129 by disulfide bridge connections.the e1 membrane glycoprotein of sindbis virus contains structural and functional domains, which are conformationally dependent on the presence of intramolecular disulfide bridges (b. a. abell and d. t. brown, j. virol. 67:5496-5501, 1993; r. p. anthony, a. m. paredes, and d. t. brown, virology 190:330-336, 1992). we have examined the disulfide bonds in e1 and have determined that the e1 membrane glycoprotein contains two separate sets of interconnecting disulfide linkages, which divide the prote ...200010982379
mutagenesis reveals a role for abp/grip binding to glur2 in synaptic surface accumulation of the ampa receptor.we studied the role of pdz proteins grip, abp, and pick1 in glur2 ampa receptor trafficking. an epitope-tagged mycglur2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. with the mutant mycglur2delta1-10, which lacks the pdz binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. however, over time, myc-glur2delta1-10 accumulated at synapses significantly less than mycglur2. notabl ...200010985351
fitting atomic models into electron-microscopy maps.combining x-ray crystallographically determined atomic structures of component domains or subunits with cryo-electron microscopic three-dimensional images at around 22 a resolution can produce structural information that is accurate to about 2.2 a resolution. in an initial step, it is necessary to determine accurately the absolute scale and absolute hand of the cryo-electron microscopy map, the former of which can be off by up to 5%. it is also necessary to determine the relative height of densi ...200010998631
adeno-associated virus type 2 rep78 induces apoptosis through caspase activation independently of p53.adeno-associated virus (aav) type 2 rep78 is a multifunctional protein required for aav dna replication, integration, and gene regulation. the biochemical activities of rep78 have been described, but the effects of rep proteins on the cell have not been characterized. we have analyzed rep-mediated cytotoxicity. we demonstrated that rep78 expression is sufficient to induce cell death and disruption of the cell cycle. cell death was found to be mediated by apoptosis. rep78 expression resulted in t ...200011000213
passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein e(rns).infection of cells with classical swine fever virus (csfv) is mediated by the interaction of envelope glycoprotein e(rns) and e2 with the cell surface. in this report we studied the role of the cell surface glycoaminoglycans (gags), chondroitin sulfates a, b, and c (cs-a, -b, and -c), and heparan sulfate (hs) in the initial binding of csfv strain brescia to cells. removal of hs from the surface of swine kidney cells (sk6) by heparinase i treatment almost completely abolished infection of these c ...200011000226
biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases.the arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a zn finger structure linked to a superfamily 1 (sf1) helicase domain. a recombinant form of nsp10, mbp-nsp10, was produced in escherichia coli as a fusion protein with the maltose-binding protein. the protein was partially purified by affinity chromatography and shown to have atpase activity that was strongly stimulated by poly(dt), poly(u), and poly(da) but not by poly(g). the prote ...200011000230
alphavirus rna genome repair and evolution: molecular characterization of infectious sindbis virus isolates lacking a known conserved motif at the 3' end of the genome.the 3' nontranslated region of the genomes of sindbis virus (sin) and other alphaviruses carries several repeat sequence elements (rses) as well as a 19-nucleotide (nt) conserved sequence element (3'cse). the 3'cse and the adjoining poly(a) tail of the sin genome are thought to act as viral promoters for negative-sense rna synthesis and genome replication. eight different sin isolates that carry altered 3'cses were studied in detail to evaluate the role of the 3'cse in genome replication. the sa ...200011000254
replicon vectors derived from sindbis virus and semliki forest virus that establish persistent replication in host cells.alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. for many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. recently, noncytopathic sindbis virus (sin) va ...200011000258
membrane proteins organize a symmetrical virus.alphaviruses are enveloped icosahedral viruses that mature by budding at the plasma membrane. according to a prevailing model maturation is driven by binding of membrane protein spikes to a preformed nucleocapsid (nc). the t = 4 geometry of the membrane is thought to be imposed by the nc through one-to-one interactions between spike protomers and capsid proteins (cps). this model is challenged here by a semliki forest virus capsid gene mutant. its cps cannot assemble into ncs, or its intermediat ...200011013211
suppressor mutations that allow sindbis virus rna polymerase to function with nonaromatic amino acids at the n-terminus: evidence for interaction between nsp1 and nsp4 in minus-strand rna synthesis.the alphavirus rna polymerase, nsp4, invariably has a tyr residue at the n-terminus. previously we reported that the n-terminal tyr residue of nsp4 of sindbis virus, the type species of the genus alphavirus, can be substituted with phe, trp, or his without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for met, were lethal or quasilethal. here we report the identification of two suppressor mutations in nsp4 (glu-191 to leu and glu-315 to gly, val, ...200011022003
rubella virus replication and links to teratogenicity.rubella virus (rv) is the causative agent of the disease known more popularly as german measles. rubella is predominantly a childhood disease and is endemic throughout the world. natural infections of rubella occur only in humans and are generally mild. complications of rubella infection, most commonly polyarthralgia in adult women, do exist; occasionally more serious sequelae occur. however, the primary public health concern of rv infection is its teratogenicity. rv infection of women during th ...200011023958
molecular strategies for interrupting arthropod-borne virus transmission by mosquitoes.arthropod-borne virus (arbovirus) infections cause a number of emerging and resurgent human and veterinary infectious diseases. traditional means of controlling arbovirus diseases include vaccination of susceptible vertebrates and mosquito control, but in many cases these have been unavailable or ineffective, and so novel strategies for disease control are needed. one possibility is genetic manipulation of mosquito vectors to render them unable to transmit arboviruses. this review describes rece ...200011023962
differential regulation of bcl-2 and bax expression in cells infected with virulent and nonvirulent strains of sindbis virus.sindbis virus is an alphavirus that infects cells in either lytic or persistent infection. in this study we examined the effects of sindbis virus on cell apoptosis and on the expression of bcl-2 and bax. of the two strains studied, sva and svni, only the neurovirulent strain, svni, induced apoptosis of astrocytes and pc-12 cells. sva, which infects cells in a persistent manner, induced up-regulation of bcl-2 mrna and bcl-2 protein, whereas svni induced an increase in bax levels. our results indi ...200011040115
ecotropic murine leukemia virus receptor is physically associated with caveolin and membrane rafts.we used a sindbis virus expression system to stably express a chimeric ecotropic murine leukemia virus (mlv) receptor gene, cat1, fused to green fluorescent protein (gfp) in bhk cells. the chimeric gene was expressed on the cell surface and functioned as an mlv receptor. using gfp as an epitope tag, we found that cat1 cross-immunoprecipitated with caveolin, a cellular protein associated non-clathrin-coated endocytic vesicles. biochemical studies showed that cat1 copurified with caveolin in a det ...200011040117
generation of subgenomic rna directed by a satellite rna associated with bamboo mosaic potexvirus: analyses of potexvirus subgenomic rna promoter.satellite rna of bamboo mosaic potexvirus (satbamv), a single-stranded positive-sense rna encoding a nonstructural protein of 20 kda (p20), depends on bamboo mosaic potexvirus (bamv) for replication and encapsidation. a full-length cdna clone of satbamv was used to examine the sequences required for the synthesis of potexvirus subgenomic rnas (sgrnas). subgenomic promoter-like sequences (sgps), 107 nucleotides (nt) upstream from the capsid protein (cp) gene of bamv-v, were inserted upstream of t ...200011044078
glycosaminoglycan sulfation requirements for respiratory syncytial virus infection.glycosaminoglycans (gags) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (rsv) infection. we evaluated the importance of sulfation, the major biosynthetic modification of gags, using an improved recombinant green fluorescent protein-expressing rsv (rgrsv) to assay infection. pretreatment of hep-2 cells with 50 mm sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgrsv infection t ...200011044095
in vivo selection of protease cleavage sites by using chimeric sindbis virus libraries.identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. one route to this end is the generation and screening of random libraries of cleavage sites. both synthetic and phage-displayed libraries have been extensively used in vitro. we describe a novel system based on recombinant sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzy ...200011044100
strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model.a systematic method was developed to assemble functional full-length genomes of large rna and dna viruses. coronaviruses contain the largest single-stranded positive-polarity rna genome in nature. the approximately 30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cdna construct. we have assembled a full-length infectious construct of transmissible gastroenteritis virus (tgev), an important pathogen in swine. using a novel approac ...200011044104
expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant venezuelan equine encephalitis virus replicon particles.rna replicon particles derived from a vaccine strain of venezuelan equine encephalitis virus (vee) were used as a vector for expression of the major envelope proteins (g(l) and m) of equine arteritis virus (eav), both individually and in heterodimer form (g(l)/m). open reading frame 5 (orf5) encodes the g(l) protein, which expresses the known neutralizing determinants of eav (u. b. r. balasuriya, j. f. patton, p. v. rossitto, p. j. timoney, w. h. mccollum, and n. j. maclachlan, virology 232:114- ...200011044106
development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression.rubella virus (rub) is a small plus-strand rna virus classified in the rubivirus genus of the family togaviridae. live, attenuated rub vaccines have been successfully used in vaccination programs for over 25 years, making rub an attractive vaccine vector. in this study, such a vector was constructed using a recently developed rub infectious cdna clone (robo). using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicate ...200011044128
nmda but not non-nmda excitotoxicity is mediated by poly(adp-ribose) polymerase.poly(adp-ribose) polymerase (parp-1), a nuclear enzyme that facilitates dna repair, may be instrumental in acute neuronal cell death in a variety of insults including, cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism, and cns trauma. excitotoxicity is thought to underlie these and other toxic models of neuronal death. different glutamate agonists may trigger different downstream pathways toward neurotoxicity. we examine the role of parp-1 in nmda- and non-nmda ...200011050121
characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside triphosphatase/helicase domain of the hepatitis c virus nonstructural 3 protein.we have produced a murine monoclonal antibody (mab), zx10, recognizing the ntpase/helicase domain of the hepatitis c virus (hcv) nonstructural 3 protein (ns3), from which we designed a single-chain variable fragment (scfv). the zx10 mab recognized a discontinuous epitope of the ntpase/helicase domain, of which the linear sequence geipfygkaipl at residues 1371 to 1382 constitutes one part. cdnas from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into ...200010618278
large-plaque mutants of sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation.laboratory strains of sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. during infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the e2 glycoprotein. recombinant viruses were constructed with these ...200010623725
mapping of genetic determinants of rubella virus associated with growth in joint tissue.rubella virus (rv) strains vary in their abilities to replicate and persist in cell cultures derived from human joint tissue (synovial cells [sc]), and this arthrotropism appears to be linked to their association with joint symptoms in vivo. in order to map the genetic determinants of arthrotropism, an infectious clone of the cendehill vaccine strain of rv was constructed, as well as two chimeric clones containing cdnas from both cendehill and therien (wild-type) strains. replacement of the enti ...200010623741
role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis.the initial steps of venezuelan equine encephalitis virus (vee) spread from inoculation in the skin to the draining lymph node have been characterized. by using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of vee infection in the first 48 h following inoculation. vee viral replicon particles, which can undergo only one round of infection, identified langerhans cells to be the initial set of cells infected b ...200010623754
perspectives for the treatment of infections with flaviviridae.the family flaviviridae contains three genera: hepacivirus, flavivirus, and pestivirus. worldwide, more than 170 million people are chronically infected with hepatitis c virus and are at risk of developing cirrhosis and/or liver cancer. in addition, infections with arthropod-borne flaviviruses (such as dengue fever, japanese encephalitis, tick-borne encephalitis, st. louis encephalitis, murray valley encephalitis, west nile, and yellow fever viruses) are emerging throughout the world. the pestiv ...200010627492
cap-independent translational enhancement of turnip crinkle virus genomic and subgenomic rnas.the presence of translational control elements and cap structures has not been carefully investigated for members of the carmovirus genus, a group of small icosahedral plant viruses with positive-sense rna genomes. in this study, we examined both the 5' and 3' untranslated regions (utrs) of the turnip crinkle carmovirus (tcv) genomic rna (4 kb) as well as the 5' utr of the coat protein subgenomic rna (1.45 kb) for their roles in translational regulation. all three utrs enhanced translation of th ...200010627518
the replication activity of influenza virus polymerase is linked to the capacity of the pa subunit to induce proteolysis.the pa subunit of the influenza virus polymerase complex is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins. the amino-terminal third of the protein is responsible for the induction of proteolysis. we mutated five potential casein kinase ii phosphorylation sites located in the amino-terminal third of the protein. mutations affecting position 157 almost completely abrogated proteolysis induction, whereas a mu ...200010627541
alpha/beta interferons potentiate virus-induced apoptosis through activation of the fadd/caspase-8 death signaling pathway.interferon (ifn) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded rna (dsrna)-dependent protein kinase, pkr. here we report that inducible overexpression of functional pkr in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of pkr were completely resistant. we determined that the mechanism of influenza virus-induced apoptosis involved death signaling t ...200010627563
the s segment of rift valley fever phlebovirus (bunyaviridae) carries determinants for attenuation and virulence in mice.unlike all the other rift valley fever virus strains (bunyaviridae, phlebovirus) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the nss nonstructural protein in the nuclei of infected cells (r. muller, j. f. saluzzo, n. lopez, t. drier, m. turell, j. smith, and m. bouloy, am. j. trop. med. hyg. 53:405-411, 1995). this defect is correlated with a large in-frame deletion in the nss coding region of the s segment of the tripartite genome. here, we sh ...200010627566
linkage of an alphavirus host-range restriction to the carbohydrate-processing phenotypes of the host cell.the sindbis virus mutant ne2g216 retains pe2 in place of e2 in its virion structure. ne2g216 is a host-range mutant that replicates with near-normal kinetics in vertebrate cells, but displays severely restricted growth in cultured mosquito cells (c6/36) due to defects in the virus maturation process. in this study we tested the hypothesis that the host-range phenotype of ne2g216 was linked to the differences in carbohydrate-processing phenotypes between vertebrate and arthropod cells. arthropod ...200010640554
biochemical consequences of a mutation that controls the cholesterol dependence of semliki forest virus fusion.the enveloped alphavirus semliki forest virus (sfv) infects cells via a low-ph-triggered membrane fusion reaction that requires cholesterol and sphingolipid in the target membrane. cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an sfv mutant that is approximately 100-fold less cholesterol dependent for infection due to a single amino acid change in the e1 spike subunit, proline 226 to serine. sensitive lipid-mixing assays here demons ...200010644331
cell recognition by foot-and-mouth disease virus that lacks the rgd integrin-binding motif: flexibility in aphthovirus receptor usage.cell surface molecules that can act as virus receptors may exert an important selective pressure on rna viral quasispecies. large population passages of foot-and-mouth disease virus (fmdv) in cell culture select for mutant viruses that render dispensable a highly conserved arg-gly-asp (rgd) motif responsible for integrin receptor recognition. here, we provide evidence that viability of recombinant fmdvs including a asp-143-->gly change at the rgd motif was conditioned by a number of capsid subst ...200010644333
cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking.canine parvovirus (cpv) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. we used a variety of approaches to define the early stages of cell entry by cpv. electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. overex ...200010644365
enhancement of tumor-specific immune response with plasmid dna replicon vectors.to enhance the immunogenicity of nucleic acid vaccines, we used plasmid dna vectors that contained replicons derived from the prototype alphavirus, sindbis, and another alphavirus, semliki forest virus. when transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating rna vector (replicon) which in turn directs the expression of a model tumor antigen. immunization with plasmid dna replicons elicited immune responses at doses 100 to 1000-fold lower than ...200010646851
part i. bcl-2 and bcl-x(l) limit apoptosis upon infection with alphavirus vectors.viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. in particular, alphavirus constructs based on sindbis virus (sv) and semliki forest virus (sfv) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (bhk) and chinese hamster ovary (c ...200010649229
photobiological properties of positively charged methylene violet analogs.o-methyl methylene violet (omemv), o-methyl bromomethylene violet (omebrmv) and o-methyl iodomethylene violet (omeimv) have been prepared in order to test their potential utility as anti-viral and anti-tumor phototoxic dyes. rates of photosensitized toxicity of kb cells with 633 nm irradiation are (x 10(-19) photon-1): 2.4, 2.2, 1.9 and 0.17 for omeimv, omebrmv, methylene violet (mv) and omemv, respectively. rates of photosensitized inactivation of sindbis virus in phosphate-buffered saline with ...200010649885
rainbow trout sleeping disease virus is an atypical alphavirus.sleeping disease (sd) is currently a matter of concern for salmonid fish farmers in most parts of the world. a viral etiology of sd has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. in salmonid-derived cell lines, the maximal rate of virus production was observed at 10 degrees c, while little virus was produced at 14 degrees c. through biochemical, physicochemical, and morphological studies, sd virus (sdv) was shown to be an enveloped vir ...200010590104
integrins alpha2beta1 and alpha4beta1 can mediate sa11 rotavirus attachment and entry into cells.most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins vp4 and vp7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. here we report that sa11 rotavirus binding to and infection of k562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection ...200010590110
in vitro transcription by the turnip yellow mosaic virus rna polymerase: a comparison with the alfalfa mosaic virus and brome mosaic virus replicases.recently, we showed that the main determinant in the trna-like structure of turnip yellow mosaic virus rna to initiate minus-strand synthesis in vitro is the 3' acca end. by mutational analysis of the 3'-terminal hairpin, we show here that only a non-base-paired acca end is functional and that the stability of the wild-type 3'-proximal hairpin is the most favorable, in that it has the lowest deltag value and a high transcription efficiency. with a nested set of rna fragments, we show that the mi ...200010590114
vaccination of macaques against pathogenic simian immunodeficiency virus with venezuelan equine encephalitis virus replicon particles.vaccine vectors derived from venezuelan equine encephalitis virus (vee) that expressed simian immunodeficiency virus (siv) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. immunization with vee replicon particles induced both humoral and cellular immune responses. four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic siv swarm, ...200010590126
alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum.we report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (den) virus. we found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prm (the intracellular glycosylated precursor of m [membrane protein]) and e (envelope protein): the proper folding of prm bearing unprocessed n-linked oligosaccharide is inefficient, and this causes delayed formation of prme heterodimer. the complexes formed between incomple ...200010590151
identification of a bovine coronavirus packaging signal.a region of the bovine coronavirus (bcv) genome that functions as a packaging signal has been cloned. the 291-nucleotide clone shares 72% homology with the region of mouse hepatitis coronavirus (mhv) gene 1b that contains the packaging signal. rna transcripts were packaged into both bcv and mhv virions when the cloned region was appended to a noncoronavirus rna. this is the first identification of a bcv packaging signal. the data demonstrate that the bcv genome contains a sequence that is conser ...200010590153
increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and trail.chemotherapeutic genotoxins induce apoptosis in epithelial-cell-derived cancer cells. the death receptor ligand trail also induces apoptosis in epithelial-cell-derived cancer cells but generally fails to induce apoptosis in nontransformed cells. we show here that the treatment of four different epithelial cell lines with the topoisomerase ii inhibitor etoposide in combination with trail (tumor necrosis factor [tnf]-related apoptosis-inducing ligand) induces a synergistic apoptotic response. the ...200010594023
active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus.the polyprotein of infectious pancreatic necrosis virus (ipnv), a birnavirus, is processed by the viral protease vp4 (also named ns) to generate three polypeptides: pvp2, vp4, and vp3. site-directed mutagenesis at 42 positions of the ipnv vp4 protein was performed to determine the active site and the important residues for the protease activity. two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pvp2-vp4 and the vp4-vp3 junctions. wild-type activity at the p ...200010666235
the in vitro-synthesized rna from a cdna clone of hepatitis e virus is infectious.hepatitis e virus (hev) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the indian subcontinent and prevalent in most of the developing parts of the world. the infection is often associated with acute liver failure and high mortality, particularly in pregnant women. in order to develop methods of intervention, it is essential to understand the biology of the virus. this is particularly important as no reliable in vitro culture system is available. we hav ...200010666275
pseudotyping of glycoprotein d-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein g enables mutant virus attachment and entry.the use of herpes simplex virus (hsv) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. because hsv glycoprotein d (gd) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gd to provide new cell recognition specificities in a manner designed to preserve gd's essential role in virus entry. in this study, we have d ...200010666285
development of a sindbis virus expression system that efficiently expresses green fluorescent protein in midguts of aedes aegypti following per os infection.a double subgenomic sindbis (dssin) virus, mre/3'2 j/gfp, was constructed to efficiently express green fluorescent protein (gfp) in the midgut of aedes aegypti following per os infection. the mre/3'2 j/gfp rna genome contained the nonstructural genes and cis-acting sequences of the dssin virus, te/3'2 j/gfp, but had the structural genes of mre16 sin virus. mre/3'2 j/gfp virus, unlike te/3'2 j/gfp virus, efficiently infected mosquitoes orally. at 1-2 days postinfection, gfp was observed as multip ...200010672072
the promise of nucleic acid vaccines.establishing the effective use of 'naked' nucleic acids as vaccines would undoubtedly be one of the most important advances in the history of vaccinology. while nucleic acids show much promise for use as vaccine vectors in experimental animals, not a single naked nucleic acid vector has been approved for use in humans. indeed, data from human clinical trials is scant: nucleic acid vaccines have not been clearly demonstrated to have any convincing efficacy in the prevention or treatment of infect ...200010673713
imaging high-resolution structure of gfp-expressing neurons in neocortex in vivo.to detect subtle changes in neuronal morphology in response to changes in experience, one must image neurons at high resolution in vivo over time scales of minutes to days. we accomplished this by infecting postmitotic neurons in rat and mouse barrel cortex with a sindbis virus carrying the gene for enhanced green fluorescent protein. visualized with 2-photon excitation laser scanning microscopy, infected neurons showed bright fluorescence that was distributed homogeneously throughout the cell, ...200011112802
alphavirus dna and particle replicons for vaccines and gene therapy.alphaviruses have several features that make them attractive as gene delivery platforms, and vectors derived principally from sindbis virus (sin), semliki forest virus (sfv), and venezuelan equine encephalitis virus (vee), are currently being developed as prophylactic and therapeutic vaccines for infectious diseases and cancer. alphavirus vectors, termed "replicons", retain the nonstructural protein genes encoding the viral replicase, that in turn programme high level cytoplasmic amplification o ...200011713818
antiviral activities in plants endemic to madagascar.in order to determine the potential of malagasy plants as sources of antiviral activities, ethanolic extracts of 11 plants, endemic to madagascar, were evaluated for antiviral activities. nine of the extracts had significant activity against herpes simplex virus (hsv), whereas only four were active against sindbis virus. five extracts: cynometra cloiselii , cynometra madagascariensis , evonymopsis longipes , ravensara retusa , and terminalia monoceros were particularly potent and could completel ...200021214437
antiviral activities in extracts of turkish medicinal plants.a total of 16 ethanol extracts of turkish medicinal plants were evaluated for antiviral activities against herpes simplex virus (hsv) and sindbis virus (sinv). extracts of galanthus elwesii and rheum ribes showed the most potent anti-hsv activities, while six other extracts had weaker activities. galanthus elwesii and leucojum aestivum were the most potent anti-sinv extracts with four others showing weaker activities. in total, five extracts were active against both viruses, three were selective ...200021214457
tom1, an arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein.host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. however, information regarding the host factors involved in this process is limited. here we report the map-based cloning of an arabidopsis thaliana gene, tom1, which is necessary for the efficient multiplication of tobamoviruses, positive-strand rna viruses infecting a wide variety of plants. the tom1 mrna is suggested to encode a 291-aa polypeptide that is predicted to be a multip ...200010944200
west nile virus - where did it come from and where might it go? 200018159286
death, autoantigen modifications, and tolerance.autoantibodies present in the serum of patients with a variety of inflammatory diseases have proven useful as diagnostic markers and as probes with which to elucidate biochemical and signaling pathways. the mechanisms governing the generation of autoantibodies remain elusive, constituting a critical missing link in our understanding of rheumatologic illnesses. several lines of experimentation in recent years have strongly implicated events surrounding cell death in this process. this review will ...200011094420
gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays.gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. these include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts.200111513755
characterization of an endogenous gene expressed in aedes aegypti using an orally infectious recombinant sindbis virus.sindbis virus expression vectors have been used successfully to express and silence genes of interest in vivo in several mosquito species, including aedes aegypti, ae. albopictus, ae. triseriatus,culex pipiens, armigeres subalbatus and anopheles gambiae. here we describe the expression of an endogenous gene, defensin, in ae. aegypti using the orally infectious sindbis virus, mre/3'2j expression vector. we optimized conditions to infect mosquito larvae per os using c6/36ae. albopictus cells infec ...200115455070
a herpes simplex viral vector expressing green fluorescent protein can be used to visualize morphological changes in high-density neuronal culture.high-density cultures of mammalian neurons offer a model system for studies of brain development, but the morphological features of individual neurons is difficult to ascertain. we show that a herpes virus vector expressing a bioluminescent protein allows detailed morphometric analyses of living neurons in complex culture environments. expression of enhanced green fluorescent protein (egfp) was constitutively driven in neurons using the herpes simplex virus amplicon system. this system allowed u ...200120811504
cis-acting rna elements at the 5' end of sindbis virus genome rna regulate minus- and plus-strand rna synthesis.alphavirus genome replication is a multistep asymmetric process. several lines of evidence suggest that the template preference of the rna replicase is regulated by proteolytic cleavage of the viral nonstructural polyprotein. cis-acting rna elements in the viral genome also play crucial roles in regulating genome replication and subgenomic rna transcription. in this report, a series of rna templates were analyzed in vitro and in vivo to define functional elements in the 5' end of the genome. the ...200111720292
in vivo transduction of central neurons using recombinant sindbis virus: golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins.a new recombinant virus which labeled the infected neurons in a golgi stain-like fashion was developed. the virus was based on a replication-defective sindbis virus and was designed to express green fluorescent protein with a palmitoylation signal (palgfp). when the virus was injected into the ventrobasal thalamic nuclei, many neurons were visualized with the fluorescence of palgfp in the injection site. the labeling was enhanced by immunocytochemical staining with an antibody to green fluoresce ...200111724897
clearance of extracellular and cell-associated amyloid beta peptide through viral expression of neprilysin in primary neurons.amyloid beta peptide (abeta), the pathogenic agent of alzheimer's disease (ad), is a physiological metabolite constantly anabolized and catabolized in the brain. we previously demonstrated that neprilysin is the major abeta-degrading enzyme in vivo. to investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating abeta levels, we expressed neprilysin in primary cortical neurons using a sindbis viral vector and examined the effect on abe ...200111726269
alphavirus vectors: development and potential therapeutic applications.alphaviruses are rna enveloped viruses that are proving their value as expression vectors. they are particularly well-suited for this role as they are easily and quickly engineered and can be used to produce high levels of proteins of interest. a promising and important use is as vaccines against disease-causing agents such as hiv. the three alphaviruses now serving as vectors are sindbis virus, semliki forest virus (sfv) and venezuelan equine encephalitis (vee) virus. sindbis virus and sfv are ...200111727528
antiherpetic activity of dimeric derivatives of netropsin. 200111727562
alphavirus nucleocapsid protein contains a putative coiled coil alpha-helix important for core assembly.the alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. this protein consists of a poorly conserved n-terminal region of unknown function and a c-terminal conserved autoprotease domain with a major role in virion formation. in this study, an 18-amino-acid conserved region, predicted to fold into an alpha-helix (helix i) and embedded in a low-complexity sequence enriched with basic and pro residues, has be ...200111119567
protective t-cell-based immunity induced in neonatal mice by a single replicative cycle of herpes simplex virus.newborns are very susceptible to infections because their immune systems are not fully developed and react to antigen exposure preferentially with unresponsiveness. uv-inactivated herpes simplex virus type 1 (hsv-1) represents such an antigen and does not induce an immune response in neonates. in contrast, protective t cells were primed in newborn mice by a single replicative cycle of disc hsv-1 given once within 24 h of birth. each of the hsv-1-primed cd4(+) or cd8(+) t cells induced in wild-ty ...200111119576
role of nf-kappab and myc proteins in apoptosis induced by hepatitis b virus hbx protein.chronic infection with hepatitis b virus (hbv) promotes a high level of liver disease and cancer in humans. the hbv hbx gene encodes a small regulatory protein that is essential for viral replication and is suspected to play a role in viral pathogenesis. hbx stimulates cytoplasmic signal transduction pathways, moderately stimulates a number of transcription factors, including several nuclear factors, and in certain settings sensitizes cells to apoptosis by proapoptotic stimuli, including tumor n ...200111119591
calpain inhibition protects against virus-induced apoptotic myocardial injury.viral myocarditis is an important cause of human morbidity and mortality for which reliable and effective therapy is lacking. using reovirus strain 8b infection of neonatal mice, a well-characterized experimental model of direct virus-induced myocarditis, we now demonstrate that myocardial injury results from apoptosis. proteases play a critical role as effectors of apoptosis. the activity of the cysteine protease calpain increases in reovirus-infected myocardiocytes and can be inhibited by the ...200111119604
alphavirus vectors for gene therapy applications.high-titer alphavirus vectors have been generated for efficient gene delivery both in vitro and in vivo. studies on cns infection via intranasal and peripheral injections with virulent and avirulent replication-competent semliki forest virus (sfv) strains has demonstrated the potential of gene delivery. replication-deficient alphavirus particles have shown high local transgene expression of a transient nature in rodent brain. alphavirus vectors have been demonstrated to induce apoptosis in infec ...200112109136
human immunodeficiency virus type 1 gag-specific vaginal immunity and protection after local immunizations with sindbis virus-based replicon particles.the majority of human immunodeficiency virus (hiv) infections occur through vaginal and rectal transmission. in seeking a safe, nonreplicating gene-delivery vector that can induce mucosal and systemic immune responses and protection, sindbis virus-based replicon particles expressing hiv-1 gag (sin-gag) were developed. in mice, after nasal or intramuscular immunization with sin-gag and vaginal challenge with vaccinia virus (vv) expressing hiv-1 gag (vv-gag), cd8(+) t cell-mediated responses were ...200111740739
interferon-stimulated response element (isre)-binding protein complex draf1 is activated in sindbis virus (hr)-infected cells.to elucidate the host cell defense mechanisms in response to sindbis viral infection, we have started to characterize interferon (ifn)-stimulated response element (isre)-binding proteins activated in infected cells that are involved in the transcriptional induction of ifn type i-inducible genes. using electromobility shift assays (emsa), we detected several protein complexes with a human ifn-stimulated gene 15 (isg15) isre in extracts from virus-infected l929 cells that were absent in extracts f ...200111747630
sindbis virus-based vectors for the study of class i antigen presentation in vitro and in vivo. 200111068754
ross river virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production.pseudotyped retroviruses have important applications as vectors for gene transfer and gene therapy and as tools for the study of viral glycoprotein function. recombinant moloney murine leukemia virus (mo-mulv)-based retrovirus particles efficiently incorporate the glycoproteins of the alphavirus ross river virus (rrv) and utilize them for entry into cells. stable cell lines that produce the rrv glycoprotein-pseudotyped retroviruses for prolonged periods of time have been constructed. the pseudot ...200111222688
inhibition of hepatitis b virus replication by the interferon-inducible mxa protein.human mxa is an alpha/beta interferon-inducible intracytoplasmic protein that mediates antiviral activity against several rna viruses. we had previously shown that overexpression of the hepatitis b virus (hbv) capsid led to selective downregulation of mxa gene expression, suggesting a mechanism by which the virus escapes from the host defense system (o. rosmorduc, h. sirma, p. soussan, e. gordien, p. lebon, m. horisberger, c. brechot and d. kremsdorf, j. gen. virol. 80:1253-1262, 1999). in the p ...200111222692
in vitro assembly of sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins.a nucleic acid-bound capsid protein dimer was previously identified using a sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. this intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. this paper presents the purification of cross-linked dimers of a trunc ...200111222705
differences in cytokine and chemokine responses during neurological disease induced by polytropic murine retroviruses map to separate regions of the viral envelope gene.infection of the central nervous system (cns) by several viruses can lead to upregulation of proinflammatory cytokines and chemokines. in immunocompetent adults, these molecules induce prominent inflammatory infiltrates. however, with immunosuppressive retroviruses, such as human immunodeficiency virus (hiv), little cns inflammation is observed yet proinflammatory cytokines and chemokines are still upregulated in some patients and may mediate pathogenesis. the present study examined expression o ...200111222710
expression of human mxa protein in mosquito cells interferes with lacrosse virus replication.human mxa protein inhibits lacrosse virus (lac virus; family bunyaviridae) replication in vertebrate cells and mxa-transgenic mice. lac virus is transmitted to humans by aedes triseriatus mosquitoes. in this report, we have shown that transfected mosquito cells expressing the human mxa cdna are resistant to lac virus but permissive for sindbis virus (family togaviridae) infection.200111222725
identification of b-cell epitope of dengue virus type 1 and its application in diagnosis of patients.using a serotype-specific monoclonal antibody (mab) of dengue virus type 1 (den-1), 15f3-1, we identified the b-cell epitope of den-1 from a random peptide library displayed on phage. fourteen immunopositive phage clones that bound specifically to mab 15f3-1 were selected. these phage-borne peptides had a consensus motif of hxyawb (a = s/t, b = k/h/r) that mimicked the sequence hkyswk, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (ns1) of den-1. among the f ...200111230414
uag readthrough in mammalian cells: effect of upstream and downstream stop codon contexts reveal different signals.translation termination is mediated through an interaction between the release factors erf1 and erf3 and the stop codon within its nucleotide context. although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.200111242562
lectin and carbohydrate affinity capture surfaces for mass spectrometric analysis of microorganisms.in a preliminary report (bundy, j. l.; fenselau, c. anal. chem 1999, 71, 1460-1463), we demonstrated the use of lectin-derivatized surfaces to capture and concentrate complex carbohydrates as well as microorganisms from sample matrixes unamenable to direct maldi mass spectrometry. here, we extend the work to include samples representative of a wider variety of microorganisms of importance to human health and of enveloped viruses. in this study, lectins were immobilized directly to a membrane sur ...200111248888
comparison of bcl-2 to a bcl-2 deletion mutant for mammalian cells exposed to culture insults.apoptosis has been found to occur in bioreactors as a result of environmental stresses. the overexpression of bcl-2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. in this study, the effectiveness of wild-type bcl-2 was compared to a bcl-2 mutant lacking the nonstructured loop domain in two commercially prominent cell lines, chinese hamster ovary (cho) and baby hamster kidney (bhk) cells. the generation of a dna "ladder" and condensation of chromatin ind ...200111257603
human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase-mediated suicide gene therapy.to evaluate human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase (hsv-tk) -mediated gene therapy, we tested the utility of different viral vectors on three human cell lines du-145, lncap, and pc-3. our viral vectors were carrying a fusion gene of hsv-tk and green fluorescent protein for accurate determination of the gene transfer rate and its contribution to the treatment outcome in each case. we observed that adenoviral and lentiviral vectors were efficient vehicl ...200111263529
sequence requirements for sindbis virus subgenomic mrna promoter function in cultured cells.the sindbis virus minimal subgenomic mrna promoter (spanning positions -19 to +5 relative to the subgenomic mrna start site) is approximately three- to sixfold less active than the fully active -98 to +14 promoter region. we identified two elements flanking the -19 to +5 region which increase its transcription to levels comparable to the -98 to +14 region. these elements span positions -40 to -20 and +6 to +14 and act synergistically to enhance transcription. nine different virus libraries were ...200111264340
role of alpha/beta interferon in venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region.venezuelan equine encephalitis virus (vee) is an important equine and human pathogen of the americas. in the adult mouse model, cdna-derived, virulent v3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. a single change (g to a) at nucleotide 3 (nt 3) of the 5' untranslated region (utr) of the v3000 genome resulted in a virus (v3043) that was avirulent in mice. the mechanism of attenuation by the v3043 mutation was stu ...200111264360
y2, the smallest of the sendai virus c proteins, is fully capable of both counteracting the antiviral action of interferons and inhibiting viral rna synthesis.an open reading frame (orf) overlapping the amino-terminal portion of the sendai virus (sev) p orf in the +1 frame produces a nested set of carboxy-coterminal proteins, c', c, y1, and y2, which are referred to collectively as the c proteins. the c proteins are extremely versatile triple-role players; they counteract the antiviral action of interferons (ifns), inhibit viral rna synthesis, and are involved in virus assembly. in this study, we established hela cell lines stably expressing the c, y1 ...200111264369
biogenesis of the semliki forest virus rna replication complex.the nonstructural (ns) proteins nsp1 to -4, the components of semliki forest virus (sfv) rna polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. all ns proteins were associated with large cytoplasmic vacuoles (cpv), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. all ns proteins were localized by immuno-electron microscopy (em) to the li ...200111264376
coupling of antibodies via protein z on modified polyoma virus-like particles.therapeutic application of virus-based delivery systems often implies a change of the tropism of these vectors. this can be achieved by insertion of polypeptides (e.g., antibody fragments) in viral coat proteins. such fusion proteins have only been used in viral vectors so far and, as part of a virus, they have not been available for a detailed biophysical characterization. we analyzed a fusion protein called vp1-z, which is based on the polyoma virus coat protein vp1 and protein z. protein z is ...200111266629
arbovirus of marine mammals: a new alphavirus isolated from the elephant seal louse, lepidophthirus macrorhini.a novel alphavirus was isolated from the louse lepidophthirus macrorhini, collected from southern elephant seals, mirounga leonina, on macquarie island, australia. the virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known australasian alphaviruses. nearly all macquarie island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified. antarctic division personnel who have worked exte ...200111287559
pharmacokinetics study of a novel chimeric single-chain variable fragment antibody against western equine encephalitis virus.a novel recombinant single-chain fragment variable (scfv) antibody against western equine encephalitis (wee) virus has been previously constructed and partially characterized. the rs10b5hufc antibody was made by fusing an anti-wee scfv to a human heavy-chain igg1 constant region. the rs10b5hufc antibody was functional in binding to wee virus in enzyme-linked immunosorbent assays (elisas), and the fc domain of the antibody was capable of effector functions, such as binding to protein g and human ...200111289221
activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing p2x2-green fluorescent protein receptors.atp-gated p2x(2) receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. we engineered functional green fluorescent protein (gfp)-tagged p2x(2) receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. atp application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher p2x(2)-gfp receptor density. redistribu ...200111296257
prevalence of sindbis virus neutralizing antibodies among swedish passerines indicates that thrushes are the main amplifying hosts.the sindbis virus uses birds as vertebrate hosts in the summer amplification cycle, and the virus is transmitted by ornithophilic culex species. previous field and experimental studies have shown that mainly passerine birds are involved in the amplification. to delineate the pattern of sindbis virus infections among passerines, we collected and sampled birds for blood at five study sites located in northern, central, and southern sweden. all study sites were lowland forested wetlands and humid f ...200111296837
non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex.the replicase polyproteins of equine arteritis virus (eav; family arteriviridae, order nidovirales) are processed by three viral proteases to yield 12 non-structural proteins (nsps). the nsp2 and nsp3 cleavage products have previously been found to interact, a property that allows nsp2 to act as a co-factor in the processing of the downstream part of the polyprotein by the nsp4 protease. remarkably, upon infection of vero cells, but not of bhk-21 or rk-13 cells, eav nsp2 is now shown to be subje ...200111297673
locations of carbohydrate sites on alphavirus glycoproteins show that e1 forms an icosahedral scaffold.there are 80 spikes on the surface of sindbis virus arranged as an icosahedral surface lattice. each spike consists of three copies of each of the glycoproteins e1 and e2. there are two glycosylation sites on e1 and two on e2. these four sites have been located by removal of the glycosylation recognition motifs using site-specific mutagenesis, followed by cryoelectron microscopy. the positions of these sites have demonstrated that e2 forms the protruding spikes and that e1 must be long and narro ...200111301008
enhancement of hepatitis c virus rna replication by cell culture-adaptive mutations.studies of the hepatitis c virus (hcv) replication cycle have been made possible with the development of subgenomic selectable rnas that replicate autonomously in cultured cells. in these replicons the region encoding the hcv structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these rnas. subsequent analyses revealed that, within selected cells, hcv rnas had acquired adaptive mutations that i ...200111312331
m-x-i motif of semliki forest virus capsid protein affects nucleocapsid assembly.alphavirus budding is driven by interactions between spike and nucleocapsid proteins at the plasma membrane. the binding motif, y-x-l, on the spike protein e2 and the corresponding hydrophobic cavity on the capsid protein were described earlier. the spike-binding cavity has also been suggested to bind an internal hydrophobic motif, m113-x-i115, of the capsid protein. in this study we found that replacement of amino acids m113 and i115 with alanines, as single or double mutations, abolished forma ...200111312332
coupling between replication and packaging of flavivirus rna: evidence derived from the use of dna-based full-length cdna clones of kunjin virus.in order to study whether flavivirus rna packaging is dependent on rna replication, we generated two dna-based kunjin virus constructs, pkun1 and pkun1dgdd, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the ns5 gene rna-polymerase motif gdd) full-length kunjin virus rnas, respectively, via nuclear transcription by cellular rna polymerase ii. as expected, transfection of pkun1 plasmid dna into bhk cells resulted in the recovery of secreted infect ...200111312333
rna is a structural element in retrovirus particles.a single retroviral protein, gag, is sufficient for virus particle assembly. while gag is capable of specifically packaging the genomic rna into the particle, this rna species is unnecessary for particle assembly in vivo. in vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant gag proteins, apparently by acting as "scaffolding" in the particle. to address the participation of rna in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack ge ...200111320254
analysis of viral glycoproteins by maldi-tof mass spectrometry.membrane glycoproteins were shown to be useful biomarkers of enveloped viruses using on-target deglycosylation and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (maldi-tof ms). sindbis virus, the prototype alpha-virus, was used as a model system. the glycoproteins and the capsid protein of the sindbis virus were successfully detected by maldi-tof ms using two solvent systems. one of them is 0.5% n-octyl glucoside/0.5% trifluoroacetic acid. the two components of thi ...200111321307
sindbis viral-mediated expression of ca2+-permeable ampa receptors at hippocampal ca1 synapses and induction of nmda receptor-independent long-term potentiation.gene manipulation in order to artificially express a particular gene in neurons in the central nervous system is a powerful tool for the analysis of brain function. sindbis viral vectors have been developed to express high levels of foreign genes in postmitotic brain neurons with little transfection of glial cells. in this study, we expressed the gene encoding the unedited glur2 (glur-b) subunit of the ampa-type glutamate receptor that forms inwardly rectifying and ca2+-permeable channels, in ra ...200111328357
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