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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
epidermal growth factor and membrane trafficking. egf receptor activation of endocytosis requires rab5a.activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. here we show that egf stimulation of nr6 cells induces a specific, rapid and transient activation of rab5a. egf also enhanced translocation of the rab5 effector, earl ...011062256
sindbis virus-based vectors for the study of class i antigen presentation in vitro and in vivo. 200111068754
a mutant allele of essential, general translation initiation factor ded1 selectively inhibits translation of a viral mrna.positive-strand rna virus genomes are substrates for translation, rna replication, and encapsidation. to identify host factors involved in these functions, we used the ability of brome mosaic virus (bmv) rna to replicate in yeast. we report herein identification of a mutation in the essential yeast gene ded1 that inhibited bmv rna replication but not yeast growth. ded1 encodes a dead (asp-glu-ala-asp)-box rna helicase required for translation initiation of all yeast mrnas. inhibition of bmv rna ...200011069307
the host range phenotype displayed by a sindbis virus glycoprotein variant results from virion aggregation and retention on the surface of mosquito cells.the sindbis virus variant ne2g216 is a pe2-containing host range mutant that is growth restricted in cultured mosquito cells (c6/36) due to inefficient release of virions from this cell type. the maturation defect of ne2g216 has been linked to the structures of n-linked oligosaccharides synthesized by arthropod cells. analysis of c6/36 cells infected with ne2g216 by transmission electron microscopy revealed the presence of dense virus aggregates within cytoplasmic vacuoles and virus aggregates a ...200011070041
survival motor neuron protein modulates neuron-specific apoptosis.spinal muscular atrophy (sma) is attributed to mutations in the smn1 gene, leading to loss of spinal cord motor neurons. the neurotropic sindbis virus vector system was used to investigate a role for the survival motor neuron (smn) protein in regulating neuronal apoptosis. here we show that smn protects primary neurons and differentiated neuron-like stem cells, but not cultured cell lines from virus-induced apoptotic death. smn also protects neurons in vivo and increases survival of virus-infect ...200011078511
broad antiviral activity in tissues of crustaceans.innate antiviral substances occur in vertebrates and may function as host defenses. virus infections are common among invertebrates, but little is known about the ability of invertebrates to control viral infections. pre-existing antiviral substances may be particularly important, since invertebrates lack the antiviral defense conferred by specific immunity. in our study, we found that tissue extracts of blue crab (callinectes sapidus), shrimp (penaeus setiferus), and crayfish (procambarus clark ...200011080539
correlation of susceptibility of immature mice to fungal infection (blastomycosis) and effector cell function.immature mice are highly susceptible to blastomycosis, which is similar to other mycoses and has parallels in humans. the murine susceptibility is noteworthy in that it persists beyond the development of resistance to other, nonfungal pathogens and the maturation of most immune functions. as the susceptibility to blastomycosis appeared to be related to an early event after infection, primary effector cell function was studied. we found that peritoneal inflammatory cells, enriched for neutrophils ...200011083802
a universal 'one-tube' rt-pcr protocol for amplifying isolates of bovine viral diarrhoea virus.the increase in the knowledge of the genetic variability of bvdv and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. this study was undertaken to develop a standard protocol for rt-pcr that allows the amplification of various parts of the genome of bvdv without the need for optimizing each individual reaction. the reaction set-up is very flexible because it ...200011085469
prolonged arthritis associated with sindbis-related (pogosta) virus infection.a follow-up study of musculoskeletal symptoms after pogosta virus infection.200011085809
interaction between complement receptor gc1qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation.hepatitis c virus (hcv) is an important human pathogen that is remarkably efficient at establishing persistent infection. the hcv core protein is the first protein expressed during the early phase of hcv infection. our previous work demonstrated that the hcv core protein suppresses host immune responses, including anti-viral cytotoxic t-lymphocyte responses in a murine model. to investigate the mechanism of hcv core-mediated immunosuppression, we searched for host proteins capable of associating ...200011086025
enhancing b- and t-cell immune response to a hepatitis c virus e2 dna vaccine by intramuscular electrical gene transfer.we describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis c virus (hcv) envelope 2 (e2) glycoprotein responses in mammals through electrical gene transfer (egt) of plasmid dna into muscle fibers. intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (hvr1) peptide mimic fused at the n terminus of the e2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, in ...200011090158
infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the e2 glycoprotein.the ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. toward this goal, we derived variants of the prototype alphavirus, sindbis virus (sin), with differential abilities to infect human dendritic cells. cloning and sequencing of the sin variant genomes revealed that the genetic determinant for human dendritic cell (dc) tropism mapped to a single amino acid substit ...200011090185
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
efficient enhancement of priming effect by intermittent treatment with interferon.to improve the efficacy of interferon (ifn) therapy for chronic hepatitis c, we proposed a therapy with twice-a-day injection of ifnbeta as the induction. to assess its biological enhancement, we compared antiviral activities in vitro using intermittent treatment schedules simulating the clinical condition. fl cells were treated with ifnbeta twice in 12 h interval (single treatment, 1000 and 0 iu/ml; double treatment, 500 iu/ml each) and challenged with sindbis virus. antiviral activities were d ...200011095955
cytopathogenesis and inhibition of host gene expression by rna viruses.many viruses interfere with host cell function in ways that are harmful or pathological. this often results in changes in cell morphology referred to as cytopathic effects. however, pathogenesis of virus infections also involves inhibition of host cell gene expression. thus the term "cytopathogenesis," or pathogenesis at the cellular level, is meant to be broader than the term "cytopathic effects" and includes other cellular changes that contribute to viral pathogenesis in addition to those chan ...011104816
c-iap1 is cleaved by caspases to produce a proapoptotic c-terminal fragment.although human c-iap1 and c-iap2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-iap1 and c-iap2 failed to protect cells from apoptosis induced by bax overexpression, tumor necrosis factor alpha treatment or sindbis virus infection. however, deletion of the c-terminal ring domains of c-iap1 and c-iap2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic ...200111106668
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 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
chronic arthritis after sindbis-related (pogosta) virus infection. 200011132212
characterization of the adomet-dependent guanylyltransferase activity that is associated with the n terminus of bamboo mosaic virus replicase.bamboo mosaic virus (bamv), a member of the potexvirus group, infects primarily members of the bambusoideae. open reading frame 1 (orf1) of bamv encodes a 155-kda polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5' end of the viral genome. to identify and characterize the enzymatic activities associated with the n-terminal domain of the bamv orf1 protein, the intact replicase and two c-terminally truncated proteins ...200111134291
recognition of nascent rna by the human la antigen: conserved and divergent features of structure and function. 200111134326
human immunodeficiency virus type 1 vpu protein affects sindbis virus glycoprotein processing and enhances membrane permeabilization.the human immunodeficiency virus type 1 (hiv-1) vpu is an integral membrane protein that forms oligomeric structures in membranes. expression of vpu using sindbis virus (sv) as a vector leads to permeabilization of plasma membrane to hydrophilic molecules and impaired maturation of wild type sv glycoproteins in bhk cells. the 6k protein is a membrane protein encoded in the sv genome that facilitates budding of virus particles and regulates transport of viral glycoproteins through the secretory p ...200111145902
sorting of marburg virus surface protein and virus release take place at opposite surfaces of infected polarized epithelial cells.marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. we have investigated here the vectorial transport of the surface protein gp of marburg virus in polarized epithelial cells. to this end, we established an mdckii cell line that was able to express gp permanently (mdck-gp). the functional integrity of gp expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. further experiments revealed that gp is transp ...200111152500
mutations in hepatitis c virus rnas conferring cell culture adaptation.as an initial approach to studying the molecular replication mechanisms of hepatitis c virus (hcv), a major causative agent of acute and chronic liver disease, we have recently developed selectable self-replicating rnas. these replicons lacked the region encoding the structural proteins and instead carried the gene encoding the neomycin phosphotransferase. although the replication levels of these rnas within selected cells were high, the number of g418-resistant colonies was reproducibly low. in ...200111152517
human papillomavirus infection requires cell surface heparan sulfate.using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (hpv-16) and hpv-33 pseudovirions. pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. carboxy-terminally deleted hpv-33 virus-like particles still bound efficiently to heparin. the kinetics of postattachment neutralization by antiseru ...200111152531
ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies.using the vesicular stomatitis virus (vsv) pseudotype system, we studied the functional properties of the ebola virus glycoprotein (gp). amino acid substitutions at the gp cleavage site, which reduce glycoprotein cleavability and viral infectivity in some viruses, did not appreciably change the infectivity of vsv pseudotyped with gp. likewise, removal of two acylated cysteine residues in the transmembrane region of gp showed no discernible effects on infectivity. although most filoviruses are be ...200111152533
hostile takeovers: viral appropriation of the nf-kappab pathway. 200111160127
polarity of human parainfluenza virus type 3 infection in polarized human lung epithelial a549 cells: role of microfilament and microtubule.human parainfluenza virus type 3 (hpiv-3) is an airborne pathogen that infects the epithelial cells of the respiratory tract. in the present study we investigated the interaction of hpiv-3 with the type ii alveolar human lung polarized epithelial a549 cells. although hpiv-3 entry and budding were bidirectional from both the apical and the basolateral domains, hpiv-3 exhibited preferential entry and release from the apical pole. while disruption of the cellular actin microfilament and microtubule ...200111160698
exposure to low ph is not required for penetration of mosquito cells by sindbis virus.it is widely held that the penetration of cells by alphaviruses is dependent on exposure to the acid environment of an endosome. the alphavirus sindbis virus replicates in both vertebrate and invertebrate cell cultures. we have found that exposure to an acid environment may not be required for infection of cells of the insect host. in this work, we investigated the effects of two agents (nh(4)cl and chloroquine), which raise the ph of intracellular compartments (lysosomotropic weak bases) on the ...200111160702
yellow fever virus encephalitis: properties of the brain-associated t-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for cd4+ lymphocytes.viral encephalitis caused by neuroadapted yellow fever 17d virus (pyf) was studied in parental and gamma interferon (ifn-gamma)-deficient (ifn-gamma knockout [gko]) c57bl/6 mice. the t-cell responses which enter the brain during acute fatal encephalitis of nonimmunized mice, as well as nonfatal encephalitis of immunized mice, were characterized for relative proportions of cd4+ and cd8+ cells, their proliferative responses, and antigen-specific expression of cytokines during stimulation in vitro. ...200111160715
enhancement of sindbis virus self-replicating rna vaccine potency by linkage of herpes simplex virus type 1 vp22 protein to antigen.recently, self-replicating and self-limiting rna vaccines (rna replicons) have emerged as an important form of nucleic acid vaccines. self-replicating rna eventually causes lysis of transfected cells and does not raise the concern associated with naked dna vaccines of integration into the host genome. this is particularly important for development of vaccines targeting proteins that are potentially oncogenic. however, the potency of rna replicons is significantly limited by their lack of intrins ...200111160740
transmission of replication-defective sindbis helper vectors encoding capsid and envelope proteins.a green fluorescent protein (gfp)-encoding derivative of the replication-defective sindbis helper vector dhbb was constructed in order to study the rate of packaging of the helper vector. the results show that despite lacking a 'packaging sequence', this vector is co-packaged about 1/300th the rate of packaging of single rnas containing a packaging sequence. this helps explain the frequent observation of recombinant, replication-competent viruses when using first generation sindbis packaging sys ...200111164486
enhancement of sindbis virus self-replicating rna vaccine potency by targeting antigen to endosomal/lysosomal compartments.self-replicating rna vaccines (rna replicons) have emerged as an attractive approach for tumor immunotherapy. rna replicons do not integrate into host chromosomes, eliminating the concern for oncogenicity associated with a dna vaccine. in this study, we used human papillomavirus type 16 (hpv-16) e7 as a model antigen and evaluated e7-specific immunity generated by a sindbis virus self-replicating rna vector, sin-rep5. three different constructs were created to target e7 antigen to different cell ...200111177561
the effects of testosterone on a viral infection in greenfinches (carduelis chloris): an experimental test of the immunocompetence-handicap hypothesis.the immunocompetence-handicap hypothesis suggests that the honesty of quality signals could be guaranteed if testosterone (t) suppresses immune function while enhancing male ornaments. in addition, it has been proposed that the cost of enhancing ornaments should be highest for males with small ornaments. recently, the assertion that t causes obligate immunosuppression has been questioned. in this study, we tested whether elevated t levels would increase susceptibility to a viral infection, and w ...200111209893
measurement of rab5 protein kinase b/akt and regulation of ras-activated endocytosis. 200111210531
viral neuroinvasion as a marker for bbb integrity following exposure to cholinesterase inhibitors.exposure to the nerve agent soman, an irreversible cholinesterase (che) inhibitor, results in changes in blood-brain barrier permeability attributed to its seizure-induced activity. however, smaller bbb changes may be independent of convulsions. such minor injury may escape detection. a nonneuroinvasive neurovirulent sindbis virus strain (svn) was used as a marker for bbb permeability. peripheral inoculation of mice with 2 x 10(3) plaque forming units (pfu) caused up to 10(5) pfu/ml viremia afte ...200111212873
vaccines against hantaviruses. 200111217404
virus-expressed, recombinant single-chain antibody blocks sporozoite infection of salivary glands in plasmodium gallinaceum-infected aedes aegypti.transgenic mosquitoes resistant to malaria parasites are being developed to test the hypothesis that they may be used to control disease transmission. we have developed an effector portion of an antiparasite gene that can be used to test malaria resistance in transgenic mosquitoes. mouse monoclonal antibodies that recognize the circumsporozoite protein of plasmodium gallinaceum can block sporozoite invasion of aedes aegypti salivary glands. an anti-circumsporozoite monoclonal antibody, n2h6d5, w ...200011220756
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
enhancement of sindbis virus self-replicating rna vaccine potency by linkage of mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene.recently, self-replicating rna vaccines (rna replicons) have emerged as an effective strategy for nucleic acid vaccine development. unlike naked dna vaccines, rna replicons eventually cause lysis of transfected cells and therefore do not raise the concern of integration into the host genome. we evaluated the effect of linking human papillomavirus type 16 e7 as a model ag to mycobacterium tuberculosis heat shock protein 70 (hsp70) on the potency of ag-specific immunity generated by a sindbis viru ...200111342644
adaptation of tick-borne encephalitis virus to bhk-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo.propagation of the flavivirus tick-borne encephalitis virus in bhk-21 cells selected for mutations within the large surface glycoprotein e that increased the net positive charge of the protein. in the course of 16 independent experiments, 12 different protein e mutation patterns were identified. these were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein e. the mutations resulted in the formation of local patches of predom ...200111356970
new immunochromatographic rapid test for diagnosis of acute puumala virus infection.a new immunochromatographic rapid test, poc puumala (erilab ltd., kuopio, finland), for detection of acute-phase puumala virus (puuv) infection was developed based on a highly purified baculovirus-expressed puuv nucleocapsid protein antigen and lateral immunodiffusion techniques. after addition of sample (5 microl of serum, plasma, or fingertip blood) and buffer, puuv-specific immunoglobulin m (igm) antibodies, if present, together with the gold-conjugated anti-human igm, formed a specific color ...200111376049
physicians' database searches as a tool for early detection of epidemics.we analyzed retrospectively the use of physician desk reference database searches to identify epidemics of tularemia, nephropathy, pogosta disease, and lyme disease and compared the searches with mandatory laboratory reports to the national infectious diseases register in finland during 1995. continuous recording of such searches may be a tool for early detection of epidemics.200111384534
deacylation of the transmembrane domains of sindbis virus envelope glycoproteins e1 and e2 does not affect low-ph-induced viral membrane fusion activity.the envelope glycoproteins e1 and e2 of sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (e1 at position 430, and e2 at positions 388 and 390). here, we investigated the in vitro membrane fusion activity of sindbis virus variants (derived from the toto 1101 infectious clone), in which the e1 c430 and/or e2 c388/390 residues had been substituted for alanines. both the e1 and e2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a stric ...200111389898
potential sources of the 1995 venezuelan equine encephalitis subtype ic epidemic.venezuelan equine encephalitis viruses (veev) belonging to subtype ic have caused three (1962-1964, 1992-1993 and 1995) major equine epizootics and epidemics. previous sequence analyses of a portion of the envelope glycoprotein gene demonstrated a high degree of conservation among isolates from the 1962-1964 and the 1995 outbreaks, as well as a 1983 interepizootic mosquito isolate from panaquire, venezuela. however, unlike subtype iab veev that were used to prepare inactivated vaccines that prob ...200111390583
trypsin cleavage stabilizes the rotavirus vp4 spike.trypsin enhances rotavirus infectivity by an unknown mechanism. to examine the structural basis of trypsin-enhanced infectivity in rotaviruses, sa11 4f triple-layered particles (tlps) grown in the absence (nontrypsinized rotavirus [ntr]) or presence (trypsinized rotavirus [tr]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. as expected, vp4 was not cleaved in ntr particles and was cleaved ...200111390607
stat1-independent regulation of gene expression in response to ifn-gamma.although stat1 is essential for cells to respond fully to ifn-gamma, there is substantial evidence that, in the absence of stat1, ifn-gamma can still regulate the expression of some genes, induce an antiviral state and affect cell growth. we have now identified many genes that are regulated by ifn-gamma in serum-starved stat1-null mouse fibroblasts. the proteins induced by ifn-gamma in stat1-null cells can account for the substantial biological responses that remain. some genes are induced in bo ...200111390994
biologic consequences of stat1-independent ifn signaling.although stat1 is required for many ifn-dependent responses, recent work has shown that ifngamma functions independently of stat1 to affect the growth of tumor cells or immortalized fibroblasts. we now demonstrate that both ifngamma and ifnalpha/beta regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from stat1-null mice. using both representational difference analysis and gene arrays, we show that ifngamma exerts its stat1-independent actions on mononuclear p ...200111390995
dna vaccination against neu reduces breast cancer incidence and metastasis in mice.the gene for her2/neu is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression correlates with increased metastasis and poor prognosis. the her2/neu gene product, a transmembrane protein kinase member of the egf receptor family, has significant potential as a tumor antigen for vaccination. we inserted the sequence for neu into a novel plasmid called elvis containing a sindbis virus replicon that reproduces multiple copies of mrna. mice vaccinated one time intramuscularly ...200111393278
virus-specific mrna capping enzyme encoded by hepatitis e virus.hepatitis e virus (hev), a positive-strand rna virus, is an important causative agent of waterborne hepatitis. expression of cdna (encoding amino acids 1 to 979 of hev nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kda protein (p110), a fraction of which was proteolytically processed to an 80-kda protein. p110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. immunopurified p110 catalyzed transfer of a methyl group from ...200111413290
fidelity of leader and trailer sequence usage by the respiratory syncytial virus and avian pneumovirus replication complexes.the specificity of usage of promoters for replication and transcription by the pneumoviruses human respiratory syncytial virus (hrsv) and avian pneumovirus (apv) was studied using minigenomes containing a reporter gene. when infectious hrsv or apv was used as helper virus, replication could occur only if both the leader and trailer regions (containing the replicative and transcriptional promoters) were derived from the helper virus. in contrast, when the hrsv replication complex was supplied fro ...200111413292
an amino acid substitution in the coding region of the e2 glycoprotein adapts ross river virus to utilize heparan sulfate as an attachment moiety.passage of ross river virus strain nb5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. sequencing of these variants identified two independent sites that might be responsible for the phenotype. we now demonstrate, using a molecular cdna clone of the wild-type t48 strain, that an amino acid substitution at residue 218 in the e2 glycoprotein can account for the phenotype. substitutions that replaced the wild-type asparagine wi ...200111413296
induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus.avian coronavirus infectious bronchitis virus (ibv) is the causative agent of chicken infectious bronchitis, an acute, highly contagious viral respiratory disease. replication of ibv in vero cells causes extensive cytopathic effects (cpe), leading to destruction of the entire monolayer and the death of infected cells. in this study, we investigated the cell death processes during acute ibv infection and the underlying mechanisms. the results show that both necrosis and apoptosis may contribute t ...200111413307
a monoclonal antibody specific for reovirus outer-capsid protein sigma3 inhibits sigma1-mediated hemagglutination by steric hindrance.reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. outer-capsid protein sigma1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. monoclonal antibody (mab) 4f2, which is specific for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (ha). to determine whether mab 4f2 inhibits ha by altering sigma1 ...200111413330
viral replicase gene products suffice for coronavirus discontinuous transcription.we have used vaccinia virus as a vector to clone a 22.5-kbp cdna that represents the 5' and 3' ends of the human coronavirus 229e (hcov 229e) genome, the hcov 229e replicase gene, and a single reporter gene (coding for green fluorescent protein [gfp]) located downstream of a regulatory element for coronavirus mrna transcription. when rna transcribed from this cdna was transfected into bhk-21 cells, a small percentage of cells displayed strong fluorescence. a region of the mrna encoding gfp was a ...200111413334
innate gastrointestinal immunity: characterization of broadly active viral inhibitors.innate viral inhibitors that are broadly active have been characterized in the serum and the nervous system, but incompletely characterized in the gastrointestinal (gi) tract. gi preparations from porcine gastric mucosa, mouse intestine, and in neuramide (a pharmaceutical product), were examined for broad antiviral activity, molecular size and mechanism of action for comparison with the previously characterized, innate inhibitors in the serum and nervous system. the gi inhibitors were found to b ...200111428242
sindbis virus-induced neuronal death is both necrotic and apoptotic and is ameliorated by n-methyl-d-aspartate receptor antagonists.virus infection of neurons leads to different outcomes ranging from latent and noncytolytic infection to cell death. viruses kill neurons directly by inducing either apoptosis or necrosis or indirectly as a result of the host immune response. sindbis virus (sv) is an alphavirus that induces apoptotic cell death both in vitro and in vivo. however, apoptotic changes are not always evident in neurons induced to die by alphavirus infection. time lapse imaging revealed that sv-infected primary cortic ...200111435592
pocket protein p130/rb2 is required for efficient herpes simplex virus type 1 gene expression and viral replication.we have reported previously that herpes simplex virus type 1 (hsv-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a g(1)-like state. infected cells were characterized by a decline in cyclin-dependent kinase 2 (cdk2) activities, loss of hyperphosphorylated retinoblastoma protein (prb), accumulation of e2f-pocket protein complexes, and failure to initiate cellular dna replication. in the present study, we investigated the role of the pocket proteins prb ...200111435596
sindbis virus-induced silencing of dengue viruses in mosquitoes.aedes aegypti were injected intrathoracically with double subgenomic sindbis (dssin) viruses with inserted sequences derived from the genome of one or more of the four dengue (den) virus serotypes. mosquitoes were highly resistant to challenge with homologous den viruses from which the effector sequences were derived, and resistance to den viruses was independent of the orientation of the effector rna. dssin viruses designed to express rna derived from the premembrane coding region of den-2 prev ...200111437918
role of antibodies in controlling alphavirus infection of neurons. 200111443874
interferon-gamma-mediated site-specific clearance of alphavirus from cns neurons.recovery from viral encephalomyelitis requires immune-mediated noncytolytic clearance from neurons by mechanisms assumed to be the same for all neurons. in alphavirus encephalomyelitis, antibody clears infectious virus from neurons in all regions of the central nervous system (cns), but cd8 t cells contribute to elimination of viral rna. to understand the role of t cells in clearance, we infected antibody knockout mice with sindbis virus. virus was cleared from spinal cord and brain stem neurons ...200111452126
characterization of new syncytium-inhibiting monoclonal antibodies implicates lipid rafts in human t-cell leukemia virus type 1 syncytium formation.we have previously shown that erythroleukemia cells (k562) transfected with vascular adhesion molecule 1 (vcam-1) are susceptible to human t-cell leukemia virus type 1 (htlv-1)-induced syncytium formation. since expression of vcam-1 alone is not sufficient to render cells susceptible to htlv-1 fusion, k562 cells appear to express a second molecule critical for htlv-induced syncytium formation. by immunizing mice with k562 cells, we have isolated four monoclonal antibodies (mabs), k5.m1, k5.m2, k ...200111462007
human herpesvirus 8 envelope glycoprotein k8.1a interaction with the target cells involves heparan sulfate.human herpesvirus-8 (hhv-8) or kaposi's sarcoma-associated herpesvirus k8.1 gene encodes for two immunogenic glycoproteins, gpk8.1a and gpk8.1b, originating from spliced messages. the 228-amino-acid (aa) gpk8.1a is the predominant form associated with the virion envelope, consisting of a 167-aa region identical to gpk8.1b and a 61-aa unique region (l. zhu, v. puri, and b. chandran, virology 262:237-249, 1999). hhv-8 has a broad in vivo and in vitro cellular tropism, and our studies showed that t ...200111462024
recombinant measles viruses expressing altered hemagglutinin (h) genes: functional separation of mutations determining h antibody escape from neurovirulence.measles virus (mv) strain cam/rb, which was adapted to growth in the brain of newborn rodents, is highly neurovirulent. it has been reported earlier that experimentally selected virus variants escaping from the monoclonal antibodies (mabs) nc32 and l77 to hemagglutinin (h) preserved their neurovirulence, whereas mutants escaping mabs k71 and k29 were found to be strongly attenuated (u. g. liebert et al., j. virol. 68:1486-1493, 1994). to investigate the molecular basis of these findings, we have ...200111462033
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