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combinatorial screening and intracellular antiviral activity of hairpin ribozymes directed against hepatitis b virus.a combinatorial screening method has been used to identify hairpin ribozymes that inhibit hepatitis b virus (hbv) replication in transfected human hepatocellular carcinoma (hcc) cells. a hairpin ribozyme library (5 x 10(5) variants) containing a randomized substrate-binding domain was used to identify accessible target sites within 3.3 kb of full-length in vitro-transcribed hbv pregenomic rna. forty potential target sites were found within the hbv pregenomic rna, and 17 sites conserved in all fo ...199910364285
nonhomologous rna recombination in bovine viral diarrhea virus: molecular characterization of a variety of subgenomic rnas isolated during an outbreak of fatal mucosal disease.four bovine viral diarrhea virus type 2 (bvdv-2) pairs consisting of cytopathogenic (cp) and noncp bvdv-2 were isolated during an outbreak of mucosal disease. comparative sequence analysis showed that the four noncp bvdv-2 isolates were almost identical. for the cp bvdv-2 isolates, viral subgenomic rnas were shown by northern blot to have a length of about 8 kb, which is about 4.3 kb shorter than the genome of noncp bvdv. cytopathogenicity and the expression of ns3 were both strictly correlated ...199910364314
neutralizing antibodies inhibit axonal spread of herpes simplex virus type 1 to epidermal cells in vitro.the ability of antibodies to interfere with anterograde transmission of herpes simplex virus (hsv) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. the number and size of viral cytopathic plaques in epidermal cells after axonal transmission from hsv type 1 (hsv-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the ...199910364346
poliovirus induces apoptosis in the mouse central nervous system.poliovirus (pv) is the etiological agent of human paralytic poliomyelitis. paralysis results from the destruction of motoneurons, a consequence of pv replication. however, the pv-induced process leading to the death of motoneurons is not well known. we investigated whether pv-induced central nervous system (cns) injury is associated with apoptosis by using mice as animal models. transgenic mice expressing the human pv receptor were infected intracerebrally with either the neurovirulent pv-1 maho ...199910364359
svmpa, a mutant of sindbis virus resistant to mycophenolic acid and ribavirin, shows an increased sensitivity to chick interferon.svmpa is a mutant of sindbis virus, selected for its ability to replicate in mycophenolic acid (mpa)-treated mosquito cells. svmpa has another phenotype: although able to replicate normally in primary cultures of chick embryo fibroblasts (cef), its replication is restricted in secondary cultures prepared from aged primary cef cultures. the mutations responsible for these phenotypes mapped to the region of the viral genome that codes for nsp1. we report here that svmpa has yet another phenotype. ...199910364507
effect of rho and adp-ribosylation factor gtpases on phospholipase d activity in intact human adenocarcinoma a549 cells.phospholipase d (pld) has been implicated as a crucial signaling enzyme in secretory pathways. two 20-kda guanine nucleotide-binding proteins, rho and adp-ribosylation factor (arf), are involved in the regulation of secretion and can activate pld in vitro. we investigated in intact (human adenocarcinoma a549 cells) the role of rhoa and arf in activation of pld by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. to express recombinant clostridium botulinum c3 exoenzyme ...199910373471
phorbol ester promotes endocytosis by activating a factor involved in endosome fusion.previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of rab5 on endosome fusion. rab5 is a small gtpase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. in its gtp-bound form, rab5 promotes endocytosis and enhances fusion among early endosomes. to determine if pma stimulates endocytosis by activating a factor required for endosome fus ...199910393811
cancer therapy using a self-replicating rna vaccine.'naked' nucleic acid vaccines are potentially useful candidates for the treatment of patients with cancer, but their clinical efficacy has yet to be demonstrated. we sought to enhance the immunogenicity of a nucleic acid vaccine by making it 'self-replicating'. we accomplished this by using a gene encoding an rna replicase polyprotein derived from the semliki forest virus, in combination with a model antigen. a single intramuscular injection of a self-replicating rna immunogen elicited antigen-s ...199910395329
inhibition of virus-induced neuronal apoptosis by bax.the bax protein is widely known as a pro-apoptotic bcl-2 family member that when overexpressed can trigger apoptosis in multiple cell types and is important for the developmental cell death of neurons. however, bax was found here to be a potent inhibitor of neuronal cell death in mice infected with sindbis virus. newborn mice, which are highly susceptible to a fatal infection with neurotropic sindbis virus, were significantly protected from neuronal apoptosis and fatal disease when infected with ...199910395331
membrane permeabilization by small hydrophobic nonstructural proteins of japanese encephalitis virus.infection with japanese encephalitis virus (jev), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. we observed that jev replication rendered infected baby hamster kidney (bhk-21) cells sensitive to the translational inhibitor hygromycin b or alpha-sarcine, to which mock-infected cells were insensitive. however, little is known about whether any jev nonstructural (ns) proteins contribute to virus-induced c ...199910400716
the furin protease cleavage recognition sequence of sindbis virus pe2 can mediate virion attachment to cell surface heparan sulfate.cell culture-adapted sindbis virus strains attach to heparan sulfate (hs) receptors during infection of cultured cells (w. b. klimstra, k. d. ryman, and r. e. johnston, j. virol. 72:7357-7366, 1998). at least three e2 glycoprotein mutations (e2 arg 1, e2 lys 70, and e2 arg 114) can independently confer hs attachment in the background of the consensus sequence sindbis virus (tr339). in the studies reported here, we have investigated the mechanism by which the e2 arg 1 mutation confers hs-dependen ...199910400721
the cc-chemokine rantes increases the attachment of human immunodeficiency virus type 1 to target cells via glycosaminoglycans and also activates a signal transduction pathway that enhances viral infectivity.we have studied the mechanisms by which the cc-chemokine rantes can enhance the infectivities of human immunodeficiency virus type 1 (hiv-1) and other enveloped viruses, when present at concentrations in excess of 500 ng/ml in vitro. understanding the underlying mechanisms might throw light on fundamental processes of viral infection, in particular for hiv-1. our principal findings are twofold: firstly, that oligomers of rantes can cross-link enveloped viruses, including hiv-1, to cells via glyc ...199910400729
selective irreversible inactivation of replicating mengovirus by nucleoside analogues: a new form of viral interference.we describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (brtu) and tubercidin (tu). at a concentration of 5 microgram/ml, brtu reversibly blocked the synthesis of cellular mrna and rrna but did not inhibit either mengovirus rna synthesis or multiplication. brtu is a potent inhibitor of adenosine kinase, and low concentrations of brtu (e.g., 0.5 microgram/ml), which did not by themse ...199910400738
identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein g.respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract disease in infants and young children worldwide. infection is mediated, in part, by an initial interaction between attachment protein (g) and a highly sulfated heparin-like glycosaminoglycan (gag) located on the cell surface. synthetic overlapping peptides derived from consensus sequences of the g protein ectodomain from both rsv subgroups a and b were tested by heparin-agarose affinity chromatography for their abi ...199910400758
the h gene of rodent brain-adapted measles virus confers neurovirulence to the edmonston vaccine strain.molecular determinants of neuropathogenesis have been shown to be present in the hemagglutinin (h) protein of measles virus (mv). an h gene insertion vector has been generated from the edmonston b vaccine full-length infectious clone of mv. using this vector, it is possible to insert complete h open reading frames into the parental (edtag) background. the h gene from a rodent brain-adapted mv strain (cam/rb) was inserted into this vector, and a recombinant virus (edtagcamh) was rescued by using ...199910400789
two-helper rna system for production of recombinant semliki forest virus particles.alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. however, present strategies for production of such particles have biosafety limitations due to the generation, by rna recombination, of replication-proficient viruses (rpvs). here we describe a new packaging system for semliki forest virus (sfv) based on a the use of a two-helper system in which the capsid and ...19999882310
endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function.proteolytic processing is required for the activation of numerous viral glycoproteins. here we show that the envelope glycoprotein from the zaire strain of ebola virus (ebo-gp) is proteolytically processed into two subunits, gp1 and gp2, that are likely covalently associated through a disulfide linkage. murine leukemia virions pseudotyped with ebo-gp contain almost exclusively processed glycoprotein, indicating that this is the mature form of ebo-gp. mutational analysis identified a dibasic moti ...19999882347
replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes.the sequences involved in the replication and packaging of transmissible gastroenteritis virus (tgev) rna have been studied. the structure of a tgev defective interfering rna of 9.7 kb (di-c) was described previously (a. mendez, c. smerdou, a. izeta, f. gebauer, and l. enjuanes, virology 217: 495-507, 1996), and a cdna with the information to encode di-c rna was cloned under the control of the t7 promoter. the molecularly cloned di-c rna was replicated in trans upon transfection of helper virus- ...19999882359
class i mhc molecule-mediated inhibition of sindbis virus replication.the threshold for systemic viral infection relies on the amplification of virus at a primary infection site. we have identified that class i mhc molecules can trigger the inhibition of replication of sindbis virus in a haplotype- and allele-specific manner. class i mhc molecules of h-2d haplotypes exhibit a strong inhibitory effect whereas h-2k haplotypes show minimal inhibition of sindbis viral replication. by a single gene transfection of h-2d class i mhc molecules, into cells that express cla ...19999886371
a balance of signaling by rho family small gtpases rhoa, rac1 and cdc42 coordinates cytoskeletal morphology but not cell survival.rho family gtpases are known to be involved in cytoskeletal reorganization. we examined the possibility that these functions may be dictated by a balance of rho family gtpase signaling. using transient viral expression of rhoa, rac1, cdc42 and their mutants, as well as c3 exoenzyme, we altered cytoskeletal organization under normal growth conditions. overexpression of wild-type or constitutively active forms of the rho family gtpases led to their respective activation phenotypes. overexpression ...19999926919
cell-specific targeting of a thymidine kinase/ganciclovir gene therapy system using a recombinant sindbis virus vector.transfer of the herpes simplex virus type i thymidine kinase (hsv-tk) gene into tumor cells using virus-based vectors in conjunction with ganciclovir (gcv) exposure provides a potential gene therapy strategy for the treatment of cancer. the possibility of using a novel targetable sindbis virus expression vector containing the hsv-tk gene was examined. baby hamster kidney (bhk) cells and several human tumor cells infected with a sindbis virus containing the hsv-tk gene showed strong expression of ...19999935240
eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization.eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (egfr) and is characterized by the presence of a novel protein:protein interaction domain, the eh domain. eps15 also stably binds the clathrin adaptor protein complex ap-2. previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. in this study we show that, upon activation of the egfr kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the pl ...19999950686
regulated expression of a sindbis virus replicon by herpesvirus promoters.we describe the use of herpesvirus promoters to regulate the expression of a sindbis virus replicon (sinrep/lacz). we isolated cell lines that contain the cdna of sinrep/lacz under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. wild-type sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and beta- ...19999971780
open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex.the replicase of equine arteritis virus (eav; family arteriviridae, order nidovirales) is expressed in the form of two polyproteins (the open reading frame 1a [orf1a] and orf1ab proteins). three viral proteases cleave these precursors into 12 nonstructural proteins, which direct both genome replication and subgenomic mrna transcription. immunofluorescence assays showed that most eav replicase subunits localize to membranes in the perinuclear region of the infected cell. using replicase-specific ...19999971782
proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication.the open reading frame (orf) 1b-encoded part of the equine arteritis virus (eav) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an orf1ab fusion protein (345 kda). four orf1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in eav-infected cells (l. c. van dinten, a. l. m. wassenaar, a. e. gorbalenya, w. j. m. spaan, and e. j. snijder, j. virol. 70:6625-6633, 1996). ...19999971783
capsid structure of simian cytomegalovirus from cryoelectron microscopy: evidence for tegument attachment sites.we have used cryoelectron microscopy and image reconstruction to study b-capsids recovered from both the nuclear and the cytoplasmic fractions of cells infected with simian cytomegalovirus (scmv). scmv, a representative betaherpesvirus, could thus be compared with the previously described b-capsids of the alphaherpesviruses, herpes simplex virus type 1 (hsv-1) and equine herpesvirus 1 (ehv-1), and of channel catfish virus, an evolutionarily remote herpesvirus. nuclear b-capsid architecture is ge ...19999971801
in vivo addition of poly(a) tail and au-rich sequences to the 3' terminus of the sindbis virus rna genome: a novel 3'-end repair pathway.alphaviruses are mosquito-transmitted rna viruses that cause important diseases in both humans and livestock. sindbis virus (sin), the type species of the alphavirus genus, carries a 11.7-kb positive-sense rna genome which is capped at its 5' end and polyadenylated at its 3' end. the 3' nontranslated region (3'ntr) of the sin genome carries many au-rich motifs, including a 19-nucleotide (nt) conserved element (3'cse) and a poly(a) tail. this 3'cse and the adjoining poly(a) tail are believed to r ...19999971825
mutant influenza viruses with a defective ns1 protein cannot block the activation of pkr in infected cells.a short model genome rna and also the genome rna of influenza a virus bearing both 5'- and 3'-terminal common sequences activated the interferon-induced double-stranded-rna-dependent protein kinase, pkr, by stimulating autophosphorylation in vitro. the activated pkr catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eif2alpha). the ns1 protein efficiently eliminated the pkr-activating activity of these rnas by binding to them. two mutant ns1 proteins, e ...19999971827
resistance of interleukin-1beta-deficient mice to fatal sindbis virus encephalitis.interleukin-1beta (il-1beta) concentrations are frequently elevated in central nervous system (cns) viral infections, but the pathophysiologic significance of such elevations is not known. to examine the role of il-1beta in cns viral pathogenesis, we compared the natural histories of il-1beta-deficient and wild-type 129 sv(ev) mice infected with a neurovirulent viral strain, neuroadapted sindbis virus (nsv). we found that the incidence of severe paralysis and death was markedly decreased in nsv- ...19999971844
the inflammatory response to nonfatal sindbis virus infection of the nervous system is more severe in sjl than in balb/c mice and is associated with low levels of il-4 mrna and high levels of il-10-producing cd4+ t cells.sjl mice are susceptible to inflammatory autoimmune diseases of the central nervous system (cns), while balb/c mice are relatively resistant. to understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of sjl and balb/c mice to infection with sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. clearance of virus was similar, but sjl mice developed a more intense inflammatory response in the ...19999973422
rubella virus-induced apoptosis varies among cell lines and is modulated by bcl-xl and caspase inhibitors.rubella virus (rv) causes multisystem birth defects in the fetuses of infected women. to investigate the cellular basis of this pathology, we examined the cytopathic effect of rv in three permissive cell lines: vero 76, rk13, and bhk21. electron microscopy and the tunel assay showed that the cytopathic effect resulted from rv-induced programmed cell death (apoptosis) in all three cell lines, but the extent of apoptosis varied among these cells. at 48 h postinfection, the rk13 cell line showed th ...199910049827
alphaviruses as tools in neurobiology and gene therapy.the broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for semliki forest virus (sfv) and sindbis virus (sin). the generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. despite the viral cytopathogenic effects, functional assays on recombinant prot ...199910071792
a brome mosaic virus intergenic rna3 replication signal functions with viral replication protein 1a to dramatically stabilize rna in vivo.brome mosaic virus (bmv), a positive-strand rna virus in the alphavirus-like superfamily, encodes two rna replication proteins. the 1a protein has putative helicase and rna-capping domains, whereas 2a contains a polymerase-like domain. saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a bmv rna3 template produced by in vivo transcription of a dna copy of rna3. although insufficient for rna3 replication, the expression of 1a protein alone results in a dramatic and specific s ...199910074107
expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides.proteolytic processing of the replicase gene product of mouse hepatitis virus (mhv) is essential for viral replication. in mhv strain a59 (mhv-a59), the replicase gene encodes two predicted papain-like proteinase (plp) domains, plp-1 and plp-2. previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cis and trans cleavage activities for plp-1. we have cloned and overexpressed the plp-1 domain in escherichia ...199910074111
primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic rna1.barley yellow dwarf luteovirus (bydv) generates three 3'-coterminal subgenomic rnas (sgrnas) in infected cells. the promoter of sgrna1 is a putative hot spot for rna recombination in luteovirus evolution. the sgrna1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of bydv genomic rna (grna) in two independent studies. our data support the former initiation site. the boundaries of the sgrna1 promoter map between nucleotides 2595 and 2692 on genomic rna. ...199910074135
rotavirus capsid protein vp5* permeabilizes membranes.proteolytic cleavage of the vp4 outer capsid spike protein into vp8* and vp5* proteins is required for rotavirus infectivity and for rotavirus-induced membrane permeability. in this study we addressed the function of the vp5* cleavage fragment in permeabilizing membranes. expressed vp5* and truncated vp5* proteins were purified by nickel affinity chromatography and assayed for their ability to permeabilize large unilamellar vesicles (luvs) preloaded with carboxyfluorescein (cf). vp5* and vp5* tr ...199910074166
differentiation of m1 myeloid precursor cells into macrophages results in binding and infection by theiler's murine encephalomyelitis virus and apoptosis.infection of susceptible mouse strains with bean, a less virulent strain of theiler's murine encephalomyelitis virus (tmev), results in immune system-mediated demyelinating lesions in the central nervous system (cns) similar to those in multiple sclerosis. since macrophages appear to carry the major detectable antigen burden in vivo, and purification of sufficient cell numbers from the cns for detailed analysis is difficult, macrophage-like cell lines provide an accessible system with which to s ...199910074176
long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis c virus.two chimpanzees (ch1535 and ch1536) became infected with hepatitis c virus (hcv) following intrahepatic inoculation with rna transcribed from a full-length cdna clone of the virus. both animals were persistently infected and have been followed for 60 weeks. they showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (ch1535) and 12 (ch1536) postinoculation (p.i.). antibody responses to structural and nonstructural ...199910074186
acute and persistent infection of human neural cell lines by human coronavirus oc43.human coronaviruses (hucv) are recognized respiratory pathogens. data accumulated by different laboratories suggest their neurotropic potential. for example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the oc43 strain of hucv (a. bonavia, n. arbour, v. w. yong, and p. j. talbot, j. virol. 71:800-806, 1997). we speculate that the neurotropism of hucv will lead to persistence within the central nervous system, as was observed for murine corona ...199910074188
mutagenic analysis of the 3' cis-acting elements of the rubella virus genome.thermodynamically predicted secondary structure analysis of the 3'-terminal 305 nucleotides (nt) of the rubella virus (rub) genome, a region conserved in all rub defective interfering rnas, revealed four stem-loop (sl) structures; sl1 and sl2 are both located in the e1 coding region, while sl3 and sl4 are within the 59-nt 3' untranslated region (utr) preceding the poly(a) tract. sl2 is a structure shown to interact with human calreticulin (cal), an autoantigen potentially involved in rub rna rep ...199910074193
infectious pancreatic necrosis virus: identification of a vp3-containing ribonucleoprotein core structure and evidence for o-linked glycosylation of the capsid protein vp2.virions of infectious pancreatic necrosis virus (ipnv) were completely disintegrated upon dialysis against salt-free buffers. direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. by using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein vp3. isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire vp3 conte ...199910074207
rapid dendritic morphogenesis in ca1 hippocampal dendrites induced by synaptic activity.activity shapes the structure of neurons and their circuits. two-photon imaging of ca1 neurons expressing enhanced green fluorescent protein in developing hippocampal slices from rat brains was used to characterize dendritic morphogenesis in response to synaptic activity. high-frequency focal synaptic stimulation induced a period (longer than 30 minutes) of enhanced growth of small filopodia-like protrusions (typically less than 5 micrometers long). synaptically evoked growth was long-lasting an ...199910082466
geographic distribution and evolution of sindbis virus in australia.the molecular epidemiology and evolution of sindbis (sin) virus in australia was examined. several sin virus strains isolated from other countries were also included in the analysis. two regions of the virus genome were sequenced including a 418 bp region of the e2 gene and a 484 bp region containing part of the junction region and the 5' end of the c gene. analysis of the nucleotide and deduced amino acid sequence data from 40 sin virus isolates clearly separated the paleoarctic/ethiopian and o ...199910092014
antiviral effect of hyperthermic treatment in rhinovirus infection.human rhinoviruses (hrv) are recognized as the major etiologic agents for the common cold. starting from the observation that local hyperthermic treatment is beneficial in patients with natural and experimental common colds, we have studied the effect of brief hyperthermic treatment (ht) on hrv replication in hela cells. we report that a 20-min ht at 45 degrees c is effective in suppressing hrv multiplication by more than 90% when applied at specific stages of the virus replication cycle. synthe ...199910103186
attenuated vesicular stomatitis viruses as vaccine vectors.we showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (vsv) expressing an influenza virus hemagglutinin (ha) protein provided complete protection from lethal challenge with influenza virus (a. roberts, e. kretzschmar, a. s. perkins, j. forman, r. price, l. buonocore, y. kawaoka, and j. k. rose, j. virol. 72:4704-4711, 1998). because some pathogenesis was associated with the vector itself, in the present study we generated new vsv vectors ...199910196265
selection of rna replicons capable of persistent noncytopathic replication in mammalian cells.the natural life cycle of alphaviruses, a group of plus-strand rna viruses, involves transmission to vertebrate hosts via mosquitoes. chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mrna translation and cell death. using engineered sindbis virus rna replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing pers ...199910196280
genetic and phenotypic changes accompanying the emergence of epizootic subtype ic venezuelan equine encephalitis viruses from an enzootic subtype id progenitor.recent studies have indicated that epizootic venezuelan equine encephalitis (vee) viruses can evolve from enzootic, subtype id strains that circulate continuously in lowland tropical forests (a. m. powers, m. s. oberste, a. c. brault, r. rico-hesse, s. m. schmura, j. f. smith, w. kang, w. p. sweeney, and s. c. weaver, j. virol. 71:6697-6705, 1997). to identify mutations associated with the phenotypic changes leading to epizootics, we sequenced the entire genomes of two subtype ic epizootic vee v ...199910196323
the cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence.semliki forest virus (sfv) and sindbis virus (sin) are enveloped alphaviruses that enter cells via low-ph-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. previous studies with cholesterol-depleted insect cells have shown that sfv requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. an sfv mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the e1 spik ...199910196324
genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells.the alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. to test this hypothesis, eastern equine encephalitis virus was passaged in bhk or mosquito cells, as well as in alternating (both) host cell passages. high and low multiplicities were used to examine the effect of defective interfering particles. clonal bhk and persistent mosquito cell infections were also evaluated. fitness was measured with one-step growth curves and competition assays, and m ...199910196330
stable alphavirus packaging cell lines for sindbis virus and semliki forest virus-derived vectors.alphavirus vectors are being developed for possible human vaccine and gene therapy applications. we have sought to advance this field by devising dna-based vectors and approaches for the production of recombinant vector particles. in this work, we generated a panel of alphavirus vector packaging cell lines (pcls). these cell lines were stably transformed with expression cassettes that constitutively produced rna transcripts encoding the sindbis virus structural proteins under the regulation of t ...199910200308
persistent hz-1 virus infection in insect cells: evidence for insertion of viral dna into host chromosomes and viral infection in a latent status.persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. in this study, the long-term association between the hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. with the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. once vira ...19999847315
processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab.replicase gene expression by the human coronavirus 229e involves the synthesis of two large polyproteins, pp1a and pp1ab. experimental evidence suggests that these precursor molecules are subject to extensive proteolytic processing. in this study, we show that a chymotrypsin-like enzyme, the virus-encoded 3c-like proteinase (3clpro), cleaves within a common region of pp1a and pp1ab (amino acids 3490 to 4068) at four sites. trans-cleavage assays revealed that polypeptides of 5, 23, 12, and 16 kda ...19999847320
porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents.porcine reproductive and respiratory syndrome virus (prrsv) is a recently described arterivirus responsible for disease in swine worldwide. comparative sequence analysis of 3'-terminal structural genes of the single-stranded rna viral genome revealed the presence of two genotypic classes of prrsv, represented by the prototype north american and european strains, vr-2332 and lelystad virus (lv), respectively. to better understand the evolution and pathogenicity of prrsv, we obtained the 12,066-ba ...19999847330
immunization of woodchucks with plasmids expressing woodchuck hepatitis virus (whv) core antigen and surface antigen suppresses whv infection.dna vaccination can induce humoral and cellular immune response to viral antigens and confer protection to virus infection. in woodchucks, we tested the protective efficacy of immune response to woodchuck hepatitis core antigen (whcag) and surface antigen (whsag) of woodchuck hepatitis virus (whv) elicited by dna-based vaccination. plasmids pwhcim and pwhsim containing whv c- or pre-s2/s genes expressed whcag and whsag in transient transfection assays. pilot experiments in mice revealed that a s ...19999847331
selection of functional variants of the ns3-ns4a protease of hepatitis c virus by using chimeric sindbis viruses.the ns3-ns4a serine protease of hepatitis c virus (hcv) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the hcv replication machinery. despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for hcv in cultured cells. we have recently described the generation of chimeric hcv-sindbis viruses whose propagatio ...19999847361
persistent infection promotes cross-species transmissibility of mouse hepatitis virus.persistent infection with mouse hepatitis virus (mhv) strain a59 in murine dbt (delayed brain tumor) cells resulted in the emergence of host range variants, designated v51a and v51b, at 210 days postinfection. these host range mutants replicated efficiently in normally nonpermissive chinese hamster ovary (cho), in human hepatocarcinoma (hepg2), and to a lesser extent in human breast carcinoma (mcf7) cell lines. little if any replication was noted in baby hamster kidney (bhk), green african monke ...19999847369
sendai virus infection induces apoptosis through activation of caspase-8 (flice) and caspase-3 (cpp32).sendai virus (sv) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. we now show that sv infection triggers an apoptotic program in target cells. incubation of infected cells with the peptide inhibitor z-vad-fmk abrogated sv-induced apoptosis, indicating that proteases of the caspase family were involved. moreover, proteolytic activation of two distinct caspases, cpp32/caspase-3 and, as shown for the first time in virus-infected cells, flice/caspase ...19999847376
new mouse model for dengue virus vaccine testing.several dengue (den) virus vaccines are in development; however, the lack of a reliable small animal model in which to test them is a major obstacle. because evidence suggests that interferon (ifn) is involved in the human anti-den virus response, we tested mice deficient in their ifn functions as potential models. intraperitoneally administered mouse-adapted den 2 virus was uniformly lethal in ag129 mice (which lack alpha/beta ifn and gamma ifn receptor genes), regardless of age. immunized mice ...19999847388
induction of manganese-superoxide dismutase in mrc-5 cells persistently infected with an alphavirus, sindbis.sindbis virus (sv), a single-stranded positive-sense rna virus, multiplies in a variety of cells and causes various outcomes of infection. as we described acute infection of sv induces stress response of small heat shock protein hsp27 and activation of mitogen-activated protein kinase (map) signaling pathway (nakatsue, t., et al., biochem. biophys. res. commun. 253, 59-64, 1998). in contrast to lytic infection in vero cells, mrc-5 cells, a human fetus lung cell line, resulted in persistent infec ...199910405336
a salmonella virulence protein that inhibits cellular trafficking.salmonella enterica requires a type iii secretion system, designated spi/ssa, to survive and proliferate within macrophages. the spi/ssa system is encoded within the spi-2 pathogenicity island and appears to function intracellularly. here, we establish that the spi-2-encoded spic protein is exported by the spi/ssa type iii secretion system into the host cell cytosol where it interferes with intracellular trafficking. in j774 macrophages, wild-type salmonella inhibited fusion of salmonella-contai ...199910406797
mosquito-borne viruses in western europe: a review.several mosquito-borne arboviruses belonging to the genera alphavirus, flavivirus, and bunyavirus have been reported to occur in mosquitoes and to infect humans and other vertebrates in western europe. these zoonotic viruses circulate in nature either in an aedes-mammal, anopheles-mammal, or culex-bird transmission cycle. infected humans normally do not contribute to the virus circulation. west nile virus (flavivirus) caused an outbreak of fever, malaise, pain in eyes and muscles, and headache a ...199910436876
pathogenesis of chimeric mhv4/mhv-a59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence.the mouse hepatitis virus (mhv) spike glycoprotein, s, has been implicated as a major determinant of viral pathogenesis. in the absence of a full-length molecular clone, however, it has been difficult to address the role of individual viral genes in pathogenesis. by using targeted rna recombination to introduce the s gene of mhv4, a highly neurovirulent strain, into the genome of mhv-a59, a mildly neurovirulent strain, we have been able to directly address the role of the s gene in neurovirulenc ...199910438865
intracellular distribution of rubella virus nonstructural protein p150.antiserum prepared against an amino-terminal fragment of rubella virus (rub) nonstructural polyprotein was used to study rub-infected vero cells. replicase protein p150 was associated with vesicles and vacuoles of endolysosomal origin and later with large, convoluted, tubular membrane structures. newly incorporated bromouridine was associated with the same structures and specifically with small membrane invaginations, spherules, indicating that these structures may be the sites of viral rna synt ...199910438871
lipoproteins account for part of the broad non-specific antiviral activity of human serum.several antiviral substances have been detected in human serum but few have been shown to possess broad antiviral activity. these broadly active antiviral molecules could be of significance as innate defense mechanisms. we have previously identified and characterized a broadly antiviral glycoprotein, uti3, which accounts for 50 antiviral units/ml of human and mammalian sera. in addition there are reports of antiviral activity of human serum apolipoprotein a-1 (apo a-1), an important constituent ...199910443533
the role of host immunocompetence in neuroinvasion of sindbis virus.viral infections of the central nervous system (cns) following peripheral inoculation of sindbis viruses were studied. the use of viral strains, which vary in their neuroinvasive and neurovirulent properties, and various strains of mice, which differ in immunocompetence, revealed several pathways of viral neuroinvasion in adult mice. a genetic-trait dependent mechanism was exhibited by the neuroinvasive viruses, showing a similar pattern in all mice strains tested. a second mechanism, dependent ...199910446650
association of a cellular 21-kda transmembrane protein (vap21) with enveloped viruses.we reported previously that the rabies virions contained a 21-kda cellular transmembrane protein (referred to as vap21) as a minor component (sagara, j. et al, microbiol. immunol. 41(12): 947-955, 1997). in this study, we further examined the possible interactions of vap21 with other enveloped viruses, including the vesicular stomatitis virus (vsv; negative-stranded rna virus), sindbis virus (positive-stranded rna virus) and herpes simplex virus type 1 (hsv-1; double-stranded dna virus). an immu ...199910449251
immunogenicity of dna vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences.novel tuberculosis dna vaccines encoding native esat-6, mpt-64, katg, or hbha mycobacterial proteins or the same proteins fused to tissue plasminogen activator (tpa) signal sequences were evaluated for their capacity to elicit humoral, cell-mediated, and protective immune responses in vaccinated mice. while all eight plasmids induced specific humoral responses, the constructs expressing the tpa fusions generally evoked higher antibody responses in vaccinated hosts. although most of the dna vacci ...199910456931
structural maturation of the transmissible gastroenteritis coronavirus.during the life cycle of the transmissible gastroenteritis coronavirus (tgev), two types of virus-related particles are detected in infected swine testis cells: large annular viruses and small dense viruses. we have studied the relationships between these two types of particles. immunoelectron microscopy showed that they are closely related, since both large and small particles reacted equally with polyclonal and monoclonal antibodies specific for tgev proteins. monensin, a drug that selectively ...199910482542
regulation of closterovirus gene expression examined by insertion of a self-processing reporter and by northern hybridization.a reporter open reading frame (orf) coding for a fusion of bacterial beta-glucuronidase (gus) with a proteinase domain (pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (byv). insertion of this reporter orf between the first and second codons of the byv orfs encoding the hsp70 homolog (hsp70h), a major capsid protein (cp), and a 20-kda protein (p20) resulted in the expression of the processed gus-pro reporter from corresponding subg ...199910482546
low-ph-dependent fusion of sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes.there is controversy as to whether the cell entry mechanism of sindbis virus (sin) involves direct fusion of the viral envelope with the plasma membrane at neutral ph or uptake by receptor-mediated endocytosis and subsequent low-ph-induced fusion from within acidic endosomes. here, we studied the membrane fusion activity of sin in a liposomal model system. fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled l ...199910482600
the severity of murray valley encephalitis in mice is linked to neutrophil infiltration and inducible nitric oxide synthase activity in the central nervous system.a study of immunopathology in the central nervous system (cns) during infection with a virulent strain of murray valley encephalitis virus (mve) in weanling swiss mice following peripheral inoculation is presented. it has previously been shown that virus enters the murine cns 4 days after peripheral inoculation, spreads to the anterior olfactory nucleus, the pyriform cortex, and the hippocampal formation at 5 days postinfection (p.i.), and then spreads throughout the cerebral cortex, caudate put ...199910482632
localization of a passively transferred human recombinant monoclonal antibody to herpes simplex virus glycoprotein d to infected nerve fibers and sensory neurons in vivo.a human recombinant monoclonal antibody to herpes simplex virus (hsv) glycoprotein d labeled with the fluorescent dye cy5 was administered to mice infected in the cornea with hsv type 1 (hsv-1). the distribution of such antibody in the corneas and trigeminal ganglia of the mice was then investigated by confocal microscopy. the antibody was detected on hsv-infected nerve fibers in the cornea--identified by colocalization with hsv antigens and the neuritic markers neurofilament, gap-43, synapsin-1 ...199910482637
antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon sindbis virus vector infection.viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. in particular, sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems. unfortunately, infection of cultured cells with sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by dna laddering and fluorescence microscopy. fortunately, it has recently been demonstr ...199910486128
alphavirus vectors for gene expression and vaccines.alphavirus expression vectors are finding novel uses in research. they are showing increasing promise as vaccines and are being developed for diagnostic assays of other viruses. some highlights over the past couple of years include improvements in packaging of replicons, targeting of sindbis virus replicons, stable cell lines that can be induced to produce replicons, and the isolation of noncytopathic variants of sindbis virus replicons. reports that alphavirus vectors can efficiently infect neu ...199910508626
gerp95, a membrane-associated protein that belongs to a family of proteins involved in stem cell differentiation.a panel of mabs was elicited against intracellular membrane fractions from rat pancreas. one of the antibodies reacted with a 95-kda protein that localizes primarily to the golgi complex or the endoplasmic reticulum (er), depending on cell type. the corresponding cdna was cloned and sequenced and found to encode a protein of 97.6 kda that we call gerp95 (golgi er protein 95 kda). the protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as s ...199910512872
palmitoylation of the intracytoplasmic r peptide of the transmembrane envelope protein in moloney murine leukemia virus.previously it was reported that the 16-amino-acid (aa) c-terminal cytoplasmic tail of moloney murine leukemia virus (momlv) transmembrane protein pr15e is cleaved off during virus synthesis, yielding the mature, fusion active transmembrane protein p15e and the 16-aa peptide (r peptide or p2e). it remains to be elucidated how the r peptide impairs fusion activity of the uncleaved pr15e. the r peptide from momlv was analyzed by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ...199910516003
identification of functional domains in the 14-kilodalton envelope protein (a27l) of vaccinia virus.the mechanism of entry of vaccinia virus (vv) into cells is still a poorly understood process. a 14-kda protein (encoded by the a27l gene) in the envelope of intracellular mature virus (imv) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. we have previously described the structural organization of the vv 14-kda protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted leu zipper-like third alpha h ...199910516016
polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3' untranslated region, thereby altering rna conformation.mouse hepatitis virus (mhv) rna transcription is regulated mainly by the leader and intergenic (ig) sequences. however, a previous study has shown that the 3' untranslated region (3'-utr) of the viral genome is also required for subgenomic mrna transcription; deletion of nucleotides (nt) 270 to 305 from the 3'-utr completely abolished subgenomic mrna transcription without affecting minus-strand rna synthesis (y.-j. lin, x. zhang, r.-c. wu, and m. m. c. lai, j. virol. 70:7236-7240, 1996), suggest ...199910516017
assignment of the multifunctional ns3 protein of bovine viral diarrhea virus during rna replication: an in vivo and in vitro study.studies on the replication of the pestivirus bovine viral diarrhea virus (bvdv) were considerably facilitated by the recent discovery of an autonomous subgenomic bvdv rna replicon (di9c). di9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins ns3, ns4a, ns4b, ns5a, and ns5b. to assess the significance of the ns3-associated nucleoside triphosphatase/helicase activity during rna replication and to explore other functional features of ...199910516027
establishment and characterization of cytopathogenic and noncytopathogenic pestivirus replicons.defective interfering particles (dis) of bovine viral diarrhea virus (bvdv) have been identified and shown to be cytopathogenic (cp) in the presence of noncytopathogenic (noncp) helper virus. moreover, a subgenomic (sg) rna corresponding in its genome structure to one of those bvdv dis (di9) was replication competent in the absence of helper virus. we report here that an sg bvdv replicon which encodes from the viral proteins only the first three amino acids of the autoprotease n(pro) in addition ...199910516051
synthesis and biological evaluation of 2-methoxy- and 2-methylthio-6-[2'-alkylamino)ethyl]-4(3h)-pyrimidinones with anti-rubella virus activity.the synthesis of a new family of antiviral compounds, 2-methoxy-, and 2-methylthio-6-[(2'-alkylamino)ethyl]-4(3h)-pyrimidinones, has been accomplished. the activity of these agents against positive strand (rubella virus and sindbis virus) and negative strand (vesicular stomatitis virus) rna viruses is reported. some of these compounds are efficient and selective inhibitors of rubella virus.199910530941
comparison of the protective efficacy of naked dna, dna-based sindbis replicon, and packaged sindbis replicon vectors expressing hantavirus structural genes in hamsters.seoul virus (seov) is a member of the hantavirus genus (family bunyaviridae) and an etiological agent of hemorrhagic fever with renal syndrome. the medium (m) and small (s) gene segments of seov encode the viral envelope glycoproteins and nucleocapsid protein, respectively. we compared the immunogenicity and protective efficacy of naked dna (pwrg7077), dna-based sindbis replicon (psin2.5), and packaged sindbis replicon vectors (psinrep5), containing either the m or s gene segment of seov in syri ...199910544095
tnfalpha, interferon, and stress response induction as a function of age-related susceptibility to fatal sindbis virus infection of mice.the age-related acquisition of resistance to fatal sindbis virus infection was examined using a molecularly cloned laboratory strain of the ar339 isolate designated trsb. trsb caused 100% mortality in mice up to 5 days of age. resistance to fatal infection developed abruptly between 5 and 9 days of age. lethal sindbis virus infection of mice inoculated at 4 days of age was characterized by high levels of virus replication, induction of high levels of interferon-alpha/beta and tnf-alpha and sever ...199910544107
an amino acid change in the exodomain of the e2 protein of sindbis virus, which impairs the release of virus from chicken cells but not from mosquito cells.in order to obtain a mutant of sindbis virus (sv) with a low methionine-resistant (lmr) phenotype, i.e., able to replicate in methionine-deprived aedes albopictus mosquito cells, standard sv (sv(std)) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. although the virus obtained by this procedure, sv(lm17), did have the desired lmr phenotype, it also appeared to have acquired a host-range phenotype. we have now characte ...199910544144
inhibition of luciferase expression in transgenic aedes aegypti mosquitoes by sindbis virus expression of antisense luciferase rna.a rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. we hypothesized that the virus expression system based on the mosquito-borne alphavirus, sindbis (togaviridae), may efficiently transcribe effector rnas that inhibit expression of a targeted mosquito gene. to test this hypothesis ...199910557332
interaction of hepatitis c virus core protein with viral sense rna and suppression of its translation.to clarify the binding properties of hepatitis c virus (hcv) core protein and its viral rna for the encapsidation, morphogenesis, and replication of hcv, the specific interaction of hcv core protein with its genomic rna synthesized in vitro was examined in an in vivo system. the positive-sense rna from the 5' end to nucleotide (nt) 2327, which covers the 5' untranslated region (5'utr) and a part of the coding region of hcv structural proteins, interacted with hcv core protein, while no interacti ...199910559281
improving proteolytic cleavage at the 3a/3b site of the hepatitis a virus polyprotein impairs processing and particle formation, and the impairment can be complemented in trans by 3ab and 3abc.the orchestrated liberation of viral proteins by 3c(pro)-mediated proteolysis is pivotal for gene expression by picornaviruses. proteolytic processing is regulated either by the amino acid sequence at the cleavage site of the substrate or by cofactors covalently or noncovalently linked to the viral proteinase. to determine the role of the amino acid sequence at cleavage sites 3a/3b and 3b/3c that are essential for the liberation of 3c(pro) from its precursors and to assess the function of the st ...199910559299
an epitope of the semliki forest virus fusion protein exposed during virus-membrane fusion.semliki forest virus (sfv) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic ph in the endocytic pathway. fusion is mediated by the spike protein e1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. we have characterized four monoclonal antibodies (mabs) specific for the acid conformation of e1. these mabs did not inhibit fusion, suggesting that they bind to an e1 region differe ...199910559317
putative rna capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a.brome mosaic virus (bmv) rna replication is directed by two virus-encoded proteins, 1a and 2a. the amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsp1, which has been implicated in capping viral rnas. in this study, we examined the enzymatic activities of bmv 1a expressed in yeast, where the protein is fully functional in rna replication. 1a methylated gtp, dgtp, and the cap analogs gpppg and gpppa, using s-adenosylmethionine (adomet) as the methyl donor. prod ...199910559320
efficient trans-complementation of the flavivirus kunjin ns5 protein but not of the ns1 protein requires its coexpression with other components of the viral replicase.successful trans-complementation of the defective kunjin virus (kun) rna fldgdd with a deletion of the rna polymerase motif gdd in the ns5 gene by using a bhk cell line, repbhk, that continuously produced a functionally active kun replication complex (rc) from replicon rna was recently reported (a. a. khromykh, m. t. kenney, and e. g. westaway, j. virol. 72:7270-7279, 1998). in order to identify whether this complementation of fldgdd rna was provided by the wild-type ns5 protein alone or with th ...199910559344
induction of apoptosis by sindbis virus occurs at cell entry and does not require virus replication.sindbis virus (sv) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. to determine the step in virus replication during which apoptosis is triggered, we used uv-inactivated sv, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for sv infection of many cells. in initial experiments, uv-inactivated neuroadapted sv (nsv) induced apoptosis in chinese hamster ...199910559347
brome mosaic virus rna replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum.the universal membrane association of positive-strand rna virus rna replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. brome mosaic virus (bmv) encodes two mutually interacting rna replication proteins: 1a, which contains rna capping and helicase-like domains, and the polymerase-like 2a protein. in cells from the natural plant hosts of bmv, 1a and 2a colocalize on the endoplasmic reticulum (er). 1a and 2a also direct bmv rna replicatio ...199910559348
infection of neonatal mice with sindbis virus results in a systemic inflammatory response syndrome.laboratory strains of viruses may contain cell culture-adaptive mutations which result in significant quantitative and qualitative alterations in pathogenesis compared to natural virus isolates. this report suggests that this is the case with sindbis virus strain ar339. a cdna clone comprising a consensus sequence of sindbis virus strain ar339 has been constructed (w. b. klimstra, k. d. ryman, and r. e. johnston, j. virol. 72:7357-7366, 1998). this clone (ptr339) regenerates a sequence predicted ...199910559357
a single nucleotide change in the 5' noncoding region of sindbis virus confers neurovirulence in rats.two pairs of sindbis virus (sv) variants that differ in their neuroinvasive and neurovirulent traits in mice have been isolated. recently, we mapped the genetic determinants responsible for neuroinvasiveness in weanling mice. here, we extend this study to newborn and adult rats and to rat neuronal cultures. remarkably, certain aspects of the pathogenesis of these strains in rats were found to be quite distinct from the mouse model. suckling rats were susceptible to all four isolates, and replica ...199910559362
infecting larval arthropods with a chimeric, double subgenomic sindbis virus vector to express genes of interest. 199910572634
ectopic gene expression and homeotic transformations in arthropods using recombinant sindbis viruses.the morphological diversity of arthropods makes them attractive subjects for studying the evolution of developmental mechanisms. comparative analyses suggest that arthropod diversity has arisen largely as a result of changes in expression patterns of genes that control development. direct analysis of how a particular gene functions in a given species during development is hindered by the lack of broadly applicable techniques for manipulating gene expression.199910574759
dna and rna-based vaccines: principles, progress and prospects.dna vaccines were introduced less than a decade ago but have already been applied to a wide range of infectious and malignant diseases. here we review the current understanding of the mechanisms underlying the activities of these new vaccines. we focus on recent strategies designed to enhance their function including the use of immunostimulatory (cpg) sequences, dendritic cells (dc), co-stimulatory molecules and cytokine- and chemokine-adjuvants. although genetic vaccines have been significantly ...199910580187
mutagenesis of the rgd motif in the yellow fever virus 17d envelope protein.the envelope protein of yellow fever virus 17d (yfv-17d) contains a solvent-exposed rgd motif, which has led to the suggestion that integrins may function as cellular receptors for yfv-17d. we found that mutating the rgd motif to rge had no effect on viral titers, whereas changing rgd to tgd, tge, tad, tae, or rgs led to reduced titers. substitution of rgd by rad or rae yielded rna genomes that replicated in mammalian cells but could not spread to neighboring cells at 37 degrees c. these mutants ...199910603320
primary megakaryocytes reveal a role for transcription factor nf-e2 in integrin alpha iib beta 3 signaling.platelet integrin alphaiibbeta3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of alphaiibbeta3 signaling remain poorly understood. to better understand this process, we established conditions to study alphaiibbeta3 signaling in primary murine megakaryocytes. unlike platelets, these platelet precursors are amenable to genetic manipulation. cytokine-stimulated bone marrow cultures produced three arbitrary populations of alpha ...199910613901
transplacental drug delivery: gene and virus delivery to the trophoblast.the development of successful strategies for delivering genes to the placenta may provide new opportunities for modifying trophoblast function in order to learn more about trophoblast physiology and to offer novel therapeutic options for complications of pregnancy that result from placental dysfunction. replication-deficient recombinant viral vectors are useful vehicles for introducing genes into cells in vitro and in vivo. recombinant adenovirus and herpes simplex virus vectors are unable to ef ...199910837747
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