TitleAbstractYear(sorted ascending)
glut4 phosphorylation and inhibition of glucose transport by dibutyryl investigate the mechanism responsible for the inhibition of glucose transport by dibutyryl camp (bt2camp), two different transporter isoforms (glut1 and glut4) and several glut1/4 chimeric transporters were expressed in chinese hamster ovary (cho) cells by using a sindbis virus expression system. bt2camp inhibited glut4-mediated 2-deoxy[3h]glucose (2dog) uptake by 50% but was without effect on glut1-mediated uptake. when the subcellular distribution of glut4 was assessed by quantitative immun ...19938393869
antiviral melanization reaction of heliothis virescens hemolymph against dna and rna viruses in vitro.1. antiviral activity of heliothis virescens larval hemolymph was determined using a cytotoxicity/virus inhibition test (tcld50) done in vero cell tissue cultures. excellent antiviral activity was found especially against herpes simplex viruses-1 and -2 and also against vesicular stomatitis, parainfluenza-3, coxsackie b3 and sindbis viruses. 2. prolonged incubation of herpes simplex virus-1 and vesicular stomatitis virus with hemolymph was virucidal and greatly reduced infectivity of the two vir ...19938395989
detection of ockelbo virus rna in skin biopsies by polymerase chain reaction.a sensitive assay based on the polymerase chain reaction for the detection of ockelbo virus rna was developed. two primer pairs from the gene coding for the e2 glycoprotein were chosen. by use of a nested strategy for the primers, as few as 1 to 10 pfu could be detected. the amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. the primer pairs allowed amplification of several ockelbo and sindbis virus isolates but discriminated between these a ...19938396582
phosphorylation and dephosphorylation events play critical roles in sindbis virus maturation.we have examined the effects of various inhibitors of protein kinases and phosphatases on sindbis virus maturation in bhk cells. 2-aminopurine, a nonspecific protein kinase inhibitor, n-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (w-7), a specific inhibitor of calmodulin/ca(2+)-dependent protein kinase, and okadaic acid (oka), a protein phosphatase inhibitor, dose-dependently inhibited sindbis virus maturation. although virus production was inhibited, the membrane glycoprotein ...19938396806
sindbis virus expression vectors: packaging of rna replicons by using defective helper rnas.since the recovery of infectious rna transcripts from full-length cdna clones, alphavirus genome rnas have been engineered to allow expression of heterologous rnas and proteins. the highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. such recombinant rnas are self-replicating (replicons) and can be introduced into cells as naked rna, but they require trans complementation to be packaged and released ...19938411346
molecular analysis of neurovirulent strains of sindbis virus that evolve during persistent infection of scid understand the role of tissue-specific adaptation and antibody-induced selectional pressures in the evolution of neurovirulent viruses, we analyzed three strains of sindbis virus isolated from the brains of persistently infected scid mice and four strains of sindbis virus isolated from the brains of scid mice with viral reactivation following immune serum treatment. for each viral isolate, we tested neurovirulence in weanling balb/c mice and sequenced regions of the e2 and e1 envelope glycopr ...19938411391
three-dimensional structure of a membrane-containing virus.the structure of sindbis virus was determined by electron cryomicroscopy. the virion contains two icosahedral shells of viral-encoded proteins separated by a membrane bilayer of cellular origin. the three-dimensional structure of the ice-embedded intact sindbis virus, reconstructed from electron images, unambiguously shows that proteins in both shells are arranged with the same icosahedral lattice of triangulation number t = 4. these studies also provide structural evidence of contact between th ...19938415660
the distribution of sindbis virus proteins in mosquito cells as determined by immunofluorescence and immunoelectron microscopy.two aedes albopictus (mosquito) subclones, c7-10 and c6/36, were examined by immunofluorescence and immunoelectron microscopy for the distribution of sindbis virus structural and non-structural proteins. both the viral glycoproteins, e1 and e2, and the non-structural proteins, nsp1 and nsp2, were found within vesicles and electron-dense, amorphous matrices associated with sindbis virus infection. the labelling patterns indicated that both replication of viral rna and production of virus particle ...19938429303
transient translocation of the cytoplasmic (endo) domain of a type i membrane glycoprotein into cellular membranes.the e2 glycoprotein of the alphavirus sindbis is a typical type i membrane protein with a single membrane spanning domain and a cytoplasmic tail (endo domain) containing 33 amino acids. the carboxyl terminal domain of the tail has been implicated as (a) attachment site for nucleocapsid protein, and (b) signal sequence for integration of the other alpha-virus membrane proteins 6k and e1. these two functions require that the carboxyl terminus be exposed in the cell cytoplasm (a) and exposed in the ...19938432728
deletion analysis of the capsid protein of sindbis virus: identification of the rna binding region.the capsid protein of sindbis virus has multiple functions in the life cycle of the virus. one essential function is to interact with the genomic rna of the virus to form the nucleocapsid. the experiments described in this article define a region of the protein that is required for binding to sindbis virus rna. the assay we used measured the binding of in vitro-translated proteins to rna on the basis of their migration with the rna during electrophoresis in an agarose gel. binding to rna showed ...19938437233
directed mutagenesis of a sindbis virus pathogenesis site.the e2 glycoprotein gene of sindbis virus contains three neutralizing antigenic sites, and examination of monoclonal antibody (mab) escape mutants of one of these, the e2c site, has suggested an association of the e2c domain with determinants of virulence in neonatal mice. codons associated with the e2c site, e2 62, 96, and 159, were subjected to site-directed mutagenesis generating a library of 25 viable virus mutants containing different amino acids at these loci. the mutants were assessed for ...19938438562
conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene.little is known about virus-host cell interactions that regulate the lytic potential of viruses during productive replication. sindbis virus (sv), a single-stranded positive-sense rna virus in the alphavirus genus (family togaviridae), results in lytic infection in most vertebrate cell lines, but persistent productive infection in post-mitotic neurons. the cellular oncogene bcl-2, which encodes an inner mitochondrial membrane protein of m(r) 26,000 (ref. 2), blocks programmed cell death (apoptos ...19938441470
assembly of functional sindbis virus rna replication complexes: requirement for coexpression of p123 and p34.a vaccinia virus transient expression system was used to determine which of the sindbis virus (sin) proteins and/or polyproteins are necessary for the formation of active replication complexes and, in particular, to analyze the role of nsp4, the putative polymerase, versus p34 in rna replication. we generated vaccinia virus recombinants in which the cdna for the entire sin nonstructural coding region as well as cdna copies of the individual nonstructural proteins (nsps) and several intermediate ...19938445716
roles of nonstructural polyproteins and cleavage products in regulating sindbis virus rna replication and transcription.using vaccinia virus to express sindbis virus (sin) nonstructural proteins (nsps) and template rnas, we showed previously that synthesis of all three viral rnas occurred only during expression of either the entire nonstructural coding region or the polyprotein precursors p123 and p34. in this report, the vaccinia virus system was used to express cleavage-defective polyproteins and nsp4 proteins containing various n-terminal extensions to directly examine the roles of the p123 and p34 polyprotein ...19938445717
refined structure of sindbis virus core protein and comparison with other chymotrypsin-like serine proteinase structures.crystal forms 2 and 3 of sindbis virus core protein have been refined to 2.8 a and 3.0 a resolution, respectively. the three independent molecular copies in the two crystal forms are essentially identical, except for regions where the molecules are involved in different crystal packing interactions. the overall polypeptide backbone fold of sindbis virus core protein is similar to other chymotrypsin-like serine proteinase structures despite a lack of significant sequence homology. detailed analys ...19938450538
potentiation of interferon-alpha in vitro antiviral activity by interferon-gamma is not abrogated by antibody to interferon-gamma.the addition of minute amounts of recombinant interferon-gamma (ifn-gamma) to recombinant ifn-alpha 2 significantly enhances its antiviral activity. although this potentiating action of ifn-gamma is destroyed by treatment with acid, it is not significantly affected by treatment with antibodies to ifn-gamma, suggesting that ifn-gamma molecules can interact with cells to potentiate ifn-alpha activity, even in the presence of neutralizing antibodies. implications of this phenomenon are multifaceted ...19938454911
inhibition of sindbis virus replication by cyclopentenone prostaglandins: a cell-mediated event associated with heat-shock protein synthesis.cyclopentenone prostaglandins (pgs) have been shown to inhibit the replication of several dna and rna viruses. here we report on the effect of prostaglandin a1 (pga1) on the multiplication of a positive strand rna virus, sindbis virus, in vero cells under one-step multiplication conditions. pga1 was found to inhibit sindbis virus production dose-dependently, and virus yield was reduced by more than 90% at the concentration of 8 micrograms/ml, which was non-toxic to the cells and did not inhibit ...19938470883
site-directed mutations in the sindbis virus e2 glycoprotein identify palmitoylation sites and affect virus budding.the assembly and budding of sindbis virus, a prototypic member of the alphavirus subgroup in the family togaviridae, requires a specific interaction between the nucleocapsid core and the membrane-embedded glycoproteins e1 and e2. these glycoproteins are modified posttranslationally by the addition of palmitic acid, and inhibitors of acylation interfere with this budding process (m.j. schlesinger and c. malfer, j. biol. chem. 257:9887-9890, 1982). this report describes the use of site-directed mu ...19938474160
the unusual uva-dependent antiviral properties of the furoisocoumarin, coriandrin.the novel furoisocoumarin, coriandrin, which was found recently to possess an interesting combination of photobiological properties, was investigated for antiviral activity in the presence and absence of uva (long-wavelength ultraviolet radiation). in contrast to results obtained with other antiviral furocoumarins, such as 8-mop (8-methoxypsoralen), coriandrin was much more phototoxic to the rna-virus sindbis virus than to the dna-virus murine cytomegalovirus, although both viruses were substant ...19938475184
[industrial pasteurization of plasma and criteria of quality].the approach followed in the design of a large-scale pasteurization treatment (60 degrees c for 10 hours in the liquid state) of fresh frozen plasma is presented. various aspects thought to influence the viral safety of such a product are discussed. they are based largely upon the fact that, although it is subjected to a specific viral inactivation treatment, this plasma does not benefit from any fractionation steps known to participate in the potential elimination of infectious agents during th ...19938476493
glut-4 nh2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration.expression of chimeras, composed of portions of two different glucose transporter isoforms (glut-1 and glut-4), in cho cells had indicated that the cytoplasmic nh2 terminus of glut-4 contains important targeting information that mediates intracellular sequestration of this isoform (piper, r. c., c. tai, j. w. slot, c. s. hahn, c. m. rice, h. huang, d. e. james. 1992. j. cell biol. 117:729-743). in the present studies, the amino acid constituents of the glut-4 nh2-terminal targeting domain have b ...19938509445
size exclusion removal of model mammalian viruses using a unique membrane system, part i: membrane qualification.we have previously described a new class of composite membrane that has the capability to efficiently remove particles, including viruses, from protein solution. the qualification of this membrane requires that it reproducibly and predictably remove model mammalian viruses. using the viresolve/70 membrane, the mammalian viruses polio, simian virus-40, sindbis, reovirus type 3 and rauscher murine leukemia virus are shown to be reproducibly removed via a sieving mechanism. mammalian virus retentio ...19938117441
size exclusion removal of model mammalian viruses using a unique membrane system, part ii: module qualification and process simulation.the performance of a fabricated device may be influenced by the characteristics of the fluid management within the device and reproducibility with which the device is manufactured. the performance of the viresolve/70 membrane is not diminished when incorporated into a fabricated module. the viresolve/70 fabricated modules are shown to reproducibly retain viruses via a sieving mechanism, independent of virus type or character, in excellent agreement with the base membrane retention coefficients r ...19938117442
molecular organization and replication of hepatitis e virus (hev).the recently characterized fecal-orally transmitted agent of hepatitis e (formerly known as enterically transmitted non-a, non-b hepatitis) has been determined to be a new type of positive strand rna virus. the complete sequencing of four different geographic isolates of the hepatitis e virus (hev) has confirmed a similar genetic organization not previously recognized in nonenveloped positive strand rna viruses. the approximately 7.5 kb rna genome (including polya tail) has nonstructural genes l ...19938219799
structure-function relationship of the small gtpase rab5.overexpression of rab5 via a sindbis virus vector resulted in a 2-3-fold stimulation of horseradish peroxidase uptake in bhk-21 cells. based on this functional assay of rab5 activity, we conducted extensive mutational analysis of the structure-function relationship of rab5. a total of 21 deletion and substitution mutations were created and their effects on rab5 activity were examined. deletion of the entire c-terminal tetrapeptide motif ccsn abolished rab5 activity. a mutant with the last three ...19938226999
virus specificity and isotype expression of intraparenchymal antibody-secreting cells during sindbis virus encephalitis in study the generation of specific antibody responses within the central nervous system (cns), we have utilized a murine model of acute viral encephalitis. when sindbis virus (sv) is injected intracerebrally into weanling mice it causes an acute non-fatal encephalitis and recovery is primarily dependent on the development of antiviral antibody. we used a modified enzyme-linked immunoassay to determine the number of antibody-secreting cells (asc) specific for sv and their ig isotype in brain, sp ...19938227306
a truncated herpes simplex virus origin binding protein which contains the carboxyl terminal origin binding domain binds to the origin of replication but does not alter its conformation.we have studied the dna binding properties of a polypeptide consisting of the carboxyl terminal 37% of ul9, the herpes simplex virus type 1 (hsv-1) origin of replication binding protein. using a sindbis virus expression system, we expressed and partially purified this truncated form of ul9 (ul9ct) which contains the site-specific dna binding domain. ul9ct specifically recognized ul9 binding sites on a 200 base pair dna fragment containing the hsv origin ori(s) and appeared to bind as a dimer to ...19938255778
expression of toxoplasma gondii p30 as fusions with glutathione s-transferase in animal cells by sindbis recombinant virus. 19938259126
reversal of the antiviral activity of ribavirin against sindbis virus in ae. albopictus mosquito cells.earlier work in our laboratory has shown that the replication of sindbis virus in aedes albopictus mosquito cells is inhibited by ribavirin (rbv) and mycophenolic acid (mpa) (sarver and stollar (1978) virology 91, 267-282; malinoski and stollar (1980) virology 102, 473-476). we report here that the antiviral effect of rbv and mpa can be reversed by depriving infected cells of methionine or isoleucine, or by treating them with fluorodeoxyuridine (fudr) or cycloleucine. we suggest that, as was the ...19938279817
formation and rearrangement of disulfide bonds during maturation of the sindbis virus e1 glycoprotein.the rigidly ordered icosahedral lattice of the sindbis virus envelope is composed of a host-derived membrane bilayer in which the viral glycoproteins e1 and e2 reside. e1-e1 interactions stabilized by intramolecular disulfide bridges play a significant role in maintaining the envelope's structural integrity (r. p. anthony and d. t. brown, j. virol. 65:1187-1194, 1991; r. p. anthony, a. m. paredes, and d. t. brown, virology 190:330-336, 1992). we have examined the acquisition of disulfide bridges ...19948289384
intracerebral cytokine mrna expression during fatal and nonfatal alphavirus encephalitis suggests a predominant type 2 t cell response.sindbis virus (sv) causes an acute encephalomyelitis in mice. a t cell-dependent inflammatory response is first detected 3 days after infection and includes t cells, b cells, and macrophages. the cytokines produced locally by intrinsic cells of the brain in response to infection and by infiltrating mononuclear cells and their contributions to outcome of infection have not been identified. semiquantitative reverse transcriptase-pcr was used to evaluate the expression of mrnas for il-1 beta, il-2, ...19948301132
mutations in the nsp1 coding sequence of sindbis virus which restrict viral replication in secondary cultures of chick embryo fibroblasts prepared from aged primary cultures.svmpa, a mutant of sindbis virus (sv), which is able to replicate in aedes albopictus cells treated with mycophenolic acid (mpa) or ribavirin, also has a host range phenotype. this phenotype is most clearly demonstrated by means of efficiency of plaquing (eop) assays on secondary chick embryo fibroblasts (cef) prepared from aged primary cef. for example, in one experiment in which standard sv (svstd) had a relative eop (eop on primary cef divided by eop on secondary cef) of 1.6 the corresponding ...19948259645
a cell line that expresses a reporter gene in response to infection by sindbis virus: a prototype for detection of positive strand rna viruses.we describe a stably transformed cell line (bhksinluc2) that contains a defective sindbis virus genome under the control of a rous sarcoma virus promoter and the luciferase gene downstream of the viral subgenomic rna promoter. this cell line expresses high levels of luciferase activity following infection with sindbis virus and provides a sensitive assay for titering variants of sindbis virus that lack the structural protein genes, in particular, sindbis virus replicons that express heterologous ...19948259675
a 55-kda protein induced in aedes albopictus (mosquito) cells by antiviral protein.we have previously shown that the antiviral protein (avp) produced by sindbis virus-infected aedes albopictus (mosquito) cells (riedel and brown, j. virol. 29, 51-60, 1979) blocks sindbis viral rna synthesis and stimulates its own production when applied to uninfected a. albopictus cells (luo and brown, virology 194, 44-49, 1993). from a virus-sensitive (wild-type) mosquito cell line (u4.4) we produced a virus-resistant cell line (l4.4) by exposing u4.4 cells to the avp. l4.4 cells constitutivel ...19948128621
interactions between sindbis virus rnas and a 68 amino acid derivative of the viral capsid protein further defines the capsid binding previous studies of encapsidation of sindbis virus rna, we identified a 570nt fragment (nt 684-1253) from the 12 kb genome that binds to the viral capsid protein with specificity and is required for packaging of sindbis virus defective interfering rnas. we now show that the capsid binding activity resides in a highly structured 132nt fragment (nt 945-1076). we had also demonstrated that a 68 amino acid peptide derived from the capsid protein retained most of the binding activity of the origin ...19948139918
[the effect of sindbis virus multiplication on gene expression of host cells].sindbis virus (sbv) infection mediated a rapid shutoff of host cellular gene expression (mrna synthesis and protein synthesis); however the synthesis of cellular rrna remained at the same level as the uninfected cells. meanwhile a cellular protein p105 was shown to be enriched in the nuclear matrix. actionmycin d treatment after virus infection resulted in an apparent reduce in the production of viral structural proteins and infectious virions. the results presented here not only demonstrated th ...19947871779
structural features of the gtp-binding defective rab5 mutants required for their inhibitory activity on endocytosis.rab5 is a ras-like small gtpase that regulates early events of endocytosis. previous work indicates that two gtp-binding defective rab5 mutants (rab5:s34n and rab5:n133i) are dominant inhibitors of endocytosis. in this report, we have initiated experiments to address the structural features necessary for the inhibitory activity of these two rab5 mutants. second-site mutations were introduced into rab5:s34n and rab5:n133i, respectively, and the resulting double mutants were expressed in cultured ...19948182071
development and small-scale production of a severely heated factor viii concentrate.the small-scale production of a severely heated factor viii concentrate is described. as starting material for the production 48 units of fresh frozen plasma from whole-blood donations or 24 units of plasma from apheresis are used. during a glycine and sodium chloride fractionation, contaminating proteins are removed from a pooled extract of single-donor cryoprecipitates. the resulting factor viii-rich precipitate is redissolved in freeze-drying buffer and this solution is desalted by diafiltrat ...19948184598
effect of monosaccharides during severe dry heat treatment of coagulation factor viii concentrates.during product development of a factor viii concentrate (dutch blood banks) the conversion from unsterilized to autoclaved freeze-drying buffer caused impaired product characteristics after severe dry heat treatment (80 degrees c for 72 h). analysis of the freeze-drying buffers showed the presence of fructose and glucose in heated buffers, resulting from hydrolysis of sucrose. the detrimental effect of glucose and fructose on solubility, yield of factor viii and color of the heat-treated product ...19948184599
the effects of alphavirus infection on neurons.sindbis virus is an alphavirus that causes encephalitis in mice. the primary target cells for central nervous system infection are neurons. the outcome of neuronal infection is dependent on the age of the mouse at the time of infection (maturity of the neuron) and the strain of virus used for infection (virulence of the virus). sindbis virus causes neuronal death by inducing apoptosis. as neurons mature, they become resistant to virus-induced apoptosis, resulting in a persistent infection. host ...19948185292
construction of rubella virus genome-length cdna clones and synthesis of infectious rna transcripts.plasmids containing a complete cdna copy of the rubella virus (rub) genomic rna were constructed. transfection into cell culture of genome-length rna transcribed in vitro from one of these cdna clones, robo102, resulted in the production of virus which preserved the genetic and phenotypic characteristics of the parental virus from which the cdna clone was derived. prior to construction of the rub genome-length cdna clones, the 5'-terminal sequence of the rub genomic rna was determined to be 5'ca ...19948189494
neurovirulent strains of alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the e2 glycoprotein.the isolation and sequence comparison of avirulent and neurovirulent strains of polio virus, alpha virus, herpes virus, immunodeficiency virus, and other viruses have identified genetic changes that are required to cause disease in the nervous system. the molecular mechanisms by which these genetic changes result in neurovirulence are unknown. an avirulent laboratory strain of the alphavirus sindbis kills most cultured cell lines not by lethal parasitism, but by inducing apoptosis or programmed ...19948197207
the organization of the spike complex of semliki forest virus.semliki forest virus (sfv) is an enveloped animal virus comprising an icosahedral nucleocapsid surrounded by a membrane containing 80 transmembrane, trimeric spikes. sfv was treated with the non-ionic detergent n-octyl beta-d-glucopyranoside (octylglucoside) and analysed by cryo-electron microscopy and image reconstruction to explore the interaction between the spikes and the capsid. comparison of the structure of detergent treated sfv (dsfv) with sfv by three-dimensional image reconstruction fr ...19948107141
comparison of the effects of sindbis virus and sindbis virus replicons on host cell protein synthesis and cytopathogenicity in bhk cells.infection of bhk cells by sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (cpe). we have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. we tested a variety of sindbis viruses and sindbis virus replicons (virus particles containing an rna that is self-r ...19948107233
regulation of sindbis virus rna replication: uncleaved p123 and nsp4 function in minus-strand rna synthesis, whereas cleaved products from p123 are required for efficient plus-strand rna synthesis.nonstructural proteins of sindbis virus, nsp1, nsp2, nsp3, and nsp4, as well as intermediate polyproteins, are produced from two precursor polyproteins, p123 and p1234, by a proteolytic enzyme encoded in the c-terminal half of nsp2. we studied the requirements for and the functions of the intermediate and mature processing products for sindbis virus rna synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various co ...19948107248
the expression of chloramphenicol acetyltransferase in aedes albopictus (c6/36) cells and aedes triseriatus mosquitoes using a double subgenomic recombinant sindbis virus.genomic rna was transcribed in vitro from the double subgenomic recombinant sindbis (sin) virus expression vector, pte/3'2j/cat, and transfected into bhk-21 cells to generate recombinant virus stocks. te/3'2j/cat virus was used to infect c6/36 (aedes albopictus) cells and adult female aedes triseriatus. when c6/36 cells were infected with te/3'2j/cat virus at a multiplicity of infection (moi) of greater than 20, 100% of the cells expressed cat. the number of cat polypeptides expressed per cell a ...19948111422
sindbis virus-induced inhibition of protein synthesis is partially reversed by medium containing an elevated potassium concentration.infection of cultured vertebrate cells by sindbis virus, an alphavirus, results in a reduction in the overall rate of protein synthesis and in selective termination (shutoff) of host-specified protein synthesis. the shutoff of host protein synthesis by sindbis virus has been temporally correlated with a decrease in intracellular k+ concentration (and an increase in intracellular na+ concentration) which occurs as a consequence of virus-mediated inhibition of the plasma membrane-associated na+/k+ ...19948113764
[genetical and biochemical studies on the metabolisms and biological roles of membrane phospholipids in mammalian cells].the genetical and biochemical approach is used as a powerful tool for the elucidation of the metabolic regulation as well as the biological significance of various constituents in living cells. this technique has been successfully applied to the membrane phospholipids in mammalian cells, actually to phosphatidylserine, sphingomyelin, and cardiolipin, all of which are rather minor constituents in membrane phospholipids. isolation and characterization of the mutant cells defective in biosyntheses ...19947853148
genetic analysis of the nsp3 region of sindbis virus: evidence for roles in minus-strand and subgenomic rna synthesis.sindbis virus nonstructural polyproteins and their cleavage products are believed to be essential components of viral rna replication and transcription complexes. although numerous studies have investigated the effect of mutations in nsp1-, nsp2-, and nsp4-coding regions on sindbis virus-specific rna synthesis, relatively little is known about the function of the region encoding nsp3. nsp3 is a phosphoprotein comprising two regions: an n-terminal portion which is highly conserved among alphaviru ...19948057460
multivalent rna probes and their use in the quantitation of multiple and non-homologous rna species immobilized onto nylon membranes. 19948065945
analysis of the molecular basis of neuropathogenesis of rna viruses in experimental animals: relevance for human disease?rna viruses with segmented genomes were the first model used for molecular analysis of viral neuropathogenesis, since they could be analysed genetically by reassortment. four viruses with non-segmented genomes have been used as models of neurovirulence and demyelinating disease: jhm coronavirus, theiler's virus, sindbis virus and semliki forest virus (sfv). virus gene expression in the central nervous system of infected animals has been measured by in situ hybridization and immunocytochemistry. ...19948072672
inactivation of free and cell-associated human immunodeficiency virus in platelet suspensions by aminomethyltrimethylpsoralen and ultraviolet has previously been reported that 40 micrograms per ml of aminomethyltrimethylpsoralen (amt) plus 2.4 to 7.2 j per cm2 of ultraviolet a (uva) light inactivated 4 to 6 log10 of several model viruses in platelet suspensions. this inactivation was achieved while satisfactory levels of platelet count, ph, morphology, aggregation, and hemostatic effectiveness were maintained.19948073484
formation and intracellular localization of hepatitis c virus envelope glycoprotein complexes expressed by recombinant vaccinia and sindbis viruses.hepatitis c virus (hcv) encodes two putative virion glycoproteins (e1 and e2) which are released from the polyprotein by signal peptidase cleavage. in this report, we have characterized the complexes formed between e1 and e2 (called e1e2) for two different hcv strains (h and bk) and studied their intracellular localization. vaccinia virus and sindbis virus vectors were used to express the hcv structural proteins in three different cell lines (hepg2, bhk-21, and pk-15). the kinetics of associatio ...19948083956
alphavirus nsp3 functions to form replication complexes transcribing negative-strand rna.the alphavirus mutant sindbis virus hr ts4, which has been assigned to the a complementation group, possessed a selective defect in negative-strand synthesis that was similar although not identical to that observed for the b complementation group mutant ts11 (y.-f. wang, s. g. sawicki, and d. l. sawicki, j. virol. 65:985-988, 1991). the causal mutation was identified as a change of a c to a u residue at nucleotide 4903 in the nsp3 open reading frame that predicted a change of ala-268 to val. thu ...19948083984
murine t-helper cell immune response to recombinant vaccinia-venezuelan equine encephalitis virus.the t-helper (th) cell immune response following immunization of c3h (h-2k) mice with a recombinant vaccinia (vac) virus (tc-5a) expressing the structural proteins (capsid, e1 and e2) of the attenuated vaccine strain (tc-83) of venezuelan equine encephalitis (vee) virus was compared with the immune response induced in mice after immunization with tc-83 virus. tc-5a virus elicited th cells that strongly recognized both vac and tc-83 viruses in in vitro lymphoblastogenesis tests. th-cell activatio ...19948085379
the configuration of sindbis virus envelope proteins is stabilized by the nucleocapsid protein.we have previously provided evidence that a strong and precise interaction of the sindbis virus nucleocapsid with the cytoplasmic tail of e2 is critical for the function of the mature virion and that changes in this association may affect infectivity (21). in this study, we examined the effects of temperature-sensitive defects in capsid protein on virus infectivity and glycoprotein function. the two sindbis virus mutants chosen were ts2 and ser180/gly183, which contain amino acid substitutions i ...19948091681
removal and inactivation of enveloped and non-enveloped viruses during the purification of a high-purity factor ix by metal chelate affinity chromatography.virus reduction during the copper chelate affinity chromatography stage used during the purification of a new high-purity factor ix (bpls 9mc) has been investigated. virus reduction for the enveloped virus sindbis was 6.5 log, a value which included approximately 2 log of inactivation due to the use of an acidic wash buffer (ph 4.4) during chromatography. in the case of the non-enveloped hepatitis-a-like poliovirus, which is acid-resistant, the virus reduction value was 4.0 log and was exclusive ...19948091741
subgenomic mrna of aura alphavirus is packaged into virions.purified virions of aura virus, a south american alphavirus related to sindbis virus, were found to contain two rna species, one of 12 kb and the other of 4.2 kb. northern (rna) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb rna was the viral genomic rna, whereas the 4.2-kb rna present in virus preparations was identical to the 26s subgenomic rna present in infected cells. the subgenomic rna is the messenger for translation of the viral structural proteins ...19947902874
lethality of pe2 incorporation into sindbis virus can be suppressed by second-site mutations in e3 and e2.sindbis virions contain two glycoproteins, e1 and e2. e2 is produced initially as a precursor, pe2, from which the amino-terminal 64 amino acids are cleaved by a cellular protease at a late stage in virion maturation. a mutation at e2 position 1 (arg to asn) was placed into sindbis virus ar339 by site-directed mutagenesis of a full-length ar339 cdna clone, ptrsb, to produce ptrsb-n. the mutation created a signal for n-linked glycosylation immediately adjacent to the pe2 cleavage signal. virions ...19947908062
deletion and duplication mutations in the c-terminal nonconserved region of sindbis virus nsp3: effects on phosphorylation and on virus replication in vertebrate and invertebrate cells.little is known concerning the function of the c-terminal nonconserved region of sindbis virus nsp3. in this report, we created a number of in-frame deletions and duplications (from 12 to 159 residues) in this region and examined their effects on sindbis virus replication. sindbis rna transcripts containing these mutations were infectious and gave rise to viable virus after transfection of chicken embryo fibroblasts (cef). in cef, the rate of virus release and accumulation of viral rnas were sim ...19947912020
mutations in an exposed domain of sindbis virus capsid protein result in the production of noninfectious virions and morphological variants.a critical event in the process of sindbis virus assembly is the interaction of the virus nucleocapsid with the target membrane which has been modified by virus envelope glycoproteins. a specific association between the endodomain of the pe2/e2 glycoprotein and an unidentified domain in the capsid protein is responsible for this association. we have examined the nature of this association by the production of a mutant which has two amino acid substitutions in a domain of the capsid protein known ...19947912022
host-cell receptors for sindbis virus.sindbis virus has a very wide host range, infecting many species of mosquitoes and other hematophagous insects and infecting many species of higher vertebrates. we have used two approaches to study host cell receptors used by sindbis virus to enter cells. anti-idiotype antibodies to neutralizing antibodies directed against glycoprotein e2 of the virus identified a 63-kda protein as a putative receptor in chicken cells. in a second approach, monoclonal antibodies identified a 67 kda protein, beli ...19947913360
new phthalocyanines for photodynamic virus inactivation in red blood cell concentrates.cationic phthalocyanines with either aluminum or silicon as the central metal were evaluated for their ability to inactivate viruses in red blood cell concentrates (rbcc) photodynamically. in addition, the virucidal potential of a substituted anionic phthalocyanine, aluminum dibenzodisulfophthalocyanine hydroxide (a1n2sb2poh) was evaluated and compared with that of the much studied anionic aluminum tetrasulfophthalocyanine hydroxide (a1pcs4oh). based on the rate of inactivation of the lipid-enve ...19947938215
antiviral assays on phytochemicals: the influence of reaction parameters.investigations of phytochemicals for antiviral activities are assuming greater importance; but little attention has been given to the influence of various reaction parameters. we found that the activities of several known antiviral phytochemicals were profoundly affected by the presence of serum components, but in different ways. thus, the terthiophene, alpha-terthienyl (alpha-t), was inhibited in a concentration-dependent manner by serum. in the case of a carboxylic acid derivative of alpha-t, ...19947938267
demonstration of sindbis virus antigen in lowicryl-embedded cultured cells by immunogold staining.eight monoclonal antibodies (mabs) were tested for the use in immunogold staining of sindbis virus (sin) antigen in cultured baby hamster kidney (bhk) cells. the antibodies were directed against the capsid (c) protein (5) or against the glycoprotein e1 (3), respectively. four out of five anti-c mabs and two out of three anti-e1 mabs reacted on frozen sections, whereas two of the anti-c antibodies and none of the anti-e1 antibodies reacted on lowicryl k4m-embedded cells. the mabs reacting, in par ...19947941845
the amino-terminal residue of sindbis virus glycoprotein e2 influences virus maturation, specific infectivity for bhk cells, and virulence in mice.the e2 glycoprotein of sindbis virus is synthesized as a precursor, pe2, which is cleaved by furin or a furin-like host cell protease at a late stage of maturation. the four-residue pe2 cleavage signal conforms to the basic amino acid-x-basic-basic motif which is present in many other viral and cellular glycoproteins which are processed by the cellular enzyme(s). in this report, we present evidence that the amino acid which immediately follows the signal, the n-terminal residue of e2, can influe ...19947966596
translation of sindbis virus mrna: effects of sequences downstream of the initiating incentive for developing the alphavirus sindbis virus as a vector for the expression of heterologous proteins is the very high level of viral structural proteins that accumulates in infected cells. although replacement of the structural protein genes by a heterologous gene should lead to an equivalent accumulation of the heterologous protein, the sindbis virus capsid protein is produced at a level 10- to 20-fold higher than that of any foreign protein. chimeric mrnas which contain the first ...19947966601
differential sensitivities of viruses in red cell suspensions to methylene blue photosensitization.previous studies explored the feasibility of using the photosensitizer methylene blue (mb) as a virucidal agent in red cell suspensions. under treatment conditions (5 microm [5 mumol/l] mb, 3.4 x 10(4) j/m2) that resulted in more than 6 log10 inactivation of vesicular stomatitis virus (vsv) or of the enveloped bacteriophage phi 6, red cell membrane alterations were observed. increased red cell ion permeability and the binding of plasma proteins to the red cell surface were the most sensitive ind ...19948023394
intracellular immunization of mosquito cells to lacrosse virus using a recombinant sindbis virus vector.a cdna of the small rna genome segment of la crosse (lac) virus was inserted, in an antisense orientation, into a double subgenomic sindbis (dssin) virus expression vector generating pte/3'2j/anti-s (15,000bp). in vitro transcription of the pte/3'2j/anti-s template generated genomic rna that was electrotransfected into bhk-21 cells to produce virus. northern blot analysis of rna isolated from infected aedes albopictus (c6/36) cells showed that the te/3'2j/anti-s virus produced a subgenomic mrna ...19948030366
common replication strategies emerging from the study of diverse groups of positive-strand rna viruses.studies using brome mosaic virus (bmv), sindbis virus and poliovirus have provided evidence that disparate groups of plant and animal positive strand rna viruses have remarkably similar replication strategies. the conservation of several functional domains within virus-encoded nonstructural proteins implies that, although the precise character of these and interacting host components varies for each virus, they employ similar mechanisms for rna replication. for (+) strand replication, similariti ...19948032249
age-dependent susceptibility to fatal encephalitis: alphavirus infection of neurons.sindbis virus encephalitis in mice provides a model for studying age-dependent susceptibility to acute viral encephalitis. the ar339 strain of sv causes fatal encephalitis in newborn mice, but weanling mice recover uneventfully. increased virulence for older mice is associated with a single amino acid change from gln to his at position 55 of the e2 glycoprotein. weanling mice with normal immune systems clear infectious virus from neurons through an antibody-mediated mechanism. this does not happ ...19948032263
nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.we have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using ross river virus-sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. a virus containing the ross river virus genome in which the capsid protein had been replaced with that from sindbis virus was almost nonviable. nucleocapsids formed in normal numbers in the infected cell, but very little ...19947508993
polypeptide requirements for assembly of functional sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand rna synthesis.proteolytic processing of the sindbis virus non-structural polyproteins (p123 and p1234) and synthesis of minus- and plus-strand rnas are highly regulated during virus infection. although their precise roles have not been defined, these polyproteins, processing intermediates or mature cleavage products (nsp1-4) are believed to be essential components of viral replication and transcription complexes. in this study, we have shown that nsp4 can function as the polymerase for both minus- and plus-st ...19947517863
recombination between sindbis virus rnas.the sindbis virus rna genome is divided into two modules--one coding for the nonstructural protein genes and the other coding for the structural protein genes. in our studies of recombination, the two parental rnas were defective in different modules. analysis of the recombinant rnas demonstrated that the parental rnas each contributed its intact module and that the crossovers occurred within the defective modules. the recombinational events giving rise to infectious virion rnas could create del ...19947518272
recombinant sindbis virus as an expression system for cell biology. 19947529867
viral safety of solvent/detergent-treated blood products.laboratory research that began in 1982 led to the licensing in the usa of a solvent/detergent (sd)-treated factor viii concentrate in 1985. the licence was granted on the basis of several factors. first, studies had demonstrated the inactivation of several marker viruses (vesicular stomatitis virus, sindbis virus, sendai virus) and other viruses such as human immunodeficiency virus (hiv), hepatitis b virus (hbv), and non-a, non-b hepatitis virus (nanbhv; now known principally to be hepatitis c v ...19947749045
hepatitis c and arboviral antibodies in the island populations of mauritius and rodrigues.a serological survey for antibodies to hepatitis c virus (hcv), dengue viruses (den), west nile virus (wn), and sindbis virus (sin) was carried out in sera of selected groups of the population of the islands of mauritius (n = 449) and rodrigues (n = 115), indian ocean. 8.3% of 564 sera were positive for anti-hcv. in mauritius, 2.1% of sera of healthy individuals were found with anti-hcv. the highest prevalence was found in sexually transmitted disease (std) patients and prison inmates with 46.2% ...19947534820
studies on the antiviral activity of protein kinase inhibitors against the replication of vesicular stomatitis virus.several derivatives of k-252a, a protein kinase inhibitor isolated from nocardiopsis sp., were investigated for their effects on the replication of vesicular stomatitis virus (vsv) in bhk-21 cell cultures. among those we tested, kt5926, which preferentially inhibits the myosin light chain kinase (mlck), suppressed the viral replication by 95-99% at 15 microm. k-252a, which inhibits a broad spectrum of cellular protein kinase, similarly affected the viral replication. other derivatives, kt5720 an ...19957550128
mosquito sensitivity to a scorpion neurotoxin expressed using an infectious sindbis virus vector.the scorpion, androctonus australis hector, produces an insect-specific toxin (aahit) encoded by the scotox gene. to assess the toxicity of aahit for mosquitoes, we have taken a novel approach to express the scotox gene in vivo. we have engineered a double subgenomic sindbis (dssin) virus that contains the scotox gene in the viral genome and intrathoracically inoculated the virus (te/3'2j/scotox) into mosquitoes (aedes aegypti, ae. triseriatus and culex pipiens), houseflies (musca domestica) and ...19957551198
double-subgenomic sindbis virus recombinants expressing immunogenic proteins of japanese encephalitis virus induce significant protection in mice against lethal jev infection.a series of double-subgenomic sindbis virus (dssin) recombinants that express cassettes encoding the immunogenic proteins of japanese encephalitis virus (jev) [prm-e, prm-e-ns1, ns1-ns2a, 80%e (encodes the amino-terminal 80% part of e), and ns1] were constructed and analyzed for their ability to confer protective immunity in mice against lethal challenge with neurovirulent jev. the cassettes were introduced into both 5' [second subgenomic promoter of the vector precedes the sin structural open r ...19957571428
myristoylation is required for the intracellular localization and endocytic function of arf6.adp-ribosylation factors (arfs) are modified by a myristate group that is covalently linked to the second glycine residue at the amino terminus. with the recent evidence that arf6 localizes to the cell periphery and plays a regulatory role in endocytic traffic, we have investigated the role of myristoylation on the membrane association, biological activity, and subcellular distribution of arf6 in intact cells. a gly2ala mutation produced a nonmyristoylated protein that failed to associate with m ...19957589240
iodine-mediated inactivation of lipid- and nonlipid-enveloped viruses in human antithrombin iii concentrate.human plasma-derived protein concentrates intended for clinical use must be treated for viral inactivation to ensure patient safety. this study explored the use of liquid iodine for inactivation of several lipid- and nonlipid-enveloped viruses in an antithrombin iii (at-iii) concentrate. iodine at levels of 0.01% to 0.02% caused between 43% and 94% loss of at-iii activity, as well as degradation of at-iii as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and wester ...19957606009
a mutant of sindbis virus which is released efficiently from cells maintained in low ionic strength medium.experiments many years ago showed that reducing the ionic strength of the medium sindbis virus-infected chick embryo fibroblasts were maintained prevented the release of progeny virus into the medium. it was proposed at that time that the susceptibility of virus release to inhibition by low ionic strength (lis) medium might be determined by the properties of the viral envelope proteins. we thought that if such were the case, it might be possible to obtain a viral mutant which is resistant to the ...19957618264
replication and tissue tropism of the alphavirus sindbis in the mosquito aedes albopictus.we have examined the replication and tissue distribution of the alphavirus sindbis in the mosquito aedes albopictus. parenteral inoculation of virus resulted in an acute infection accompanied by rapid virus replication and a persistent infection, during which total virus production was reduced. acute and persistent phase virus rna synthesis, virus production, and organ-specific distribution of infection were determined over an 18-day incubation period. organs were classified as refractory (ovari ...19957676618
effects of anti-e2 monoclonal antibody on sindbis virus replication in at3 cells expressing bcl-2.antibodies directed to sindbis virus (sv) envelope protein e2 are able to control virus replication in vivo and in persistently infected cultures of neurons in vitro. we investigated the mechanisms by which anti-e2 monoclonal antibody (mab) alters virus replication by using at3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2. treatment of sv-infected at3-bcl-2 cells with anti-e2 mab g5 for 2 h decreased the rate of virus release for 6 to 8 h after removal of the antibod ...19957474120
antiviral activity of alpha interferon in sindbis virus-infected cells is restored by anti-e2 monoclonal antibody treatment.pretreatment of at3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2 (at3-bcl-2 cells) with alpha interferon (ifn-alpha) affected replication of a virulent strain of sindbis virus (sv) but did not protect against virus-induced cell death. treatment of cells with ifn-alpha late during infection affected ongoing sv replication very little. previous studies have shown that cross-linking of the viral glycoprotein e2 with antibody delays the inhibition of k+ influx by improvi ...19957474167
putative receptor binding sites on alphaviruses as visualized by cryoelectron microscopy.the structures of sindbis virus and ross river virus complexed with fab fragments from monoclonal antibodies have been determined from cryoelectron micrographs. both antibodies chosen for this study bind to regions of the virions that have been implicated in cell-receptor recognition and recognize epitopes on the e2 glycoprotein. the two structures show that the fab fragments bind to the outermost tip of the trimeric envelope spike protein. hence, the same region of both the sindbis virus and ro ...19957479858
[the distribution of sbv nonstructural protein 2 (nsp2) in host cell].the distribution of sbv nsp2 has been observed by means of immunolabelling electron microscopy. the results showed that sbv nsp2 existed both in the cytoplasm and in the nucleus of host cell. when the cdna of sbv nsp2 was transfected in host cell, the nsp2 also accumulates in the nucleus.19957483582
equine arteritis virus subgenomic rna transcription: uv inactivation and translation inhibition studies.the expression of the genetic information of equine arteritis virus (eav), an arterivirus, involves the synthesis of six subgenomic (sg) mrnas. these are 5' and 3' coterminal since they are composed of a leader and a body sequence, which are identical to the 5' and 3' ends of the genome, respectively. previously, it has been suggested that cis-splicing of a genome-length precursor rna is involved in their synthesis. this was reevaluated in a comparative analysis of the sg rna synthesis of eav, t ...19957491761
genesis of sindbis virus by in vivo recombination of nonreplicative rna precursors.genetically engineered rna transcripts coding for various sindbis virus (sin) genes were used to study structure and sequence requirements of rna recombination in bhk cells. three different groups of rna transcripts were made: (i) rnas which retain the ability to replicate and which carry sequences coding for either viral polymerase or viral structural proteins; (ii) rnas which lack the complete 3' end of the sin genome and thus are incapable of replicating; and (iii) rnas which lack the complet ...19957494243
evolution of the sindbis virus subgenomic mrna promoter in cultured cells.transcription of the subgenomic mrna of alphaviruses initiates at an internal site, called the promoter, which is highly conserved. to determine the functional significance of this conservation, we used an approach that randomizes positions -13 to -9 of the promoter to generate a library containing all possible sequences within this region, including the wild-type sequence. viruses in the mixed population with more-efficient promoters were selected for during passaging in mammalian (bhk-21) cell ...19957494287
host-dependent evolution of the sindbis virus promoter for subgenomic mrna synthesis.alphaviruses are alternately transmitted between arthropod and vertebrate hosts. in each host, the virus transcribes a subgenomic mrna that encodes the viral structural proteins which encapsidate the genome to form progeny virions. transcription initiates at an internal site called the promoter. to determine if promoter utilization varies in mammalian versus mosquito cells, we used these cells as hosts to select for active promoters among a library of different mutant promoters. compared with th ...19957494288
the expression of chloramphenicol acetyltransferase in mosquitoes and mosquito cells using a packaged sindbis replicon system.sindbis (sin) replicon virus was used to express chloramphenicol acetyltransferase (cat) in aedes albopictus (c6/36) cells and aedes triseriatus mosquitoes. rna transcribed in vitro from a sin replicon plasmid (psinrep5/cat) and from sin virus helper constructs (pdh-eb or pdh(26s)5'sin) was coelectroporated into bhk-21 cells to generate replicon viruses, designated rep5/cat/eb and rep5/cat/26s. c6/36 cells infected with rep5/cat/eb or rep5/cat/26s virus at a multiplicity of infection of 3, expre ...19957498436
expression of the rubella virus structural proteins by an infectious sindbis virus assess the potential of an infectious sindbis virus vector (dssin) as a eukaryotic expression vector, dssin recombinants which contain each of the rubella virus (rub) structural proteins (c, e2, and e1) individually were constructed. expression of the rub proteins by each dssin recombinant was robust and processing was similar to that observed when these proteins were expressed using other vectors. the c and e2 recombinants, each of which contains a cassette of less than 1,000 nts, were stabl ...19957503703
isolation of the anthropogenic compound fluoranthene in a screening of chinese medicinal plants for antiviral compounds.thirty-one species of medicinal plants used in the treatment of diseases of viral origin in yunnan province of china were assayed for inhibition of sindbis and murine cytomegalovirus in mammalian cell cultures. sixteen species displayed antiviral activity. a compound, which exhibited long wavelength uv-mediated antiviral activity, was isolated from leaves and twigs of elsholtzia ciliata (lamiaceae) using bioassay-guided fractionation and identified as the polycyclic aromatic hydrocarbon, fluoran ...19957753931
a sindbis virus mrna polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo.the direct intramuscular delivery of naked plasmid dna has been demonstrated to allow expression of encoded heterologous genes in the target myocytes. the method has been employed to elicit immunization based upon delivery of antigen encoding plasmid dna. for application in the context of achieving anti-tumor immunization against antigenic transforming oncoproteins, delivery of plasmid dnas encoding these molecules would create significant potential safety hazards. as an alternative to dna polyn ...19957784202
assessment of fusogenic properties of influenza virus hemagglutinin deacylated by site-directed mutagenesis and hydroxylamine treatment.influenza virus hemagglutinin (ha) subtype h7 expressed from a baculovirus vector in insect cells requires cysteine residues for palmitoylation. mutant ha devoid of fatty acids shows hemagglutinating and hemolytic activities almost identical to those of the acylated wild-type ha (wt). using a membrane mixing assay (r18), neither the kinetics nor the ph dependence of fusion induced by wt or mutant ha was significantly different from virus-induced fusion. ha-induced fusion of insect cells with hum ...19957793071
molecular genetic manipulation of mosquito vectors.despite their central role in disease transmission, relatively little is known of the molecular biology of arthropod vectors. modern molecular approaches will undoubtedly provide considerable information about gene regulation and expression in vectors and consequently a much better understanding of the biology and molecular biology of vectors. such knowledge is essential for developing effective control strategies for vector-borne diseases. in this review, we focus upon techniques and approaches ...19957810990
intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant sindbis virus.a single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. to see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. the variable genes and interchain ...19957815482
reaction in alphavirus mrna capping: formation of a covalent complex of nonstructural protein nsp1 with 7-methyl-gmp.after the start of transcription, the 5' ends of eukaryotic mrna molecules are modified by the addition of a guanylyl residue to form a cap structure, g(5')ppp(5')n. the guanylyltransferase (gtp:mrna guanylyltransferase, ec reaction responsible for cap formation usually proceeds via a covalent enzyme-gmp intermediate. we have studied the alphavirus-specific guanylyltransferase by incubating lysates from semliki forest and sindbis virus-infected cells with [alpha-32p]gtp, using vaccinia ...19957831320
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