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the african swine fever virus prenyltransferase is an integral membrane trans-geranylgeranyl-diphosphate synthase.in a previous study, it was shown that the protein encoded by the gene b318l of african swine fever virus (asfv) is a trans-prenyltransferase that catalyzes in vitro the condensation of farnesyl diphosphate and isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and longer chain prenyl diphosphates (alejo, a., yáñez, r. j., rodríguez, j. m., viñuela, e., and salas, m. l. (1997) j. biol. chem. 272, 9417-9423). to investigate the in vivo function of the viral enzyme, we have determine ...199910364254
characterization of an equine arteritis virus replicase mutant defective in subgenomic mrna synthesis.equine arteritis virus (eav) is a positive-stranded rna virus that synthesizes a 5'- and 3'-coterminal nested set of six subgenomic mrnas. these mrnas all contain a common leader sequence which is derived from the 5' end of the genome. subgenomic mrna transcription and genome replication are directed by the viral replicase, which is expressed in the form of two polyproteins and subsequently processed into smaller nonstructural proteins (nsps). during the recent construction of an eav infectious ...199910364273
in vitro assembly of alphavirus cores by using nucleocapsid protein expressed in escherichia coli.the production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the golgi apparatus. these components associate during the budding process to produce the mature virion. the nucleocapsid proteins of sindbis virus and ross river virus have been produced in a t7-based escherichia coli expression system and purified. in the presence of single-stranded but not double- ...199910364277
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
a visual screen of a gfp-fusion library identifies a new type of nuclear envelope membrane protein.the nuclear envelope (ne) is a distinct subdomain of the er, but few membrane components have been described that are specific to it. we performed a visual screen in tissue culture cells to identify proteins targeted to the ne. this approach does not require assumptions about the nature of the association with the ne or the physical separation of ne and er. we confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subco ...010402458
removal of viruses from human intravenous immune globulin by 35 nm nanofiltration.viral safety is an important prerequisite for clinical immunoglobulin preparations. a common manufacturing practice is to utilize several virus removal/inactivation process steps to ensure the safety of human intravenous immunoglobulin (ivig). in this regard, we examined the use of planova 35 nm filters to reduce potential loads of both non-enveloped and enveloped viruses prior to end-stage solvent detergent treatment. the nanofiltration process was validated for removal of a variety of envelope ...199810403036
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
vaccines timeline. interview by philip cohen. 199910559053
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
arf6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting.activation of rac1, a member of the rho family of gtpases, is associated with multiple cellular responses, including membrane ruffling and focal complex formation. the mechanisms by which rac1 is coupled to these functional responses are not well understood. it was recently shown that arf6, a gtpase implicated in cytoskeletal alterations and a membrane recycling pathway, is required for rac1-dependent phagocytosis in macrophages (q. zhang et al., j. biol. chem. 273:19977-19981, 1998). to determi ...010567541
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
adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs.viruses are cellular parasites. the linkage between viral and host functions makes the study of a viral life cycle an important key to cellular functions. a deeper understanding of many aspects of viral life cycles has emerged from coordinated molecular and structural studies carried out with a wide range of viral pathogens. structural studies of viruses by means of cryo-electron microscopy and three-dimensional image reconstruction methods have grown explosively in the last decade. here we revi ...199910585969
rainbow trout sleeping disease virus is an atypical alphavirus.sleeping disease (sd) is currently a matter of concern for salmonid fish farmers in most parts of the world. a viral etiology of sd has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. in salmonid-derived cell lines, the maximal rate of virus production was observed at 10 degrees c, while little virus was produced at 14 degrees c. through biochemical, physicochemical, and morphological studies, sd virus (sdv) was shown to be an enveloped vir ...200010590104
integrins alpha2beta1 and alpha4beta1 can mediate sa11 rotavirus attachment and entry into cells.most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins vp4 and vp7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. here we report that sa11 rotavirus binding to and infection of k562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection ...200010590110
in vitro transcription by the turnip yellow mosaic virus rna polymerase: a comparison with the alfalfa mosaic virus and brome mosaic virus replicases.recently, we showed that the main determinant in the trna-like structure of turnip yellow mosaic virus rna to initiate minus-strand synthesis in vitro is the 3' acca end. by mutational analysis of the 3'-terminal hairpin, we show here that only a non-base-paired acca end is functional and that the stability of the wild-type 3'-proximal hairpin is the most favorable, in that it has the lowest deltag value and a high transcription efficiency. with a nested set of rna fragments, we show that the mi ...200010590114
vaccination of macaques against pathogenic simian immunodeficiency virus with venezuelan equine encephalitis virus replicon particles.vaccine vectors derived from venezuelan equine encephalitis virus (vee) that expressed simian immunodeficiency virus (siv) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. immunization with vee replicon particles induced both humoral and cellular immune responses. four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic siv swarm, ...200010590126
alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum.we report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (den) virus. we found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prm (the intracellular glycosylated precursor of m [membrane protein]) and e (envelope protein): the proper folding of prm bearing unprocessed n-linked oligosaccharide is inefficient, and this causes delayed formation of prme heterodimer. the complexes formed between incomple ...200010590151
identification of a bovine coronavirus packaging signal.a region of the bovine coronavirus (bcv) genome that functions as a packaging signal has been cloned. the 291-nucleotide clone shares 72% homology with the region of mouse hepatitis coronavirus (mhv) gene 1b that contains the packaging signal. rna transcripts were packaged into both bcv and mhv virions when the cloned region was appended to a noncoronavirus rna. this is the first identification of a bcv packaging signal. the data demonstrate that the bcv genome contains a sequence that is conser ...200010590153
increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and trail.chemotherapeutic genotoxins induce apoptosis in epithelial-cell-derived cancer cells. the death receptor ligand trail also induces apoptosis in epithelial-cell-derived cancer cells but generally fails to induce apoptosis in nontransformed cells. we show here that the treatment of four different epithelial cell lines with the topoisomerase ii inhibitor etoposide in combination with trail (tumor necrosis factor [tnf]-related apoptosis-inducing ligand) induces a synergistic apoptotic response. the ...200010594023
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
characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside triphosphatase/helicase domain of the hepatitis c virus nonstructural 3 protein.we have produced a murine monoclonal antibody (mab), zx10, recognizing the ntpase/helicase domain of the hepatitis c virus (hcv) nonstructural 3 protein (ns3), from which we designed a single-chain variable fragment (scfv). the zx10 mab recognized a discontinuous epitope of the ntpase/helicase domain, of which the linear sequence geipfygkaipl at residues 1371 to 1382 constitutes one part. cdnas from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into ...200010618278
large-plaque mutants of sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation.laboratory strains of sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. during infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the e2 glycoprotein. recombinant viruses were constructed with these ...200010623725
mapping of genetic determinants of rubella virus associated with growth in joint tissue.rubella virus (rv) strains vary in their abilities to replicate and persist in cell cultures derived from human joint tissue (synovial cells [sc]), and this arthrotropism appears to be linked to their association with joint symptoms in vivo. in order to map the genetic determinants of arthrotropism, an infectious clone of the cendehill vaccine strain of rv was constructed, as well as two chimeric clones containing cdnas from both cendehill and therien (wild-type) strains. replacement of the enti ...200010623741
role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis.the initial steps of venezuelan equine encephalitis virus (vee) spread from inoculation in the skin to the draining lymph node have been characterized. by using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of vee infection in the first 48 h following inoculation. vee viral replicon particles, which can undergo only one round of infection, identified langerhans cells to be the initial set of cells infected b ...200010623754
perspectives for the treatment of infections with flaviviridae.the family flaviviridae contains three genera: hepacivirus, flavivirus, and pestivirus. worldwide, more than 170 million people are chronically infected with hepatitis c virus and are at risk of developing cirrhosis and/or liver cancer. in addition, infections with arthropod-borne flaviviruses (such as dengue fever, japanese encephalitis, tick-borne encephalitis, st. louis encephalitis, murray valley encephalitis, west nile, and yellow fever viruses) are emerging throughout the world. the pestiv ...200010627492
cap-independent translational enhancement of turnip crinkle virus genomic and subgenomic rnas.the presence of translational control elements and cap structures has not been carefully investigated for members of the carmovirus genus, a group of small icosahedral plant viruses with positive-sense rna genomes. in this study, we examined both the 5' and 3' untranslated regions (utrs) of the turnip crinkle carmovirus (tcv) genomic rna (4 kb) as well as the 5' utr of the coat protein subgenomic rna (1.45 kb) for their roles in translational regulation. all three utrs enhanced translation of th ...200010627518
the replication activity of influenza virus polymerase is linked to the capacity of the pa subunit to induce proteolysis.the pa subunit of the influenza virus polymerase complex is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins. the amino-terminal third of the protein is responsible for the induction of proteolysis. we mutated five potential casein kinase ii phosphorylation sites located in the amino-terminal third of the protein. mutations affecting position 157 almost completely abrogated proteolysis induction, whereas a mu ...200010627541
alpha/beta interferons potentiate virus-induced apoptosis through activation of the fadd/caspase-8 death signaling pathway.interferon (ifn) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded rna (dsrna)-dependent protein kinase, pkr. here we report that inducible overexpression of functional pkr in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of pkr were completely resistant. we determined that the mechanism of influenza virus-induced apoptosis involved death signaling t ...200010627563
the s segment of rift valley fever phlebovirus (bunyaviridae) carries determinants for attenuation and virulence in mice.unlike all the other rift valley fever virus strains (bunyaviridae, phlebovirus) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the nss nonstructural protein in the nuclei of infected cells (r. muller, j. f. saluzzo, n. lopez, t. drier, m. turell, j. smith, and m. bouloy, am. j. trop. med. hyg. 53:405-411, 1995). this defect is correlated with a large in-frame deletion in the nss coding region of the s segment of the tripartite genome. here, we sh ...200010627566
modulation of nucleotide sensitivity of atp-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase.atp-sensitive potassium channels (k(atp) channels) regulate cell excitability in response to metabolic changes. k(atp) channels are formed as a complex of a sulfonylurea receptor (surx), a member of the atp-binding cassette protein family, and an inward rectifier k(+) channel subunit (kir6.x). membrane phospholipids, in particular phosphatidylinositol (pi) 4,5-bisphosphate (pip(2)), activate k(atp) channels and antagonize atp inhibition of k(atp) channels when applied to inside-out membrane patc ...010639183
linkage of an alphavirus host-range restriction to the carbohydrate-processing phenotypes of the host cell.the sindbis virus mutant ne2g216 retains pe2 in place of e2 in its virion structure. ne2g216 is a host-range mutant that replicates with near-normal kinetics in vertebrate cells, but displays severely restricted growth in cultured mosquito cells (c6/36) due to defects in the virus maturation process. in this study we tested the hypothesis that the host-range phenotype of ne2g216 was linked to the differences in carbohydrate-processing phenotypes between vertebrate and arthropod cells. arthropod ...200010640554
biochemical consequences of a mutation that controls the cholesterol dependence of semliki forest virus fusion.the enveloped alphavirus semliki forest virus (sfv) infects cells via a low-ph-triggered membrane fusion reaction that requires cholesterol and sphingolipid in the target membrane. cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an sfv mutant that is approximately 100-fold less cholesterol dependent for infection due to a single amino acid change in the e1 spike subunit, proline 226 to serine. sensitive lipid-mixing assays here demons ...200010644331
cell recognition by foot-and-mouth disease virus that lacks the rgd integrin-binding motif: flexibility in aphthovirus receptor usage.cell surface molecules that can act as virus receptors may exert an important selective pressure on rna viral quasispecies. large population passages of foot-and-mouth disease virus (fmdv) in cell culture select for mutant viruses that render dispensable a highly conserved arg-gly-asp (rgd) motif responsible for integrin receptor recognition. here, we provide evidence that viability of recombinant fmdvs including a asp-143-->gly change at the rgd motif was conditioned by a number of capsid subst ...200010644333
cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking.canine parvovirus (cpv) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. we used a variety of approaches to define the early stages of cell entry by cpv. electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. overex ...200010644365
enhancement of tumor-specific immune response with plasmid dna replicon vectors.to enhance the immunogenicity of nucleic acid vaccines, we used plasmid dna vectors that contained replicons derived from the prototype alphavirus, sindbis, and another alphavirus, semliki forest virus. when transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating rna vector (replicon) which in turn directs the expression of a model tumor antigen. immunization with plasmid dna replicons elicited immune responses at doses 100 to 1000-fold lower than ...200010646851
part i. bcl-2 and bcl-x(l) limit apoptosis upon infection with alphavirus vectors.viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. in particular, alphavirus constructs based on sindbis virus (sv) and semliki forest virus (sfv) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (bhk) and chinese hamster ovary (c ...200010649229
photobiological properties of positively charged methylene violet analogs.o-methyl methylene violet (omemv), o-methyl bromomethylene violet (omebrmv) and o-methyl iodomethylene violet (omeimv) have been prepared in order to test their potential utility as anti-viral and anti-tumor phototoxic dyes. rates of photosensitized toxicity of kb cells with 633 nm irradiation are (x 10(-19) photon-1): 2.4, 2.2, 1.9 and 0.17 for omeimv, omebrmv, methylene violet (mv) and omemv, respectively. rates of photosensitized inactivation of sindbis virus in phosphate-buffered saline with ...200010649885
use of vegetable-derived tween 80 for virus inactivation by solvent/detergent treatment. 199910652181
active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus.the polyprotein of infectious pancreatic necrosis virus (ipnv), a birnavirus, is processed by the viral protease vp4 (also named ns) to generate three polypeptides: pvp2, vp4, and vp3. site-directed mutagenesis at 42 positions of the ipnv vp4 protein was performed to determine the active site and the important residues for the protease activity. two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pvp2-vp4 and the vp4-vp3 junctions. wild-type activity at the p ...200010666235
the in vitro-synthesized rna from a cdna clone of hepatitis e virus is infectious.hepatitis e virus (hev) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the indian subcontinent and prevalent in most of the developing parts of the world. the infection is often associated with acute liver failure and high mortality, particularly in pregnant women. in order to develop methods of intervention, it is essential to understand the biology of the virus. this is particularly important as no reliable in vitro culture system is available. we hav ...200010666275
pseudotyping of glycoprotein d-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein g enables mutant virus attachment and entry.the use of herpes simplex virus (hsv) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. because hsv glycoprotein d (gd) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gd to provide new cell recognition specificities in a manner designed to preserve gd's essential role in virus entry. in this study, we have d ...200010666285
development of a sindbis virus expression system that efficiently expresses green fluorescent protein in midguts of aedes aegypti following per os infection.a double subgenomic sindbis (dssin) virus, mre/3'2 j/gfp, was constructed to efficiently express green fluorescent protein (gfp) in the midgut of aedes aegypti following per os infection. the mre/3'2 j/gfp rna genome contained the nonstructural genes and cis-acting sequences of the dssin virus, te/3'2 j/gfp, but had the structural genes of mre16 sin virus. mre/3'2 j/gfp virus, unlike te/3'2 j/gfp virus, efficiently infected mosquitoes orally. at 1-2 days postinfection, gfp was observed as multip ...200010672072
the promise of nucleic acid vaccines.establishing the effective use of 'naked' nucleic acids as vaccines would undoubtedly be one of the most important advances in the history of vaccinology. while nucleic acids show much promise for use as vaccine vectors in experimental animals, not a single naked nucleic acid vector has been approved for use in humans. indeed, data from human clinical trials is scant: nucleic acid vaccines have not been clearly demonstrated to have any convincing efficacy in the prevention or treatment of infect ...200010673713
an outbreak of west nile fever among migrants in kisangani, democratic republic of congo.in february 1998, an outbreak of acute febrile illness was reported from the kapalata military camp in kisangani, the democratic republic of congo. the illness was characterized by an acute onset of fever associated with severe headache, arthralgia, backache, neurologic signs, abdominal pain, and coughing. in 1 individual, hemorrhagic manifestations were observed. the neurologic signs included an altered level of consciousness, convulsions, and coma. malaria was initially suspected, but the pati ...199910674664
efficient gene delivery into adult cardiomyocytes by recombinant sindbis virus.somatic gene therapy as a potential strategy for the treatment of myocardial diseases relies on an efficient gene transfer into cardiac muscle cells. the difficulty of delivering genes into adult cardiomyocytes exists not only in vivo but also in primary culture systems. therefore, possibilities for ex vivo gene transfer and the in vitro study of physiological processes by reverse genetics are limited. we investigated the potential of an alphavirus-based vector system to transduce adult rat card ...199910682322
adaptive mutations in sindbis virus e2 and ross river virus e1 that allow efficient budding of chimeric viruses.alphavirus glycoproteins e2 and e1 form a heterodimer that is required for virus assembly. we have studied adaptive mutations in e2 of sindbis virus (sin) and e1 of ross river virus (rr) that allow these two glycoproteins to interact more efficiently in a chimeric virus that has sin e2 but rr e1. these mutations include k129e, k131e, and v237f in sin e2 and s310f and c433r in rr e1. although rr e1 and sin e2 will form a chimeric heterodimer, the chimeric virus is almost nonviable, producing abou ...200010684281
genetics of mosquito vector competence.mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. efforts to control mosquito-borne diseases have been impeded, in part, by the development of drug-resistant parasites, insecticide-resistant mosquitoes, and environmental concerns over the application of insecticides. therefore, there is a need to develop novel disease control strategies that can complement or replace existing control methods. one such strategy is to generate pathogen-resi ...200010704476
functional correlation of gaba(a) receptor alpha subunits expression with the properties of ipscs in the developing thalamus.gaba(a) receptor alpha1 and alpha2 subunits are expressed differentially with ontogenic period in the brain, but their functional roles are not known. we have recorded gaba(a) receptor-mediated ipscs from laterodorsal (ld) thalamic relay neurons in slices of rat brain at various postnatal ages and found that decay times of evoked ipscs and spontaneous miniature ipscs undergo progressive shortening during the first postnatal month. with a similar time course, expression of transcripts and protein ...200010704495
human immunodeficiency virus type 1 vpr induces apoptosis through caspase activation.human immunodeficiency virus type 1 (hiv-1) vpr is a 96-amino-acid protein that is found associated with the hiv-1 virion. vpr induces cell cycle arrest at the g(2)/m phase of the cell cycle, and this arrest is followed by apoptosis. we examined the mechanism of vpr-induced apoptosis and found that hiv-1 vpr-induced apoptosis requires the activation of a number of cellular cysteinyl aspartate-specific proteases (caspases). we demonstrate that ectopic expression of anti-apoptotic viral proteins, ...200010708425
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