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protease inhibitors as antiviral agents.currently, there are a number of approved antiviral agents for use in the treatment of viral infections. however, many instances exist in which the use of a second antiviral agent would be beneficial because it would allow the option of either an alternative or a combination therapeutic approach. accordingly, virus-encoded proteases have emerged as new targets for antiviral intervention. molecular studies have indicated that viral proteases play a critical role in the life cycle of many viruses ...19989767059
characterization of la crosse virus rna in autopsied central nervous system tissues.a reverse transcription-pcr (rt-pcr) technique was used to detect la crosse (lac) virus rna in the central nervous system (cns) tissues of two patients who died of lac encephalitis in 1960 and 1978. viral rna was readily detected by rt-pcr although the tissues had been stored frozen for up to 37 years. lac virus was detected in the cerebral cortex but not in other cns tissues. rt-pcr allowed detection of replicative forms of the virus, indicating that the virus was actively replicating in the sp ...19989774588
probing the potential glycoprotein binding site of sindbis virus capsid protein with dioxane and model building.alphavirus budding from the plasma membrane is initiated by the specific interaction of the nucleocapsid with the cytoplasmic domain of the glycoprotein e2. it was proposed (lee et al., structure 4:531-541, 1996) that binding of the capsid protein residues 108 to 110 (the "n-terminal arm" residues) to a hydrophobic pocket on the surface of the neighboring capsid protein in the crystal structure mimics the binding of the e2 c-terminal residues into this pocket. in addition, structural comparisons ...19989779796
fractalkine and cx3cr1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.leukocyte migration into sites of inflammation involves multiple molecular interactions between leukocytes and vascular endothelial cells, mediating sequential leukocyte capture, rolling, and firm adhesion. in this study, we tested the role of molecular interactions between fractalkine (fkn), a transmembrane mucin-chemokine hybrid molecule expressed on activated endothelium, and its receptor (cx3cr1) in leukocyte capture, firm adhesion, and activation under physiologic flow conditions. immobiliz ...19989782118
human biliary glycoproteins function as receptors for interspecies transfer of mouse hepatitis virus.a variant mouse hepatitis virus (mhv), designated mhv-h2, was isolated by serial passage in mixed cultures of permissive dbt cells and nonpermissive syrian hamster kidney (bhk) cells. mhv-h2 replicated efficiently in hamster, mouse, primate kidney (vero, cos 1, cos 7), and human adenocarcinoma (hrt) cell lines but failed to replicate in porcine testicular (st), feline kidney (crfk), and canine kidney (mdck) cells. to understand the molecular basis for coronavirus cross-species transfer into huma ...19989782263
effect of clostridium difficile toxin a on human colonic lamina propria cells: early loss of macrophages followed by t-cell apoptosis.we have previously shown that clostridium difficile toxin a induces detachment of human colonic epithelial cells from the basement membrane and subsequent cell death by apoptosis. because these cells require adhesion-dependent signalling from the extracellular matrix for survival, their detachment from the basement membrane by other means also induces apoptosis. the role of toxin a in the induction of apoptosis therefore remains to be determined. in addition, sensitivities to c. difficile toxin ...19989784558
cyclin-dependent kinases participate in death of neurons evoked by dna-damaging agents.previous reports have indicated that dna-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. accordingly, it has become important to understand the signaling events that control this process. we recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by dna damage. consequently, we examined whether cyclin-dependent kinase inhibitors (ckis) and dominant-negativ ...19989786955
rna virus vectors: where are we and where do we need to go? 19989788984
noncytopathic sindbis virus rna vectors for heterologous gene expression.infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. recombinant sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. we recently selected sindbis replicons capable of persistent, noncytopathic growth in bhk cells and describe here a new generation of ...19989789028
a review of alphavirus replication in neurons.alphaviruses are important causes of mosquito-borne viral encephalitis. the prototype alphavirus, sindbis virus, causes encephalomyelitis in mice. the primary target cell for nervous system infection is the neuron. thus, sindbis virus infection of mice provides a model system for studying virus-neuron interactions. the outcome of infection is dependent on the maturity of the targeted neurons and on the strain of sindbis virus used for infection. most sindbis virus strains can induce programmed c ...19989809307
recombinant herpes simplex virus type 1 engineered for targeted binding to erythropoietin receptor-bearing cells.the utility of recombinant herpes simplex virus type 1 (hsv-1) vectors may be expanded by manipulation of the virus envelope to achieve cell-specific gene delivery. to this end, an hsv-1 mutant virus deleted for glycoprotein c (gc) and the heparan sulfate binding domain of gb (kgbpk-gc-) was engineered to encode different chimeric proteins composed of n-terminally truncated forms of gc and the full-length erythropoietin hormone (epo). biochemical analyses demonstrated that one gc-epo chimeric mo ...19989811702
human immunodeficiency virus induces a dual regulation of bcl-2, resulting in persistent infection of cd4(+) t- or monocytic cell lines.this work aims at characterizing the interplay between human immunodeficiency virus type 1 (hiv-1) and the antiapoptotic cellular protein bcl-2 responsible for a persistent infection in lymphoblastoid t (j.jhan) or monocytic (u937) cells. we report that the kinetics of bcl-2 protein level during the establishment of a chronic infection is biphasic, characterized by a transient decrease followed by restoration to the initial level. the extent and duration of this transient decrease were inversely ...19989811703
viral glycoproteins accumulate in newly formed annulate lamellae following infection of lymphoid cells by human herpesvirus 6.ultrastructural analysis of hsb-2 t-lymphoid cells and human cord blood mononuclear cells infected with human herpesvirus 6 revealed the presence, in the cell cytoplasm, of annulate lamellae (al), which were absent in uninfected cells. time course analysis of the appearance of al following viral infection showed that no al were visible within the first 72 h postinfection and that their formation correlated with the expression of the late viral glycoprotein gp116. the requirement of active viral ...19989811708
cytoplasmic domain of sendai virus hn protein contains a specific sequence required for its incorporation into virions.in the assembly of paramyxoviruses, interactions between viral proteins are presumed to be specific. the focus of this study is to elucidate the protein-protein interactions during the final stage of viral assembly that result in the incorporation of the viral envelope proteins into virions. to this end, we examined the specificity of hn incorporation into progeny virions by transiently transfecting hn cdna genes into sendai virus (sv)-infected cells. sv hn expressed from cdna was efficiently in ...19989811709
antiapoptotic but not antiviral function of human bcl-2 assists establishment of japanese encephalitis virus persistence in cultured cells.upon infection of japanese encephalitis virus (jev), baby hamster kidney (bhk-21) and chinese hamster ovary (cho) cells were killed by a mechanism involved in apoptosis. while readily established in a variety of cell lines, jev persistence has never been successfully instituted in bhk-21 and cho cells. since stable expression of human bcl-2 in bhk-21 cells has been shown to delay jev-induced apoptosis, in this study we investigated whether jev persistence could be established in such cells. when ...19989811720
formation and amplification of a novel tombusvirus defective rna which lacks the 5' nontranslated region of the viral genome.defective interfering (di) rnas of tomato bushy stunt virus (tbsv) are small, subgenomic, helper-dependent replicons that are believed to be generated primarily by aberrant events during replication of the plus-sense rna genome. prototypical tbsv di rnas contain four noncontiguous segments (regions i through iv) derived from the 5' nontranslated region (ntr) (i), an internal section (ii), and the 3'-terminal portion (iii and iv) of the viral genome. we have studied the formation of these molecul ...19989811726
distinct biology of kaposi's sarcoma-associated herpesvirus from primary lesions and body cavity lymphomas.the dna sequence for kaposi's sarcoma-associated herpesvirus was originally detected in kaposi's sarcoma biopsy specimens. since its discovery, it has been possible to detect virus in cell lines established from aids-associated body cavity-based b-cell lymphoma and to propagate virus from primary kaposi's sarcoma lesions in a human renal embryonic cell line, 293. in this study, we analyzed the infectivity of kaposi's sarcoma-associated herpesvirus produced from these two sources. viral isolates ...19989811747
identification and characterization of the escherichia coli-expressed rna-dependent rna polymerase of bamboo mosaic virus.bamboo mosaic virus (bamv), a member of the potexvirus group, infects primarily members of the bambusoideae. the open reading frame 1 (orf1) of bamv encodes a 155-kda polypeptide that was postulated to be involved in the replication and the formation of cap structure at the 5' end of the viral genome. to characterize the activities associated with the 155-kda viral protein, it was expressed in escherichia coli bl21(de3) cells with thioredoxin, hexahistidine, and s. tag fused consecutively at its ...19989811749
alphavirus replicase protein nsp1 induces filopodia and rearrangement of actin filaments.expression of the nsp1 protein of semliki forest virus and sindbis virus in cultured cells induced filopodia-like extensions containing nsp1 but not f actin. the actin stress fibers disappeared, whereas vimentin, keratin, and tubulin networks remained intact. the effects of nsp1 were dependent on its palmitoylation but not on its enzymatic activities and were also observed in virus-infected cells.19989811773
capping and methylation of mrna by purified recombinant vp4 protein of bluetongue virus.the core of bluetongue virus (btv) is a multienzyme complex composed of two major proteins (vp7 and vp3) and three minor proteins (vp1, vp4, and vp6) in addition to the viral genome. the core is transcriptionally active and produces capped mrna from which all btv proteins are translated, but the relative role of each core component in the overall reaction process remains unclear. previously we showed that the 76-kda vp4 protein possesses guanylyltransferase activity, a necessary part of the rna ...19989811835
detection and quantitation of human respiratory syncytial virus (rsv) using minigenome cdna and a sindbis virus replicon: a prototype assay for negative-strand rna viruses.we describe here a novel approach for detecting and quantitating human respiratory syncytial virus (rsv) based on expression of a reporter gene from an rsv minigenome. bhk cells were cytoplasmically transformed with a noncytopathic sindbis virus replicon expressing t7 rna polymerase. these cells were then cotransfected with t7 expression plasmids that contain the cdna of an rsv minigenome and the genes for rsv nucleocapsid proteins n, p, and l. the minigenome contains a reporter gene such as lac ...19989813215
nuclear import and the evolution of a multifunctional rna-binding protein.la (ss-b) is a highly expressed protein that is able to bind 3'-oligouridylate and other common rna sequence/structural motifs. by virtue of these interactions, la is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an rna-folding protein or rna chaperone. we have recently characterized the nuclear import pathway of the s. cerevisiae la, lhp1p. the soluble transport factor, or karyopherin, that mediates the import of lhp1p is kap108p/sxm ...19989817748
characterization of diverse viral vector preparations, using a simple and rapid whole-virion dot-blot method.a number of different viruses have been adapted as gene transfer vectors, including retroviruses, adenoviruses, adenoassociated viruses (aavs), herpes simplex virus, sv40 viruses, and alphaviruses (both semliki forest and sindbis viruses). one of the major rate-limiting and time-consuming steps in the characterization of these vectors is the process of determining the viral vector titers. in addition, there is no "universal" method that can be used to rapidly estimate the titer and the utility o ...19989829539
overexpression of chicken interferon regulatory factor-1 (ch-irf-1) induces constitutive expression of mhc class i antigens but does not confer virus resistance to a permanent chicken fibroblast cell line.the chicken fibroblast cell line c32 has been transfected with the chicken homolog (ch-irf-1) of the mammalian transcription factor irf-1. stable transfectants were generated, constitutively overexpressing ch-irf-1 mrna and protein. cells overexpressing ch-irf-1 showed enhanced constitutive expression of mhc class i (b-f, beta-microglobulin) antigens. with increasing number of passages cells with normal b-f iv surface antigen expression accumulated. in the revertants, the amount of ch-irf-1 mrna ...19989831662
a neuron-specific gene transfer by a recombinant defective sindbis virus.we examined the possibility that sindbis virus, an alpha virus with a single-stranded rna genome, would be applied for neuronal gene transfer. the recombinant defective sindbis viruses were constructed by replacing the structural genes of sindbis virus with genes encoding beta-galactosidase (rdsind-lacz) or enhanced green fluorescent protein (rdsind-egfp). in neuron-glia cocultures prepared from the neocortex, hippocampus, and striatum, egfp or beta-galactosidase was expressed selectively in neu ...19989838041
virus maturation by budding.enveloped viruses mature by budding at cellular membranes. it has been generally thought that this process is driven by interactions between the viral transmembrane proteins and the internal virion components (core, capsid, or nucleocapsid). this model was particularly applicable to alphaviruses, which require both spike proteins and a nucleocapsid for budding. however, genetic studies have clearly shown that the retrovirus core protein, i.e., the gag protein, is able to form enveloped particles ...19989841669
persistent hz-1 virus infection in insect cells: evidence for insertion of viral dna into host chromosomes and viral infection in a latent status.persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. in this study, the long-term association between the hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. with the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. once vira ...19999847315
processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab.replicase gene expression by the human coronavirus 229e involves the synthesis of two large polyproteins, pp1a and pp1ab. experimental evidence suggests that these precursor molecules are subject to extensive proteolytic processing. in this study, we show that a chymotrypsin-like enzyme, the virus-encoded 3c-like proteinase (3clpro), cleaves within a common region of pp1a and pp1ab (amino acids 3490 to 4068) at four sites. trans-cleavage assays revealed that polypeptides of 5, 23, 12, and 16 kda ...19999847320
porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents.porcine reproductive and respiratory syndrome virus (prrsv) is a recently described arterivirus responsible for disease in swine worldwide. comparative sequence analysis of 3'-terminal structural genes of the single-stranded rna viral genome revealed the presence of two genotypic classes of prrsv, represented by the prototype north american and european strains, vr-2332 and lelystad virus (lv), respectively. to better understand the evolution and pathogenicity of prrsv, we obtained the 12,066-ba ...19999847330
immunization of woodchucks with plasmids expressing woodchuck hepatitis virus (whv) core antigen and surface antigen suppresses whv infection.dna vaccination can induce humoral and cellular immune response to viral antigens and confer protection to virus infection. in woodchucks, we tested the protective efficacy of immune response to woodchuck hepatitis core antigen (whcag) and surface antigen (whsag) of woodchuck hepatitis virus (whv) elicited by dna-based vaccination. plasmids pwhcim and pwhsim containing whv c- or pre-s2/s genes expressed whcag and whsag in transient transfection assays. pilot experiments in mice revealed that a s ...19999847331
selection of functional variants of the ns3-ns4a protease of hepatitis c virus by using chimeric sindbis viruses.the ns3-ns4a serine protease of hepatitis c virus (hcv) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the hcv replication machinery. despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for hcv in cultured cells. we have recently described the generation of chimeric hcv-sindbis viruses whose propagatio ...19999847361
persistent infection promotes cross-species transmissibility of mouse hepatitis virus.persistent infection with mouse hepatitis virus (mhv) strain a59 in murine dbt (delayed brain tumor) cells resulted in the emergence of host range variants, designated v51a and v51b, at 210 days postinfection. these host range mutants replicated efficiently in normally nonpermissive chinese hamster ovary (cho), in human hepatocarcinoma (hepg2), and to a lesser extent in human breast carcinoma (mcf7) cell lines. little if any replication was noted in baby hamster kidney (bhk), green african monke ...19999847369
sendai virus infection induces apoptosis through activation of caspase-8 (flice) and caspase-3 (cpp32).sendai virus (sv) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. we now show that sv infection triggers an apoptotic program in target cells. incubation of infected cells with the peptide inhibitor z-vad-fmk abrogated sv-induced apoptosis, indicating that proteases of the caspase family were involved. moreover, proteolytic activation of two distinct caspases, cpp32/caspase-3 and, as shown for the first time in virus-infected cells, flice/caspase ...19999847376
new mouse model for dengue virus vaccine testing.several dengue (den) virus vaccines are in development; however, the lack of a reliable small animal model in which to test them is a major obstacle. because evidence suggests that interferon (ifn) is involved in the human anti-den virus response, we tested mice deficient in their ifn functions as potential models. intraperitoneally administered mouse-adapted den 2 virus was uniformly lethal in ag129 mice (which lack alpha/beta ifn and gamma ifn receptor genes), regardless of age. immunized mice ...19999847388
pogosta disease: clinical observations during an outbreak in the province of north karelia, finland.to characterize the clinical picture of pogosta disease.19989851265
alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-trna-protein transferase, a component of the n-end rule pathway.the n-end rule relates the in vivo half-life of a protein to the identity of its n-terminal residue. the underlying ubiquitin-dependent proteolytic system, called the n-end rule pathway, is organized hierarchically: n-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-trna-protein transferase (r-transferase), to arginine, a primary destabilizing residue. we isolated cdna encoding the ...19999858543
[the mutiplication of sindbis virus and host bhk-21 cell apoptosis].the process of apoptosis of bhk-21 cells induced by sindbis virus (sbv) infection is reported here in details. the nuclear dna cleavage can be first detected at 6 h after sbv infection, followed by the chromatin margination and condensation at 12 h and the dna ladder can be detected at 24 h after infection. the details of apoptosis body's formation can be revealed by electron microscopy: first, the outer nuclear membrane protrudes where the condensed chromatin accumulates, then the chromatin get ...19979863210
evaluation of recombinant dense granule antigen 7 (gra7) of toxoplasma gondii for detection of immunoglobulin g antibodies and analysis of a major antigenic domain.dense granule protein 7 (gra7) of toxoplasma gondii was expressed in escherichia coli as a fusion protein. the leader peptide contained a 25-amino-acid mouse tumor necrosis factor fragment and six histidyl residues. after purification by metal chelate affinity chromatography, the antigen was evaluated in an enzyme-linked immunosorbent assay for detection of immunoglobulin g (igg). for two sets of igg-positive human serum samples, obtained from routine screening, an overall sensitivity of 81% was ...19999874659
acute infection of sindbis virus induces phosphorylation and intracellular translocation of small heat shock protein hsp27 and activation of p38 map kinase signaling pathway.in general, viral infection is supposed to induce stress responses in the host cell. however, very few detailed observations about virus-induced stress responses have been reported. here we investigated specific stress responses in vero cells infected with sindbis virus (sv), a single-stranded rna virus, acute infection with which is known to cause apoptotic cell death in the host cells. prior to the onset of apoptosis, p38 mitogen-activated protein kinase (mapk) and c-jun nh2-terminal kinases ( ...19989875220
broad-spectrum antiviral activity and mechanism of antiviral action of the fluoroquinolone derivative k-12.the fluoroquinolone derivatives have been shown to inhibit human immunodeficiency virus (hiv) replication at the transcriptional level. we confirmed the anti-hiv activity of the most potent congener, 8-difluoromethoxy-1-ethyl-6-fluoro-1,4-dihydro-7-[4-(2- methoxyphenyl)-1-piperazinyl]-4-quinolone-3-carboxylic acid (k-12), in both acutely and chronically infected cells. k-12 was active against different strains of hiv-1 (including azt- and ritonavir-resistant hiv-1 strains), hiv-2 and simian immu ...19989875393
recombination of nonreplicative rna precursors of sindbis virus in infected cells overexpressing murine-inducible nitric oxide synthase.the sindbis virus-based sinrep5 expression system is one of the most efficient vectors for gene transfer leading to fast and high expression of the gene of interest. this system was used to transfect vascular endothelial and smooth muscle cells using murine inducible nitric oxide synthase (minos) as a reporter gene. infection of both cell types leads to high expression levels of minos. in addition, the harvested supernatant of these infected cells was used for further rounds of infections, demon ...19989878569
two-helper rna system for production of recombinant semliki forest virus particles.alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. however, present strategies for production of such particles have biosafety limitations due to the generation, by rna recombination, of replication-proficient viruses (rpvs). here we describe a new packaging system for semliki forest virus (sfv) based on a the use of a two-helper system in which the capsid and ...19999882310
endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function.proteolytic processing is required for the activation of numerous viral glycoproteins. here we show that the envelope glycoprotein from the zaire strain of ebola virus (ebo-gp) is proteolytically processed into two subunits, gp1 and gp2, that are likely covalently associated through a disulfide linkage. murine leukemia virions pseudotyped with ebo-gp contain almost exclusively processed glycoprotein, indicating that this is the mature form of ebo-gp. mutational analysis identified a dibasic moti ...19999882347
replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes.the sequences involved in the replication and packaging of transmissible gastroenteritis virus (tgev) rna have been studied. the structure of a tgev defective interfering rna of 9.7 kb (di-c) was described previously (a. mendez, c. smerdou, a. izeta, f. gebauer, and l. enjuanes, virology 217: 495-507, 1996), and a cdna with the information to encode di-c rna was cloned under the control of the t7 promoter. the molecularly cloned di-c rna was replicated in trans upon transfection of helper virus- ...19999882359
class i mhc molecule-mediated inhibition of sindbis virus replication.the threshold for systemic viral infection relies on the amplification of virus at a primary infection site. we have identified that class i mhc molecules can trigger the inhibition of replication of sindbis virus in a haplotype- and allele-specific manner. class i mhc molecules of h-2d haplotypes exhibit a strong inhibitory effect whereas h-2k haplotypes show minimal inhibition of sindbis viral replication. by a single gene transfection of h-2d class i mhc molecules, into cells that express cla ...19999886371
a balance of signaling by rho family small gtpases rhoa, rac1 and cdc42 coordinates cytoskeletal morphology but not cell survival.rho family gtpases are known to be involved in cytoskeletal reorganization. we examined the possibility that these functions may be dictated by a balance of rho family gtpase signaling. using transient viral expression of rhoa, rac1, cdc42 and their mutants, as well as c3 exoenzyme, we altered cytoskeletal organization under normal growth conditions. overexpression of wild-type or constitutively active forms of the rho family gtpases led to their respective activation phenotypes. overexpression ...19999926919
cell-specific targeting of a thymidine kinase/ganciclovir gene therapy system using a recombinant sindbis virus vector.transfer of the herpes simplex virus type i thymidine kinase (hsv-tk) gene into tumor cells using virus-based vectors in conjunction with ganciclovir (gcv) exposure provides a potential gene therapy strategy for the treatment of cancer. the possibility of using a novel targetable sindbis virus expression vector containing the hsv-tk gene was examined. baby hamster kidney (bhk) cells and several human tumor cells infected with a sindbis virus containing the hsv-tk gene showed strong expression of ...19999935240
eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization.eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (egfr) and is characterized by the presence of a novel protein:protein interaction domain, the eh domain. eps15 also stably binds the clathrin adaptor protein complex ap-2. previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. in this study we show that, upon activation of the egfr kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the pl ...19999950686
regulated expression of a sindbis virus replicon by herpesvirus promoters.we describe the use of herpesvirus promoters to regulate the expression of a sindbis virus replicon (sinrep/lacz). we isolated cell lines that contain the cdna of sinrep/lacz under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. wild-type sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and beta- ...19999971780
open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex.the replicase of equine arteritis virus (eav; family arteriviridae, order nidovirales) is expressed in the form of two polyproteins (the open reading frame 1a [orf1a] and orf1ab proteins). three viral proteases cleave these precursors into 12 nonstructural proteins, which direct both genome replication and subgenomic mrna transcription. immunofluorescence assays showed that most eav replicase subunits localize to membranes in the perinuclear region of the infected cell. using replicase-specific ...19999971782
proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication.the open reading frame (orf) 1b-encoded part of the equine arteritis virus (eav) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an orf1ab fusion protein (345 kda). four orf1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in eav-infected cells (l. c. van dinten, a. l. m. wassenaar, a. e. gorbalenya, w. j. m. spaan, and e. j. snijder, j. virol. 70:6625-6633, 1996). ...19999971783
capsid structure of simian cytomegalovirus from cryoelectron microscopy: evidence for tegument attachment sites.we have used cryoelectron microscopy and image reconstruction to study b-capsids recovered from both the nuclear and the cytoplasmic fractions of cells infected with simian cytomegalovirus (scmv). scmv, a representative betaherpesvirus, could thus be compared with the previously described b-capsids of the alphaherpesviruses, herpes simplex virus type 1 (hsv-1) and equine herpesvirus 1 (ehv-1), and of channel catfish virus, an evolutionarily remote herpesvirus. nuclear b-capsid architecture is ge ...19999971801
in vivo addition of poly(a) tail and au-rich sequences to the 3' terminus of the sindbis virus rna genome: a novel 3'-end repair pathway.alphaviruses are mosquito-transmitted rna viruses that cause important diseases in both humans and livestock. sindbis virus (sin), the type species of the alphavirus genus, carries a 11.7-kb positive-sense rna genome which is capped at its 5' end and polyadenylated at its 3' end. the 3' nontranslated region (3'ntr) of the sin genome carries many au-rich motifs, including a 19-nucleotide (nt) conserved element (3'cse) and a poly(a) tail. this 3'cse and the adjoining poly(a) tail are believed to r ...19999971825
mutant influenza viruses with a defective ns1 protein cannot block the activation of pkr in infected cells.a short model genome rna and also the genome rna of influenza a virus bearing both 5'- and 3'-terminal common sequences activated the interferon-induced double-stranded-rna-dependent protein kinase, pkr, by stimulating autophosphorylation in vitro. the activated pkr catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eif2alpha). the ns1 protein efficiently eliminated the pkr-activating activity of these rnas by binding to them. two mutant ns1 proteins, e ...19999971827
resistance of interleukin-1beta-deficient mice to fatal sindbis virus encephalitis.interleukin-1beta (il-1beta) concentrations are frequently elevated in central nervous system (cns) viral infections, but the pathophysiologic significance of such elevations is not known. to examine the role of il-1beta in cns viral pathogenesis, we compared the natural histories of il-1beta-deficient and wild-type 129 sv(ev) mice infected with a neurovirulent viral strain, neuroadapted sindbis virus (nsv). we found that the incidence of severe paralysis and death was markedly decreased in nsv- ...19999971844
the inflammatory response to nonfatal sindbis virus infection of the nervous system is more severe in sjl than in balb/c mice and is associated with low levels of il-4 mrna and high levels of il-10-producing cd4+ t cells.sjl mice are susceptible to inflammatory autoimmune diseases of the central nervous system (cns), while balb/c mice are relatively resistant. to understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of sjl and balb/c mice to infection with sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. clearance of virus was similar, but sjl mice developed a more intense inflammatory response in the ...19999973422
rubella virus-induced apoptosis varies among cell lines and is modulated by bcl-xl and caspase inhibitors.rubella virus (rv) causes multisystem birth defects in the fetuses of infected women. to investigate the cellular basis of this pathology, we examined the cytopathic effect of rv in three permissive cell lines: vero 76, rk13, and bhk21. electron microscopy and the tunel assay showed that the cytopathic effect resulted from rv-induced programmed cell death (apoptosis) in all three cell lines, but the extent of apoptosis varied among these cells. at 48 h postinfection, the rk13 cell line showed th ...199910049827
alphaviruses as tools in neurobiology and gene therapy.the broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for semliki forest virus (sfv) and sindbis virus (sin). the generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. despite the viral cytopathogenic effects, functional assays on recombinant prot ...199910071792
a brome mosaic virus intergenic rna3 replication signal functions with viral replication protein 1a to dramatically stabilize rna in vivo.brome mosaic virus (bmv), a positive-strand rna virus in the alphavirus-like superfamily, encodes two rna replication proteins. the 1a protein has putative helicase and rna-capping domains, whereas 2a contains a polymerase-like domain. saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a bmv rna3 template produced by in vivo transcription of a dna copy of rna3. although insufficient for rna3 replication, the expression of 1a protein alone results in a dramatic and specific s ...199910074107
expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides.proteolytic processing of the replicase gene product of mouse hepatitis virus (mhv) is essential for viral replication. in mhv strain a59 (mhv-a59), the replicase gene encodes two predicted papain-like proteinase (plp) domains, plp-1 and plp-2. previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cis and trans cleavage activities for plp-1. we have cloned and overexpressed the plp-1 domain in escherichia ...199910074111
primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic rna1.barley yellow dwarf luteovirus (bydv) generates three 3'-coterminal subgenomic rnas (sgrnas) in infected cells. the promoter of sgrna1 is a putative hot spot for rna recombination in luteovirus evolution. the sgrna1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of bydv genomic rna (grna) in two independent studies. our data support the former initiation site. the boundaries of the sgrna1 promoter map between nucleotides 2595 and 2692 on genomic rna. ...199910074135
rotavirus capsid protein vp5* permeabilizes membranes.proteolytic cleavage of the vp4 outer capsid spike protein into vp8* and vp5* proteins is required for rotavirus infectivity and for rotavirus-induced membrane permeability. in this study we addressed the function of the vp5* cleavage fragment in permeabilizing membranes. expressed vp5* and truncated vp5* proteins were purified by nickel affinity chromatography and assayed for their ability to permeabilize large unilamellar vesicles (luvs) preloaded with carboxyfluorescein (cf). vp5* and vp5* tr ...199910074166
differentiation of m1 myeloid precursor cells into macrophages results in binding and infection by theiler's murine encephalomyelitis virus and apoptosis.infection of susceptible mouse strains with bean, a less virulent strain of theiler's murine encephalomyelitis virus (tmev), results in immune system-mediated demyelinating lesions in the central nervous system (cns) similar to those in multiple sclerosis. since macrophages appear to carry the major detectable antigen burden in vivo, and purification of sufficient cell numbers from the cns for detailed analysis is difficult, macrophage-like cell lines provide an accessible system with which to s ...199910074176
long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis c virus.two chimpanzees (ch1535 and ch1536) became infected with hepatitis c virus (hcv) following intrahepatic inoculation with rna transcribed from a full-length cdna clone of the virus. both animals were persistently infected and have been followed for 60 weeks. they showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (ch1535) and 12 (ch1536) postinoculation (p.i.). antibody responses to structural and nonstructural ...199910074186
acute and persistent infection of human neural cell lines by human coronavirus oc43.human coronaviruses (hucv) are recognized respiratory pathogens. data accumulated by different laboratories suggest their neurotropic potential. for example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the oc43 strain of hucv (a. bonavia, n. arbour, v. w. yong, and p. j. talbot, j. virol. 71:800-806, 1997). we speculate that the neurotropism of hucv will lead to persistence within the central nervous system, as was observed for murine corona ...199910074188
mutagenic analysis of the 3' cis-acting elements of the rubella virus genome.thermodynamically predicted secondary structure analysis of the 3'-terminal 305 nucleotides (nt) of the rubella virus (rub) genome, a region conserved in all rub defective interfering rnas, revealed four stem-loop (sl) structures; sl1 and sl2 are both located in the e1 coding region, while sl3 and sl4 are within the 59-nt 3' untranslated region (utr) preceding the poly(a) tract. sl2 is a structure shown to interact with human calreticulin (cal), an autoantigen potentially involved in rub rna rep ...199910074193
infectious pancreatic necrosis virus: identification of a vp3-containing ribonucleoprotein core structure and evidence for o-linked glycosylation of the capsid protein vp2.virions of infectious pancreatic necrosis virus (ipnv) were completely disintegrated upon dialysis against salt-free buffers. direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. by using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein vp3. isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire vp3 conte ...199910074207
rapid dendritic morphogenesis in ca1 hippocampal dendrites induced by synaptic activity.activity shapes the structure of neurons and their circuits. two-photon imaging of ca1 neurons expressing enhanced green fluorescent protein in developing hippocampal slices from rat brains was used to characterize dendritic morphogenesis in response to synaptic activity. high-frequency focal synaptic stimulation induced a period (longer than 30 minutes) of enhanced growth of small filopodia-like protrusions (typically less than 5 micrometers long). synaptically evoked growth was long-lasting an ...199910082466
geographic distribution and evolution of sindbis virus in australia.the molecular epidemiology and evolution of sindbis (sin) virus in australia was examined. several sin virus strains isolated from other countries were also included in the analysis. two regions of the virus genome were sequenced including a 418 bp region of the e2 gene and a 484 bp region containing part of the junction region and the 5' end of the c gene. analysis of the nucleotide and deduced amino acid sequence data from 40 sin virus isolates clearly separated the paleoarctic/ethiopian and o ...199910092014
antiviral effect of hyperthermic treatment in rhinovirus infection.human rhinoviruses (hrv) are recognized as the major etiologic agents for the common cold. starting from the observation that local hyperthermic treatment is beneficial in patients with natural and experimental common colds, we have studied the effect of brief hyperthermic treatment (ht) on hrv replication in hela cells. we report that a 20-min ht at 45 degrees c is effective in suppressing hrv multiplication by more than 90% when applied at specific stages of the virus replication cycle. synthe ...199910103186
attenuated vesicular stomatitis viruses as vaccine vectors.we showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (vsv) expressing an influenza virus hemagglutinin (ha) protein provided complete protection from lethal challenge with influenza virus (a. roberts, e. kretzschmar, a. s. perkins, j. forman, r. price, l. buonocore, y. kawaoka, and j. k. rose, j. virol. 72:4704-4711, 1998). because some pathogenesis was associated with the vector itself, in the present study we generated new vsv vectors ...199910196265
selection of rna replicons capable of persistent noncytopathic replication in mammalian cells.the natural life cycle of alphaviruses, a group of plus-strand rna viruses, involves transmission to vertebrate hosts via mosquitoes. chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mrna translation and cell death. using engineered sindbis virus rna replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing pers ...199910196280
genetic and phenotypic changes accompanying the emergence of epizootic subtype ic venezuelan equine encephalitis viruses from an enzootic subtype id progenitor.recent studies have indicated that epizootic venezuelan equine encephalitis (vee) viruses can evolve from enzootic, subtype id strains that circulate continuously in lowland tropical forests (a. m. powers, m. s. oberste, a. c. brault, r. rico-hesse, s. m. schmura, j. f. smith, w. kang, w. p. sweeney, and s. c. weaver, j. virol. 71:6697-6705, 1997). to identify mutations associated with the phenotypic changes leading to epizootics, we sequenced the entire genomes of two subtype ic epizootic vee v ...199910196323
the cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence.semliki forest virus (sfv) and sindbis virus (sin) are enveloped alphaviruses that enter cells via low-ph-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. previous studies with cholesterol-depleted insect cells have shown that sfv requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. an sfv mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the e1 spik ...199910196324
genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells.the alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. to test this hypothesis, eastern equine encephalitis virus was passaged in bhk or mosquito cells, as well as in alternating (both) host cell passages. high and low multiplicities were used to examine the effect of defective interfering particles. clonal bhk and persistent mosquito cell infections were also evaluated. fitness was measured with one-step growth curves and competition assays, and m ...199910196330
stable alphavirus packaging cell lines for sindbis virus and semliki forest virus-derived vectors.alphavirus vectors are being developed for possible human vaccine and gene therapy applications. we have sought to advance this field by devising dna-based vectors and approaches for the production of recombinant vector particles. in this work, we generated a panel of alphavirus vector packaging cell lines (pcls). these cell lines were stably transformed with expression cassettes that constitutively produced rna transcripts encoding the sindbis virus structural proteins under the regulation of t ...199910200308
inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration.we previously established that nf-kappab dna binding activity is required for sindbis virus (sv)-induced apoptosis. to investigate whether sv induces nuclear translocation of nf-kappab via the proteasomal degradation pathway, we utilized mg132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microm mg132 completely abrogated sv-induced nf-kappab nuclear activity at early time points after infection. parallel measures of cell viability 48 h after sv infection revealed ...199810200512
virus reduction in the preparation of intravenous immune globulin: in vitro experiments.while immune globulins for intravenous administration (igiv) have an excellent record with respect to virus safety, concern regarding these preparations has been raised by reports of transmission of hepatitis c virus (hcv) to patients treated with igiv and the presence of genetic material for hcv in igiv preparations.199910204586
packaging of aednv-gfp transducing virus by expression of densovirus structural proteins from a sindbis virus expression system.genetic recombination resulting in the production of wild-type infectious virus is an obstacle in the current system for producing densovirus transducing particles. in order to eliminate this problem, a double subgenomic sindbis virus (te/3'2j/vp) was engineered that expresses the structural proteins (vps) of aedes densonucleosis virus (aednv) from the second subgenomic promoter. expression of aednv vps from te/3'2j/vp was confirmed by northern analysis of rna from infected c6/36 (aedes albopict ...199910208920
binding of tumour necrosis factor-alpha (tnf-alpha) to tnf-ri induces caspase(s)-dependent apoptosis in human cholangiocarcinoma cell lines.cholangiocarcinoma (cca), a tumour of the bile duct epithelium, occurs with a higher incidence in south-east asian countries than in europe and north america. the prognosis is poor, due to the unavailability of early diagnosis and the tumours being relatively resistant to chemotherapy. in the present study one of the fatal routes of this tumour was studied. this death was stimulated by tnf-alpha. tnf-alpha at a concentration of 760 pg/ml and 100 pg/ml in the presence of 1 microgram/ml actinomyci ...199910209503
endoprotease pace4 is ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain.pace4 is a member of the eukaryotic subtilisin-like endoprotease family. the expression of human pace4 in rpe.40 cells (furin-null mutants derived from chinese hamster ovary k1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. expression of pace4 in these cells failed to restore wild-type sensitivity to pseudomonas exotoxin a. co-expression of human pace4 ...199910215603
seroprevalence of dengue, chikungunya and sindbis virus infections in german aid workers.vector-borne virus infections were studied in 670 german overseas aid workers who had spent an average of 37.7 months in tropical areas of africa and asia. antibodies to dengue viruses (den) were detected by indirect immunofluorescence assay in 43/670 (6.4%) aid workers. of these 43, 41 (95.3%) were also positive for antibodies to dengue by haemagglutination inhibition assay. the highest seroprevalence was in aid workers returning from thailand (19.4%), benin (14.8%) and burkina faso (9.2%). ant ...199910219635
decreased intracellular superoxide levels activate sindbis virus-induced apoptosis.infection of many cultured cell types with sindbis virus (sv), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. however, the upstream signals by which sv activates downstream apoptotic effectors, including caspases, remain unclear. here we report that in at-3 prostate carcinoma cells, sv infection decreases superoxide (o-2) levels within minutes of infection as monitored by an aconitase activity assay. this sv-induced decrease in o-2 levels appears to act ...199910224137
genetic interaction of flavivirus nonstructural proteins ns1 and ns4a as a determinant of replicase function.nonstructural protein 1 (ns1) of yellow fever virus (yf) is a glycoprotein localized to extracytoplasmic compartments within infected cells. we have previously shown that ns1 can be supplied in trans and is required for viral rna replication, a process thought to occur in membrane-bound cytoplasmic complexes. here we report that the ns1 gene from a related virus, dengue virus (den), is unable to function in the process of yf rna replication. this virus-specific incompatibility leads to a lack of ...199910233920
mutational analysis, using a full-length rubella virus cdna clone, of rubella virus e1 transmembrane and cytoplasmic domains required for virus release.we report on the construction of a full-length cdna clone, pbrm33, derived from wild-type rubella virus m33 strain. the rna transcripts synthesized in vitro from pbrm33 are highly infectious, and the viruses produced retain the phenotypic characteristics of the parental m33 virus in growth rate and plaque size. this cdna clone was used to study the role of e1 transmembrane and cytoplasmic domains in virus assembly by site-directed mutagenesis. three different alanine substitutions were introduce ...199910233921
dengue virus structural differences that correlate with pathogenesis.the understanding of dengue virus pathogenesis has been hampered by the lack of in vitro and in vivo models of disease. the study of viral factors involved in the production of severe dengue, dengue hemorrhagic fever (dhf), versus the more common dengue fever (df), have been limited to indirect clinical and epidemiologic associations. in an effort to identify viral determinants of dhf, we have developed a method for comparing dengue type 2 genomes (reverse transcriptase pcr in six fragments) dir ...199910233934
human cd46 enhances nitric oxide production in mouse macrophages in response to measles virus infection in the presence of gamma interferon: dependence on the cd46 cytoplasmic domains.cd46 is a transmembrane complement regulatory protein widely expressed on nucleated human cells. laboratory-adapted strains of measles virus (mv) bind to the extracellular domains of cd46 to enter human cells. the cytoplasmic portion of cd46 consists of a common juxtamembrane region and different distal sequences called cyt1 and cyt2. the biological functions of these cytoplasmic sequences are unknown. in this study, we show that expression of human cd46 with the cyt1 cytoplasmic domain in mouse ...199910233938
characterization of a chinese hamster ovary cell line developed by retroviral insertional mutagenesis that is resistant to sindbis virus infection.the alphavirus sindbis virus (sv) has a wide host range and infects many types of cultured cells in vitro. the outcome of infection is dependent on the strain of virus used for infection and the properties of the cells infected. to identify cellular determinants of susceptibility to sv infection we mutagenized chinese hamster ovary (cho) cells by retroviral insertion with a vector containing the neomycin resistance gene that allowed selection for integration into transcriptionally active genes. ...199910233953
oligomerization within virions and subcellular localization of human immunodeficiency virus type 1 integrase.previous biochemical and genetic evidence indicated that the functional form of retroviral integrase protein (in) is a multimer. a direct demonstration of in oligomerization during the infectious cycle was, however, missing, due to the absence of a sensitive detection method. we describe here the generation of infectious human immunodeficiency virus type 1 (hiv-1) viral clones carrying in protein tagged with highly antigenic epitopes. in this setting, we could readily visualize in both in produc ...199910233971
infection of primary cultures of human kupffer cells by dengue virus: no viral progeny synthesis, but cytokine production is evident.we investigated the ability of dengue virus to invade human primary kupffer cells and to complete its life cycle. the virus effectively penetrated kupffer cells, but the infection did not result in any viral progeny. dengue virus-replicating kupffer cells underwent apoptosis and were cleared by phagocytosis. infected kupffer cells produced soluble mediators that could intervene in dengue virus pathogenesis.199910233989
reducing cytotoxicity induced by sindbis viral vectors.sindbis virus has been recognized as a potentially useful virus vector for gene therapy. in an effort to improve its utility and provide cell-targeting capability to gene therapy vectors, we recently developed sindbis virus vectors possessing chimeric envelopes with cell-specific targeting ability [k. ohno et al. nature biotechnol 15:763-767, 1997; k. sawai et al. biochem biophys res commun 248:315-323, 1998]. however, a residual problem associated with sindbis virus vectors is the apoptotic eff ...199910329021
mechanisms of t-cell activation by human t-cell lymphotropic virus type i.the interactions between human t-cell lymphotropic virus type i (htlv-i) and the cellular immune system can be divided into viral interference with functions of the infected host t cell and the subsequent interactions between the infected t cell and the cellular immune system. htlv-i-mediated activation of the infected host t cell is induced primarily by the viral protein tax, which influences transcriptional activation, signal transduction pathways, cell cycle control, and apoptosis. these prop ...199910357853
recombinant semliki forest virus and sindbis virus efficiently infect neurons in hippocampal slice cultures.gene transfer into nervous tissue is a powerful tool for the analysis of gene function. by using a rat hippocampal slice culture preparation, we show here that semliki forest virus (sfv) and sindbis virus (sin) vectors are useful for the effective infection of neurons. the stratum pyramidale and/or the granular cell layer were injected with recombinant virus encoding beta-galactosidase (lacz) or green fluorescent protein (gfp). by using low concentrations of injected sfv-lacz or sin-lacz, we det ...199910359835
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
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