PMID(sorted ascending)
lentivirus cross-reactive determinants present in the capsid protein of equine infectious anaemia this study we used immune sera from equine infectious anaemia virus (eiav)-infected horses which uniquely display broad reactivity with different lentivirus capsid proteins (ca) to characterize the cross-reactive determinants of lentivirus ca proteins. in particular, the role of the major homology region (mhr) of lentivirus ca proteins in this serological cross-reactivity was evaluated using both equine immune serum and murine monoclonal antibodies (mabs) directed against the mhr segment of d ...19947510329
alternative modes of polymerization distinguish the subunits of equine infectious anemia virus reverse transcriptase.a comparative study of recombinant 51- and 66-kda subunits comprising equine infectious anemia virus reverse transcriptase (eiav rt) is reported. both polypeptides sedimented as stable homodimers (molecular mass, 102 and 132 kda, respectively) when analyzed by rate sedimentation through glycerol gradients. consistent with their dimer composition, each preparation displayed considerable levels of both rna- and dna-dependent dna polymerase activity on different homopolymeric template/primer combin ...19947510690
a versatile synthetic peptide-based elisa for identifying antibody epitopes.a simple, versatile and very inexpensive procedure for cross-linking synthetic peptides to the polystyrene surfaces of micro-well assay plates for use in elisa was developed. the method is based on the use of poly-l-lysine (pll) as the anchor protein for synthetic peptides which were then easily and covalently linked to the pll using glutaraldehyde. the synthetic peptides used for the study were based on the amino acid sequence of the equine infectious anemia virus (eiav) envelope sequence and e ...19947513733
screening anti-hiv chinese materia medica with hiv and equine infectious anemic virus reverse transcriptase.the inhibitory activities of human immunodeficiency virus reverse transcriptase (hiv-rt) inhibitors pfa and suramin against hiv-rt and equine infectious anemic virus reverse transcriptase (eiav-rt) were studied in this paper. the 50% inhibitory concentration (ic50) of hiv-rt and eiav-rt treated by pfa and suramin were 0.2 mumol, 9.8 mumol and 17 mumol, 19.9 mumol, respectively. more than thirty chinese medicines, including recipes, herbs, extracts of traditional materia medica and isolated compo ...19947515133
structure of the equine infectious anemia virus tat protein.trans-activator (tat) proteins regulate the transcription of lentiviral dna in the host cell genome. these rna binding proteins participate in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (hiv) or the equine infectious anemia virus (eiav). the consensus rna binding motifs [the trans-activation responsive element (tar)] of hiv-1 as well as eiav tat proteins are well characterized. the structure of the 75-amino acid eiav tat protein in solution was determine ...19947515512
envelope sequence variation, neutralizing antibodies, and primate lentivirus persistence.studies in ungulate lentivirus systems clearly indicate that neutralization escape variants emerge over time in chronically infected animals. studies in the eiav system, in particular, have provided strong evidence that the humoral branch of the immune system is at least one selective force acting on an array of viral variants. in previous studies with the ungulate lentiviruses, molecularly cloned virus was never used, and plaque-purified virus was only sometimes used; the genetic determinants r ...19947523031
mechanism of inhibition of hiv reverse transcriptase by toxiusol, a novel general inhibitor of retroviral and cellular dna polymerases.toxiusol, a natural product isolated from the red sea sponge toxiclona toxius, has been shown to be a potent inhibitor of various viral reverse transcriptases (rt) [i.e., of human immunodeficiency virus (hiv-1), equine infectious anemia virus, and murine leukemia virus] and cellular dna polymerases (i.e., of dna polymerases alpha and beta and escherichia coli dna polymerase i). a thorough investigation of the mode of inhibition was conducted with hiv-1 rt-associated dna polymerase activity. the ...19957532006
incorporation of uracil into viral dna correlates with reduced replication of eiav in macrophages.the retrovirus equine infectious anemia virus (eiav) encodes a dutpase situated between reverse transcriptase and integrase. we have described the inability of eiav with a 270-bp dutpase deletion, delta du eiav, to replicate to wild-type (wt) levels in equine macrophages (d. s. threadgill, w. k. steagall, m. t. flaherty, f. j. fuller, s. t. perry, k. e. rushlow, s. f. j. legrice, and s. l. payne, j. virol. 67, 2592-2600, 1993). here we describe the construction of a second dutpase-deficient viru ...19957542416
production of monoclonal antibodies in horses. 19957550692
analysis of cross reactivity of retrovirus proteases using a vaccinia virus-t7 rna polymerase-based expression system.we have used the vaccinia virus-t7 rna polymerase-based expression system for studies on the activity of proteases from various retroviruses on homologous and heterologous gag polyproteins in eukaryotic cells. proteases from human immunodeficiency virus (hiv) types 1 and 2, equine infectious anaemia virus, human t cell leukaemia virus type 1 and human spumavirus were produced and were shown to cleave their cognate gag substrates produced in trans. analysis of cross reactivity revealed that lenti ...19957561754
helix-stabilizing agent, cc-1065, enhances suppression of translation by an antisense oligodeoxynucleotide.the antitumor antibiotic cc-1065 is known to bind at selected sequences in the minor groove of duplex nucleic acids and to hyperstabilize the duplexes against thermal melting. these properties suggested that cc-1065 may enhance translation inhibition by antisense oligonucleotides directed against a specific mrna. a 585 bp mrna transcript containing the equine infectious anemia virus (eiav) s2 gene and a portion of the env gene was prepared. also, a complementary 20 mer antisense oligodeoxynucleo ...19957580119
dutpase from the retrovirus equine infectious anemia virus: high-level expression in escherichia coli and purification.deoxyuridine 5'-triphosphate nucleotidohydrolase (dutpase, ec catalyzes the hydrolysis of dutp to dump and pyrophosphate, and plays important roles in nucleotide metabolism and dna replication. the dutpase gene of the retrovirus equine infectious anemia virus (eiav) was cloned and overexpressed in escherichia coli using the t7 rna polymerase expression system. the recombinant vector (pet-3a/edu), constructed by mutagenic pcr, was transformed into e. coli bl21 (de3) plyss cells, resulti ...19957663176
the tendency of lentiviral open reading frames to become a-rich: constraints imposed by viral genome organization and cellular trna availability.human immunodeficiency virus type 1 (hiv-1) and other lentiviridae demonstrate a strong preference for the a-nucleotide, which can account for up to 40% of the viral rna genome. the biological mechanism responsible for this nucleotide bias is currently unknown. the increased a-content of these viral genomes corresponds to the typical use of synonymous codons by all members of the lentiviral family (hiv, siv, biv, fiv, caev, eiav, visna) and the human spuma retrovirus, but not by other retrovirus ...19957666442
high prevalence of serum antibodies to equine infectious anemia virus reverse transcriptase.the immunogenicity of the equine infectious anemia virus (eiav) reverse transcriptase (rt) was examined by immunoblot assay with recombinant eiav rt. all of the 19 sera from eiav-infected horses tested contained antibodies that recognized eiav rt and directly inhibited the polymerase activity of the enzyme. an examination of sera obtained sequentially from two experimentally infected animals revealed that anti-rt antibodies arise early in infection and increase in level. the appearance of the an ...19937678974
fidelity of dna synthesis exhibited in vitro by the reverse transcriptase of the lentivirus equine infectious anemia virus.the lentivirus equine infectious anemia virus (eiav) shows high genetic variations. to gain insight into the relative contribution of the reverse transcription process to the eiav mutation rate, the accuracy of dna synthesis catalyzed in vitro by the reverse transcriptase (rt) of eiav was determined. since the rt of eiav shows a relatively high sequence homology with other lentiviral rts, most notable being the rts of human immunodeficiency viruses (hivs), type 1 and type 2, it was of interest t ...19937687876
expression and mutational analysis of the reverse transcriptase of the lentivirus equine infectious anemia virus.the reverse transcriptase of equine infectious anemia virus (eiav) shows sequence similarity with the reverse transcriptases of other lentiviruses, particularly with those of human immunodeficiency viruses types 1 and 2 (hiv-1 and hiv-2). we have constructed a plasmid that when introduced into e. coli induces the synthesis of substantial quantities of the nearly authentic eiav reverse transcriptase. the viral and bacterially expressed reverse transcriptases are similar in their molecular weights ...19937694581
replication in vitro and in vivo of an equine infectious anemia virus mutant deficient in dutpase an important enzyme in dna synthesis, dutpase is present in a wide variety of organisms and viruses and has been identified as a component of the equine infectious anemia virus (eiav) pol gene. the role of eiav dutpase, designated du, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of eiav. a deletion mutant that was deficient in dutpase activity was constructed, and its replication kinetics was examined in fetal equine kidney (f ...19957707512
structural studies of hiv-1 tat protein.tat (trans-activator) proteins are early rna binding proteins regulating lentiviral transcription. these proteins are necessary components in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (hiv) or the equine infectious anemia virus (eiav). tat proteins are thus ideal targets for drugs intervening with lentiviral growth. the consensus rna binding motif (tar, trans-activation responsive element) of hiv-1 is well characterized. structural features of the 86 am ...19957723010
identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral tat proteins.transcriptional activation of hiv-1 gene expression by the viral tat protein requires the interaction of a cellular cofactor with the tat activation domain. this domain has been shown to consist of the cysteine-rich and core motifs of hiv-1 tat and is functionally conserved in the distantly related tat proteins of hiv-2 and eiav. using the yeast two-hybrid system, we have identified a novel human gene product, termed ht2a, that specifically and precisely binds to the activation domain of hiv-1 t ...19957778269
application of cycle dideoxy fingerprinting to screening heterogeneous populations of the equine infectious anemia virus.nucleotide sequence heterogeneity in a population of the equine infectious anemia virus (eiav) was investigated using a modification of the dideoxy fingerprinting (ddf) technique. pcr-amplified regions of the gag gene from eiav isolates were ligated into plasmid vectors and used to transform bacteria. the single dideoxynucleotide sequencing step was performed using plasmid dna prepared from individual bacterial colonies using an 35s end-labeled primer and taq dna polymerase. analysis of the prod ...19947818901
the aspartic proteinase of equine infectious anaemia virus. 19947821563
biological activity and intracellular location of the tat protein of equine infectious anemia virus.the tat protein of equine infectious anemia virus (eiav) was synthesized in escherichia coli using the inducible expression plasmid, pet16b, which contains a his.tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. yields of up to 20 mg of tat were obtained from 10(11) bacterial cells. the recombinant tat protein was shown to potently trans-activate the eiav long terminal repeat (ltr) following its introduction into canine ...19947821797
phosphorothioate oligonucleotides inhibit the replication of lentiviruses and type d retroviruses, but not that of type c retroviruses.phosphorothioate analogs of oligodeoxynucleotides at a concentration of 2 microm protected himalayan tahr cells from infection by caprine arthritis encephalitis virus (caev) and equine dermis cells from infection by equine infectious anemia virus (eiav). the characteristics of this inhibition against these lentiviruses are similar to those previously described for the inhibition of hiv-1 in ath8 cells [17]. thus, the 28-mer homo-oligomer of cytidine [s-(dc)28] was at least as effective as three ...19947826217
molecular dynamics simulation of equine infectious anemia virus tat protein in water and in 40% trifluoroethanol.two molecular dynamics (md) simulations were performed in order to increase the understanding of the dependence of protein conformation on solvent environment. the protein used for these simulations is the transcriptional activator of the equine infectious anemia virus (eiav-tat). the structure of this protein has been determined by nuclear magnetic resonance (nmr) in aqueous solution (willbold et al., science 264, 1584 (1994)) and in 40% (v/v) trifluoroethanol (tfe) (sticht et al., eur. j. bioc ...19947848558
enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein.serial passage of the prototype (pr) cell-adapted wyoming strain of equine infectious anemia virus (eiav) in fetal donkey dermal (fdd) rather than fetal horse (designated fetal equine kidney [fek]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in fdd cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. this neutralization-sensitive variant was designated the fdd strain. although there were d ...19957853482
lentivirus tat proteins specifically associate with a cellular protein kinase, tak, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of rna polymerase ii: candidate for a tat cofactor.efficient replication of human immunodeficiency virus types 1 and 2 (hiv-1 and hiv-2) requires the virus transactivator proteins known as tat. in order to understand the molecular mechanisms involved in tat transactivation, it is essential to identify the cellular target(s) of the tat activation domain. using an in vitro kinase assay, we previously identified a cellular protein kinase activity, tat-associated kinase (tak), that specifically binds to the activation domains of tat proteins. here i ...19957853496
structural analysis of the principal immunodominant domain of the feline immunodeficiency virus transmembrane the transmembrane envelope glycoprotein (tm) of lentiviruses, including human immunodeficiency virus type 1 (hiv-1) and feline immunodeficiency virus (fiv), two cysteine residues, conserved in most retroviruses, are thought to form a loop containing five to seven amino acids. these elements make up a b-cell epitope recognized by nearly 100% of sera from infected patients or animals, designated the principal immunodominant domain (pid). the pid amino acid sequences are highly conserved between ...19957884857
delineating minimal protein domains and promoter elements for transcriptional activation by lentivirus tat proteins.lentivirus tat proteins comprise a novel class of rna-binding transcriptional activators that are essential for viral replication. in this study, we performed a series of protein fusion experiments to delineate the minimal protein domains and promoter elements required for tat action. we show that a 15-amino-acid region of equine infectious anemia virus (eiav) tat protein, when fused to the gal4 or lexa dna binding domain, can activate transcription in appropriate promoter contexts. in the natur ...19957884911
use of an elisa test in the eradication of an equine infectious anaemia enzyme linked immunosorbent assay (elisa) test and the classic agar gel immunodiffusion (agid) test were used as diagnostic methods in the eradication of a focus of equine infectious anaemia from a herd of 86 horses. the elisa test proved to be more sensitive, detecting positive animals earlier than the agid test. a group of 16 animals positive only by elisa also became positive to the agid when retested one month later, except for 2 animals which showed clinical signs of the disease and died ...19947941030
trifluoroethanol stabilizes a helix-turn-helix motif in equine infectious-anemia-virus trans-activator protein.the solution structure of the 75-amino-acid trans-activator (tat) protein of the equine infectious-anemia virus in trifluoroethanol-containing solution was determined by two-dimensional and three-dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear-over-hauser-enhancement distance restraints, and restrained molecular-dynamics simulations. in contrast to the recently determined structure of this protein in trifluoroethanol-free ph 6.3 solution, the hydrophobic ...19947957222
chemiluminescent activation of the antiviral activity of hypericin: a molecular flashlight.hypericin is a naturally occurring photosensitizer that displays potent antiviral activity in the presence of light. the absence of light in many regions of the body may preclude the use of hypericin and other photosensitizers as therapeutic compounds for the treatment of viral infections in vivo. the chemiluminescent oxidation of luciferin by the luciferase from the north american firefly photinus pyralis was found to generate sufficiently intense and long-lived emission to induce antiviral act ...19947991618
nmr structure of a biologically active peptide containing the rna-binding domain of human immunodeficiency virus type 1 tat.the tat protein of human immunodeficiency virus type 1 enhances transcription by binding to a specific rna element on nascent viral transcripts. binding is mediated by a 10-amino acid basic domain that is rich in arginines and lysines. here we report the three-dimensional peptide backbone structure of a biologically active 25-mer peptide that contains the human immunodeficiency virus type 1 tat basic domain linked to the core regulatory domain of another lentiviral tat--i.e., that from equine in ...19948058789
the immunopathogenesis of equine infectious anemia virus. 19948067050
monocyte maturation controls expression of equine infectious anemia vivo, equine infectious anemia virus (eiav) replicates in tissues rich in macrophages, and it is widely believed that the tissue macrophage is the principal, if not sole, cell within the host that replicates virus. no viral replication has been detected in circulating peripheral blood monocytes. however, proviral dna can be detected in these cells, and monocytes may serve as a reservoir for the virus. in this study, an in vitro model was developed to clarify the role of monocyte maturation in ...19948083967
major histocompatibility complex-restricted cd8+ cytotoxic t lymphocytes from horses with equine infectious anemia virus recognize env and gag/pr proteins.cytotoxic t lymphocytes (ctl) can control some viral infections and may be important in the control of lentiviruses, including human immunodeficiency virus type 1. since there is limited evidence for an in vivo role of ctl in control of lentiviruses, dissection of immune mechanisms in animal lentiviral infections may provide needed information. horses infected with equine infectious anemia virus (eiav) a lentivirus, have acute plasma viremia which is terminated in immunocompetent horses. viremic ...19948107209
posttranscriptional effector domains in the rev proteins of feline immunodeficiency virus and equine infectious anemia systematically dissecting the rev proteins of feline immunodeficiency virus (fiv) and equine infectious anemia virus (eiav), we have identified within each a short peptide that is functionally interchangeable with the effector domains found in rev-like proteins from other retroviruses. the active sequences from fiv and eiav differ in several respects from other known effectors and may represent a distinct class of effector domain.19948107262
characterization of infectious molecular clones of equine infectious anaemia virus.we have recovered five infectious molecular clones of the lentivirus equine infectious anaemia virus (eiav). the clones were recovered from fetal equine kidney (fek) cells infected with a virulent, cell culture-adapted virus stock (designated pv) and have been characterized at a molecular level. each clone has unique envelope and long terminal repeat (ltr) sequences. we further investigated ltr sequence variation in the pv stock using pcr amplification to obtain additional ltr clones from infect ...19948113766
enhancement of eiav replication and disease by immunization with a baculovirus-expressed recombinant envelope surface glycoprotein.the potential for antibody-dependent enhancement of replication of macrophage/monocyte tropic viruses has posed a significant problem in the development of vaccines for several animal and human viruses and has raised significant concern in the design of potential aids vaccines. using the previously described equine infectious anemia virus/shetland pony system as a model for hiv-1 vaccine development, we have evaluated the efficacy of a recombinant subunit vaccine containing a baculovirus-express ...19948116252
in vivo replicative status and envelope heterogeneity of equine infectious anemia virus in an inapparent carrier.the distribution and replicative status of equine infectious anemia virus (eiav) dna in the tissues of a well-characterized inapparent carrier horse were established by using the pcr technique. the eiav pol region could be amplified in all of the tissues tested, including the cerebellum and periventricular tissue, at concentrations approximately 10(5)-fold less than in the same tissue from an acutely infected horse. further analysis of the eiav genome, with primer pairs diagnostic for sequential ...19948139056
expression of functional protease and subviral particles by vaccinia virus containing equine infectious anaemia virus gag and 5' pol genes.cells infected with vaccinia viruses expressing the equine infectious anaemia virus (eiav) gag gene (vgag) or gag plus the 5' pol encoding protease (vgag/pr) were evaluated with monoclonal antibody to a p26 capsid protein linear epitope (qeiskfltd). both recombinant viruses expressed gag precursor protein (55k) whereas only vgag/pr expressed a detectable gag-pol fusion protein (82k) with a functional protease, shown by subviral particles containing processed p26. horses inoculated with vgag/pr p ...19948151302
equine infectious anemia virus trans-regulatory protein rev controls viral mrna stability, accumulation, and alternative splicing.the cis- and trans-acting components of the rev regulatory pathway employed by equine infectious anemia virus (eiav) to regulate and coordinate viral gene expression were examined in complementation experiments. viral protein expression and mrna expression were compared in cells transiently transfected with wild-type or mutant proviruses in combination with rev expression plasmids. mutation of the predicted rev gene abolished gag protein synthesis, and this defect was complemented, in trans, by ...19948151775
cellular and viral specificity of equine infectious anemia virus tat transactivation.lentiviruses vary in their dependence on a functional tat gene during their viral life cycle. to begin to understand the viral and cellular parameters controlling equine infectious anemia virus (eiav) transactivation, we investigated tat function and tat and ltr structural requirements necessary for successful transactivation. eiav tat expression was required for detection of viral antigens from a full-length provirus. the level of transactivation by eiav tat as measured by ltr-cat assays correl ...19948178449
identification of the activation domain of equine infectious anemia virus rev.several members of the lentivirus family of complex retroviruses have been shown to encode proteins that are functionally equivalent to the rev posttranscriptional regulatory protein of human immunodeficiency virus type 1 (hiv-1). furthermore, the domain organization of hiv-1 rev, featuring a highly basic n-terminal rna binding domain and a leucin-rich c-terminal effector domain, has also been shown to be highly conserved among rev proteins derived from not only the primate but also the ovine an ...19938230455
the origin of human immunodeficiency virus type-1 rev gene. an evolutionary hypothesis.the rev protein of human immunodeficiency virus type-1 is an rna-binding posttranscriptional transregulator encoded by an accessory gene that is distinct from retroviral oncogenes and whose origin is unclear. we hypothesize that the rev gene was generated by duplication of a viral rna segment having a secondary-structure that evolved into the rev-responsive element (rre). this hypothesis is based on the following findings. first, accumulated data on functional mapping of rev, tat, and the transm ...19948307167
development of the coggins test. 19948313705
structural features of the trans-activation response rna element of equine infectious anemia virus.a 25-nucleotide rna with the sequence of the trans-activation response (tar) element of equine infectious anemia virus (eiav) was analyzed by biochemical methods and by one- and two-dimensional nmr spectroscopy. nmr, nuclease probing, and polyacrylamide gel migration rates show that the rna consists of an a-helical stem capped by two non-watson-crick u-g base pairs and a compact four-nucleotide loop. the loop is stabilized by base stacking, with loop nucleotides c12 and c15 stacked upon u11 and ...19938381023
translation of equine infectious anemia virus bicistronic tat-rev mrna requires leaky ribosome scanning of the tat ctg initiation codon.we have examined the translational regulation of the equine infectious anemia virus (eiav) bicistronic tat-rev mrna. site-directed mutagenesis of the tat leader region followed by expression of the tat-rev cdna both in vitro and in transiently transfected cells established that tat translation is initiated exclusively at a ctg codon. increasing the efficiency of tat translation by altering the ctg initiator to atg resulted in a dramatic decrease in translation of the downstream (rev) cistron, in ...19938382305
lentivirus envelope sequences and proviral genomes are stabilized in escherichia coli when cloned in low-copy-number plasmid vectors.a promoter-selection vector (pkk232-8) was used to identify sequences with strong escherichia coli promoter activity positioned near the start of the envelope-encoding genes (env) of two lentiviruses, simian immunodeficiency virus (siv) and equine infectious anemia virus (eiav). for eiav, cloning the cryptic promoter sequences together with downstream sequences encoding the envelope glycoprotein (gp90) in moderate- to high-copy-number (hcn) plasmid vectors, such as pbr322 or puc, resulted in rea ...19938382658
physical and functional characterization of transcriptional control elements in the equine infectious anemia virus promoter.equine infectious anemia virus (eiav) is a lentivirus that causes a chronic disease of horses characterized by cyclic episodes of fever, anemia, and viremia. although the genome and promoter of eiav are much less complex than those of its relatives the primate immunodeficiency viruses, the cellular proteins that activate and regulate transcription of eiav have not yet been identified. in this report, we show by electrophoretic mobility shift assays and dnase i footprinting that the eiav promoter ...19938383228
a comparison of elisa, fast-elisa and gel diffusion tests for detecting antibody to equine infectious anaemia virus.sera of sixteen horses with clinical signs of eia from six different outbreaks and sera of 100 uninfected horses were used to validate an elisa for eia diagnosis. the antigen used was a recombinant protein derived from the amino-terminal portion of the transmembrane envelope protein of eia (gp45). reactivity between positive and negative sera could be clearly distinguished. comparison with the traditional agar gel immunodiffusion test (commonly called the coggins test) showed that the elisa was ...19938383374
studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates.the proteinase of the equine infectious anemia virus (eiav), a lentivirus closely related to human immunodeficiency virus (hiv), was purified from concentrated virus. the specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the gag and gag-pol polyproteins. the length of the substrate binding pocket was found to be 1-2 residues longer than that of hiv proteinases. although the eiav and hiv proteinases cleaved most of the peptides at t ...19938384879
molecular model of equine infectious anemia virus proteinase and kinetic measurements for peptide substrates with single amino acid substitutions.a molecular model has been built of the equine infectious anemia virus (eiav) proteinase on the basis of the crystal structures of the related rous sarcoma virus (rsv) and human immunodeficiency virus (hiv) proteinases. the 104 residue long eiav proteinase has 30 identical and 11 similar amino acids compared to those in hiv-1 proteinase and 25 identical and 18 similar amino acids compared to rsv proteinase. the overall structure is predicted to be close to that of hiv-1 proteinase. two regions s ...19938384880
a review of techniques for the serologic diagnosis of equine infectious anemia. 19938385498
characterization of equine infectious anemia virus dutpase: growth properties of a dutpase-deficient mutant.the putative dutpase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (eiav) to produce a recombinant delta dupol escherichia coli expression cassette and a delta du proviral clone. expression of the recombinant delta dupol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and dna polymerase activity gels. transfection of delta du provirus into feline (fea) cells resulted in production ...19938386267
mutagenesis of eiav tat reveals structural features essential for transcriptional activation and tar element recognition.certain members of the lentivirus subfamily of retroviruses encode unique transcriptional activator (tat) proteins that modify the transcription complex after binding to the 5' end of nascent viral mrna. the tat proteins are modular, containing rna-binding and activation domains that can be exchanged between different tat proteins or replaced with heterologous protein fragments. while there is considerable sequence conservation among the divergent tat proteins, there are also some structural dif ...19938389074
genetic analysis of the cofactor requirement for human immunodeficiency virus type 1 tat function.the tat protein of human immunodeficiency virus type 1 is a potent transcriptional trans activator of the viral long terminal repeat promoter element. tat function requires the direct interaction of tat with a cis-acting viral rna target sequence termed the trans-activation response (tar) element and has also been proposed to require at least one cellular cofactor. we have used a genetic approach to attempt to experimentally define the role of the cellular cofactor in tat function and tar bindin ...19938389901
the pu.1/spi-1 proto-oncogene is a transcriptional regulator of a lentivirus promoter.the enhancer unit present in the retrovirus equine infectious anemia virus (eiav) was previously shown to contain binding sites for proteins belonging to mdbp, pea2, ap-1, and ets families. the eiav ets motif matches the consensus sequence for both pea3- and pu.1-binding sites. here, we show by gel shift analysis that pu.1, present in nuclear extracts from monocyte and b-lymphocyte cell lines, binds to oligonucleotides containing the eiav ets element. hela cells transiently transfected with a pu ...19938389910
structural and functional characterization of rev-like transcripts of equine infectious anemia virus.three cdna clones representing structurally distinct transcripts were isolated from a cdna library prepared from cells infected with equine infectious anemia virus (eiav) by using a probe representing the s3 open reading frame, which is thought to encode rev. one species, designated p2/2, contained four exons and was identical to a previously described polycistronic mrna that encodes tat. this transcript was predicted to also direct the synthesis of a truncated form of the transmembrane protein ...19938394464
sequence-specific resonance assignments of the 1h-nmr spectra of a synthetic, biologically active eiav tat protein.the equine infectious anemia virus (eiav) trans-activating (tat) protein is a close homologue of the human immunodeficiency virus (hiv) tat protein. both of these proteins bind to an rna trans-activation responsive element (tar). we synthesized chemically a protein with the sequence of the 75 amino acid tat protein from eiav. the chemically synthesized protein was shown to be biologically active. circular dichroism (cd) and 1h nuclear magnetic resonance (nmr) spectroscopy were used to structural ...19938395203
equine infectious anemia.the ability of eiav to persistently infect horses in the face of a profound immune response by the host makes it a potentially devastating disease for the horse population of the united states. its ability to evade host immune defenses by lying dormant in apparently healthy animals and by rapidly changing its antigenic determinants is proving to be a major obstacle to vaccine development. because most infected horses appear clinically normal and a large proportion of horses in this country remai ...19938395326
protein interactions with dna elements in variant equine infectious anemia virus enhancers and their impact on transcriptional activity.the long terminal repeats (ltrs) from various cloned equine infectious anemia virus (eiav) proviruses differ significantly, but all contain cis-acting dna elements identical to mdbp-, pea2-, ap-1-, and pu.1 (ets)-binding sites. a prototype eiav ltr would contain one of each of these conserved elements. the ltr variations originate from the insertion of novel sequences between the pea2 and ap-1 elements in the transcriptional enhancer unit. viewed in this way, the ltr from provirus clone lambda 1 ...19938411361
analysis of multiple mrnas from pathogenic equine infectious anemia virus (eiav) in an acutely infected horse reveals a novel protein, ttm, derived from the carboxy terminus of the eiav transmembrane protein.transcription of pathogenic equine infectious anemia virus (eiav) in an acutely infected horse was examined by using the polymerase chain reaction and nucleotide sequencing. four spliced transcripts were identified in liver tissue, in contrast to the multiplicity of alternatively spliced messages reported for in vitro-propagated human immunodeficiency virus, simian immunodeficiency virus, and, to a lesser extent, eiav. nucleotide sequence analysis demonstrated that three of these mrnas encode kn ...19938419648
equine infectious anemia virus replication is upregulated during differentiation of blood monocytes from acutely infected horses.equine infectious anemia virus is a lentivirus that replicates in mature tissue macrophages of horses. ponies were infected with equine infectious anemia virus. during febrile episodes, proviral dna was detectable, but viral mrna was not detectable. as cultured blood monocytes from these ponies differentiated into macrophages, viral expression was upregulated. in situ hybridization confirmed that viral transcription occurred in mature macrophages.19968523576
fine specificity of equine infectious anaemia virus gp90-specific antibodies associated with protective and enhancing immune responses in experimentally infected and immunized ponies.equine infectious anaemia virus (eiav) provides a model for examining the natural immunological control of a persistent lentivirus infection and for evaluating the efficacy of various vaccine strategies. as an initial characterization of antibody responses associated with protective or enhancing immune responses elicited by experimental infections or vaccinations, we have utilized synthetic peptide elisa to characterize the fine specificity of antibodies to linear determinants of the eiav surfac ...19968601778
specific derivatization of the active site tyrosine in dutpase perturbs ligand binding to the active site.selective modification of one (of three) tyrosine residue per enzyme monomer leads to inactivation of dutpase of the retrovirus equine infectious anemia virus (eiav). the substrate dump and the cofactor mg2+ protect against inactivation and modification, in agreement with the study on e. coli dutpase (vertessy et al. (1994) biochim. biophys. acta 1205, 146-150). amino acid analyses of nitrated dutpases confirmed tyr-selectivity of modification. the nitrated residue in e. coli dutpase was identif ...19968604980
isolation of an 11-kda protein associated with the topoisomerase i activity from equine infectious anemia virus.we have previously demonstrated the presence of topoisomerase i (topo i) activity in purified retroviral particles (i.e., human immunodeficiency virus type 1, equine infectious anemia virus-eiav and moloney murine leukemia virus). in our present work, an attempt was made to determine the nature and origin of the protein that is associated with this activity. for that purpose we have isolated the topo i activity from equine infectious anemia virus cores and showed that a major protein band of an ...19968607786
a common mechanism for the enhancement of mrna 3' processing by u3 sequences in two distantly related lentiviruses.the protein coding regions of all retroviral pre-mrnas are flanked by a direct repeat of r-u5 sequences. in many retroviruses, the r-u5 repeat contains a complete core poly(a) site-composed of a highly conserved aauaaa hexamer and a gu-rich downstream element. a mechanism that allows for the bypass of the 5' core poly(a) site and the exclusive use of the 3' core poly(a) site must therefore exist. in human immunodeficiency virus type 1 (hiv-1), sequences within the u3 region appear to play a key ...19968627681
interactions among sr proteins, an exonic splicing enhancer, and a lentivirus rev protein regulate alternative splicing.we examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mrna of equine infectious anemia virus (eiav). this bicistronic mrna contains four exons; exons 1 and 2 encode tat, and exons 3 and 4 encode rev. in the absence of rev expression, the four-exon mrna is synthesized exclusively, but when rev is expressed, exon 3 is skipped to produce an mrna that contains only exons 1, 2, and 4. we identify a purine-ri ...19968628299
comparative studies on the substrate specificity of avian myeloblastosis virus proteinase and lentiviral proteinases.the retroviral proteinase (pr) seems to play crucial roles in the viral life cycle, therefore it is an attractive target for chemotherapy. previously we studied the specificity of human immunodeficiency virus (hiv) type 1 and type 2 as well as equine infectious anemia virus prs using oligopeptide substrates. here a similar approach is used to characterize the specificity of avian myeloblastosis virus (amv) pr and to compare it with those of the previously characterized lentiviral prs. all peptid ...19968636100
nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus rev proteins: identification of a family of transferable nuclear export signals.the human immunodeficiency virus type 1 rev trans activator binds directly to unspliced viral mrna in the nucleus and activates its transport to the cytoplasm. in additon to the sequences that confer rna binding and nuclear localization, rev has a carboxy-terminal region, the activation domain, whose integrity is essential for biological activity. because it has been established that rev constitutively exits and reenters the nucleus and that the activation domain is required for nuclear exit, it ...19968642662
involvement of c-terminal structural elements of equine infectious anemia virus reverse transcriptase in dna polymerase and ribonuclease h order to investigate the modes of dna synthesis supported by the 66 and 51 kda subunits of equine infectious anemia virus reverse transcriptase (eiav rt), recombinant p66 polypeptides containing a modified ribonuclease h (rnase h) domain were purified and evaluated. defined heteropolymeric template-primer combinations and high-resolution gel electrophoresis provided a qualitative evaluation of dna polymerase and rnase h activities, while dnase i footprinting revealed features of replication c ...19968648620
genomic quasispecies associated with the initiation of infection and disease in ponies experimentally infected with equine infectious anemia virus.equine infectious anemia virus (eiav) provides a uniquely dynamic system in which to study the mechanism and role of genomic variation in lentiviral persistence and pathogenesis. we have used a shetland pony model of infection to investigate the association of specific long terminal repeat (ltr) and env gene genomic sequences with the initiation of infection and the onset of disease. we analyzed viral rna isolated from a pathogenic stock of virus (eiav pv) and from plasma taken during the first ...19968648664
inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of rev.the major 5' splice site of equine infectious anemia virus (eiav) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag aug. our results show that the presence of this 5' splice site on the eiav gag mrna decreases gag production 30- to 60-fold. this is caused by inefficient nuclear mrna export and inefficient mrna utilization. inhibition could be overcome by providing human immunodeficiency virus type 1 rev/rev-responsive element, ...19968648699
is eiav tat protein a homeodomain? 19968658146
lymphoid leukosis viruses, their recognition as 'persistent' viruses and comparisons with certain other retroviruses of veterinary importance.diseases caused by lymphoid leukosis virus (llv), a retrovirus, take a long time after infection to develop and have a wide variety of pathological manifestations. this long latent period is characteristic of 'persistent virus infections'. disease produced by llv infection and its underlying mechanisms is compared with 'persistent' infections caused by other retroviruses in birds and mammals of veterinary importance. the diseases considered for comparison are those caused by reticuloendotheliosi ...19968693704
detection of equine infectious anemia viral rna in plasma samples from recently infected and long-term inapparent carrier animals by pcr.control of equine infectious anemia (eia) is currently based on detection of anti-eia virus (eiav) antibodies. however, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. we developed a reverse transcriptase nested pcr (rt-npcr) assay for the detection of viral gag gene sequences in plasma from eiav-infected horses. the ability of rt-npcr to detect field strains of eiav was investigated by assay ...19968735102
inhibition of hiv-1 replication by uridine-3'-spiroxirane, a new type of reverse transcriptase inhibitor. 19968736903
rna structure is a critical determinant of poly(a) site recognition by cleavage and polyadenylation specificity factor.sequence conservation among mammalian poly(a) sites is limited to the sequence aauaaa, coupled with an amorphous downstream u- or gu-rich region. since these sequences may also occur within the coding region of mrnas, additional information must be required to define authentic poly(a) sites. several poly(a) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. the human immunodeficiency virus type 1, equine infecti ...19968756653
structural studies of the equine infectious anemia virus trans-activator protein.trans-activator (tat) proteins are necessary components for the completion of the t replication cycle of lentiviruses. the three-dimensional structure of the equine infectious anemia virus (eiav) tat protein (e-tat) was studied with cd spectroscopy, nmr spectroscopy, and restrained molecular-dynamics calculations. no stable elements of regular secondary structure were detected, but the sequence regions responsible for nucleic acid binding showed helix-forming tendency, e-tat exhibits a flexible ...19968797834
a morphometric study of bone marrow megakaryocytes in foals infected with equine infectious anemia virus.morphometric evaluation of bone marrow core biopsies was used to determine megakaryocyte (mk) numbers and mk size in nine foals with equine infectious anemia virus (eiav)-induced thrombocytopenia. both immunocompetent normal foals and foals with severe combined immunodeficiency (scid) were used. platelet counts were made three times weekly following viral infection. bone marrow core biopsies were taken from the ilium of each foal prior to experimental infection, immediately after the onset of th ...19968801716
initiation of (-) strand dna synthesis from trna(3lys) on lentiviral rnas: implications of specific hiv-1 rna-trna(3lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.initiation of minus (-) strand dna synthesis was examined on templates containing r, u5, and primer-binding site regions of the human immunodeficiency virus type 1 (hiv-1), feline immunodeficiency virus (fiv), and equine infectious anemia virus (eiav) genomic rna. dna synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic trna(3lys), and (iii) natural trna(3lys), by the reverse transcriptases of hiv-1, fiv, eiav, simian immunodeficiency ...19968816751
stability of equine infectious anemia virus in aedes aegypti (diptera: culicidae), stomoxys calcitrans (diptera:muscidae), and tabanus fuscicostatus (diptera:tabanidae) stored at -70 degrees c.equine infectious anemia virus (eiav) was injected intrathoracically into aedes aegypti, stomoxys calcitrans, and tabanus fuscicostatus, and fed to ae. aegypti in suspensions of either artificial blood of eagle's minimum essential medium. insects were stored at -70 degrees c for up to 9 months before testing for the presence of eiav. the viral tissue culture titers detected from stored insects were similar to those from insects tested at time 0.19968827617
structure of equine infectious anemia virus proteinase complexed with an inhibitor.equine infectious anemia virus (eiav), the causative agent of infectious anemia in horses, is a member of the lentiviral family. the virus-encoded proteinase (pr) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. the x-ray structure of a complex of the 154g mutant of eiav pr with the inhibitor hby-793 was solved at 1.8 a resolution and refined to a crystallographic r-factor of 0.136. the molecule is a dimer ...19968844837
characterization of protective and enhancing immune responses to equine infectious anemia virus resulting from experimental vaccines. 19968882322
a primary production deficit in the thrombocytopenia of equine infectious anemia.the purpose of this study was to identify the mechanisms responsible for the thrombocytopenia that develops following infection of horses by the lentivirus equine infectious anemia virus (eiav). immunocompetent arabian foals and arabian foals with severe combined immunodeficiency (scid), which lack functional b and t lymphocytes, were experimentally infected with eiav. levels of viremia and a number of clinical and hematologic parameters were examined prior to and following infection. thrombocyt ...19968892906
aqueous solution structure of a hybrid lentiviral tat peptide and a model of its interaction with hiv-1 tar rna.human immunodeficiency virus, type 1, (hiv-1) encodes a transactivating regulatory protein, called tat, which is required for efficient transcription of the viral genome. tat acts by binding to a specific rna stem-loop element, called tar, on nascent viral transcripts. the specificity of binding is principally determined by residues in a short, highly basic domain of tat. the structure in aqueous solution of a biologically active peptide, comprised of the ten-amino acid hiv-1 tat basic domain li ...19968906885
expression, characterisation and mutagenesis of the aspartic proteinase from equine infectious anaemia virus.the gene encoding the proteinase from equine infectious anaemia virus (eiav) was cloned and expressed in escherichia coli. the recombinant eiav proteinase was purified to homogeneity and shown to have the ability to process polyprotein and synthetic peptide substrates of human immunodeficiency virus (hiv) origin with an efficiency that can approach that exhibited by hiv proteinase. eiav proteinase, however, was not susceptible to inhibition by a wide variety of inhibitors of hiv-1 proteinase, in ...19968917470
analysis of the long terminal repeat from a cytopathic strain of equine infectious anemia virus.sequential passage of the tissue culture-adapted prototype strain of eiav in fetal donkey dermal (fdd) cell cultures generated a virus stock which exhibits cytopathic effects in fdd cell cultures. in this study, the effects of the long terminal repeat (ltr) region on virus replication and cytopathogenicity were examined. the fdd-adapted virus ltr was found to contain a number of base pair mutations and a large insertion within the u3 region in comparison with the previously characterized ltr, la ...19968918926
retroviruses and systemic lupus some animal models of autoimmune diseases the roles of exogenous and endogenous retroviruses are clearly defined. in ungulates caprine arthritis encephalitis virus, equine infectious anemia virus or maedi-visna virus infections cause a well-defined autoimmune disease and the appearance of seropositivity of the animals is of diagnostic value. likewise, in mrl lpr/lpr mice insertion of a retrotransposon into the fas gene could clearly be shown to cause survival of autoreactive lymphocytes. desp ...19968930671
biochemical characterization of recombinant equine infectious anemia virus integrase.the integrase from equine infectious anemia virus (elav) was expressed in escherichia coli as a polyhistidine fusion protein. the protein was purified under native and denaturing conditions using one-step nickel-affinity chromatography. the purified denatured protein was refolded in the presence of detergent. in vitro 3' processing and dna strand transfer activities were analyzed under mg(2+)- and mn(2+)-dependent reaction conditions. both protein preparations were similarly active. only one vir ...19968936591
phorbol ester stimulation of equine macrophage cultures alters expression of equine infectious anemia virus.equine infectious anemia virus (eiav) is a lentivirus that replicates predominantly in mature tissue macrophages. viral expression is strongly influenced by the state of differentiation of the host cell. while blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. activation of mature macrophages infected with eiav might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid ...19968972047
maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process.equine infectious anemia virus (eiav) provides a natural model system by which immunological control of lentivirus infections may be studied. to date, no detailed study addressing in parallel both the humoral and cellular immune responses induced in horses upon infection by eiav has been conducted. therefore, we initiated the first comprehensive characterization of the cellular and humoral immune responses during clinical progression from chronic disease to inapparent stages of eiav infection. u ...19979094660
hiv and human endogenous retroviruses: an hypothesis with therapeutic implications.the enzyme dutp pyrophosphatase (dutpase, ec is essential for cellular dna replication and cell viability by virtue of its role in reducing the availability of dutp as a substrate for dna polymerases. several members of the onco- and lentivirus families of retroviruses encode dutpases and mutant strains of these viruses defective in this enzyme exhibit suboptimal replication kinetics. among the lentiviruses there exists a surprising phylogenetic discontinuity in the distribution of dut ...19969104494
insertions, duplications and substitutions in restricted gp90 regions of equine infectious anaemia virus during febrile episodes in an experimentally infected horse.we have studied a horse which exhibited typical clinical signs of disease when experimentally infected with a non-adapted virulent strain of equine infectious anaemia virus (eiav), designated v70. five viruses (f1v, f2v, f3v, f4v and f5v) were recovered during periodic febrile episodes. cross-neutralization tests revealed that all of these variants and the parental v70 were antigenically distinct. sequencing of their full-length env gp90 genes and gp45 5' sequences revealed novel mutations at a ...19979129653
control of equine infectious anemia virus is not dependent on adcc mediating antibodies.horses infected with equine infectious anemia virus (eiav) have recurrent episodes of viremia which are eventually controlled, but the immune mechanisms have not been identified. antibodies were detected to the surface of eiav-infected cells within 1 month postinfection and remained for at least 3.5 years postinfection. these antibodies recognized cell surface-exposed envelope (env) glycoproteins, but could not mediate antibody dependent cellular cytotoxicity (adcc) using eiav-wsu5-infected equi ...19979143283
cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus.the gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (eiav) was cloned from eiav infected serum, expressed in e. coli, and the resultant protein purified to electrophoretic homogeneity. the protein was expressed in a soluble form and was purified by conventional protein separation methods. when analyzed by sds-page, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kda monomer. recombinant p26 (rp26), therefore, do ...19979165100
characterization and mutational studies of equine infectious anemia virus dutpase.the macrophage tropic lentivirus, equine infectious anemia virus (eiav), encodes a dutpase in the pol gene that is required for efficient replication in macrophages. two naturally occurring variants of the enzyme were expressed as recombinant proteins in escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. biochemical and enzymatic analyses of these preparations suggest th ...19979187238
localized sequence heterogeneity in the long terminal repeats of in vivo isolates of equine infectious anemia virus.the role of in vivo long terminal repeat (ltr) sequence variation of the lentivirus equine infectious anemia virus (eiav) has not been explored. in this study, we investigated the heterogeneity found in the ltr sequences from seven eiav-seropositive horses: three horses with clinical disease and four horses without any detectable signs of disease. ltr sequences were targeted in this study because the ltr u3 enhancer region of tissue culture-derived isolates has been identified as one of the few ...19979188555
in vivo dynamics of equine infectious anemia viruses emerging during febrile episodes: insertions/duplications at the principal neutralizing domain.equine infectious anemia virus (eiav) is a good model for studying mechanisms generating escaped retrovirus variants. we previously sequenced the entire gp90-encoding region of 22 cdna clones obtained from five antigenically distinct isolates (f1v to f5v) recovered during febrile episodes in horse 493 experimentally infected with the japanese virulent eiav strain v70. the results showed that the mutations occurred in the principal neutralizing domain (pnd) by insertions/duplications. in this stu ...19979188568
mutations in hiv reverse transcriptase which alter rnase h activity and decrease strand transfer efficiency are suppressed by hiv nucleocapsid protein.structural studies of authentic hiv reverse transcriptase (rt) suggest a role for the p51 carboxyl terminus in forming an active rnase h conformation [rodgers, d. w., gamblin, s. j., harris, b. a., ray, s., culp, j. s., hellmig, b., woolf, d. j., debouck, c. & harrison, s. c. (1995) proc. natl. acad. sci. usa 92, 1222-1226]. we have purified mutant rt heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. these "selectively deleted" heterodimers have been ana ...19979192628
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