proteins related to the nedd4 family of ubiquitin protein ligases interact with the l domain of rous sarcoma virus and are required for gag budding from cells.the late assembly (l) domain of retrovirus gag, required in the final steps of budding for efficient exit from the host cell, is thought to mediate its function through interaction with unknown cellular factors. here, we report the identification of the nedd4-like family of e3 ubiquitin protein ligases as proteins that specifically interact with the rous sarcoma virus (rsv) l domain in vitro and in vivo. we screened a chicken embryo cdna expression library by using a peptide derived from the rsv ...200111562473
arp/warp and molecular replacement.the aim of arp/warp is improved automation of model building and refinement in macromolecular crystallography. once a molecular-replacement solution has been obtained, it is often tedious to refine and rebuild the initial (search) model. arp/warp offers three options to automate that task to varying extents: (i) autobuilding of a completely new model based on phases calculated from the molecular-replacement solution, (ii) updating of the initial model by atom addition and deletion to obtain an i ...200111567158
molecular mechanism of sequence-specific termination of lentiviral replication.the central termination sequence (cts) terminates (+) strand dna synthesis in certain lentiviruses. the molecular mechanism underlying this event, catalyzed by equine infectious anemia virus reverse transcriptase (eiav rt), was evaluated by pre-steady-state kinetic techniques. time courses in nucleotide incorporation using several dna substrates were biphasic, consistent with release of enzyme from extended dna being the rate-limiting step for turnover. while the burst amplitude reflecting the a ...200111580289
rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral this report it is demonstrated for the first time that rabies-g envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. after delivery of rabies-g pseudotyped equine infectious anaemia virus (eiav) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. this retrograde transpor ...200111590128
immune reconstitution prevents continuous equine infectious anemia virus replication in an arabian foal with severe combined immunodeficiency: lessons for control of lentiviruses.acute infection with equine infectious anemia virus (eiav), a lentivirus of horses, results in a persistent high-level viremia in arabian foals affected with severe combined immunodeficiency (scid). this observation argues against the idea that the transient nature of acute lentiviral viremia is solely a function of viral population dynamics. to extend these studies, eiav-specific immune reconstitution was attempted prior to eiav challenge in two scid foals, using adoptively transferred virus-st ...200111683583
cis-acting sequences may contribute to size variation in the surface glycoprotein of bovine immunodeficiency virus.genetic recombination is an important mechanism of retrovirus variation and diversity. size variation in the surface (su) glycoprotein, characterized by duplication and insertion, has been observed during in vivo infection with several lentiviruses, including bovine immunodeficiency virus (biv), equine infectious anaemia virus (eiav) and human immunodeficiency virus type 1. these duplication/insertion events are thought to occur through a mechanism of template switching/strand transfer during re ...200111714975
envelope glycoprotein cytoplasmic domains from diverse lentiviruses interact with the prenylated rab acceptor.lentivirus envelope glycoproteins have unusually long cytoplasmic domains compared to those of other retroviruses. to identify cellular binding partners of the simian immunodeficiency virus (siv) envelope transmembrane protein (gp41) cytoplasmic domain (cd), we performed a yeast two-hybrid screen of a phytohemagglutinin-activated human t-cell cdna library with the siv gp41 cd. the majority of positive clones (50 of 54) encoded the prenylated rab acceptor (pra1). pra1 is a 21-kda protein associat ...200211739697
the role of international transport of equine semen on disease transmission.despite the numerous benefits of having the capability to transport semen internationally, there are serious potential ramifications if that semen is contaminated with a communicable disease. bacteria: many commensal bacteria colonize the exterior of the stallion penis and are not regarded as pathogenic. they may be cultured from an ejaculate. alterations of the normal bacterial flora on the exterior genitalia may cause the growth of opportunistic bacteria such as klebsiella pneumonia, pseudomon ...200111744267
comparison of gene transfer efficiencies and gene expression levels achieved with equine infectious anemia virus- and human immunodeficiency virus type 1-derived lentivirus vectors.this report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (hiv) and equine infectious anemia virus (eiav) lentiviral vectors in a variety of human cell types in vitro. eiav and hiv vectors transduced equivalent numbers of proliferating and g1/s- and g2/m-arrested cells, and both had very low efficiencies of transduction into g0-arrested cells. analysis of the levels of both the enhanced green fluorescent protein (egfp) and ...200211773424
functional replacement and positional dependence of homologous and heterologous l domains in equine infectious anemia virus replication.we have previously demonstrated by gag polyprotein budding assays that the gag p9 protein of equine infectious anemia virus (eiav) utilizes a unique ypdl motif as a late assembly domain (l domain) to facilitate release of the budding virus particle from the host cell plasma membrane (b. a. puffer, l. j. parent, j. w. wills, and r. c. montelaro, j. virol. 71:6541-6546, 1997). to characterize in more detail the role of the ypdl l domain in the eiav life cycle, we have examined the replication prop ...200211799151
structure of equine infectious anemia virus matrix protein.the gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the n-terminal membrane-binding domain forming the matrix protein (ma). the 2.8-a resolution crystal structure of ma of equine infectious anemia virus (eiav), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the mas of human immunodeficiency virus type 1 (hiv-1) and simian immunodeficiency virus (siv). however, u ...200211799182
budding of equine infectious anemia virus is insensitive to proteasome inhibitors.the only retrovirus protein required for the budding of virus-like particles is the gag protein; however, recent studies of rous sarcoma virus (rsv) and human immunodeficiency virus have suggested that modification of gag with ubiquitin (ub) is also required. as a consequence, the release of these viruses is reduced in the presence of proteasome inhibitors, which indirectly reduce the levels of free ub within the cell. here we show that the budding of equine infectious anemia virus (eiav) from i ...200211861830
equine infectious anemia virus and the ubiquitin-proteasome system.some retroviruses contain monoubiquitinated gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. we examined equine infectious anemia virus (eiav) particles and found that approximately 2% of the p9(gag) proteins are monoubiquitinated, demonstrating that this gag protein interacts with an ubiquitinating activity. different types of proteasome inhibitors were used to determine if pr ...200211861870
design, production, safety, evaluation, and clinical applications of nonprimate lentiviral vectors. 200211883086
oncoretroviral and lentiviral vector-mediated gene therapy.oncoretroviral vectors and lentiviral vectors offer the potential for long-term gene expression by virtue of their stable chromosomal integration and lack of viral gene expression. consequently, their integration allows passage of the transgene to all progeny cells, which makes them particularly suitable for stem cell transduction. however, a disadvantage of oncoretroviral vectors based on moloney murine leukemia virus (momlv) is that cell division is required for transduction and integration, t ...200211883092
nonprimate lentiviral vectors. 200211892254
lipopeptide stimulation of mhc class i-restricted memory cytotoxic t lymphocytes from equine infectious anemia virus-infected horses.the immunogenicity of equine infectious anemia virus (eiav) gag and env equine leukocyte alloantigen (ela)-a5.1, -a9, and -a1 restricted cytotoxic t lymphocyte (ctl) epitopes synthesized on multiple antigenic peptide (map) system coupled to tripalmitoyl-s-glycerylcysteine (p3c) was evaluated in vitro. p3c-map-peptide-stimulated peripheral blood mononuclear cells (pbmcs) from horses, chronically infected with eiav, had memory ctl (ctlm) similar to that of pbmcs stimulated with either the minimal ...200211906769
identification of broadly recognized, t helper 1 lymphocyte epitopes in an equine lentivirus.equine infectious anaemia virus (eiav) is a horse lentivirus causing lifelong, persistent infection. during acute infection, cd8(+) cytotoxic t lymphocytes (ctl) are probably involved in terminating plasma viraemia. however, only a few eiav ctl epitopes, restricted to fewer horse major histocompatibility complex (mhc) class i alleles, are known. as interferon-gamma (ifn-gamma)-secreting cd4(+), t helper 1 (th1) lymphocytes promote ctl activity and help maintain memory ctl, identifying broadly re ...200211918691
new methods to titrate eiav-based lentiviral vectors.ideally, gene transfer vectors used in clinical protocols should only express the gene of interest. so far most vectors have contained marker genes to aid their titration. we have used quantitative real-time pcr to titrate equine infectious anemia virus (eiav) vectors for gene therapy applications. viral rna was isolated from vector preparations and analyzed in a one-step rt-pcr reaction in which reverse transcription and amplification were combined in one tube. the pcr assay of vector stocks wa ...200211991747
transient immune suppression of inapparent carriers infected with a principal neutralizing domain-deficient equine infectious anaemia virus induces neutralizing antibodies and lowers steady-state virus replication.the genetic variation of equine infectious anaemia virus (eiav) clearly affects the antigenic properties of the viral envelope; however, effects on immunogenicity remain undefined, although widely assumed. here, the immunogenicity is reported of a novel, neutralization-resistant, pony-isolate envelope eiav(pv564deltapnd) that contains a 14-residue deletion in the designated principal neutralizing domain (pnd) of the gp90 protein. two ponies inoculated with a chimeric virus, eiav(deltapnd), conta ...200212029150
further characterization of equine foamy virus reveals unusual features among the foamy viruses.foamy viruses (fvs) are nonpathogenic, widely spread complex retroviruses which have been isolated in nonhuman primates, cattle, cats, and more recently in horses. the equine foamy virus (efv) was isolated from healthy horses and was characterized by molecular cloning and nucleotide sequence analysis. here, to further characterize this new fv isolate, the location of the transcriptional cap and poly(a) addition sites as well as the main splice donor and acceptor sites were determined, demonstrat ...200212072521
gene transduction efficiency in cells of different species by hiv and eiav vectors.the ability of human immunodeficiency virus (hiv)- and equine infectious anaemia virus (eiav)-based vectors to transduce cell lines from a range of species was compared. both vectors carried the vesicular stomatitis virus g (vsv-g) envelope protein and encoded an enhanced green fluorescent protein (egfp) gene driven by a human cytomegalovirus (cmv) early promoter. immunostaining for viral core proteins and vsv-g was used to demonstrate that the hiv and eiav vector preparations contained similar ...200212085241
multiple rna splicing and the presence of cryptic rna splice donor and acceptor sites may contribute to low expression levels and poor immunogenicity of potential dna vaccines containing the env gene of equine infectious anemia virus (eiav).the env gene is an excellent candidate for inclusion in any dna-based vaccine approach against equine infectious anemia virus (eiav). unfortunately, this gene is subjected to mutational pressure in e. coli resulting in the introduction of stop codons at the 5' terminus unless it is molecularly cloned using very-low-copy-number plasmid vectors. to overcome this problem, a mammalian expression vector was constructed based on the low-copy-number plg338-30 plasmid. this permitted the production of f ...200212135633
development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (eiav).a single-tube reverse transcriptase-polymerase chain reaction (rt-pcr) using a fluorogenic real-time pcr detection method is described for the quantitation of equine infectious anemia virus (eiav) rna in the plasma of equids. to compensate for variations inherent in sample preparation a multiplex real-time rt-pcr system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral rna molecules. detection of eiav rna was linear fr ...200212176154
human immunodeficiency virus type 1 reverse transcription is stimulated by tat from other lentiviruses.the tat gene is required by hiv-1 for efficient reverse transcription and this function of tat can be distinguished from its role in transcription by rna polymerase ii using tat point mutations that abrogate each function independently. the mechanism of tat's role in reverse transcription, however, is not known, nor is it known whether this role is conserved among trans-activating factors in other retroviruses. here we examine the abilities of heterologous viral trans-activating proteins from je ...200212350353
equine infectious anemia virus envelope evolution in vivo during persistent infection progressively increases resistance to in vitro serum antibody neutralization as a dominant phenotype.equine infectious anemia virus (eiav) infection of horses is characterized by well-defined waves of viremia associated with the sequential evolution of distinct viral populations displaying extensive envelope gp90 variation; however, a correlation of in vivo envelope evolution with in vitro serum neutralization phenotype remains undefined. therefore, the goal of the present study was to utilize a previously defined panel of natural variant eiav envelope isolates from sequential febrile episodes ...200212368301
the role of retroviral dutpases in replication and virulence.several retroviruses, including equine infectious anemia virus (eiav), visna virus, caprine arthritis-encephalitis virus (caev) and feline immunodeficiency virus (fiv) encode dutpase. the role of this enzyme in the replication of these viruses has been scrutinized, with particular emphasis on potential roles for dutpase in virulence and viral mutation rate. overall, the results of these studies have indicated a central role for dutpase in facilitating productive viral replication in non-dividing ...200112374097
multicistronic lentiviral vector-mediated striatal gene transfer of aromatic l-amino acid decarboxylase, tyrosine hydroxylase, and gtp cyclohydrolase i induces sustained transgene expression, dopamine production, and functional improvement in a rat model of parkinson's disease.parkinson's disease (pd) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. this loss leads to complete dopamine depletion in the striatum and severe motor impairment. it has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (eiav) gives rise to highly efficient and sustained transduction of neurons in the rat brain. therefore, a dopamine replacement strategy using eiav has been i ...200212451130
eiav, caev and other lentivirus vector systems.lentiviruses that infect non-primates make up a diverse collection of viruses. although these viruses have some features in common with hiv and other primate viruses, differences in genome organization and viral gene function have made the successful derivation of vectors from non-primate lentiviruses unpredictable. this chapter discusses the construction and application of gene transfer systems derived from four non-primate lentiviruses including equine infectious anemia virus (eiav), caprine a ...200112465465
cytotoxic t lymphocytes and neutralizing antibody in the control of equine infectious anemia virus. 200212513924
response of ela-a1 horses immunized with lipopeptide containing an equine infectious anemia virus ela-a1-restricted ctl epitope to virus challenge.lipopeptide containing an ela-a1-restricted cytotoxic t lymphocyte (ctl) epitope from the envelope surface unit (su) protein of the eiav(wsu5) strain was used to immunize three horses having the ela-a1 haplotype. peptide-specific ela-a1-restricted ctl were induced in all three horses, although these were present transiently in pbmc. these horses were further immunized with lipopeptide containing the corresponding ctl epitope from the eiav(pv) strain. then, the three immunized horses and three no ...200312531649
characterization of a cytolytic strain of equine infectious anemia virus.a novel strain of equine infectious anemia virus (eiav) called vma-1c that rapidly and specifically killed infected equine fibroblasts (ed cells) but not other infectible cell lines was established. this strain was generated from an avirulent, noncytopathic strain of eiav, ma-1. studies with this new cytolytic strain of virus have permitted us to define viral parameters associated with eiav-induced cell killing and begin to explore the mechanism. vma-1c infection resulted in induction of rapid c ...200312551976
restriction of multiple divergent retroviruses by lv1 and ref1.the mouse gene fv1 encodes a saturable restriction factor that selectively blocks infection by n-tropic or b-tropic murine leukemia virus (mlv) strains. despite the absence of an fv1 gene, a similar activity is present in humans that blocks n-mlv infection (ref1). moreover, some non-human primate cell lines express a potentially related inhibitor of hiv-1 and/or sivmac infection (lv1). here, we examine the spectrum of retrovirus-restricting activities expressed by human and african green monkey ...200312554640
a functional genetic approach suggests a novel interaction between the human immunodeficiency virus type 1 (hiv-1) tat protein and hiv-1 tar rna in vivo.human immunodeficiency virus type 1 (hiv-1) tat and human cyclin t1 form a complex and together recognize the viral tar rna element with specificity. using hiv-1/equine infectious anaemia virus tar chimeras, we show that in addition to the well-characterized interaction with the bulge, tat recognizes the distal stem and the loop of tar. these data support previously proposed, but unproven, molecular models.200312604811
retroviruses have differing requirements for proteasome function in the budding process.proteasome inhibitors reduce the budding of human immunodeficiency virus types 1 (hiv-1) and 2, simian immunodeficiency virus, and rous sarcoma virus. to investigate this effect further, we examined the budding of other retroviruses from proteasome inhibitor-treated cells. the viruses tested differed in their gag organization, late (l) domain usage, or assembly site from those previously examined. we found that proteasome inhibition decreased the budding of murine leukemia virus (plasma membrane ...200312610113
the use of fluorescence polarization assays for the detection of infectious diseases.fluorescence polarization assays (fpas) have been shown to have great utility in the detection of infectious diseases. examples are presented of the use of o-polysaccharides (opss) for the detection of antibodies in serum, whole milk and whole blood to gram negative organisms (brucella spp., salmonella spp.). the use of proteins and peptides are also described for the detection of mycobacterium bovis and equine infectious anemia virus. fluorescence polarization inhibition assays (fpias) are disc ...200312678702
long-term replacement of a mutated nonfunctional cns gene: reversal of hypothalamic diabetes insipidus using an eiav-based lentiviral vector expressing arginine vasopressin.due to the complexity of brain function and the difficulty in monitoring alterations in neuronal gene expression, the potential of lentiviral gene therapy vectors to treat disorders of the cns has been difficult to fully assess. in this study, we have assessed the utility of a third-generation equine infectious anemia virus (eiav) in the brattleboro rat model of diabetes insipidus, in which a mutation in the arginine vasopressin (avp) gene results in the production of nonfunctional mutant avp pr ...200312718901
analysis of gene transfer and expression in skeletal muscle using enhanced eiav lentivirus vectors.skeletal muscle is an attractive target tissue for gene therapy involving both muscle and nonmuscle disorders. hiv-1-based vectors transduce mature skeletal muscle; however, the use of these vectors for human gene therapy may be limited by biosafety concerns. in this study, we investigated gene transfer using lentivirus vectors based on the equine infectious anemia virus (eiav) in skeletal muscle in vitro and in vivo. eiav vectors transduce proliferating and differentiating c2c12 mouse muscle ce ...200312718906
role of the reverse transcriptase, nucleocapsid protein, and template structure in the two-step transfer mechanism in retroviral recombination.template switching during reverse transcription promotes recombination in retroviruses. efficient switches have been measured in vitro on hairpin-containing rna templates by a two-step mechanism. pausing of the reverse transcriptase (rt) at the hairpin base allowed enhanced cleavage of the initial donor rna template, exposing regions of the cdna and allowing the acceptor to base pair with the cdna. this defines the first or docking step. the primer continued synthesis on the donor, transferring ...200312801926
a nuclear kinesin-like protein interacts with and stimulates the activity of the leucine-rich nuclear export signal of the human immunodeficiency virus type 1 rev protein.the rev protein of human immunodeficiency virus type 1 (hiv-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced hiv mrnas containing the rev response element (rre). in a yeast two-hybrid screen of a hela cell-derived cdna expression library for human factors interacting with the rev leucine-rich nuclear export sequence (nes), we identified a kinesin-like protein, rebp (rev/rex effector binding protein), highly homologous to kid, the carboxy-terminal 75-residue ...200312805422
a live attenuated equine infectious anemia virus proviral vaccine with a modified s2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses.previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (eiav) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature a ...200312805423
characterization of eiav ltr variability and compartmentalization in various reservoir tissues of long-term inapparent carrier ponies.dynamic genomic variation resulting in changes in envelope antigenicity has been established as a fundamental mechanism of persistence by equine infectious anemia virus (eiav), as observed with other lentiviruses, including hiv-1. in addition to the reported changes in envelope sequences, however, certain studies indicate the viral ltr as a second variable eiav gene, with the enhancer region being designated as hypervariable. these observations have lead to the suggestion that ltr variation may ...200312832214
equine infectious anemia virus utilizes host vesicular protein sorting machinery during particle release.a final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the gag protein known as a late domain. recently, human immunodeficiency virus type 1 (hiv-1) and moloney murine leukemia virus (m-mulv) were shown to require components of the cellular vacuolar protein sorting (vps) machinery for efficient viral release. hiv-1 interacts with the vps pathway via an association of hiv-1 gag with tsg101, a component of the cellular complexes involved in vps. equin ...200312857913
long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice.inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. to this end, high-dose attenuated vsv-g pseudotyped equine infectious anaemia virus (eiav) encoding beta-gala ...200312858188
equine infectious anemia in mules: virus isolation and pathogenicity studies.there appears to be a lack of information concerning responses of mules to natural infection or experimental inoculation with equine infectious anemia virus (eiav). in the present study eiav was isolated from mules, for the first time, and its pathogenicity in naturally infected and experimentally inoculated animals was investigated. two naturally infected (a and b) and three eiav free mules (c, d and e) were used for this purpose. mule a developed clinical signs, whereas mule b remained asympto ...200312860076
[expression and immunogenicity of equine infectious anemia virus membrane protein gp90].membrane protein gp90 of china equine infectious anemia virus (eiav) vaccine strain (dlv) and its parental wild type ln strain were expressed with bac-to-bac baculovirus expression system and balb/c mice were inoculated with purified protein, thereby to explore the availability of protein for differential diagnosis and potential for preparing genetically engineered vaccine.200312870025
presentation and binding affinity of equine infectious anemia virus ctl envelope and matrix protein epitopes by an expressed equine classical mhc class i molecule.control of a naturally occurring lentivirus, equine infectious anemia virus (eiav), occurs in most infected horses and involves mhc class i-restricted, virus-specific ctl. two minimal 12-aa epitopes, env-rw12 and gag-gw12, were evaluated for presentation by target cells from horses with an equine lymphocyte ag-a1 (ela-a1) haplotype. fifteen of 15 presented env-rw12 to ctl, whereas 11 of 15 presented gag-gw12. to determine whether these epitopes were presented by different molecules, mhc class i ...200312902502
selecting peptides to optimize th1 responses to an equine lentivirus using hla-dr binding motifs and defined hiv-1 th peptides.three moderately to broadly recognized equine infectious anemia virus (eiav) peptides that contained helper t-lymphocyte (th) 1 epitopes were previously identified. although lipopeptide immunization was only weakly immunostimulatory in a preliminary study, as measured by t-lymphocyte proliferation responses, it was of interest to define additional broadly recognized th1 epitopes to include in future immunization trials. using broadly cross-reactive and conserved th epitopes known in the related ...200312942208
epitope specificity is critical for high and moderate avidity cytotoxic t lymphocytes associated with control of viral load and clinical disease in horses with equine infectious anemia virus.equine infectious anemia virus (eiav) is a lentivirus that causes persistent infections in horses. we hypothesized that high-avidity ctl specific for nonvariable epitopes might be associated with low viral load and minimal disease in eiav-infected horses. to test this hypothesis, memory ctl (ctlm) responses were analyzed in two infected horses with high plasma viral loads and recurrent disease (progressors), and in two infected horses with low-to-undetectable viral loads and mild disease (nonpro ...200312954220
enhancement of equine infectious anemia virus virulence by identification and removal of suboptimal nucleotides.pathogenicity was reportedly restored to an avirulent molecular clone of equine infectious anemia virus (eiav) by substitution of 3' sequences from the pathogenic variant strain (eiav(pv)). however, the incidence of disease in horses/ponies was found to be significantly lower (p = 0.016) with the chimeric clone (eiav(uk)) than with eiav(pv). this was attributable to 3' rather than 5' regions of the proviral genome, where eiav(uk) differs from the consensus eiav(pv) sequence by having a 68-bp dup ...200312954224
characterization of rna elements that regulate gag-pol ribosomal frameshifting in equine infectious anemia virus.synthesis of gag-pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a -1 direction to read through the stop codon in the gag reading frame. it is generally believed that a slippery sequence and a downstream rna structure are required for the programmed -1 ribosomal frameshifting. however, the mechanism regulating the gag-pol frameshifting remains poorly understood. in this report, we have defined specific mrna elements required for sufficien ...200312970412
experimental studies on equine infectious anemia (eia). iv. alteration of blood in mouse inoculated with the mouse-fixed equine infectious anemia virus and re-transmission test of the virus to a pony. 196013683955
equine infectious anaemia in two groups of horses. ii. 196514282005
aip1/alix is a binding partner for hiv-1 p6 and eiav p9 functioning in virus and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission. the primary late assembly (l) domain in the p6 region of hiv-1 gag mediates the detachment of the virion by recruiting host tsg101, a component of the class e vacuolar protein sorting (vps) machinery. we now show that hiv gag p6 contains a second region involved in l domain function that binds aip1, a homolog of the yeast class e vps protein bro1. further, aip1 interacts wi ...200314505569
the protein network of hiv release requires tsg101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (mvb). to test whether other proteins involved in mvb biogenesis (the class e proteins) also participate in hiv release, we identified 22 candidate human class e proteins. these proteins were connected into a coherent network by 43 different protein-protein interactions, with aip1 playing a key role in linking complexes that act early (tsg101/escrt-i) and late (chmp4/escrt-iii) in ...200314505570
divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins.the release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (l domain), encoded by essential ptap, ppxy, or ypdl sequence motifs. ptap-type l domains recruit one of three endosomal sorting complexes required for transport (escrt-i). however, subsequent events in viral budding are poorly defined, and neither ypdl nor ppxy-type l domains require escrt-i. here, we show that escrt-i and other class e vacuolar protein sorting (vps) fact ...200314519844
functional analysis of the interaction of the human immunodeficiency virus type 1 rev nuclear export signal with its cofactors.human immunodeficiency virus type 1 (hiv-1) rev-mediated nuclear export of viral rnas involves the interaction of its leucine-rich nuclear export sequence (nes) with nuclear cofactors. in yeast two-hybrid screens of a human lymph node derived cdna expression library, we identified the human nucleoporin nup98 as a highly specific and potent interactor of the rev nes. using an extensive panel of nuclear export positive and negative mutants of the functionally homologous ness of the hiv-1 rev, huma ...200314554087
cellular specificity of hiv-1 replication can be controlled by ltr sequences.two well-established determinants of retroviral tropism are envelope sequences that regulate entry and ltr sequences that can regulate viral expression in a cell-specific manner. studies with human immunodeficiency virus-1 (hiv-1) have demonstrated that tropism of this virus maps primarily to variable envelope sequences. studies have demonstrated that t cell and macrophage-specific transcription factor binding motifs exist in the upstream region of the ltr u3; however, the ability of the core en ...200314554095
subpopulations of equine infectious anemia virus rev coexist in vivo and differ in phenotype.lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. the quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, rev, over the course of disease in ponies ex ...200314581549
post-entry restriction of retroviral infections.pathogenic retroviruses have driven the evolution of several dominant-acting mechanisms able to block infection and protect the host. these are exemplified by the mouse gene fv1, which encodes a gag-like protein able to protect against murine leukemia virus (mlv) infection. the block is saturable, occurs after reverse transcription and is directed against the viral capsid gene. several other mammalian species are also able to block mlv infection with the same capsid specificity. a human gene wit ...200314598564
conservation of the human immunodeficiency virus type 1 gp120 v1/v2 stem/loop structure in the equine infectious anemia virus (eiav) gp90. 200314601590
important role for the ca-nc spacer region in the assembly of bovine immunodeficiency virus gag protein.lentiviral gag proteins contain a short spacer sequence that separates the capsid (ca) from the downstream nucleocapsid (nc) domain. this short spacer has been shown to play an important role in the assembly of human immunodeficiency virus type 1 (hiv-1). we have now extended this finding to the ca-nc spacer motif within the gag protein of bovine immunodeficiency virus (biv). mutation of this latter spacer sequence led to dramatic reductions in virus production, which was mainly attributed to th ...200414694086
late domain-dependent inhibition of equine infectious anemia virus budding.the gag proteins of a number of different retroviruses contain late or l domains that promote the release of virions from the plasma membrane. three types of l domains have been identified to date: pro-thr-ala-pro (ptap), pro-pro-x-tyr, and tyr-pro-asp-leu. it has previously been demonstrated that overexpression of the n-terminal, e2-like domain of the endosomal sorting factor tsg101 (tsg-5') inhibits human immunodeficiency virus type 1 (hiv-1) release but does not affect the release of the pppy ...200414694104
differential effects of actin cytoskeleton dynamics on equine infectious anemia virus particle production.retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. previous studies have shown that retroviral gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. using equine infectious anemia virus (eiav), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. first, virion production was reduced when eiav-infected cells we ...200414694119
transduction patterns of pseudotyped lentiviral vectors in the nervous system.we have developed a non-primate-based lentiviral vector based on the equine infectious anemia virus (eiav) for efficient gene transfer to the central and peripheral nervous systems. previously we have demonstrated that pseudotyping lentiviral vectors with the rabies virus glycoprotein confers retrograde axonal transport to these vectors. in the present study we have successfully produced high-titer eiav vectors pseudotyped with envelope glycoproteins from rhabdovirus vesicular stomatitis virus ( ...200414741783
attempts to adapt equine infectious anemia virus to small animals. 195014792980
influence of long terminal repeat and env on the virulence phenotype of equine infectious anemia virus.the molecular clones pspeiav19 and p19/wenv17 of equine infectious anemia virus (eiav) differ in env and long terminal repeats (ltrs) and produce viruses (eiav(19) and eiav(17), respectively) of dramatically different virulence phenotypes. these constructs were used to generate a series of chimeric clones to test the individual contributions of ltr, surface (su), and transmembrane (tm)/rev regions to the disease potential of the highly virulent eiav(17). the ltrs of eiav(19) and eiav(17) differ ...200414963146
pu.1 binding to ets motifs within the equine infectious anemia virus long terminal repeat (ltr) enhancer: regulation of ltr activity and virus replication in macrophages.binding of the transcription factor pu.1 to its dna binding motif regulates the expression of a number of b-cell- and myeloid-specific genes. the long terminal repeat (ltr) of macrophage-tropic strains of equine infectious anemia virus (eiav) contains three pu.1 binding sites, namely an invariant promoter-proximal site as well as two upstream sites. we have previously shown that these sites are important for eiav ltr activity in primary macrophages (w. maury, j. virol. 68:6270-6279, 1994). since ...200415016863
neuroprotection in a rat parkinson model by gdnf gene therapy using eiav vector.vectors based on lentiviruses are opening up new approaches for the treatment of neurodegenerative diseases. currently, the equine infectious anaemia virus (eiav) vector is one of the most attractive gene delivery systems with respect to neuronal tropism. the aim was to validate eiav-lentiviral vectors as a gene delivery system for neurotrophic factor genes in an animal model of parkinson's disease. eiav carrying the glial cell line-derived neurotrophic factor (gdnf) gene was unilaterally inject ...200415076720
highly efficient eiav-mediated in utero gene transfer and expression in the major muscle groups affected by duchenne muscular dystrophy.gene therapy for duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. in addition, the prenatal onset of disease complicates postnatal gene therapy. we have therefore proposed a fetal approach to overcome these barriers. we have applied beta-galactosidase expressing equine infectious anaemia virus ...200415141156
vegf delivery with retrogradely transported lentivector prolongs survival in a mouse als model.amyotrophic lateral sclerosis (als) causes adult-onset, progressive motor neuron degeneration in the brain and spinal cord, resulting in paralysis and death three to five years after onset in most patients. als is still incurable, in part because its complex aetiology remains insufficiently understood. recent reports have indicated that reduced levels of vascular endothelial growth factor (vegf), which is essential in angiogenesis and has also been implicated in neuroprotection, predispose mice ...200415164063
cis-acting and trans-acting modulation of equine infectious anemia virus alternative rna splicing.equine infectious anemia virus (eiav), a lentivirus distantly related to hiv-1, encodes regulatory proteins, eiav tat (etat) and rev (erev), from a four-exon mrna. exon 3 of the tat/rev mrna contains a 30-nucleotide purine-rich element (pre) which binds both erev and sf2/asf, a member of the sr family of rna splicing factors. to better understand the role of this element in the regulation of eiav pre-mrna splicing, we quantified the effects of mutation or deletion of the pre on exon 3 splicing i ...200415165825
hiv/aids in china. vaccine development with a distinctly chinese flavor. 200415178780
[combination immunization with eiav env protein expressed by recombinant baculovirus and recombinant vaccinia virus containing env gene].to develop a novel vaccine candidate of equine infectious anemia virus(eiav).200415207082
equine infectious anemia virus (eiav): what has hiv's country cousin got to tell us?equine infectious anemia virus (eiav) is a lentivirus, of the retrovirus family, with an almost worldwide distribution, infecting equids. it causes a persistent infection characterized by recurring febrile episodes associating viremia, fever, thrombocytopenia, and wasting symptoms. the disease is experimentally reproducible by inoculation of shetland ponies or horses with eiav pathogenic strains. among lentiviruses, eiav is unique in that, despite a rapid virus replication and antigenic variatio ...200415236678
recent insights into hiv-1 vif.the lentiviruses, including hiv-1 (but excluding equine infectious anemia virus), encode a viral infectivity factor (vif) protein. circumstantial evidence suggested that vif acts to neutralize an inhibitory host defense mechanism, but progress in the field was limited because the identity of the cellular target was unknown. the recent identification of the elusive host cell factor let loose a flood of advances. these findings have revealed a novel innate defense mechanism against retroviruses. i ...200415245742
design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression.adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. we have designed a portfolio of latest generation self-inactivating human (hiv-derived) and non-human (eiav-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (amys(deltas), eyfp, samy, s ...200415258250
efficient generation of transgenic pigs using equine infectious anaemia virus (eiav) derived vector.traditional methods of transgene delivery in livestock are inefficient. recently, human immunodeficiency virus (hiv-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. we now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. we used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder anim ...200415280048
cervical spinal cord delivery of a rabies g protein pseudotyped lentiviral vector in the sod-1 transgenic mouse. invited submission from the joint section meeting on disorders of the spine and peripheral nerves, march 2004.lentiviral vectors may constitute a vehicle for long-term therapeutic gene expression in the spinal cord. in amyotrophic lateral sclerosis, spinal cord sclerosis and altered axonal transport pose barriers to therapeutic gene distribution. in the present study the authors characterize gene expression distribution and the behavioral impact of the rabies g (rabg) protein pseudotyped lentiviral vector eiav.lacz through cervical spinal cord injection in control and cu/zn superoxide dismutase-1 (sod-1 ...200415291033
adaptive immunity is the primary force driving selection of equine infectious anemia virus envelope su variants during acute infection.equine infectious anemia virus (eiav) is a lentivirus that causes persistent infection in horses. the appearance of antigenically distinct viral variants during recurrent viremic episodes is thought to be due to adaptive immune selection pressure. to test this hypothesis, we evaluated envelope su cloned sequences from five severe combined immunodeficient (scid) foals infected with eiav. within the su hypervariable v3 region, 8.5% of the clones had amino acid changes, and 6.4% had amino acid chan ...200415308724
ctl from eiav carrier horses with diverse mhc class i alleles recognize epitope clusters in gag matrix and capsid proteins.cytotoxic t lymphocytes (ctl) are important for controlling equine infectious anemia virus (eiav). because gag matrix (ma) and capsid (ca) are the most frequently recognized proteins, the hypothesis that ctl from eiav-infected horses with diverse mhc class i alleles recognize epitope clusters (ec) in these proteins was tested. four ec were identified by ctl from 15 horses and 8 of these horses had diverse mhc class i alleles. two of the eight had ctl to ec1, six to ec2, five to ec3, and four to ...200415327905
leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection.neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (eiav). this report describes a case of clinically severe neurologic disease in a pony experimentally infected with eiav. this pony did not have fever or anemia, which are the characteristic clinical signs of disease. the histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. polymerase chain reaction and in situ hybridization data showed that the brain le ...200415347829
development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections.among animal lentiviruses, feline immunodeficiency virus (fiv), equine infectious anaemia virus (eiav) and small ruminant lentiviruses (srlv) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia. the detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life. capsid antigen (ca) is the major viral core protein and specific antibodies ...200415350735
lentiviral vectors for treating and modeling human cns disorders.vectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. these vectors are opening up new approaches for the treatment of neurological diseases such as parkinson's disease (pd), huntington's disease (hd), and motor neuron diseases (mnds). numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. lenti ...200415352068
non-primate eiav-based lentiviral vectors as gene delivery system for motor neuron diseases.motor neuron diseases such as amyotrophic lateral sclerosis (als) and spinal muscular atrophy (sma) are neurodegenerative diseases, which cause progressive paralysis and premature death in affected adults and children. the treatment rational for these diseases is to halt or delay the degeneration of motor neurons but to date there are no effective drugs. this may however change with recent advances in gene therapy using lentiviral vectors. these vectors can transfer genes to motor neurons with h ...200415384941
serological method using recombinant s2 protein to differentiate equine infectious anemia virus (eiav)-infected and eiav-vaccinated horses.we recently reported a highly protective attenuated live virus vaccine for equine infectious anemia virus (eiav) based on a proviral construct (eiavukdeltas2) with a genetically engineered mutation in the viral s2 gene that eliminates expression of this accessory protein. while the eiavukdeltas2 vaccine provides protection from detectable infection by experimental challenge with highly virulent virus, the potential for commercial application of this vaccine is complicated by the fact that horses ...200415539516
optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer.gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. optimized hiv-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. however, the use of hiv-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. we report that vectors based on the non-primate equine infectious anemia virus (eiav) transduce a variety of human hematopoietic ce ...200515550928
molecular analysis of the proviral dna of equine infectious anemia virus in mules in greece.molecular analysis of the regulatory and structurally important genetic segments of equine infectious anemia virus (eiav) in mules is presented. we have previously reported clinicopathological and laboratory findings in mules infected with eiav, both naturally and after experimental inoculation. in this study the fragment coding for integrase, gp90, tat and the fusion domain of gp45 of the proviral genome from these animals was sequenced and compared with one another and with that of eiav strain ...200515567035
comparison of commercial enzyme-linked immunosorbent assays and agar gel immunodiffusion tests for the serodiagnosis of equine infectious anemia.the purpose of this study was to estimate the performance characteristics (accuracy, detection limit, and precision) of commercially available enzyme-linked immunosorbent assay (elisa) and agar gel immunodiffusion (agid) kits in comparison with a reference agid kit for the detection of equine infectious anemia (eia) antibodies in horses for regulatory use in canada. a total of 285 positive and 315 negative samples by the reference agid were tested blindly on 2 other agid and 4 elisa kits. commer ...200415581219
lentiviral-mediated delivery of bcl-2 or gdnf protects against excitotoxicity in the rat hippocampus.nutrient deprivation during ischemia leads to severe insult to neurons causing widespread excitotoxic damage in specific brain regions such as the hippocampus. one possible strategy for preventing neurodegeneration is to express therapeutic proteins in the brain to protect against excitotoxicity. we investigated the utility of equine infectious anemia virus (eiav)-based vectors as genetic tools for delivery of therapeutic proteins in an in vivo excitotoxicity model. the efficacy of these vectors ...200515585409
lentivector-mediated smn replacement in a mouse model of spinal muscular atrophy.spinal muscular atrophy (sma) is a frequent recessive autosomal disorder. it is caused by mutations or deletion of the telomeric copy of the survival motor neuron (smn) gene, leading to depletion in smn protein levels. the treatment rationale for sma is to halt or delay the degeneration of motor neurons, but to date there are no effective drug treatments for this disease. we have previously demonstrated that pseudotyping of the nonprimate equine infectious anemia virus (using the lentivector gen ...200415599397
specificity of serum neutralizing antibodies induced by transient immune suppression of inapparent carrier ponies infected with a neutralization-resistant equine infectious anemia virus envelope has been previously reported that transient corticosteroid immune suppression of ponies experimentally infected with a highly neutralization resistant envelope variant of equine infectious anemia virus (eiav), designated eiav(deltapnd), resulted in the appearance of type-specific serum antibodies to the infecting eiav(deltapnd) virus. the current study was designed to determine if this induction of serum neutralizing antibodies was associated with changes in the specificity of envelope determ ...200515604441
differential effects of virulent and avirulent equine infectious anemia virus on macrophage cytokine expression.equine infectious anemia virus (eiav) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia, and viremia. most infected equid eventually bring the virus under immunological control. we recently reported the development of an equine-specific ribonuclease protection assay (rpa) to quantitate mrna levels of 10 cytokines. using this newly developed rpa, we now show significant differences in cytokine induction in equine monocyte-derived macrophages (emdm ...200515661161
modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy.genetic modification of dendritic-cell (dc) function is an attractive approach to treat disease, either using mature dcs (mdcs) to immunize patients, or immature dcs (idcs) to induce tolerance. viral vectors are efficient at transducing dcs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of dcs. adenovirus (ad), human immunodeficiency virus (hiv), equine anemia virus (eiav), and moloney murine leukemia virus (mmlv) all up-regulat ...200515671441
su proteins from virulent and avirulent eiav demonstrate distinct biological properties.biologic activity of equine infectious anemia virus (eiav) surface (su) glycoprotein was assayed in a mouse model. recombinant su from virulent eiav17 (su17), administered intraperitoneally to mouse pups, induced dose-dependent diarrheal responses similar to those reported for siv su (virology 277 (2000) 250). su17 caused fluid accumulation without histological lesions in mouse intestinal loops, induced chloride secretory currents in ussing chambers and increased inositol 1,4,5 triphosphate (ip3 ...200515708599
discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy.among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. we previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (eiav), based on mutation of the viral s2 accessory gene, elicited protection from detectable infection by virulent virus challenge (f. li e ...200515708986
amino acid preferences for a critical substrate binding subsite of retroviral proteases in type 1 cleavage sites.the specificities of the proteases of 11 retroviruses representing each of the seven genera of the family retroviridae were studied using a series of oligopeptides with amino acid substitutions in the p2 position of a naturally occurring type 1 cleavage site (val-ser-gln-asn-tyr pro-ile-val-gln; the arrow indicates the site of cleavage) in human immunodeficiency virus type 1 (hiv-1). this position was previously found to be one of the most critical in determining the substrate specificity differ ...200515767422
in vivo evaluation of an eiav vector for the systemic genetic delivery of therapeutic antibodies.lentiviral-based vectors hold great promise as gene delivery vehicles for the treatment of a wide variety of diseases. we have previously reported the development of a nonprimate lentiviral vector system based on the equine infectious anaemia virus (eiav), which is able to efficiently transduce dividing and nondividing cells both in vitro and in vivo. here, we report on the application of eiav vectors for the systemic delivery of an antibody fusion protein designed for the treatment of cancer. t ...200515772687
attenuation of dna replication by hiv-1 reverse transcriptase near the central termination sequence.previous pre-steady-state kinetic studies of equine infectious anemia virus-1 (eiav) reverse transcriptase (rt) showed two effects of dna substrates containing the central termination sequence (cts) on the polymerization reaction: reduction of burst amplitude in single nucleotide addition experiments and accumulation of termination products during processive dna synthesis [berdis, a. j., stetor, s. r., le grice, s. f. j., and barkley, m. d. (2001) biochemistry 40, 12140-12149]. the present study ...200515807528
evolution of the equine infectious anemia virus long terminal repeat during the alteration of cell tropism.equine infectious anemia virus (eiav) is a lentivirus with in vivo cell tropism primarily for tissue macrophages; however, in vitro the virus can be adapted to fibroblasts and other cell types. tropism adaptation is associated with both envelope and long terminal repeat (ltr) changes, and findings strongly suggest that these regions of the genome influence cell tropism and virulence. furthermore, high levels of genetic variation have been well documented in both of these genomic regions. however ...200515827180
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