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host and viral traits predict zoonotic spillover from mammals.the majority of human emerging infectious diseases are zoonotic, with viruses that originate in wild mammals of particular concern (for example, hiv, ebola and sars). understanding patterns of viral diversity in wildlife and determinants of successful cross-species transmission, or spillover, are therefore key goals for pandemic surveillance programs. however, few analytical tools exist to identify which host species are likely to harbour the next human virus, or which viruses can cross species ...201728636590
equine infectious anemia prevalence in feral donkeys from northeast brazil.equine infectious anemia virus (eiav) is an important cause of morbidity and mortality throughout the world. although the virus infects all members of the equidae the vast majority of studies have been conducted in horses (equus caballus) with comparatively little information available for other equid species. brazil has one of the most abundant donkey (e. asinus) populations of any nation although the economic importance of these animals is declining as transportation becomes increasingly mecha ...201728460747
protective effects of broadly neutralizing immunoglobulin against homologous and heterologous equine infectious anemia virus infection in horses with severe combined immunodeficiency.using the equine infectious anemia virus (eiav) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (scid). however, in vivo selection of a neutralization-resistant envelope variant occurred. here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. overall, protection correlated with the breadth and potency of neutrali ...201121543497
dynamics of escrt protein recruitment during retroviral assembly.the escrt (endosomal sorting complex required for transport) complexes and associated proteins mediate membrane scission reactions, such as multivesicular body formation, the terminal stages of cytokinesis and retroviral particle release. these proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the atpase vps4a. however, these events have never been observed in living cells, and their dynamics are unknown. by quantifying th ...201121394083
molecular detection, epidemiology, and genetic characterization of novel european field isolates of equine infectious anemia virus.the application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of european field isolates of equine infectious anemia virus (eiav) has not been widely reported. as a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in italy, 24 farms with a history of exposure to this disease were included in this study. new pcr-based methods were developed, which, especially in the case o ...201021084503
lentiviral vif degrades the apobec3z3/apobec3h protein of its mammalian host and is capable of cross-species activity.all lentiviruses except equine infectious anemia virus (eiav) use the small accessory protein vif to counteract the restriction activity of the relevant apobec3 (a3) proteins of their host species. prior studies have suggested that the vif-a3 interaction is species specific. here, using the apobec3h (z3)-type proteins from five distinct mammals, we report that this is generally not the case: some lentiviral vif proteins are capable of triggering the degradation of both the a3z3-type protein of t ...201020519393
[infectious anemia in belgium]. 201020415032
an lypsl late domain in the gag protein contributes to the efficient release and replication of rous sarcoma virus.the efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (escrt machinery) normally involved in the biogenesis of multivesicular bodies and in cytokinesis. retroviruses and other enveloped viruses recruit the escrt machinery through three classes of short amino acid consensus sequences termed late domains: pt/sap, ppxy, and lypx(n)l. the major late domain of rous sarcoma virus (rsv) has been mapped to a pppy ...201020392845
equine infectious anemia viral vector-mediated codelivery of endostatin and angiostatin driven by retinal pigmented epithelium-specific vmd2 promoter inhibits choroidal neovascularization.equine infectious anemia virus (eiav) is a nonprimate lentivirus that does not cause human disease. subretinal injection into mice of a recombinant eiav lentiviral vector in which lacz is driven by a cmv promoter (eiav cmv lacz) resulted in rapid and strong expression of lacz in retinal pigmented epithelial (rpe) cells and some other cells including ganglion cells, resulting in the presence of 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside within the optic nerve. substitution of the rpe-spe ...200920377369
[receptors for animal retroviruses].diseases caused by animal retroviruses have been recognized since 19th century in veterinary field. most livestock and companion animals have own retroviruses. to disclose the receptors for these retroviruses will be useful for understanding retroviral pathogenesis, developments of anti-retroviral drugs and vectors for human and animal gene therapies. of retroviruses in veterinary field, receptors for the following viruses have been identified; equine infectious anemia virus, feline immunodefici ...200920218331
defects in cellular sorting and retroviral assembly induced by gga overexpression.we previously demonstrated that overexpression of golgi-localized, gamma-ear containing, arf-binding (gga) proteins inhibits retrovirus assembly and release by disrupting the function of endogenous adp ribosylation factors (arfs). gga overexpression led to the formation of large, swollen vacuolar compartments, which in the case of gga1 sequestered hiv-1 gag.200919788741
viral load and clinical disease enhancement associated with a lentivirus cytotoxic t lymphocyte vaccine regimen.effective dna-based vaccines against lentiviruses will likely induce ctl against conserved viral proteins. equine infectious anemia virus (eiav) infects horses worldwide, and serves as a useful model for lentiviral immune control. although attenuated live eiav vaccines have induced protective immune responses, dna-based vaccines have not. in particular, dna-based vaccines have had limited success in inducing ctl responses against intracellular pathogens in the horse. we hypothesized that priming ...200919368787
in vivo evolution of the gp90 gene and consistently low plasma viral load during transient immune suppression demonstrate the safety of an attenuated equine infectious anemia virus (eiav) vaccine.to study the in vivo evolution of the attenuated chinese equine infectious anemia virus (eiav) vaccine, viral gp90 gene variation and virus replication in immunosuppressed hosts were investigated. the results showed that after vaccination, the gp90 gene followed an evolutionary trend of declining diversity. the trend coincided with the maturation of immunity to eiav, and eventually, the gp90 gene became highly homologous. the sequences of these predominant quasispecies were consistently detected ...200919363668
[equine infectious anemia (eia)].equine infectious anemia (eia) is a reportable, eradicable epizootic disease caused by the equine lentivirus of the retrovirus family which affects equids only and occurs worldwide. the virus is transmitted by blood, mainly by sanguivorous insects. the main symptoms of the disease are pyrexia, apathy, loss of body condition and weight, anemia, edema and petechia. however, infected horses can also be inapparent carriers without any overt signs. the disease is diagnosed by serological tests like t ...200919333901
[comparison of proviral genomes between the chinese eiav donkey leukocyte-attenuated vaccine and its parental virulent strain].the donkey leukocyte-attenuated vaccine of equine infectious anemia virus (eiav) was the first lentiviral vaccine that induced solid protection from the infection of virulent strains. to elucidate the mechanism of increased immunogenicity and attenuated virulence of the vaccine, the proviral genomic dna of an eiav vaccine strain, eiav(dlv121) was analyzed and compared with the genome of a parental virulent strain eiav(dv117). full length viral genomic dnas were amplified as two segments by la-pc ...200819226953
hiv-1 exploits importin 7 to maximize nuclear import of its dna genome.nuclear import of the hiv-1 reverse transcription complex (rtc) is critical for infection of non dividing cells, and importin 7 (imp7) has been implicated in this process. to further characterize the function of imp7 in hiv-1 replication we generated cell lines stably depleted for imp7 and used them in conjunction with infection, cellular fractionation and pull-down assays.200919193229
genetic variation in the long terminal repeat associated with the transition of chinese equine infectious anemia virus from virulence to avirulence.a highly virulent strain of equine infectious anemia virus (eiav) lost its fatal virulence but retained the desired antigens during serial passage over 130 generations in leukocytes in vitro. we compared the long terminal repeat (ltr) sequences of the different generations and found that three stable genetic variations occurred in the transcriptional start site, the initial base of tar, and the pre-mrna cleavage site at the r-u5 boundary, respectively. these three mutations happened at the infle ...200919130201
replication of equine infectious anemia virus in engineered mouse nih 3t3 cells.we employed the equine lentivirus equine infectious anemia virus (eiav) to investigate the cellular restrictions for lentivirus replication in murine nih 3t3 cells. the results of these studies demonstrate that nih 3t3 cells expressing the eiav receptor elr1 and equine cyclin t1 supported productive replication of eiav and produced infectious virions at levels similar to those found in a reference permissive equine cell line. the studies presented here demonstrate, for the first time, differenti ...200919073738
structural insights into the cyclin t1-tat-tar rna transcription activation complex from eiav.the replication of many retroviruses is mediated by a transcriptional activator protein, tat, which activates rna polymerase ii at the level of transcription elongation. tat interacts with cyclin t1 of the positive transcription-elongation factor p-tefb to recruit the transactivation-response tar rna, which acts as a promoter element in the transcribed 5' end of the viral long terminal repeat. here we present the structure of the cyclin box domain of cyclin t1 in complex with the tat protein fro ...200819029897
eiav vector-mediated co-delivery of endostatin and angiostatin driven by the rpe-specific vmd2 promoter inhibits choroidal neovascularization.equine infectious anemia virus (eiav) is a non-primate lentivirus that does not cause human disease. subretinal injection in mice of a recombinant eiav lentiviral vector in which lacz is driven by a cmv promoter (eiav cmv lacz) resulted in rapid and strong expression of lacz in retinal pigmented epithelial (rpe) cells and some other cells including ganglion cells resulting in x-gal within the optic nerve. substitution of the rpe-specific vmd2 promoter for the cmv promoter resulted in prolonged ( ...200818840069
effect of two synthetic peptides mimicking conserved regions of equine infectious anemia virus proteins gp90 and gp45 upon cytokine mrna expression.gp90 and gp45 synthetic peptides, which mimic conserved sequences of native viral proteins, are recognized by antibodies to equine infectious anemia virus (eiav) in asymptomatic carrier horses and generate humoral and cellular responses in immunized mice. cytokine mrna levels were evaluated in equine peripheral blood mononuclear cells (pbmcs) after in vitro stimulation with gp90 and gp45 with the aim of determining the cytokine profile associated with the proliferative response. stimulation inde ...200818825485
biochemical characterization of a recombinant trim5alpha protein that restricts human immunodeficiency virus type 1 replication.the rhesus monkey intrinsic immunity factor trim5alpha(rh) recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (hiv-1) and equine infectious anemia virus (eiav), and inhibits the accumulation of viral reverse transcripts. however, direct interactions between restricting trim5alpha proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. to facilitate structural and mechanistic studies of retrovira ...200818799573
analysis of the eiav rev-responsive element (rre) reveals a conserved rna motif required for high affinity rev binding in both hiv-1 and eiav.a cis-acting rna regulatory element, the rev-responsive element (rre), has essential roles in replication of lentiviruses, including human immunodeficiency virus (hiv-1) and equine infection anemia virus (eiav). the rre binds the viral trans-acting regulatory protein, rev, to mediate nucleocytoplasmic transport of incompletely spliced mrnas encoding viral structural genes and genomic rna. because of its potential as a clinical target, rre-rev interactions have been well studied in hiv-1; however ...200818523581
correction of the disease phenotype in the mouse model of stargardt disease by lentiviral gene therapy.autosomal recessive stargardt disease (stgd1) is a macular dystrophy caused by mutations in the abca4 (abcr) gene. the disease phenotype that is most recognized in stgd1 patients, and also in the abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. here, we tested whether delivery of the normal (wt) human abca4 gene to the subretinal space of the abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the l ...200818463687
development and characterization of an equine infectious anemia virus env-pseudotyped reporter virus.we developed a replication-defective reporter virus pseudotyped with the envelope glycoprotein of equine infectious anemia virus (eiav). the in vitro host range and neutralization phenotype of eiav env-pseudotyped virus were similar to those of replication-competent virus. an eiav env pseudovirus will improve antigenic characterization of viral variants and evaluation of lentivirus vaccines.200818448619
[establishment of a 293-cell line containing luciferase reporter for eiav receptor and ltr functions].to accurately and conveniently detect neutralizing antibodies and receptor binding affinities of different equine infectious anemia virus (eiav) strains, the cdna of eiav receptor, elr1, was cloned and inserted in an eukaryotic expression vector pcdna3.1(+). this recombinant plasmid was designated as pelr1. the 293 cell line was transiently transfected with pelr1 and the expression of elr1 on transfected cells was verified by western blot and indirect immunofluorescence assay (ifa). furthermore, ...200718271266
structural and functional studies of alix interactions with ypx(n)l late domains of hiv-1 and eiav.retrovirus budding requires short peptide motifs (late domains) located within the viral gag protein that function by recruiting cellular factors. the ypx(n)l late domains of hiv and other lentiviruses recruit the protein alix (also known as aip1), which also functions in vesicle formation at the multivesicular body and in the abscission stage of cytokinesis. here, we report the crystal structures of alix in complex with the ypx(n)l late domains from hiv-1 and eiav. the two distinct late domains ...200818066081
development of photoreceptor-specific promoters and their utility to investigate eiav lentiviral vector mediated gene transfer to photoreceptors.we wanted to investigate the ability of recombinant equine infectious anemia virus (eiav) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells.200717963276
comparative requirements for the restriction of retrovirus infection by trim5alpha and trimcyp.the restriction factors, trim5alpha in most primates and trimcyp in owl monkeys, block infection of various retroviruses soon after virus entry into the host cell. rhesus monkey trim5alpha (trim5alpha rh) inhibits human immunodeficiency virus (hiv-1) and feline immunodeficiency virus (fiv) more potently than human trim5alpha (trim5alpha hu). trimcyp restricts infection of hiv-1, simian immunodeficiency virus of african green monkeys (siv agm) and fiv. early after infection, trimcyp, like trim5al ...200717920096
envelope variation as a primary determinant of lentiviral vaccine efficacy.lentiviral envelope antigenic variation and associated immune evasion are believed to present major obstacles to effective vaccine development. although this perception is widely assumed by the scientific community, there is, to date, no rigorous experimental data assessing the effect of increasing levels of lentiviral env variation on vaccine efficacy. it is our working hypothesis that env is, in fact, a primary determinant of vaccine effectiveness. we previously reported that a successful expe ...200717846425
gag genetic heterogeneity of equine infectious anemia virus (eiav) in naturally infected horses in canada.gag genetic heterogeneity of equine infectious anemia virus (eiav) variants in naturally infected horses in canada was studied since very limited information is available on the variability of eiav gag sequences in public database. a phylogenetic analysis based on 414nts of gag gene sequences amplified by a nested polymerase chain reaction (pcr) revealed the distinct divergence of these variants compared to other published strains in a corresponding region. significant predicted amino acid seque ...200717767972
cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells.to determine the viability and potential usefulness of cryopreserved human primary cultured corneal endothelial cells by characterizing their morphology, gene expression, and ability for genetic modification by the lentiviral vector equine infectious anemia virus (eiav).200717591873
cyclin box structure of the p-tefb subunit cyclin t1 derived from a fusion complex with eiav tat.the positive transcription elongation factor b (p-tefb) is an essential regulator of viral gene expression during the life cycle of human immunodeficiency virus type 1 (hiv-1). its cyclin t1 subunit forms a ternary complex with the viral transcriptional transactivator (tat) protein and the transactivation response (tar) rna element thereby activating cyclin dependent kinase 9 (cdk9), which stimulates transcription at the level of chain elongation. we report the structure of the cyclin box domain ...200717540406
envelope-specific t-helper and cytotoxic t-lymphocyte responses associated with protective immunity to equine infectious anemia virus.equine infectious anemia virus (eiav) infection of horses provides a valuable model for examining the natural immunological control of lentivirus infection and disease and the mechanisms of protective and enhancing vaccine immunity. we have previously hypothesized that the eiav envelope (env) proteins gp90 and gp45 are major determinants of vaccine efficacy, and that the development of protective immunity by attenuated viral vaccines may be associated with the progressive redirection of immune r ...200717374779
correlation between the induction of th1 cytokines by an attenuated equine infectious anemia virus vaccine and protection against disease progression.the equine infectious anemia virus (eiav) donkey-leukocyte attenuated vaccine (dlv) has been used to protect against equine infectious anaemia (eia) disease for several decades in china. the attenuated mechanism and immunological protective mechanisms remain to be elucidated. to identify responses that correlate with the protection against disease, we immunized horses with dlv, followed by challenge with an eiav wild-type strain ln. all vaccinated horses were asymptomatic and had a low level of ...200717325374
oviduct-specific expression of two therapeutic proteins in transgenic hens.recent advances in avian transgenesis have led to the possibility of utilizing the laying hen as a production platform for the large-scale synthesis of pharmaceutical proteins. ovalbumin constitutes more than half of the protein in the white of a laid egg, and expression of the ovalbumin gene is restricted to the tubular gland cells of the oviduct. here we describe the use of lentiviral vectors to deliver transgene constructs comprising regulatory sequences from the ovalbumin gene designed to di ...200717259305
a single amino acid difference within the alpha-2 domain of two naturally occurring equine mhc class i molecules alters the recognition of gag and rev epitopes by equine infectious anemia virus-specific ctl.although ctl are critical for control of lentiviruses, including equine infectious anemia virus, relatively little is known regarding the mhc class i molecules that present important epitopes to equine infectious anemia virus-specific ctl. the equine class i molecule 7-6 is associated with the equine leukocyte ag (ela)-a1 haplotype and presents the env-rw12 and gag-gw12 ctl epitopes. some ela-a1 target cells present both epitopes, whereas others are not recognized by gag-gw12-specific ctl, sugge ...200617082657
lentiviral vector expressing retinoic acid receptor beta2 promotes recovery of function after corticospinal tract injury in the adult rat spinal cord.spinal cord injury often results in permanent and devastating neurological deficits and disability. this is due to the limited regenerative capacity of neurones in the central nervous system (cns). we recently demonstrated that a transcription factor retinoic acid receptor beta2 (rarbeta2) promoted axonal regeneration in adult sensory neurones located peripherally. however, it is not known if rarbeta2 can promote axonal regeneration in cortical neurones of the cns. here, we demonstrate that deli ...200616984961
the integration profile of eiav-based vectors.lentiviral vectors based on equine infectious anemia virus (eiav) stably integrate into dividing and nondividing cells such as neurons, conferring long-term expression of their transgene. the integration profile of an eiav vector was analyzed in dividing hek293t cells, alongside an hiv-1 vector as a control, and compared to a random dataset generated in silico. a multivariate regression model was generated and the influence of the following parameters on integration site selection determined: (a ...200616950499
long terminal repeats are not the sole determinants of virulence for equine infectious anemia virus.the long terminal repeats (ltrs) of equine infectious anemia virus donkey leukocyte-attenuated virus (eiav-dla) were substituted with those of the wild-type eiav-l (wt eiav-l, the parent virus of eiav-dla). the resulting chimeric plasmid was designated pok-ltr dla/l. purified pok-ltr dla/l was transfected into monocyte-derived macrophage (mdm) cultures prepared from eiav-negative, heparinized whole blood from a donkey. eighth-passage cell cultures developed the typical cytopathogenic effects (cp ...200716932982
[elevation of ifn-gamma transcription level in peripheral blood mononuclear cells of eiav vaccinated horses].to evaluate the relationship between the transcriptional level of ifn-gamma mrna in peripheral blood mononuclear cells (pbmc) and immune protective response driven by inoculated horses with donkey leukocyte attenuated vaccine of eiav(dlv), and to elucidate the immune mechanism of dlv.200616806002
eiav vector-mediated delivery of endostatin or angiostatin inhibits angiogenesis and vascular hyperpermeability in experimental cnv.we evaluated the efficacy of equine infectious anaemia virus (eiav)-based lentiviral vectors encoding endostatin (eiav.endostatin) or angiostatin (eiav.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laser-induced model of choroidal neovascularisation (cnv). equine infectious anaemia virus.endostatin, eiav.angiostatin or control (eiav.null) vectors were administered into the subretinal space of c57bl/6j mice. two weeks after laser injury cnv areas and the degree of ...200616572190
characterization of functional domains of equine infectious anemia virus rev suggests a bipartite rna-binding domain.equine infectious anemia virus (eiav) rev is an essential regulatory protein that facilitates expression of viral mrnas encoding structural proteins and genomic rna and regulates alternative splicing of the bicistronic tat/rev mrna. eiav rev is characterized by a high rate of genetic variation in vivo, and changes in rev genotype and phenotype have been shown to coincide with changes in clinical disease. to better understand how genetic variation alters rev phenotype, we undertook deletion and m ...200616571801
immunodiffusion studies of purified equine infectious anemia virus.antigenicity of purified equine infectious anemia (eia) virus was examined by immunodiffusion against sera obtained from horses experimentally infected with eia virus. the purified virus reacted with the infected horse serum, and virus-specific precipitating antibody was demonstrated. furthermore, it was found that purified eia virus reacted against the serum of horses infected with all strains of eia virus which were antigenically different from one another. from the result, group-specific comp ...197116557982
recruitment of the adaptor protein 2 complex by the human immunodeficiency virus type 2 envelope protein is necessary for high levels of virus release.the envelope (env) protein of human immunodeficiency virus type 2 (hiv-2) and the hiv-1 vpu protein stimulate the release of retroviral particles from human cells that restrict virus production, an activity that we call the enhancement of virus release (evr). we have previously shown that two separate domains in the hiv-2 envelope protein are required for this activity: a glycine-tyrosine-x-x-hydrophobic (gyxxtheta) motif in the cytoplasmic tail and an unmapped region in the ectodomain of the pr ...200616501101
do alix and alg-2 really control endosomes for better or for worse?alix/aip1 (alg-2-interacting protein x/apoptosis-linked-gene-2-interacting protein 1) is an adaptor protein that was first described for its capacity to bind to the calcium-binding protein alg-2 (apoptosis-linked gene 2), the expression of which seemed necessary for cell death. over-expression of truncated forms of alix blocks caspase-dependent and -independent mechanisms of cell death. numerous observations in yeast and in mammalian cells suggest that alix controls the making of and trafficking ...200616354163
restriction of feline immunodeficiency virus by ref1, lv1, and primate trim5alpha proteins.the ref1 and lv1 postentry restrictions in human and monkey cells have been analyzed for lentiviruses in the primate and ungulate groups, but no data exist for the third (feline) group. we compared feline immunodeficiency virus (fiv) to other restricted (human immunodeficiency virus type 1 [hiv-1], equine infectious anemia virus [eiav]) and unrestricted (nb-tropic murine leukemia virus [nb-mlv]) retroviruses across wide ranges of viral inputs in cells from multiple primate and nonprimate species ...200516306589
endocytosis and a low-ph step are required for productive entry of equine infectious anemia virus.recently, it has become evident that entry of some retroviruses into host cells is dependent upon a vesicle-localized, low-ph step. the entry mechanism of equine infectious anemia virus (eiav) has yet to be examined. here, we demonstrate that wild-type strains of eiav require a low-ph step for productive entry. lysosomotropic agents that inhibit the acidification of internal vesicles inhibited productive entry of eiav. the presence of ammonium chloride (30 mm), monensin (30 microm), or bafilomyc ...200516282447
evaluation of high functional avidity ctl to gag epitope clusters in eiav carrier horses.cytotoxic t lymphocytes (ctl) are critical for lentivirus control including eiav. since ctl from most eiav carrier horses recognize gag epitope clusters (ec), the hypothesis that carrier horses would have high functional avidity ctl to optimal epitopes in gag ec was tested. twenty-two optimal ec epitopes were identified; two in ec1, six in ec2, and seven each in ec3 and 4. however, only five of nine horses had high functional avidity ctl (<or=11 nm) recognizing six epitopes in ec; four in relati ...200516139857
comparison of hiv- and eiav-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.the use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. vectors based on alternative lentiviruses would theoretically be safer than hiv-1-based vectors and could also be used in hiv-positive patients, minimizing the risk of generating replication-competent virus. here we report the use of third-generation equine infectious anemia virus (eiav)- and hiv-1-based vecto ...200516099415
lymphocyte proliferation responses induced to broadly reactive th peptides did not protect against equine infectious anemia virus challenge.the effect of immunization with five lipopeptides, three containing t-helper (th) epitopes and two with both th and cytotoxic t-lymphocyte (ctl) epitopes, on equine infectious anemia virus (eiav) challenge was evaluated. peripheral blood mononuclear cells from eiav lipopeptide-immunized horses had significant proliferative responses to th peptides compared with those preimmunization, and the responses were attributed to significant responses to peptides gag from positions 221 to 245 (gag 221-245 ...200516085917
a tumor necrosis factor receptor family protein serves as a cellular receptor for the macrophage-tropic equine lentivirus.characterization of cellular receptors for human, simian, and feline immunodeficiency viruses that are tropic for lymphocytes and macrophages have revealed a common theme of a sequential binding of viral envelope proteins with two coreceptors to mediate virus infection of target cells. in contrast to these dual tropic immunodeficiency viruses, the ungulate lentiviruses, including equine infectious anemia virus (eiav), exclusively infect cells of the monocyte-macrophage lineage to cause progressi ...200515985554
comparison of hiv-1 and eiav-based lentiviral vectors in corneal transduction.in this study we compare the ability of self-inactivating human immunodeficiency virus 1 (hiv-1) and equine infectious anaemia virus (eiav)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. both vectors were pseudotyped with vesicular stomatitis virus-g (vsv-g) envelope and contained marker transgenes under the control of an internal cmv promoter. for specificity of action, the heterologous promoter in the eiav-vector was exchanged ...200515939034
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
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
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
specificity of serum neutralizing antibodies induced by transient immune suppression of inapparent carrier ponies infected with a neutralization-resistant equine infectious anemia virus envelope strain.it 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
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
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
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
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
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
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
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
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
attempts to adapt equine infectious anemia virus to small animals. 195014792980
aip1/alix is a binding partner for hiv-1 p6 and eiav p9 functioning in virus budding.hiv-1 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
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
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
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
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
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
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
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
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
design, production, safety, evaluation, and clinical applications of nonprimate lentiviral vectors. 200211883086
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
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
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
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
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
functional roles of equine infectious anemia virus gag p9 in viral budding and infection.previous studies utilizing gag polyprotein budding assays with transfected cells reveal that the equine infectious anemia virus (eiav) gag p9 protein provides a late assembly function mediated by a critical y(23)p(24)d(25)l(26) motif (l-domain) to release viral particles from the plasma membrane. to elucidate further the role of eiav p9 in virus assembly and replication, we have examined the replication properties of a defined series of p9 truncation and site-directed mutations in the context of ...200111559809
binding sites for rev and asf/sf2 map to a 55-nucleotide purine-rich exonic element in equine infectious anemia virus rna.the equine infectious anemia virus (eiav) rev protein (erev) negatively regulates its own synthesis by inducing alternative splicing of its mrna. this bicistronic mrna contains four exons; exons 1 and 2 encode tat, and exons 3 and 4 encode rev. when rev is expressed, exon 3 is skipped to produce an mrna that contains only exons 1, 2, and 4. the interaction of erev with its cis-acting rna response element, the rre, is also essential for nuclear export of intron-containing viral mrnas that encode ...200111278454
differential responses of equus caballus and equus asinus to infection with two pathogenic strains of equine infectious anemia virus.most in vivo studies with equine infectious anemia virus (eiav) have been performed in horses and ponies (equus caballus) with little published information available detailing the clinical responses of donkeys (equus asinus) to infection with this virus. consequently, donkeys were inoculated with two strains of eiav (eiav(pv) and eiav(wy)) which have been documented to produce disease in e. caballus. four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with eiav(pv) and one ...200111230932
antigenic drift of viruses within a host: a finite site model with demographic stochasticity.we theoretically study the antigenic drift of viruses within an infected host, as observed in human immunodeficiency virus (hiv) and equine infectious anemia virus (eiav) infections, assuming that a finite number of antigen-determining sites at the viral envelop gene are responsible for the specific immune response. the pattern of antigen evolution becomes more complex than that predicted from the previous one-dimensional antigen space models. if the viral growth rate is sufficiently large, the ...200011029069
an in vitro transcription system that recapitulates equine infectious anemia virus tat-mediated inhibition of human immunodeficiency virus type 1 tat activity demonstrates a role for positive transcription elongation factor b and associated proteins in the mechanism of tat activation.equine infectious anemia virus (eiav) activates transcription via a tat protein, a tar element, and the equine elongation factor positive transcription elongation factor b (p-tefb). in human cells, eiav tat (etat) can inhibit the ability of human immunodeficiency virus type 1 (hiv-1) tat (htat) to activate transcription from the hiv-1 long terminal repeat, demonstrating that eiav tat can interact nonproductively with human p-tefb. to study the mechanism of eiav tat and hiv-1 tat activation, we d ...200010964778
general effect of sam68 on rev/rex regulated expression of complex retroviruses.we have previously demonstrated that overexpression of sam68 functionally substitutes for, as well as synergizes with, hiv-1 rev in rre-mediated gene expression and virus replication. in addition, c-terminal deletion mutants of sam68 exhibit a transdominant negative phenotype in hiv replication. we now report that sam68 also enhances the activities of rev-like proteins of other complex retroviruses (e.g. htlv-1 and eiav) on their respective rna targets. furthermore, we demonstrate that sam68 can ...200010962565
mutations occurring during serial passage of japanese equine infectious anemia virus in primary horse macrophages.an attenuated equine infectious anemia virus (eiav), named v26, was previously obtained after 50 passages of the japanese virulent strain v70 in primary macrophage culture. to clarify the differences between both viruses, their full-length sequences were determined. there were higher mutations in s2 (6.15% amino acid difference) and ltr (10.7% nucleotide difference). the presumed initiation codon of the s2 gene was absent from the sequence of v26. there was a large insertion within the long-term ...200010930666
immune responses and viral replication in long-term inapparent carrier ponies inoculated with equine infectious anemia virus.persistent infection of equids by equine infectious anemia virus (eiav) is typically characterized by a progression during the first year postinfection from chronic disease with recurring disease cycles to a long-term asymptomatic infection that is maintained indefinitely. the goal of the current study was to perform a comprehensive longitudinal analysis of the course of virus infection and development of host immunity in experimentally infected horses as they progressed from chronic disease to ...200010846078
binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mrnas.in addition to facilitating the nuclear export of incompletely spliced viral mrnas, equine infectious anemia virus (eiav) rev regulates alternative splicing of the third exon of the tat/rev mrna. in the presence of rev, this exon of the bicistronic rna is skipped in a fraction of the spliced mrnas. in this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for rev binding and transport of incompletely spliced rna. the presence of a purine-rich exon spli ...200010779344
suboptimal splice sites of equine infectious anaemia virus control rev responsiveness.the rev protein of equine infectious anaemia virus (eiav) was shown previously to stimulate the expression of a heterologous cat reporter gene when the 3' half of the eiav genome was present downstream in cis. however, computer analysis could not reveal the existence of a stable rna secondary structure that could be analogous to the rev-responsive element of other lentiviruses. in the present study, the inhibitory rna element designated the cis-acting repressing sequence (crs) has been localized ...200010769069
aminoglycoside-arginine conjugates that bind tar rna: synthesis, characterization, and antiviral activity.regulation of hiv gene expression is crucially dependent on binding of the trans-activator protein, tat, to the trans-activation response rna element, tar, found at the 5' end of all hiv-1 transcripts. tat-tar interaction is mediated by a short arginine-rich domain of the protein. disruption of this interaction could, in theory, create a state of complete viral latency. a new class of small-molecule peptidomimetic tar rna binders, conjugates of aminoglycosides and arginine, was recently designed ...200010715103
structural and biochemical studies of retroviral proteases.retroviral proteases form a unique subclass of the family of aspartic proteases. these homodimeric enzymes from a number of viral sources have by now been extensively characterized, both structurally and biochemically. the importance of such knowledge to the development of new drugs against aids has been, to a large extent, the driving force behind this progress. high-resolution structures are now available for enzymes from human immunodeficiency virus types 1 and 2, simian immunodeficiency viru ...200010708846
canine cyclin t1 rescues equine infectious anemia virus tat trans-activation in human cells.human immunodeficiency virus-1 tat protein and human cyclin t1 mediate transcriptional activation by enhancing the elongation efficiency of rna polymerase ii. activation of transcription of the related equine infectious anemia virus (eiav) requires a similar protein known as etat, which does not function in human cells. expression of equine cyclin t1 in human cells rescues etat function, suggesting a general mechanism of transcription activation among lentiviruses. here we present the cloning of ...200010683321
effects of long terminal repeat sequence variation on equine infectious anemia virus replication in vitro and in vivo.the long terminal repeat (ltr) is reported to be one of the most variable portions of the equine infectious anemia virus (eiav) genome. to date, however, no information is available on the effects of observed sequence variations on viral replication properties, despite a widespread assumption of the biological importance of eiav ltr variation. eiav ltr sequence variability is confined mostly to a small portion of the enhancer within the u3 segment of the ltr. analysis of published eiav ltr seque ...199910544113
long terminal repeat sequences of equine infectious anaemia virus are a major determinant of cell tropism.the wyoming strain of equine infectious anaemia virus (eiav) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. in contrast, wyoming-derived fibroblast-adapted eiav strains (malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines fea and cf2th. wyoming and malmquist viruses differ extensively both in long terminal repeat (ltr) and envelope region sequences. we have c ...199910092016
increased interleukin-6 activity in the serum of ponies acutely infected with equine infectious anaemia virus.seven ponies were infected with the virulent wild-type wyoming strain of equine infectious anaemia virus (eiav). infection status was monitored by serum reverse transcriptase activity, rectal temperature, and complete blood count. preinfection serum and serum obtained during the initial febrile episode following infection were assayed for interleukin 6 (il-6) activity. postinfection il-6 activity was significantly increased as compared to preinfection values. the magnitude of increase in il-6 wa ...199910088717
photoactivated virucidal properties of tridentate 2,2'-dihydroxyazobenzene and 2-salicylideneaminophenol platinum pyridine complexes.the potent photoactivated virucidal activity of tridentate 2,2'-dihydroxyazobenzene- and 2-salicylideneaminophenol platinum pyridine complexes 1, 2, 4, 6, 7, 9, and 10 against enveloped viruses (e.g., eiav, hiv, and hsv) is described.199910021936
identification of retroviral late domains as determinants of particle size.retroviral gag proteins, in the absence of any other viral products, induce budding and release of spherical, virus-like particles from the plasma membrane. gag-produced particles, like those of authentic retrovirions, are not uniform in diameter but nevertheless fall within a fairly narrow distribution of sizes. for the human immunodeficiency virus type 1 (hiv-1) gag protein, we recently reported that elements important for controlling particle size are contained within the c-terminal region of ...19999971814
analysis of the polymerization kinetics of homodimeric eiav p51/51 reverse transcriptase implies the formation of a polymerase active site identical to heterodimeric eiav p66/51 reverse transcriptase.homodimeric eiav p51/51 and heterodimeric eiav p66/51 reverse transcriptase were purified in order to compare the different modes of dna synthesis supported by the enzymes. analysis of the dimerization behavior of the eiav enzymes indicates that the dimer stability of eiav reverse transcriptase enzymes is higher than that of their hiv-1 reverse transcriptase counterparts. eiav p51/51 polymerizes dna distributively whereas dna synthesis by eiav p66/51 is processive. steady-state and pre-steady-st ...19989724526
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