表达马传染性贫血病毒受体和荧光素酶报告基因的293细胞系的建立

为了在体外精确、简便地测定马传染性贫血病毒(EIAV)的中和抗体和研究不同毒株与受体的亲和性,克隆了马慢病毒受体1(ELR1)cDNA并插入真核表达载体pcDNA3.1( ),构建了表达载体pELR1。该载体瞬时转染293细胞后,经Western blot和间接免疫荧光(IFA)检测,确认了ELR1的表达。在pELR1质粒的基础上,插入EIAV疫苗株前病毒基因组转录调控区LTR以及萤火虫荧光素酶报告基因(Luc)构建了表达载体pELR1-LTR-Luc,并转染293细胞,建立了ELR1-LTR-Luc(293-E)细胞系。该细胞系能稳定表达ELR1基因,并且能在LTR的调控下表达萤火虫荧光素酶基因。用1000TCID50的EIAV驴胎皮肤细胞疫苗株D18V13接种该细胞,24h后检测其荧光素酶活性是未接毒对照的3.15倍。同时用IFA检测证明了病毒在细胞内的增殖。EIAV强毒株L21的接毒试验显示,ELR1-LTR(293-E)细胞的萤火虫荧光素酶活性与该毒株的接毒量在10-2~10-7稀释范围内呈正相关。该细胞系传35代后,外源基因的表达特征未发生改变。该细胞系的建立为进一步开展EIAV与细胞受体相互作用以及中和抗体评价等研究奠定了重要基础。     

Mapping of equine lentivirus receptor 1 residues critical for equine infectious anemia virus envelope binding

The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complementary exchanges of the respective cysteine-rich domains (CRD) between the ELR1 and murine HveA proteins revealed CRD1 as the predominant determinant of functional gp90 binding to ELR1 and also to a chimeric murine HveA protein expressed on the surface of transfected Cf2Th cells. Mutations of individual amino acids in the CRD1 segment of ELR1 and murine HveA indicated the Leu70 in CRD1 as essential for functional binding of EIAV gp90 and for virus infection of transduced Cf2Th cells. The specificity of the EIAV SU binding domain identified for the ELR1 receptor is fundamentally identical to that reported previously for functional binding of feline immunodeficiency virus SU to its coreceptor CD134, another TNFR protein. These results indicate unexpected common features of the specific mechanisms by which diverse lentiviruses can employ TNFR proteins as functional receptors.     

An equine infectious anemia virus variant superinfects cells through novel receptor interactions

Wild-type strains of equine infectious anemia virus (EIAV) prevent superinfection of previously infected cells. A variant strain of virus that spontaneously arose during passage, EIAV_vMA-1c, can circumvent this mechanism in some cells, such as equine dermis (ED) cells, but not in others, such as equine endothelial cells. EIAV_vMA-1c superinfection of ED cells results in a buildup of unintegrated viral DNA and rapid killing of the cell monolayer. Here, we examined the mechanism of resistance that is used by EIAV to prevent superinfection and explored the means by which EIAV_vMA-1c overcomes this restriction. We found that the cellular receptor used by EIAV, equine lentivirus receptor 1 (ELR1), remains on the surface of cells chronically infected with EIAV, suggesting that wild-type EIAV interferes with superinfection by masking ELR1. The addition of soluble wild-type SU protein to the medium during infection blocked infection by wild-type strains of virus, implicating SU as the viral protein responsible for interfering with virion entry into previously infected cells. Additionally, interference of wild-type EIAV binding to ELR1 by the addition of either anti-ELR1 antibodies or the ELR1 ectodomain prevented entry of the wild-type strains of EIAV into two permissive cell populations. Many of these same interference treatments prevented EIAV_vMA-1c infection of endothelial cells but only modestly affected the ability of EIAV_vMA-1c to enter and kill previously infected ED cells. These findings indicate that EIAV_vMA-1c retains the ability to use ELR1 for entry and suggest that this virus can interact with an additional, unidentified receptor to superinfect ED cells.     

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, differential levels of restriction for EIAV and human immunodeficiency virus type 1 (HIV-1) replication in murine cells and suggest that these differences can be exploited to reveal critical virus-cell interactions required for HIV-1 assembly and budding of lentivirus particles.     

Identification and characterization of a common B-cell epitope on EIAV capsid proteins

Applied Microbiology and Biotechnology - The equine infectious anemia virus (EIAV) capsid protein (p26) is one of the major immunogenic proteins during EIAV infection and is widely used for the...     

Structural insight into equine lentivirus receptor 1

Equine lentivirus receptor 1 (ELR1) has been identified as a functional cellular receptor for equine infectious anemia virus (EIAV). Herein, recombinant ELR1 and EIAV surface glycoprotein gp90 were respectively expressed in Drosophila melanogaster S2 cells, and purified to homogeneity by Ni-NTA affinity chromatography and gel filtration chromatography. Gel filtration chromatography and analytical ultracentrifugation (AUC) analyses indicated that both ELR1 and gp90 existed as individual monomers in solution and formed a complex with a stoichiometry of 1:1 when mixed. The structure of ELR1 was first determined with the molecular replacement method, which belongs to the space group P4₂2₁2 with one molecule in an asymmetric unit. It contains eight antiparallel β-sheets, of which four are in cysteine rich domain 1 (CRD1) and two are in CRD2 and CRD3, respectively. Alignment of ELR1 with HVEM and CD134 indicated that Tyr61, Leu70, and Gly72 in CRD1 of ELR1 are important residues for binding to gp90. Isothermal titration calorimetry (ITC) experiments further confirmed that Leu70 and Gly72 are the critical residues.     

Translation of equine infectious anemia virus bicistronic tat-rev mRNA requires leaky ribosome scanning of the tat CTG initiation codon.

We have examined the translational regulation of the equine infectious anemia virus (EIAV) bicistronic tat-rev mRNA. Site-directed mutagenesis of the tat leader region followed by expression of the tat-rev cDNA both in vitro and in transiently transfected cells established that tat translation is initiated exclusively at a CTG codon. Increasing the efficiency of tat translation by altering the CTG initiator to ATG resulted in a dramatic decrease in translation of the downstream (rev) cistron, indicating that leaky scanning of the tat CTG initiation codon permitted translation of the downstream rev cistron. Since the tat leader sequences precede the major EIAV splice donor and are therefore present at the 5' termini of both spliced and unspliced viral mRNAs, the expression of all EIAV structural and regulatory proteins is dependent on leaky scanning of the tat initiator.     

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, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.     

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 cell death, enhanced fusogenic activity, and increased rates of spread in equine fibroblasts compared to other strains of EIAV. The highly cytolytic nature of vMA-1c suggested that this strain might be superinfecting equine fibroblasts. Receptor interference studies demonstrated that prior infection of equine fibroblasts with EIAV did not alter the ability of vMA-1c to infect and kill these cells. In similar studies in a canine fibroblast cell line, receptor interference did occur. vMA-1c infection of equine fibroblasts was also associated with large quantities of unintegrated viral DNA, a well-established hallmark of retroviral superinfection. Cloning of the vMA-1c genome identified nucleotide changes that would result in at least one amino acid change in all viral proteins. A chimeric infectious molecular clone containing the vMA-1c tat, S2, and env open reading frames recapitulated most of the characteristics of vMA-1c, including superinfection, fibroblast killing, and fusogenic activity. In summary, in vitro selection for a strain of EIAV that rapidly killed cells resulted in the generation of a virus that was able to superinfect these cells, presumably by the use of a novel mechanism of cell entry. This phenotype mapped to the 3' half of the genome.     

Antigenic analysis of equine infectious anemia virus (EIAV) variants by using monoclonal antibodies: epitopes of glycoprotein gp90 of EIAV stimulate neutralizing antibodies.

Monoclonal antibodies produced against the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV), a lentivirus, were studied for reactivity with the homologous prototype and 16 heterologous isolates. Eighteen hybridomas producing monoclonal antibodies (MAbs) were isolated. Western blot (immunoblot) analyses indicated that 10 were specific for the major envelope glycoprotein (gp90) and 8 for the transmembrane glycoprotein (gp45). Four MAbs specific to epitopes of gp90 neutralized prototype EIAV infectivity. These neutralizing MAbs apparently reacted with variable regions of the envelope gp90, as evidenced by their unique reactivity with the panel of isolates, suggesting recognition of at least three different neutralization epitopes. The conformation of these epitopes appears to be continuous, as they resisted treatment with sodium dodecyl sulfate and reducing reagents. Monoclonal antibodies that reacted with conserved epitopes on gp90 or gp45 failed to neutralize EIAV. Our data also demonstrated that there was a large spectrum of possible EIAV serotypes and confirmed that antigenic variation occurs with high frequency in EIAV. Moreover, the data showed that variation is a rapid and random process, as no pattern of variant evolution was evident by comparison of 13 isolates from parallel infections. These results represent the first production of neutralizing MAbs specific for a lentivirus glycoprotein and document alterations in one or more neutralization epitopes of the major surface glycoprotein among sequential isolates of EIAV recovered during persistent infection.     

马传染性贫血强/弱毒嵌合病毒的体外构建

马传染性贫血病毒(equine infectious anemia virus,EIAV)引起马传染性贫血(简称马传贫),导致马持续性感染和反复病毒血症[1].EIAV与人免疫缺陷病毒Ⅰ型(HIV-1)同属反转录病毒科慢病毒属,二者有很多相似的特性[2].在反转录病毒前病毒基因组两端含有长末端重复序列(long terminal repeat,LTR).LTR含有真核启动子,其中含有病毒转录调控顺式作用位点,病毒编码的反式作用因子与其结合后可以反式激活基因的表达,对病毒基因的表达和其它生命活动起重要调控作用[3,4].因此,LTR序列的变异可能会引起病毒转录和复制方式的改变,进而引起其细胞嗜性和致病性的改变[5,6].为了探讨LTR在EIAV病毒复制和转录过程中的作用,并进一步研究EIAV的致病和免疫机制,用EIAV强毒L株LTR置换了以前构建的EIAV DLA(弱毒)感染性分子克隆中的LTR,构建了马传贫强/弱毒嵌合分子克隆,并获得了具有感染性的强/弱毒嵌合病毒.     

Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production

We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on these observations, we sought in the current study to identify the precise defect in K30 virion infection of permissive equine dermal (ED) cells. The results of these experiments clearly demonstrated that K30 virions entered target ED cells and produced early (minus-strand strong-stop) and late (Gag) viral DNA products as efficiently as did the replication-competent E32 mutant and parental EIAV(UK) viruses. However, in contrast to the replication-competent E32 mutant and parental viruses, infection with K30 mutant virus failed to produce detectable two-long-terminal-repeat DNA circles, stable integrated provirus, virus-specific Gag mRNA expression, or intracellular viral protein expression. Taken together, these data demonstrate that the K30 mutant is defective in the ability to produce sufficient nuclear viral DNA to establish a productive infection in ED cells. Thus, these observations indicate for the first time that the EIAV Gag p9 protein performs a critical role in viral DNA production and processing to provirus during EIAV infection, in addition to its previously defined role in viral budding mediated by the p9 L domain.     

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 a reference infectious molecular proviral clone, EIAV(uk). Characterization of these p9 proviral mutants revealed new functional properties of p9 in EIAV replication, not previously elucidated by Gag polyprotein budding assays. The results of these studies demonstrated that only the N-terminal 31 amino acids of a total of 51 residues in the complete p9 protein were required to maintain replication competence in transfected equine cells; proviral mutants with p9 C-terminal truncations of 20 or fewer amino acids remained replication competent, while mutants with truncations of 21 or more residues were completely replication defective. The inability of the defective p9 proviral mutations to produce infectious virus could not be attributed to defects in Gag polyprotein expression or processing, in virion RT activity, or in virus budding. While proviral replication competence appeared to be associated with the presence of a K(30)K(31) motif and potential ubiquitination of the EIAV p9 protein, mutations of these lysine residues to methionines produced variant proviruses that replicated as well as the parental EIAV(uk) in transfected ED cells. Thus, these observations reveal for the first time that EIAV p9 is not absolutely required for virus budding in the context of proviral gene expression, suggesting that other EIAV proteins can at least in part mediate late budding functions previously associated with the p9 protein. In addition, the data define a function for EIAV p9 in the infectivity of virus particles, indicating a previously unrecognized role for this Gag protein in EIAV replication.     

Systematic epitope analysis of the p26 EIAV core protein

The major core protein of equine infectious anemia virus (EIAV), p26, is one of the primary immunogenic structural proteins during a persistent infection of horses and is highly conserved among antigenically variants of viral isolates. In order to investigate its immune profile in more detail for a better diagnostic, an epitope mapping was carried out by means of two libraries of overlapping peptide fragments prepared by simultaneous and parallel SPPS on derivatized cellulose membranes (SPOT synthesis). Polyclonal equine sera from infected horses were used for the biological assay. Particularly two promising continuous epitopes (NAMRHL and MYACRD) were localized on the C-terminal extreme of p26, region 194-222. A cyclic synthetic fragment of 29 amino acid residues containing the identified epitopes was designed and studied. A significant conformational change towards a helical structure was observed when the peptide was cyclized by a bridge between Cys198 and Cys218. This observation correlated with an improvement of its ability to be recognized by specific antibodies in an EIA (Enzyme-linked Immunosorbent assay). These results suggest that the conformationally restricted synthetic antigen adequately mimics the native structure of this region of p26 core protein.     

Development of photoreceptor-specific promoters and their utility to investigate EIAV lentiviral vector mediated gene transfer to photoreceptors

BACKGROUND: 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. METHODS: Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line. The optimal promoters from this screen were then cloned into the recombinant EIAV vector for evaluation in vivo following subretinal delivery into mice. RESULTS: All promoters maintained a photoreceptor-specific expression profile in vitro and the gene expression was further enhanced in combination with the IRBP enhancer. The use of IRBP-combined RHO or PDE promoters showed modest but exclusive expression in photoreceptors following subretinal delivery to mice. By contrast an EIAV vector containing the cytomegalovirus (CMV) promoter drove reporter gene expression in both photoreceptors and retinal pigment epithelium. CONCLUSIONS: It may be possible to use recombinant EIAV vectors containing photoreceptor-specific promoters to drive therapeutic gene expression to treat a range of retinal degenerative diseases where the photoreceptor cell is the primary disease target.