The S2 Gene of Equine Infectious Anemia Virus Is Dispensable for Viral Replication In Vitro

Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAV_UK. Various EIAV_UK mutants lacking S2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAV_UK mutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAV S2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro.     

Equine infectious anemia virus entry occurs through clathrin-mediated endocytosis

Entry of wild-type lentivirus equine infectious anemia virus (EIAV) into cells requires a low-pH step. This low-pH constraint implicates endocytosis in EIAV entry. To identify the endocytic pathway involved in EIAV entry, we examined the entry requirements for EIAV into two different cells: equine dermal (ED) cells and primary equine endothelial cells. We investigated the entry mechanism of several strains of EIAV and found that both macrophage-tropic and tissue culture-adapted strains utilize clathrin-coated pits for entry. In contrast, a superinfecting strain of EIAV, EIAV_vMA-1c, utilizes two mechanisms of entry. In cells such as ED cells that EIAV_vMA-1c is able to superinfect, viral entry is pH independent and appears to be mediated by plasma membrane fusion, whereas in cells where no detectable superinfection occurs, EIAV_vMA-1c entry that is low-pH dependent occurs through clathrin-coated pits in a manner similar to wild-type virus. Regardless of the mechanism of entry being utilized, the internalization kinetics of EIAV is rapid with 50% of cell-associated virions internalizing within 60 to 90 min. Cathepsin inhibitors did not prevent EIAV entry, suggesting that the low-pH step required by wild-type EIAV is not required to activate cellular cathepsins.     

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.     

Biological Characterization of Rev Variation in Equine Infectious Anemia Virus

Sequence analysis identified significant variation in the second exon of equine infectious anemia virus (EIAV) rev. Functional analysis indicated that limited amino acid variation in Rev significantly altered the export activity of the protein but did not affect Rev-dependent alternative splicing. EIAV Rev can mediate export through two independent cis-acting Rev-responsive elements (RREs), and differences among Rev variants were more pronounced when both RREs were present. Variation in Rev may be an important mechanism for regulation of virus replication in vivo and may contribute to changes in clinical disease.     

High-Efficiency Rescue of Equine Infectious Anemia Virus from a CMV-Driven Infectious Clone

<正>Dear Editor,Equine infectious anemia virus (EIAV) belongs to the macrophage-tropic lentiviruses family and infects mainly equines, including horses, donkeys and mules. EIAV shares many similar characteristics in its viral biology and hostvirus immune regulation with other lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), and has been accepted as a     

Disease Induction by Virus Derived from Molecular Clones of Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and rev sequences.     

The surface envelope protein gene region of equine infectious anemia virus is not an important determinant of tropism in vitro.

Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms.     

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.     

Genomic quasispecies associated with the initiation of infection and disease in ponies experimentally infected with equine infectious anemia virus.

Equine infectious anemia virus (EIAV) provides a uniquely dynamic system in which to study the mechanism and role of genomic variation in lentiviral persistence and pathogenesis. We have used a Shetland pony model of infection to investigate the association of specific long terminal repeat (LTR) and env gene genomic sequences with the initiation of infection and the onset of disease. We analyzed viral RNA isolated from a pathogenic stock of virus (EIAV PV) and from plasma taken during the first disease episode from two ponies infected with EIAV PV. Overall sequence variation within gp90 was low in EIAV PV and only slightly higher in plasma virus samples isolated from ponies during the first disease episode. However, a high proportion of mutations were localized to the principal neutralizing domain in EIAV PV and to the principal neutralizing domain and the gp90 hypervariable region in the two pony-derived samples. The rate of fixation of mutations was analyzed and determined to be approximately 4 x 10(-2) mutations per site per year. Sequence diversity within the U3 region of the LTR was extremely low, which suggested that the previously reported hypervariability of this region may be a consequence of selection for replication of EIAV in different host cells. The predominant EIAV PV env and LTR sequences were used to construct chimeric viruses so that the contribution of these sequences to viral pathogenicity could be examined. The chimeras replicated in cultured equine monocytes to the same extent as the parental nonpathogenic virus and did not cause disease in Shetland ponies by 120 days postinfection, suggesting that the EIAV genomic determinants of pathogenesis are complex.     

马传染性贫血病毒N-连接糖基化回复突变的感染性克隆构建

根据马传贫强毒株EIAV-L和疫苗株EIAV-FDD表面蛋白gp90的N-连接糖基化的变化规律,采用PCR定点突变的方法,对全长感染性克隆pLGFD3-8上的N-连接糖基化的差异区域进行改造后,构建成含有3个N-连接糖基化位点突变的感染性克隆pLGNl91N236N246.将其转染驴胎皮肤细胞(FDD),通过用逆转录酶活性、间接免疫荧光和RT-PCR方法检测而确定其感染性.结果表明,在FDD细胞中盲传三代后,在细胞培养物中可检测到逆转录酶活性,RT-PCR和间接免疫荧光检测均呈阳性,电镜下见到典型的EIAV颗粒.这一结果可能对N-连接糖基化在我国马传贫弱毒疫苗致弱机理的作用研究而奠定良好的基础.     

马传染性贫血病毒基因非编码区LTR嵌合克隆的构建

在已有全长感染性克隆pLGFD3 8 和pD70344 的基础上,根据马传贫弱毒疫苗致弱过程中不同代次毒株LTR序列的分析,在LTR U3区选取特定的酶切位点对EIAV非编码区LTR基因进行了部分替换。将替换的全基因克隆转染驴胎皮肤细胞(FDD)并以驴白细胞(DL)传代,用逆转录酶活性检测、RT PCR方法及Real time RT PCR验证其感染性。结果发现,其衍生病毒感染上述两种细胞均出现明显的细胞病变;细胞培养上清可检测到RT酶活性和RT PCR阳性。电镜下可见大量典型的EIAV颗粒。pLGFD9 12 嵌合克隆衍生病毒与其父本克隆衍生病毒pLGFD3 8具有相似的复制水平,pLGFD9 12嵌合克隆衍生病毒在DL细胞上复制水平略高于FDD细胞。此结果为进一步深入研究LTR对马传染性贫血病毒复制水平和毒力的影响奠定了基础。     

中国株EIAV S2基因逆向突变感染性克隆的构建及体外感染性评价

以中国株EIAV的驴强毒株EIAVDV115与疫苗株EIAVFDDV15的S2基因变化规律为依据,利用反向遗传技术对EIAV弱毒疫苗株全基因组感染性克隆pFDDV3-8的S2基因区进行逆向突变,构建了含有强毒株EIAVDV115S2基因区内4个稳定点突变的克隆质粒pFDDVS2r1-3-4-5,将该质粒转染FDD后进行体外继代盲传,经RT-PCR、逆转录酶活性、间接免疫荧光等检测后,显示经EIAV克隆质粒pFDDVS2r1-3-4-5转染的FDD盲传3代后,可在转染的FDD细胞培养物的上清液中检测到EIAV逆转录酶活性;该细胞培养物经RT-PCR和间接免疫荧光检测均呈EIAV阳性;在电镜下可观察到典型EIAV粒子。证明已成功拯救经EIAVS2基因逆向突变后的衍生病毒vpFDDVS2r1-3-4-5。比较vpFDDVS2r1-3-4-5衍生病毒与亲本克隆衍生病毒的复制动力学,表明前者的复制比后者略滞后。以上结果提示,对疫苗株S2基因的突变未明显影响EIAV的体外复制。     

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.     

Plasma Membrane-Targeted Raf Kinase Activates NF-κB and Human Immunodeficiency Virus Type 1 Replication in T Lymphocytes

Increasing evidence points to a role of the mitogenic Ras/Raf/MEK/ERK signaling cascade in regulation of human immunodeficiency virus type 1 (HIV-1) gene expression. Stimulation of elements of this pathway leads to transactivation of the HIV-1 promoter. In particular, the NF-κB motif in the HIV long terminal repeat (LTR) represents a Raf-responsive element in fibroblasts. Regulation of the Raf kinase in T cells differs from findings with a variety of cell lines that the catalytic domain of Raf (Raf_Δ26–303) shows no activity. In this study, we restored the activity of the kinase in T cells by fusing its catalytic domain to the CAAX motif (-Cx) of Ras, thus targeting the enzyme to the plasma membrane. Constitutive activity of Raf was demonstrated by phosphorylation of mitogen-activated protein kinase kinase (MEK) and endogenous mitogen-activated protein kinase 1/2 (ERK1/2) in A3.01 T cells transfected with Raf_Δ26–303-Cx. Membrane-targeted Raf also stimulates NF-κB, as judged by κB-dependent reporter assays and enhanced NF-κB p65 binding on band shift analysis. Moreover, we found that active Raf transactivates the HIV_NL4-3 LTR in A3.01 T lymphocytes and that dominant negative Raf (C4) blocked 12-O-tetradecanoylphorbol-13-acetate induced transactivation. When cotransfected with infectious HIV_NL4-3 DNA, membrane-targeted Raf induces viral replication up to 10-fold over basal levels, as determined by the release of newly synthesized p24^gag protein. Our study clearly demonstrates that the activity of the catalytic domain of Raf in A3.01 T cells is dependent on its cellular localization. The functional consequences of active Raf in T lymphocytes include not only NF-κB activation and transactivation of the HIV_NL4-3 LTR but also synthesis and release of HIV particles.