Caughman G B, Lewis J B, Smith R H, Harty R N, O'Callaghan D J
Department of Oral Biology/Microbiology, Medical College of Georgia, Augusta 30912-1126, USA.
J Virol. 1995 May;69(5):3024-32. doi: 10.1128/JVI.69.5.3024-3032.1995.
During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro-translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.
在裂解感染期间，马疱疹病毒1型（EHV-1）的立即早期（IE）基因（IR1）产生两种转录本：一种单一的、剪接后的6.0 kb IE mRNA和一种3' 共末端的4.4 kb早期mRNA（IR2）。先前的研究表明，瞬时表达的IR1和IR2基因产物是有效的转录调节因子：IR1蛋白能够反式激活代表性的EHV-1早期和晚期启动子，而IR1蛋白和缺少IR1氨基酸残基1至322的IR2产物都能反式抑制IR1启动子。在本研究中，开发了针对主要IE蛋白IE1的单克隆抗体（MAb），对其检测IR1和IR2产物的能力进行了表征，并用于检查细胞外病毒粒子中是否存在与IE1相关的蛋白，以及确定EHV-1感染细胞中IR1和IR2蛋白的合成及细胞内分布。结果表明：（i）代表三个非竞争性表位结合组的抗IE1 MAb与多种IE蛋白种类反应，也与一种被鉴定为推定的IR2基因产物的146 kDa早期蛋白反应；（ii）三个反应性表位定位于IR1中跨越氨基酸323至552的区域；（iii）抗IE1 MAb与144 kDa体外翻译的IR2产物以及与大小相似的瞬时表达的IR2产物反应；（iv）少量的IE1和146 kDa蛋白与成熟病毒粒子的核衣壳-被膜部分相关；（v）在裂解感染细胞的免疫荧光测定中，IR1-IR2基因产物在感染后1至2小时之间首次可检测到，呈离散的、点状的核内病灶；（vi）随着感染的进展，核内反应性增加并重新分布到与活跃病毒DNA合成位点相对应的大的、染色强烈的核区室中；（vii）在感染后约5小时首次观察到的纤维状以及更广泛的细胞质染色在整个感染过程中增加；（viii）虽然病毒DNA合成是核内逐步重新分布所必需的，但IR1-IR2蛋白的细胞质积累发生在早期感染事件之后。
