Winter N, D'Halluin J C
Laboratoire de Virologie Moléculaire, INSERM U. 233, Lille, France.
J Virol. 1991 Oct;65(10):5250-9. doi: 10.1128/JVI.65.10.5250-5259.1991.
The IVa2 gene is located between 16 and 11.3 map units on the left strand of the adenovirus type 5 (Ad5) genome. The coded RNA contains an intron of 277 nucleotides. To determine whether protein IVa2 is synthetized during productive infection and to obtain an immunological reagent to study its function, we prepared antibodies directed to 414 amino acids of protein IVa2 fused to the N-terminal domain of Staphylococcus aureus protein A. Western immunoblot analysis of viral proteins demonstrates that protein IVa2 is a minor component of mature viral particles and that it is also present in assembly intermediates and young virions. Thus, contrary to a previous report (H. Persson, B. Mathisen, L. Philipson, and U. Pettersson, Virology 93:198-208, 1979), protein IVa2 is not related to the 50-kDa polypeptide, a scaffolding protein present in assembly intermediates. The biosynthesis of protein IVa2 during productive infection was examined. Time course studies using immunofluorescence analysis with polyclonal antibodies targeted to protein IVa2 revealed that this protein is first synthesized at 12 h in a few cells exhibiting very striking fluorescence. Synthesis continues until at least 24 h postinfection. When hydroxyurea is added, protein IVa2 is not detected. In cells infected with mutant H5 ts125, blocked at the nonpermissive temperature (40 degrees C) in viral DNA replication, protein IVa2 is overexpressed. These results suggest that protein IVa2 synthesis requires cellular rather than viral DNA replication. RNase protection assay results indicate that hydroxyurea inhibits protein IVa2 synthesis at the transcriptional level. Thus, overexpression of protein IVa2 in H5 ts125-infected cells may be regulated at the translational level.
IVa2基因位于5型腺病毒（Ad5）基因组左链上16至11.3个图距单位之间。编码的RNA含有一个277个核苷酸的内含子。为了确定在增殖性感染期间是否合成了IVa2蛋白，并获得一种免疫试剂来研究其功能，我们制备了针对与金黄色葡萄球菌蛋白A的N端结构域融合的IVa2蛋白414个氨基酸的抗体。对病毒蛋白的Western免疫印迹分析表明，IVa2蛋白是成熟病毒颗粒的次要成分，并且也存在于组装中间体和年轻病毒粒子中。因此，与先前的报道（H. Persson、B. Mathisen、L. Philipson和U. Pettersson，《病毒学》93:198 - 208，1979）相反，IVa2蛋白与50 kDa多肽无关，50 kDa多肽是一种存在于组装中间体中的支架蛋白。研究了增殖性感染期间IVa2蛋白的生物合成。使用针对IVa2蛋白的多克隆抗体进行免疫荧光分析的时间进程研究表明，该蛋白在感染后12小时首先在少数细胞中合成，这些细胞呈现出非常明显的荧光。合成持续到至少感染后24小时。当加入羟基脲时，未检测到IVa2蛋白。在感染了突变体H5 ts125的细胞中，在非允许温度（40℃）下病毒DNA复制受阻，IVa2蛋白过度表达。这些结果表明，IVa2蛋白的合成需要细胞而非病毒DNA复制。核糖核酸酶保护分析结果表明，羟基脲在转录水平上抑制IVa2蛋白的合成。因此，H5 ts125感染细胞中IVa2蛋白的过度表达可能在翻译水平上受到调控。
