Noiman S, Yaniv A, Tsach T, Miki T, Tronick S R, Gazit A
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.
Virology. 1991 Oct;184(2):521-30. doi: 10.1016/0042-6822(91)90422-8.
Nucleotide sequence analysis of a cDNA library of EIAV-infected canine cells established a complex pattern of gene expression, characterized by alternatively spliced polycistronic transcripts. The EIAV tat gene product was shown to be encoded by at least three species of mRNA which differed in their ability to trans-activate the EIAV LTR upon expression in canine cells. The most active cDNA was monocistronic, consisting of three exons. The most abundant cDNA in the library contained four exons and was identical to a polycistronic transcript previously described (Noiman et al., 1990b) which contains open frames for Tat, putative Rev, and truncated transmembrane proteins. Products consistent in size with those predicted for these last two proteins could be detected in in vitro translation experiments. The third Tat message, another four-exon form, also potentially encodes an amino terminally truncated transmembrane protein. In vitro mutagenesis experiments and analysis of subgenomic and partial cDNA clones confirmed and extended previous findings that S1 sequences are essential for trans-activation and that Tat translation initiates at a non-AUG codon either in the full-length Tat message or in the genomic S1 open reading frame. The Tat protein (8 kDa) was detected in cells transfected with a Tat cDNA construct and in canine cells persistently infected with EIAV. The Tat activity of polycistronic mRNAs was lower than that of the monocistronic form, suggesting that the expression of the EIAV trans-activator may be subject to several levels of posttranscriptional control.
对感染马传染性贫血病毒(EIAV)的犬类细胞的cDNA文库进行核苷酸序列分析,确立了一种复杂的基因表达模式,其特征为可变剪接的多顺反子转录本。结果表明,EIAV tat基因产物由至少三种mRNA编码,这些mRNA在犬类细胞中表达时,在反式激活EIAV长末端重复序列(LTR)的能力上存在差异。活性最高的cDNA是单顺反子的,由三个外显子组成。文库中最丰富的cDNA包含四个外显子,与先前描述的一个多顺反子转录本相同(Noiman等人,1990b),该转录本包含Tat、假定的Rev和截短的跨膜蛋白的开放阅读框。在体外翻译实验中可以检测到与这最后两种蛋白质预测大小一致的产物。第三种Tat信使RNA,也是另一种四外显子形式,也可能编码一个氨基末端截短的跨膜蛋白。体外诱变实验以及亚基因组和部分cDNA克隆的分析证实并扩展了先前的发现,即S1序列对于反式激活至关重要,并且Tat翻译在全长Tat信使RNA或基因组S1开放阅读框中的一个非AUG密码子处起始。在用Tat cDNA构建体转染的细胞以及持续感染EIAV的犬类细胞中检测到了Tat蛋白(8 kDa)。多顺反子mRNA的Tat活性低于单顺反子形式,这表明EIAV反式激活因子的表达可能受到转录后多个水平的调控。