Ling P, Kinchington P R, Sadeghi-Zadeh M, Ruyechan W T, Hay J
Department of Microbiology, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
J Virol. 1992 Jun;66(6):3690-8. doi: 10.1128/JVI.66.6.3690-3698.1992.
Three transcripts map to the varicella-zoster virus (VZV) open reading frame (ORF) 67, which encodes glycoprotein IV (gpIV). All of these transcripts are polyadenylated and are transcribed from left to right towards the genomic terminal short repeats. Previous Northern (RNA) blot analyses suggested that the most abundant of these transcripts (1.65 kb) might code for gpIV. We performed S1 nuclease protection and primer extension assays and determined that the 5' terminus of the 1.65-kb transcript maps 91 bp upstream from the gpIV initiation codon. An AT-rich region (ATAAA), -28 bp from the cap site, is a potential TATA box, and at -71 bp there is a consensus CCAAT box motif. The 3' end of the 1.65-kb transcript is 20 bp downstream of two overlapping polyadenylation signals, AATAAA and ATTAAA, and just downstream of the 3' terminus is a GU-rich sequence. These results are reminiscent of data from our analysis of the VZV gpV gene, confirming that VZV appears able to use unusual TATA box motifs. Many canonical TATA sequences are present upstream from these VZV transcriptional start sites but, apparently, are not used. We tested sequences upstream from the gpIV cap site for promoter activity in transient expression experiments by cloning a DNA fragment (+63 to -343 bp) into pCAT3M, which contains a chloramphenicol acetyltransferase reporter gene. This clone showed little constitutive promoter activity but was activated more than 200-fold by infection with VZV and 5-fold with herpes simplex virus. The two known VZV transactivating genes (those for ORF 4 and ORF 62) were tested for their abilities to activate expression from the gpIV promoter by using their cognate promoters. The ORF 4 gene was minimally active, whereas the ORF 62 gene gave twofold induction; both genes, acting together, gave fivefold induction. However, replacement of the IE62 promoter with the immediate-early cytomegalovirus promoter in the ORF 62 construct gave over 40-fold induction of chloramphenicol acetyltransferase activity under the gpIV promoter in the same assay.
三个转录本定位于水痘-带状疱疹病毒（VZV）开放阅读框（ORF）67，该开放阅读框编码糖蛋白IV（gpIV）。所有这些转录本都进行了多聚腺苷酸化，并且从左至右朝着基因组末端短重复序列转录。先前的Northern（RNA）印迹分析表明，这些转录本中最丰富的（1.65 kb）可能编码gpIV。我们进行了S1核酸酶保护和引物延伸试验，并确定1.65 kb转录本的5'末端位于gpIV起始密码子上游91 bp处。一个富含AT的区域（ATAAA），距帽位点-28 bp，是一个潜在的TATA框，在-71 bp处有一个共有CCAAT框基序。1.65 kb转录本的3'末端在两个重叠的多聚腺苷酸化信号AATAAA和ATTAAA下游20 bp处，3'末端下游紧接着是一个富含GU的序列。这些结果让人想起我们对VZV gpV基因分析的数据，证实VZV似乎能够使用不寻常的TATA框基序。许多典型的TATA序列存在于这些VZV转录起始位点的上游，但显然未被使用。我们通过将一个DNA片段（+63至-343 bp）克隆到含有氯霉素乙酰转移酶报告基因的pCAT3M中，在瞬时表达实验中测试了gpIV帽位点上游序列的启动子活性。该克隆显示出很少的组成型启动子活性，但通过VZV感染激活了200多倍，通过单纯疱疹病毒感染激活了5倍。使用它们的同源启动子测试了两个已知的VZV反式激活基因（ORF 4和ORF 62的基因）激活gpIV启动子表达的能力。ORF 4基因活性最低，而ORF 62基因诱导了两倍；两个基因共同作用诱导了五倍。然而，在相同的试验中，在ORF 62构建体中用立即早期巨细胞病毒启动子替换IE62启动子，在gpIV启动子下氯霉素乙酰转移酶活性诱导了40多倍。
