Kinchington P R, Vergnes J P, Defechereux P, Piette J, Turse S E
Department of Ophthalmology, University of Pittsburgh, Pennsylvania 15213.
J Virol. 1994 Jun;68(6):3570-81. doi: 10.1128/JVI.68.6.3570-3581.1994.
Four of the 68 varicella-zoster virus (VZV) unique open reading frames (ORFs), i.e., ORFs 4, 61, 62, and 63, encode proteins that influence viral transcription and are considered to be positional homologs of herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins. In order to identify the elements that regulate transcription of VZV ORFs 4 and 63, the encoded mRNAs were mapped in detail. For ORF 4, a major 1.8-kb and a minor 3.0-kb polyadenylated [poly(A)+] RNA were identified, whereas ORF 63-specific probes recognized 1.3- and 1.9-kb poly(A)+ RNAs. Probes specific for sequences adjacent to the ORFs and mapping of the RNA 3' ends indicated that the ORF 4 RNAs were 3' coterminal, whereas the RNAs for ORF 63 represented two different termination sites. S1 nuclease mapping and primer extension analyses indicated a single transcription initiation site for ORF 4 at 38 bp upstream of the ORF start codon. For ORF 63, multiple transcriptional start sites at 87 to 95, 151 to 153, and (tentatively) 238 to 243 bp upstream of the ORF start codon were identified. TATA box motifs at good positional locations were found upstream of all mapped transcription initiation sites. However, no sequences resembling the TAATGARAT motif, which confers IE regulation upon HSV-1 IE genes, were found. The finding of the absence of this motif was supported through analyses of the regulatory sequences of ORFs 4 and 63 in transient transfection assays alongside those of ORFs 61 and 62. Sequences representing the promoters for ORFs 4, 61, and 63 were all stimulated by VZV infection but failed to be stimulated by coexpression with the HSV-1 transactivator Vmw65. In contrast, the promoter for ORF 62, which contains TAATGARAT motifs, was activated by VZV infection and coexpression with Vmw65. These results extend the transcriptional knowledge for VZV and suggest that ORFs 4 and 63 contain regulatory signals different from those of the ORF 62 and HSV-1 IE genes.
68个水痘带状疱疹病毒（VZV）独特的开放阅读框（ORF）中的4个，即ORF 4、61、62和63，编码影响病毒转录的蛋白质，被认为是单纯疱疹病毒1型（HSV-1）立即早期（IE）蛋白的位置同源物。为了确定调节VZV ORF 4和63转录的元件，对编码的mRNA进行了详细定位。对于ORF 4，鉴定出一种主要的1.8 kb和一种次要的3.0 kb多聚腺苷酸化[poly(A)+]RNA，而ORF 63特异性探针识别出1.3 kb和1.9 kb的poly(A)+ RNA。针对与ORF相邻序列的特异性探针以及RNA 3'末端的定位表明，ORF 4的RNA在3'端共末端，而ORF 63的RNA代表两个不同的终止位点。S1核酸酶图谱分析和引物延伸分析表明，ORF 4在ORF起始密码子上游38 bp处有一个单一的转录起始位点。对于ORF 63，在ORF起始密码子上游87至95、151至153以及（暂定）238至243 bp处鉴定出多个转录起始位点。在所有定位的转录起始位点上游均发现了位置合适的TATA盒基序。然而，未发现类似于赋予HSV-1 IE基因IE调节作用的TAATGARAT基序的序列。通过在瞬时转染试验中分析ORF 4和63以及ORF 61和62的调控序列，支持了未发现该基序这一发现。代表ORF 4、61和63启动子的序列均受到VZV感染的刺激，但与HSV-1反式激活因子Vmw65共表达时未受到刺激。相反，含有TAATGARAT基序的ORF 62启动子被VZV感染以及与Vmw65共表达激活。这些结果扩展了对VZV转录的认识，并表明ORF 4和63包含与ORF 62和HSV-1 IE基因不同的调控信号。
