Matsumura T, Smith R H, O'Callaghan D J
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130.
Virology. 1993 Apr;193(2):910-23. doi: 10.1006/viro.1993.1200.
DNA sequence and transcriptional analyses were performed on the region of the equine herpesvirus type 1 (EHV-1) genome (KyA strain) (map units 0.129 to 0.152) encoding open reading frames (ORFs) 15 and 16. ORF16 encodes a homolog of glycoprotein C of HSV-1 (herpes simplex virus type 1), while ORF15 corresponds in position to HSV-1 UL45 but exhibits no significant homology at the amino acid level. Sequence analyses revealed that the EHV-1 gC ORF of 468 amino acids and ORF15 of 227 amino acids mapped at nucleotides (nt) 716 to 2119 and 2397 to 3077, respectively (relative to the 5' end of the BamHI recognition site at map unit 0.152). ORF15 exhibited significant homology (69% identity) at the amino acid level to the EHV-4 gene located 3' of the EHV-4 gC homolog. Sequence analyses identified potential CAAT and TATA boxes for the EHV-1 gC ORF and a TATA box for ORF15. While no consensus polyadenylation signal was detected between ORFs 15 and 16, two polyadenylation signals were detected 3' of ORF15. Northern blot and S1 nuclease analyses were used to map and characterize the gC and ORF15 mRNAs, and metabolic inhibitors were used to identify the kinetic class of these two genes. The results revealed that gC is a gamma-1 gene which encodes a 2.8-kb mRNA, while ORF15 is a gamma-2 gene encoding a 0.9-kb mRNA which is 3' coterminal with the gC transcript. The gC and ORF15 mRNAs were shown by S1 nuclease analyses to initiate approximately 34 and 26 nucleotides downstream of their respective TATA boxes and to have a common termination site 18 to 20 nucleotides downstream of a consensus polyadenylation signal. Comparative sequence analysis revealed that the KyA strain gC protein differs in only three amino acid residues from the gC protein of the EHV-1 Ab4 and T431 strains, and one of the three amino acid differences occurred within a segment of six contiguous amino acids showing high degree of hydrophilicity in the gC molecule. Further comparative sequence analysis revealed that KyA strain genome has a major deletion in the region of ORF17 which lies 5' of gC in the Ab4 strain. This finding that 1038 base pairs (bp) of the 1203-bp ORF17 is deleted indicates that ORF17 is nonessential for EHV-1 replication in cell culture. To examine the regulation of the EHV-1 gC gene, transient transfection assays using CAT (chloramphenicol acetyltransferase) reporter gene constructs of gC were performed.(ABSTRACT TRUNCATED AT 400 WORDS)
对马疱疹病毒1型（EHV - 1）（KyA株）基因组（图谱单位0.129至0.152）中编码开放阅读框（ORF）15和16的区域进行了DNA序列和转录分析。ORF16编码单纯疱疹病毒1型（HSV - 1）糖蛋白C的同源物，而ORF15在位置上对应于HSV - 1 UL45，但在氨基酸水平上无显著同源性。序列分析表明，EHV - 1的gC ORF含468个氨基酸，ORF15含227个氨基酸，分别定位在核苷酸（nt）716至2119和2397至3077处（相对于图谱单位0.152处BamHI识别位点的5'端）。ORF15在氨基酸水平上与位于EHV - 4 gC同源物3'端的EHV - 4基因具有显著同源性（69%同一性）。序列分析确定了EHV - 1 gC ORF的潜在CAAT和TATA框以及ORF15的TATA框。虽然在ORF15和16之间未检测到共有多聚腺苷酸化信号，但在ORF15的3'端检测到两个多聚腺苷酸化信号。采用Northern印迹和S1核酸酶分析来定位和表征gC和ORF15 mRNA，并使用代谢抑制剂来确定这两个基因的动力学类别。结果表明，gC是一个γ - 1基因，编码一个2.8 kb的mRNA，而ORF15是一个γ - 2基因，编码一个0.9 kb的mRNA，其3'端与gC转录本共末端。S1核酸酶分析表明，gC和ORF15 mRNA分别在各自TATA框下游约34和26个核苷酸处起始，并在共有多聚腺苷酸化信号下游18至20个核苷酸处有一个共同的终止位点。比较序列分析表明，KyA株gC蛋白与EHV - 1 Ab4和T431株的gC蛋白仅在三个氨基酸残基上不同，这三个氨基酸差异之一发生在gC分子中六个连续氨基酸的一段区域内，该区域显示出高度亲水性。进一步的比较序列分析表明，KyA株基因组在ORF17区域有一个主要缺失，该区域在Ab4株中位于gC的5'端。这一发现表明，1203 bp的ORF17中有1038个碱基对（bp）被删除，这表明ORF17对于EHV - 1在细胞培养中的复制不是必需的。为了研究EHV - 1 gC基因的调控，使用gC的氯霉素乙酰转移酶（CAT）报告基因构建体进行了瞬时转染试验。（摘要截短于400字）
