单纯疱疹病毒Ⅰ型新开放读码框UL57的鉴定  

Identification of a New Open Reading Frame UL57 in Herpes Simplex Virus Type 1

作　　者：佘明敏 蒋海芳 陈晓湘[1] SHE Mingmin;JIANG Haifang;CHEN Xiaoxiang(The Department of Medical Microbiology and Immunology,Guangzhou Medical University,Guangzhou 511436,China)

机构地区：[1]广州医科大学病原生物学与免疫学教研室,广州511436

出　　处：《病毒学报》2019年第1期51-57,共7页Chinese Journal of Virology

基　　金：广州市属高校科研项目(项目号:1201620304);题目:HSV-1潜伏感染期ATF3的表达及其在病毒潜伏/激活中的机制研究~~

摘　　要：单纯疱疹病毒1型(Herpes simplex virus type 1,HSV-1)潜伏感染期间LATs的活跃转录可能与其启动子与增强子两侧的CTCF结合序列有关。本研究对位于UL56下游与LAT启动子上游之间并与CTCF结合序列重叠存在的一个新开放读码框(本研究中命名为UL57)进行了鉴定。首先利用HSV-1(F)细菌人工染色体(HSV-BAC)系统构建重组病毒HSV-EGFP-UL57,将EGFP序列插入UL57 5’端;然后分别通过Northern Blot和Western Blot检测EGFP标记的UL57的转录和表达;同时构建敲除UL57的重组病毒HSV-ΔUL57,观察UL57对病毒增殖的影响。结果显示,重组病毒HSV-EGFP-UL57感染HEp-2细胞17h后,EGFP探针检测到两条转录产物,其中1.8kb转录产物与预测大小相符;使用放线菌酮(Cycloheximide,CHX)阻断病毒即刻早期蛋白/早期蛋白合成后,UL57转录受到明显抑制。重组病毒HSV-EGFP-UL57感染Vero细胞后,9h可见融合蛋白表达,24h表达明显;融合蛋白分子量与预测大小(58kD)一致。病毒生长曲线显示,重组病毒HSV-EGFP-UL57及HSV-ΔUL57在Vero细胞中的增殖水平与HSV-1(F)基本一致。本研究表明,在HSV-1基因组(GenBank:GU734771.1)UL56下游与LAT启动子上游之间存在一个新开放读码框UL57(116 921bp～117 799bp),UL57可以进行转录,且其转录受病毒即刻早期蛋白/早期蛋白调控;转录产物可以翻译出融合蛋白,但表达水平较低。删除UL57对病毒增殖无明显影响。In HSV-1 genome, the CTCF-binding sites flanking the LAT promoter and enhancer play potential roles in the active transcription of LAT gene during latency. This study indentified a new open reading frame UL57 overlapping the clustered CTCF-binding sites between UL56 and LAT promoter. HSV-BAC system was used to construct mutants. The mutant HSV-EGFP-UL57 was designed to carry EGFP coding sequence at the 5’ terminus of UL57. Northern Blot results revealed 2 transcripts of this mutant by EGFP probe 17 hrs postinfection of HEp-2 cells. The larger transcript coincided with the predicted size(1.8 kb).UL57 transcription was inhibited when the expression of α proteins and β proteins were blocked by cycloheximide pretreatment. Western blot results identified the expression of UL57 fusion protein by anti-EGFP mAb 9 hrs post infection of Vero cells and the expression increased until 24 hrs post-infection. The molecular weight of fusion protein also coincided with the predicted size(58 kD). Another UL57-deleted mutant HSV-ΔUL57 was constructed to determine the effect of UL57 on viral replication. The viral growth curve showed no significant difference between the mutants HSV-EGFP-UL57, HSV-ΔUL57 and HSV-1(F) in Vero cells. These results demonstrated a new open reading frame UL57(116,921 bp ~117,799 bp) located between UL56 and the LAT promoter in HSV-1 genome(GenBank: GU734771.1). Transcription of UL57 was regulated by the viral a proteins and β proteins. Expression of UL57 produced a 58 kD fusion protein. Deletion of UL57 showed no influence on viral replication.

关 键 词：单纯疱疹 单纯疱疹病毒1型(HSV-1) UL57 开放读码框 基因转录 基因表达 

分 类 号：R373.9[医药卫生—病原生物学] R511[医药卫生—基础医学]