单纯疱疹病毒Ⅰ型新开放读码框UL57的鉴定
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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.
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.
引文
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[6] Post L E, Conley A J, Mocarski E S, Roizman B. Cloning of reiterated and nonreiterated herpes simplex virus 1sequences as BamHI fragments[J]. Proc Natl Acad Sci USA, 1980, 77:4201-4205.
[7] Knipe D M, Howley P. Fields Virology[M], Lippincott Williams&Wilkins, 2013:1830.
[8] Amelio A L,McAnany P K,and Bloom D C. A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcript region binds CCCTC-binding factor and displays enhancer-blocking and silencing activities[J]. J Virol, 2006, 80:2358-2368.
[9] Chen Q, Lin L,Smith S,Huang J, Berger S L,and Zhou J. CTCF-dependent chromatin boundary element between the latency-associated transcript and ICPO promoters in the herpes simplex virus type 1 genome[J]. J Virol,2007, 81:5192-5201.
[10] Ertel M K, Cammarata A L, Hron R J, and Neumanna D M. CTCF occupation of the herpes simplex virus 1 genome is disrupted at early times postreactivation in a transcription-dependent manner[J]. J Virol, 2012, 86:12741-12759.
[2] Umbach J L,Kramer M F,Jurak I,Karnowski H W,Coen D M, Cullen B R. MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs[J]. Nature, 2008, 454:780-783.
[3] Ejercito P M, Kieff E D, Roizman B. Characterization of herpes simplex virus strains differing in their effects on social behaviour of infected cells[J]. J Gen Virol,1968, 2:357-364.
[4] Ye G J, Roizman B. The essential protein encoded by the UL31 gene of herpes simplex virus 1 depends for its stability on the presence of UL34 protein[J]. Proc Natl Acad Sci USA,2000, 97:11002-11007.
[5] Horsburgh B C, Hubinette M M, Tufaro F. Genetic manipulation of herpes simplex virus using bacterial artificial chromosomes[J]. Methods Enzymol,1999,306:337-352.
[6] Post L E, Conley A J, Mocarski E S, Roizman B. Cloning of reiterated and nonreiterated herpes simplex virus 1sequences as BamHI fragments[J]. Proc Natl Acad Sci USA, 1980, 77:4201-4205.
[7] Knipe D M, Howley P. Fields Virology[M], Lippincott Williams&Wilkins, 2013:1830.
[8] Amelio A L,McAnany P K,and Bloom D C. A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcript region binds CCCTC-binding factor and displays enhancer-blocking and silencing activities[J]. J Virol, 2006, 80:2358-2368.
[9] Chen Q, Lin L,Smith S,Huang J, Berger S L,and Zhou J. CTCF-dependent chromatin boundary element between the latency-associated transcript and ICPO promoters in the herpes simplex virus type 1 genome[J]. J Virol,2007, 81:5192-5201.
[10] Ertel M K, Cammarata A L, Hron R J, and Neumanna D M. CTCF occupation of the herpes simplex virus 1 genome is disrupted at early times postreactivation in a transcription-dependent manner[J]. J Virol, 2012, 86:12741-12759.