CORNEAL HSV-1: A NOVEL VIRULENCE/REACTIVATION GENE

摘要：（Abstract）。复发性单纯疱疹病毒1型（HSV-1） 感染是病毒性失明的主要原因。LAT，唯一的HSV-1基因 以前认为在延迟期间是活动的，对于高效的 HSV-1的再活化。PI最近发现了一种新的HSV-1基因（AL） 像LAT一样，在潜伏期也是转录活跃的， 因此是参与潜伏-再激活循环的候选者。AL是 与LAT反义并且与LAT启动子和LAT的5'末端重叠。的 AL-RNA是聚腺苷酸化的，含有ORF。删除大部分AL-ORF 和LAT的相应区域，产生了具有增加的毒力的突变体 和减少自发再激活，表明（1）AL和/或LAT是 参与自发再激活，和（2）AL-蛋白发挥作用， 毒性。具体目标包括：1. AL基因的精细定位和表征， RNA和蛋白质。作图将采用RT-PCR、RPA引物延伸、RACE和 组织培养和兔HSV-1 RNA的cDNA克隆和序列分析 三叉神经节将制备抗AL蛋白抗体并用于确认 一种AL蛋白的存在将确定AL表达的时间。2. 确定AL的功能。AL-/LAT+突变体将被构建和分析 在家兔中观察对毒力和自发再活化的影响。自从AL 和LAT重叠，敲除一个基因而不改变另一个基因的功能 相当复杂.方法包括：（a）AL-ORF ATG中的单核苷酸变化 敲除AL蛋白而不改变LAT RNA结构;（B）最小LAT 启动子改变以阻断LAT转录而不改变AL蛋白 氨基酸序列;和（c）从替代位点表达AL蛋白 在AL-/LAT+和AL-/LAT-突变体中。这些的构建和分析 互补的AL-/LAT+和AL+/LAT-突变体应该允许我们确定 AL是否参与自发再激活和/或毒力。

DESCRIPTION: (from abstract). Recurrent herpes simplex virus type 1 (HSV-1) infection is a major cause of viral induced blindness. LAT, the only HSV-1 gene previously thought active during latency, is important for efficient reactivation of HSV-1. The PI has recently discovered a novel HSV-1 gene (AL) that, like LAT, is also transcriptionally active during latency, and is therefore a candidate for involvement in the latency-reactivation cycle. AL is antisense to LAT and overlaps the LAT promoter and the 5' end of LAT. The AL-RNA is polyadenylated and contains an ORF. Deletion of most of the AL-ORF and the corresponding region of LAT, produced a mutant with increased virulence and decreased spontaneous reactivation, suggesting that (1) AL and/or LAT are involved in spontaneous reactivation, and (2) AL-protein plays a role in virulence. The specific aims include: 1. Fine map and characterize the AL gene, RNA, and protein. Mapping will employ RT-PCR, RPA primer extension, RACE and cDNA cloning and sequencing, of HSV-1 RNA from tissue culture and rabbit trigeminal ganglia. Anti-AL protein antibody will be made and used to confirm the presence of an AL-protein. The time of AL expression will be determined. 2. Determine the function of AL. AL-/LAT+ mutants will be constructed and analyzed in rabbits for the effect on virulence and spontaneous reactivation. Since AL and LAT overlap, knocking out one gene without altering function of the other is complex. Approaches include: (a) single nucleotide changes in the AL-ORF ATG that knockout AL-protein without altering LAT RNA structure; (b) minimal LAT promoter alterations to block LAT transcription without altering the AL-protein amino acid sequence; and (c) expression of AL-protein from an alternative site in AL-/LAT+ and AL-/LAT- mutants. The construction and analysis of these complementary AL-/LAT+ and AL+/LAT- mutants should allow us to determine whether AL is involved in spontaneous reactivation and/or virulence.