CORNEAL HSV-1: A NOVEL VIRULENCE/REACTIVATION GENE

描述：(摘自摘要)。复发性单纯疱疹病毒1型(HSV-1) 感染是病毒致盲的主要原因。后来，唯一的HSV-1基因 以前被认为在延迟期间处于活动状态，这对于提高效率很重要 HSV-1的重新激活。皮尤中心最近发现了一个新的单纯疱疹病毒1型基因(AL)。 它和LAT一样，在潜伏期也是转录活跃的，而且是 因此，有可能参与延迟-重新激活周期。艾尔是 与LAT反义，并与LAT启动子和LAT的5‘端重叠。这个 Al-RNA是多腺化的，含有一个开放阅读框架。大部分AL-ORF的缺失 和LAT的相应区域，产生了一个毒力增加的突变体 并降低自发再激活，提示(1)AL和/或LAT是 参与自发再激活，以及(2)AL-蛋白在 致命性。具体目标包括：1.对AL基因进行精细定位和特征分析， 核糖核酸和蛋白质。作图将使用RT-PCR、RPA引物延伸、RACE和 组织培养和兔单纯疱疹病毒1型RNA的克隆及序列分析 三叉神经节。将制备抗AL蛋白抗体并用于确认 AL蛋白的存在。AL表达的时间将确定。2. 确定AL的功能。将构建和分析Al-/LAT+突变体 对兔的毒力和自发复活的影响。自AL以来 和LAT重叠，敲除一个基因而不改变另一个基因的功能 是很复杂的。方法包括：(A)AL-ORF ATG中的单核苷酸变化 在不改变LAT RNA结构的情况下敲除AL蛋白；(B)最小的LAT 在不改变AL蛋白的情况下阻止LAT转录的启动子改变 氨基酸序列；和(C)来自替代位点的AL蛋白的表达 在AL-/LAT+和AL-/LAT-突变体中。这些概念的构建和分析 互补的AL-/LAT+和AL+/LAT-突变应该能让我们确定 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.