Gussow Anne M, Giordani Nicole V, Tran Robert K, Imai Yumi, Kwiatkowski Dacia L, Rall Glenn F, Margolis Todd P, Bloom David C
Department of Molecular Genetics and Microbiology, Box 100266, University of Florida College of Medicine, Gainesville, FL 32610-0266, USA.
J Virol. 2006 Oct;80(19):9414-23. doi: 10.1128/JVI.00530-06.
To study the regulation of herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) expression and processing in the absence of other cis and trans viral functions, a transgenic mouse containing the region encompassing the LAT promoter (LAP1) and the LAT 5' exon through the 2.0-kb intron was created. LAT expression was detectable by reverse transcriptase PCR (RT-PCR) in a number of tissues, including the dorsal root ganglia (DRG), trigeminal ganglia (TG), brain, skin, liver, and kidney. However, when the accumulation of the 2.0-kb LAT intron was analyzed at the cellular level by in situ hybridization, little or no detectable accumulation was observed in the brain, spinal cord, kidney, or foot, although the 2.0-kb LAT intron was detected at high levels (over 90% of neurons) in the DRG and TG. Northern blot analysis detected the stable 2.0-kb LAT intron only in the sensory ganglia. When relative amounts of the spliced and unspliced LAT within the brain, liver, kidney, spinal cord, TG, and DRG were analyzed by real-time RT-PCR, splicing of the 2.0-kb LAT intron was significantly more efficient in the sensory ganglia than in other tissues. Finally, infection of both transgenic mice and nontransgenic littermates with HSV-1 revealed no differences in lytic replication, establishment of latency, or reactivation, suggesting that expression of the LAT transgene in trans has no significant effect on those functions. Taken together, these data indicate that the regulation of expression and processing of LAT RNA within the mouse is highly cell-type specific and occurs in the absence of other viral cis- and trans-acting factors.
为了研究在不存在其他顺式和反式病毒功能的情况下单纯疱疹病毒1型(HSV-1)潜伏相关转录物(LAT)的表达和加工调控,构建了一种转基因小鼠,其包含涵盖LAT启动子(LAP1)和LAT 5'外显子直至2.0 kb内含子的区域。通过逆转录酶PCR(RT-PCR)在包括背根神经节(DRG)、三叉神经节(TG)、脑、皮肤、肝脏和肾脏在内的多种组织中可检测到LAT表达。然而,当通过原位杂交在细胞水平分析2.0 kb LAT内含子的积累时,在脑、脊髓、肾脏或足部几乎未观察到可检测到的积累,尽管在DRG和TG中检测到高水平(超过90%的神经元)的2.0 kb LAT内含子。Northern印迹分析仅在感觉神经节中检测到稳定的2.0 kb LAT内含子。当通过实时RT-PCR分析脑、肝脏、肾脏、脊髓、TG和DRG中剪接和未剪接LAT的相对量时,2.0 kb LAT内含子在感觉神经节中的剪接效率明显高于其他组织。最后,用HSV-1感染转基因小鼠和非转基因同窝小鼠,在裂解复制、潜伏建立或再激活方面未发现差异,这表明LAT转基因的反式表达对这些功能没有显著影响。综上所述,这些数据表明小鼠体内LAT RNA的表达和加工调控具有高度的细胞类型特异性,并且在不存在其他病毒顺式和反式作用因子的情况下发生。