Chen X, Schmidt M C, Goins W F, Glorioso J C
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
J Virol. 1995 Dec;69(12):7899-908. doi: 10.1128/JVI.69.12.7899-7908.1995.
Herpes simplex virus type 1 (HSV-1) establishes latency in human sensory ganglia, during which time the viral genome is transcriptionally silent with the exception of the latency-associated transcripts (LATs). The most abundant LAT is a 2-kb RNA whose biosynthesis is poorly characterized. The 2-kb LAT may be a primary transcript, or its synthesis may involve splicing and/or other forms of processing. Two potential RNA polymerase II promoters (LAP1 and LAP2) upstream of the 2-kb LAT 5' end have been identified. To investigate the role played by LAP1 and LAP2 in the synthesis of the 2-kb LAT under lytic and latent conditions, we analyzed HSV-1 mutants which contain deletions of one or both of these promoters. During lytic infection in cell culture, the cis elements critical for the normal accumulation of the 2-kb LAT were mapped to LAP2, while LAP1 sequences were largely dispensable. The 5' ends of the major 2-kb LATs produced by the wild-type and LAP deletion viruses were examined by primer extension analysis and were all found to be identical (+/- 2 bp). The accumulation of the 2-kb LAT during latent infections of murine trigeminal ganglia was examined by Northern (RNA) blot and by reverse transcription-PCR. In contrast to the results found in lytic infections, the critical cis elements needed for 2-kb LAT accumulation during latency were mapped to LAP1. Deletion of LAP1 resulted in a 500-fold reduction in 2-kb LAT accumulation, whereas deletion of LAP2 resulted in only a 2- to 3-fold reduction. Deletion of both LAP1 and LAP2 resulted in undetectable levels of the 2-kb LAT. Our results indicate that both LAP1 and LAP2 are critical for 2-kb LAT expression but under different conditions. LAP1 is essential for LAT expression during latency, while LAP2 is primarily responsible for LAT expression in lytic infections in cell culture. LAP1 and LAP2 may prove to be functionally independent promoter elements that control 2-kb LAT expression during different stages of HSV-1 infections.
单纯疱疹病毒1型（HSV-1）在人类感觉神经节中建立潜伏感染，在此期间，除了潜伏相关转录本（LATs）外，病毒基因组转录沉默。最丰富的LAT是一种2 kb的RNA，其生物合成特征尚不明确。2 kb的LAT可能是初级转录本，或者其合成可能涉及剪接和/或其他形式的加工。在2 kb LAT 5'端上游已鉴定出两个潜在的RNA聚合酶II启动子（LAP1和LAP2）。为了研究LAP1和LAP2在裂解和潜伏条件下2 kb LAT合成中所起的作用，我们分析了包含这些启动子之一或两者缺失的HSV-1突变体。在细胞培养中的裂解感染期间，对2 kb LAT正常积累至关重要的顺式元件定位于LAP2，而LAP1序列在很大程度上是可有可无的。通过引物延伸分析检测了野生型和LAP缺失病毒产生的主要2 kb LAT的5'端，发现它们均相同（±2 bp）。通过Northern（RNA）印迹和逆转录PCR检测了小鼠三叉神经节潜伏感染期间2 kb LAT的积累。与裂解感染中的结果相反，潜伏期间2 kb LAT积累所需的关键顺式元件定位于LAP1。LAP1的缺失导致2 kb LAT积累减少500倍，而LAP2的缺失仅导致2至3倍的减少。LAP1和LAP2两者的缺失导致无法检测到2 kb LAT的水平。我们的结果表明，LAP1和LAP2对于2 kb LAT的表达都很关键，但在不同条件下。LAP1对于潜伏期间的LAT表达至关重要，而LAP2主要负责细胞培养中裂解感染期间的LAT表达。LAP1和LAP2可能被证明是在HSV-1感染的不同阶段控制2 kb LAT表达的功能独立的启动子元件。
