Baker Institute of Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
J Virol. 2022 Mar 9;96(5):e0219121. doi: 10.1128/jvi.02191-21. Epub 2022 Jan 12.
To determine the role of ICP22 in transcription, we performed precise nuclear run-on followed by deep sequencing (PRO-seq) and global nuclear run-on with sequencing (GRO-seq) in cells infected with a viral mutant lacking the entire ICP22-encoding α22 (US1/US1.5) gene and a virus derived from this mutant bearing a restored α22 gene. At 3 h postinfection (hpi), the lack of ICP22 reduced RNA polymerase (Pol) promoter proximal pausing (PPP) on the immediate early α4, α0, and α27 genes. Diminished PPP at these sites accompanied increased Pol processivity across the entire herpes simplex virus 1 (HSV-1) genome in GRO-seq assays, resulting in substantial increases in antisense and intergenic transcription. The diminished PPP on α gene promoters at 3 hpi was distinguishable from effects caused by treatment with a viral DNA polymerase inhibitor at this time. The ICP22 mutant had multiple defects at 6 hpi, including lower viral DNA replication, reduced Pol activity on viral genes, and increased Pol activity on cellular genes. The lack of ICP22 also increased PPP release from most cellular genes, while a minority of cellular genes exhibited decreased PPP release. Taken together, these data indicate that ICP22 acts to negatively regulate transcriptional elongation on viral genes in part to limit antisense and intergenic transcription on the highly compact viral genome. This regulatory function directly or indirectly helps to retain Pol activity on the viral genome later in infection. The longstanding observation that ICP22 reduces RNA polymerase II (Pol II) serine 2 phosphorylation, which initiates transcriptional elongation, is puzzling because this phosphorylation is essential for viral replication. The current study helps explain this apparent paradox because it demonstrates significant advantages in negatively regulating transcriptional elongation, including the reduction of antisense and intergenic transcription. Delays in elongation would be expected to facilitate the ordered assembly and functions of transcriptional initiation, elongation, and termination complexes. Such limiting functions are likely to be important in herpesvirus genomes that are otherwise highly transcriptionally active and compact, comprising mostly short, intronless genes near neighboring genes of opposite sense and containing numerous 3'-nested sets of genes that share transcriptional termination signals but differ at transcriptional start sites on the same template strand.
为了确定 ICP22 在转录中的作用，我们在感染缺失整个 ICP22 编码α22（US1/US1.5）基因的病毒突变体和源自该突变体的携带恢复的α22 基因的病毒的细胞中进行了精确的核转录物连续进动（PRO-seq）和全局核转录物连续进动测序（GRO-seq）。在感染后 3 小时（hpi），缺乏 ICP22 减少了即时早期α4、α0 和α27 基因上 RNA 聚合酶（Pol）启动子近端暂停（PPP）。在 GRO-seq 测定中，这些位点的 PPP 减少伴随着整个单纯疱疹病毒 1（HSV-1）基因组中 Pol 进程性的增加，导致反义转录物和基因间转录物的大量增加。在 3 hpi 时，α 基因启动子上 PPP 的减少与此时用病毒 DNA 聚合酶抑制剂处理引起的效应不同。在 6 hpi 时，ICP22 突变体有多个缺陷，包括病毒 DNA 复制减少、病毒基因上 Pol 活性降低以及细胞基因上 Pol 活性增加。缺乏 ICP22 还增加了大多数细胞基因上 PPP 的释放，而少数细胞基因上 PPP 的释放减少。综上所述，这些数据表明，ICP22 负调控病毒基因的转录延伸，部分原因是限制高度紧凑的病毒基因组上的反义转录物和基因间转录物。这种调节功能直接或间接有助于在感染后期保留病毒基因组上的 Pol 活性。长期以来的观察表明，ICP22 减少 RNA 聚合酶 II（Pol II）丝氨酸 2 磷酸化，这是转录延伸的起始，这令人费解，因为这种磷酸化对病毒复制至关重要。当前的研究有助于解释这种明显的悖论，因为它证明了在负调控转录延伸方面具有显著优势，包括减少反义转录物和基因间转录物。延伸的延迟预计将有利于转录起始、延伸和终止复合物的有序组装和功能。在其他方面高度转录活跃且紧凑的疱疹病毒基因组中，这种限制功能可能很重要，因为它们主要由短的、无内含子的基因组成，这些基因靠近相反极性的邻近基因，并且包含许多 3'-嵌套基因集，这些基因集共享转录终止信号，但在同一模板链上的转录起始位点不同。
