Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
PLoS Pathog. 2019 Jun 17;15(6):e1007884. doi: 10.1371/journal.ppat.1007884. eCollection 2019 Jun.
In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.
与人类细胞相反，已知很少有 HSV-1 基因被剪接，尽管共享相同的前体 mRNA 处理机制。在这里，通过对感染 HSV-1 和缺失感染性细胞培养蛋白 27 (ICP27) 的 HSV-1 突变病毒的细胞中剪接接头的全局分析，我们鉴定了数百个新的替代剪接接头，这些接头映射到先前已知的 HSV-1 剪接基因和先前未知的剪接基因，其中大多数改变了病毒基因的编码潜力。基于 RNA-Seq 和 RT-PCR 的这些新的选择性剪接基因的定量和定性剪接效率分析表明，只有当 ICP27 缺失时，这些新的剪接位点的剪接才是有效的；而在野生型 HSV-1 感染的细胞中，这些新的剪接接头的剪接在很大程度上以基因/序列特异性方式被沉默，这表明 ICP27 不仅促进 ICP27 靶向转录物的积累，而且通过抑制选择性剪接确保正确的功能编码序列。此外，ICP27 通过抑制剪接和激活新鉴定的内含子中的近端多聚腺苷酸化信号 (PAS) 来切换主要病毒神经毒力因子 ICP34.5 的表达，揭示了病毒基因表达的新调节机制。因此，通过病毒 IE 蛋白 ICP27，HSV-1 共同利用剪接和多聚腺苷酸化机制在裂解感染过程中实现最佳病毒基因表达。另一方面，在潜伏感染期间当 ICP27 缺失时，HSV-1 可能利用宿主剪接机制来限制随机激活的抗原性病毒基因的表达，以实现免疫逃避。
