Herpes simplex virus 1 expressing GFP-tagged virion host shutoff (vhs) protein uncouples the activities of degradation and nuclear retention of the infected cell transcriptome
Herpes simplex virus 1 expressing GFP-tagged virion host shutoff (vhs) protein uncouples the activities of degradation and nuclear retention of the infected cell transcriptome
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单纯疱疹病毒 1 表达 GFP 标记的病毒颗粒宿主关闭 (vhs) 蛋白，解开受感染细胞转录组的降解和核保留活动
DOI：
10.1101/2022.01.04.475014
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Wise E
中科院分区：
文献类型：
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作者：
Wise E
病毒体宿主关闭蛋白（vhs）是由单纯疱疹病毒1型（HSV 1）编码的核糖核酸内切酶。Vhs导致感染细胞环境的许多变化，有利于晚期（L）病毒蛋白的翻译：细胞mRNA被降解，立即早期（IE）和早期（E）病毒转录物与polyA结合蛋白（PABPC 1）一起被隔离在细胞核中，dsRNA被降解以帮助抑制PKR依赖性应激反应。为了进一步了解vhs的细胞生物学，我们构建了一种表达vhs的病毒，该病毒在其C-末端用GFP标记。当首次表达时，vhs-GFP定位于单核簇，随后它与其结合伴侣VP 16共定位并相互作用，并被包装成病毒体。尽管vhs-GFP在单独表达时保持活性，但它在感染期间未能降解mRNA或重新定位PABPC 1，而病毒转录水平与vhs敲除病毒相似。PKR磷酸化在vhs-GFP感染的细胞中也增强，与降解dsRNA的失败一致。然而，mRNA FISH显示，在Wt而不是Δvhs感染中，IE和E，而不是L转录物在晚期保留在vhs-GFP感染的细胞核中。此外，一个典型的细胞转录本，这是通常高度敏感的vhs降解，也保留在细胞核中。这些结果表明，感染细胞转录组的vhs诱导的核保留依赖于vhs表达，但不依赖于其核糖核酸内切酶活性，使vhs的这两种功能解偶联。重要性与许多病毒一样，单纯疱疹病毒1（HSV 1）表达核糖核酸内切酶，即病毒体宿主关闭（vhs）蛋白，其调节受感染细胞的RNA环境并促进病毒蛋白翻译的经典级联。它通过引起一些mRNA分子的降解和其他mRNA分子的核保留来实现这一点。在这里，我们描述了一种病毒表达的VHS标记在其C-末端与绿色荧光蛋白（GFP），并表明，VHS-GFP融合蛋白保留了天然VHS的物理特性，但不诱导mRNA的降解。尽管如此，vhs-GFP保持了将感染细胞转录组捕获在细胞核中的能力，首次证明mRNA降解不是vhs对细胞核转录组影响的先决条件。因此，这种病毒解除了vhs的核滞留和降解活动，提供了对vhs在感染过程中的新认识。
Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). Vhs causes a number of changes to the infected cell environment that favour translation of late (L) virus proteins: cellular mRNAs are degraded, immediate-early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C-terminus with GFP. When first expressed, vhs-GFP localised to juxtanuclear clusters, and later it colocalised and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Δvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. Moreover, a representative cellular transcript which is ordinarily highly susceptible to vhs degradation, was also retained in the nucleus. These results reveal that the vhs-induced nuclear retention of the infected cell transcriptome is dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs.ImportanceLike many viruses, herpes simplex virus 1 (HSV1) expresses an endoribonuclease, the virion host shutoff (vhs) protein, which regulates the RNA environment of the infected cell and facilitates the classical cascade of virus protein translation. It does this by causing the degradation of some mRNA molecules and the nuclear retention of others. Here we describe a virus expressing vhs tagged at its C-terminus with green fluorescent protein (GFP) and show that the vhs-GFP fusion protein retains the physical properties of native vhs, but does not induce the degradation of mRNA. Nonetheless, vhs-GFP maintains the ability to trap the infected cell transcriptome in the nucleus, proving for the first time that mRNA degradation is not a prerequisite for vhs effects on the nuclear transcriptome. This virus has therefore uncoupled the nuclear retention and degradation activities of vhs, providing new understanding of vhs during infection.